input
stringlengths 600
32.7k
| label
stringclasses 3
values |
|---|---|
" curcumin is herbal compound that has been shown to have anticancer effects in preclinical andclinical studies the anticancer effects of curcumin include inhibiting the carcinogenesis inhibiting angiogenesisand inhibiting tumour growth this study aims to determine the clinical effects of curcumin in different types ofcancers using systematic review approachmethods a systematic review methodology is adopted for undertaking detailed analysis of the effects of curcuminin cancer therapy the results presented in this paper is an outcome of extracting the findings of the studiesselected from the s published in international databases including sid magiran iranmedex irandoc googlescholar sciencedirect scopus pubmed and web of science isi these databases were thoroughly searched andthe relevant publications were selected based on the plausible keywords in accordance with the study aims asfollows prevalence curcumin clinical features cancerresults the results are derived based on several clinical studies on curcumin consumption with chemotherapydrugs highlighting that curcumin increases the effectiveness of chemotherapy and radiotherapy which results inimproving patients survival time and increasing the expression of antimetastatic proteins along with reducingtheir side effects the comprehensive systematic review presented in this paper confirms that curcumin reduces the sideeffects of chemotherapy or radiotherapy resulting in improving patients quality of life a number of studiesreported that curcumin has increased patient survival time and decreased tumor markers levelkeywords prevalence curcumin clinical feature cancer systematic review research over the past years has significantly increased our understanding of the molecular genetic basisof cancer it is now well known that cancer is caused bya set of molecular genetic changes that lead to loss ofgrowth control and cellular differentiation resulting in correspondence nsalarikumsacir masoudmohammadi1989yahoocom5department of biostatistics school of health kermanshah university ofmedical sciences kermanshah iran7department of nursing school of nursing and midwifery kermanshahuniversity of medical sciences kermanshah iranfull list of author information is available at the end of the uncontrollable cell growth that eventually leads to tumorformation more than half of all cancers occur in developingcountries including those located in southern americaand asia nearly threequarters of people of these countries are classified into low or middleincome categoriesthe cancer survival rates in developing countries aregenerally onethird ofthe patients in the developedcountries there are million new cases of cancereach year with million new cancer cases in the developed countries and million in developing countries in the next decades cancer will be one of the leading the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cmansouri bmc cancer page of causes ofillness worldwide and the number of newcases of various types of cancer is expected to rise to million by furthermore cancer is predicted to bethe leading cause of death by given that cancerstatistics are on the rise and their treatments are costlyit is very crucial to find effective and economically viablemethods for patients in low and middle income countries therefore this study is motivated by using effectiveand relatively cheap treatments for cancer therapy asystematic review of the clinical studies on curcumin useand its effectiveness in inhabiting and treating varioustypes of cancer is carried out to obtain comprehensiveinformation aboutthe curcumin effects on cancertherefore a structured review of all published sand other relevant documents on the use of curcuminfor cancer therapy creates a more complete picture ofcurcumin effects on cancer patients from different angles in the process of this review only evidence from thestudies with highest quality are selected to gather information and derive on curcumin effects andeffectiveness at various stages of cancer therapyamong the medical herbs flavonoids are a large subgroup of the family of natural polyphenolic compoundsthat are the result of secondary metabolism in plants in recent years research has shown that flavonoids havebeen very effective in the prevention and control of common diseases complex such as cancer cardiovasculardiseases alzheimers stroke diabetes osteoporosisand rheumatoid arthritis furthermore there are robustevidence of antiviral antiinflammatory and antiallergic effects of flavonoidsin the recent years the use and effectiveness of medicinal herbs in treatment of various diseases has been received enormous attention huge research efforts weremade on extraction and examination of the properties ofthe herbal compounds in the treatment of different typesof diseases eg cancers and providing detailed mechanisms of drug performance of these compounds amongst the wide range of the medical herbs curcuminis an effective ingredient of turmeric plant with the scientific name of longa curcuma chemical name ofdiferuloylmethane and the chemical formula of c21h20 o6 as illustrated in fig curcumin makes up between to of turmericcompounds and is considered as the main cause of yellowgolden colour of turmeric and it has also been identified as responsible for many ofthe properties ofturmeric [ ] however curcumin has low inherenttoxicity and various properties with great impact and applications on a wide range of pharmacological developmentsantiinflammatoryantimicrobial and anticancer drugs []antioxidantincludingcurcumin has been shown to have preventive and therapeutic effects on various types of cancers the findingsfrom several studies suggest that curcumin compound canprevent the formation and spread of tumors or reduce theirsize it was shown that curcumin can inhibit the formationof cancer and spread the cancerous cells by exerting antiangiogenic effects inducing apoptosis and interfering withthe cell proliferation cycle [ ] curcumin exerts itsanticancer effects through a variety of mechanisms curcumin inhibits and suppresses the proliferation of a widerange of cancer cells which exerts its effects by reducingthe modulation of antiapoptotic gene products activatingcaspase and upregulating cancersuppressive genes such asp53 [] recent studies confirm the preventive andtherapeutic effects of curcumin on various types of cancersindicating that it can prevent or reduce the formation orspread of tumors curcumin inhibits tumor invasion by reducing the modification of matrix metalloproteasesmmps the cell surface adhesive molecules nfκ ap1tnfα lox and cox2 chemokines growth factorsher2 and egfr inhibits nterminal activity and tyrosine kinase protein [ ] curcumin inhibits angiogenesisin some tumors by suppressing angiogeniccytokines such as il6 il23 and il1 [] due tothe strong relationship between inflammation and cancerthe antiinflammatory effects of curcumin would well resultin its antitumor effects it was reported that curcumin hasprevented the development of several types of cancer by reducing the production of mediators of the inflammatoryprocess such as cox2 lipoxygenase inos and relatedcytokines one of the possible mechanisms for suppressing tumor proliferation is the chemical inhibitor effectof curcumin as a result topical use of curcumin considerably inhibits inflammation due to tetradecanoylphorbol13fig the chemical expansion of curcumin by coreldraw graphics suite 0cmansouri bmc cancer page of acetate 12o tpa hyperplasia cell proliferation odcactivity production of active oxygen species oxidativedna changes and papillomavirus formation [] multiple human gastrointestinal cell interactions with curcumininhibit lipid peroxidation inhibit cox2 expression inhibitpge2 production and increase glutathionestransferaseenzyme levels [ ] the other mechanism of the anticancer effects of curcumin is due to its interference in thecell cycle and reduction in cdk expression cdks are actually serine threonine kinases that control cell cycle progression furthermore curcumin inhibits the stat3phosphorylation which is responsible for signalling carcinogenic pathways given that cancer statistics are on the rise and theirtreatments are quite costly it is very crucial to find someeffective methods and economically viable for low andmiddleincome patients therefore this paper providesupto date evidence and findings of clinical studies onthe effects of curcumin contributions in tumor cells survival and metastasis using a systematic review approachmethodssystematic review approach is adopted for undertakingthis study by extracting the findings of the relevant studies selected from the s published in national andinternational databases including sid magiran iranmedex irandoc google scholar sciencedirect scopuspubmed and web of science isi these databases werethoroughly searched and the relevant publication records were selected based on the plausible keywords inaccordance with the aim of this study as follows prevalence curcumin clinical features cancerthe selection of relevant studies for the systematic review and the output quality control process involvedseveral steps first all related s were collectedbased on the search keywords mentioned in the nextstep the specifications including the name of thejournal and authors were hidden and the full text of thes were made available to the reviewers each was investigated independently by two reviewersmm shr and if an was excluded in the studyfig the flowchart on the stages of including the studies in the systematic review prisma 0cmansouri bmc cancer page of detailed rationale were give accordingly in the case ofdisagreement between the two reviewers the wasjudged by a third reviewer in this paper all studies related to clinical investigations of curcumin use and impacts at varioustreatment weresystematically examined without any time constraintsand according to prisma guidelines fig stages of cancer selection criterias with the following characteristics were selectedfor metaanalysis original research s clinical trialstudies s that their full text and data are availableand studies that examined the clinical effects of curcumin in various types of cancers we prepared a list of s specifications based on prisma includingthe researchers name the title the year and placeof the study sample size and number of patients duration of study dosage of the drug and the result of theintervention table including review papers exclusion criteriastudiessystematic reviewmetaanalysis cohort casecontrol crosssectional descriptive and those which didnt present samples fromcancer patients and those which conducted with secondary data were excluded from the review duplicate publication and multiple publicationssamepopulation will be removed using citation managementsoftware endnote version x7 for windows thomsonreutersfrom thequality assessmentthe quality of the selected s was evaluated basedon criteria outlined by the consort checklist the lastconsort statement published in included items the consort statement has been shown to improve the scientific quality of rct reporting [ ]each was blindly assessed by two independentevaluators mm shr the result of each item wasassessed by yes point or no point and some itemswere assessed as not applicable due to the features ofstudies accordingly the maximum quality score of was considered and papers with a score of less than were considered to have low quality and thus they wereexcluded from the studycurcumin role in the prevention of cancersfree radicals and toxic products resulted from oxidativestress play an important role in the early stages of cancerformation therefore compounds that have antioxidanteffects can be helpful in preventing cancer formationcurcumin has the property of trapping free radicals andthus can play a crucial role in inhibiting the onset ofcancer several cellular and preclinical studies havereported that curcumin inhibits dna damage caused byoxidative factors such as ionizing radiation by inhibitingfree radicals and active oxygen species the nfkappab plays an important role in the formation of nitricoxide synthase and oxidative stress and as a resultcauses cancer curcumin suppresses the onset ofcancer by inhibiting nfkappab from formation [ ]curcumin was reported to be effective on liver enzymescytochrome p450 which has an imminent role in theoxidation and detoxification of toxins it also inhibits thephase i enzymes that is involved in the production oftoxic metabolites and carcinogens furthermore curcumin activates the phase ii enzymes which plays a crucialrole in detoxification of toxic metabolites curcumin prevent tumor formation and growth by inhibitingand activating these two enzymes phase i ii effect of curcumin on metastasis angiogenesis andinflammation in cancer cellsangiogenesis is the process of new blood vessel formation from preexisting vessels that is dependent on aprice equilibrium between antiangiogenic and angiogenicfactors however under pathological conditions for example tumor growth this tight regulation becomes lostwhich can result in tumor metastasis many gene products that are produced by different cells have a role inangiogenesis process hypoxia usually occurs in tumorsites in order to overcome to hypoxia tumor cells regulate and control the expression of genes related to angiogenesis cell cycle metastasis and drug resistance usinghypoxiainducible factor hif1 hif1 was first recognized as a transcription factor involved in hypoxiainduced erythropoietin expression this factor has beenpresented as a main transcription regulator for thesemolecules [ ] several studies have shown thathif1 activation of genes including vascular endothelialvegf angiopoietin1 ang1 andgrowth factorangiopoietin2 ang2 nfkb etc induced angiogenesis in the tumor cells furthermore the activation ofgenes such as insulinlike growth factor igf2 transforming growth factor a tgfa and mapk and pi3ksignalling pathway will also enable the survival proliferation and metastasis of tumor cells hif1 by activating genes involved in angiogenesis and also activatessignalling pathways associated with cell survival and proliferation plays an important role in the stability andgrowth of tumors as above mentioned hif1α is apotent activator of angiogenesis and curcumin inhibitsits expression ap1 is a transcription factor that is activated in response to hypoxia which is the principlephysiological stimulus that induces angiogenesis it isalso involved in the conversion of epithelial cells to mesenchymal cells which is the primary stage of metastasisand causes the expression of mmp and upa urokinase 0cmansouri bmc cancer page of table examines the characteristics extracted in the studiesauthors name yearcountry duration ofdosage of the drughejazi2013 belcaro2014 iranitalystudy years g per daynumber ofpatientsresultscurcumin reduces the severity of urinary symptoms mg with soy lecithin curcumin reduced side effectsbayetrobert2010 france months to mgryam2013 kanai2011 hemati2011 garcea2005 sharma2001 sharma2004 usajapaniranukukukcruzcorrea m2006 usa years months months months months months g g g to mg per day g per day g per day g per dayyu he2010 china days g per daydurgaprasad2005 india weeks g per daydhillon2008 usa g per dayide2010 japan months g per daygolombick2009 australia months g per daypolasa1992 hastak1997 indiaindia days g per day months g per daycheng2001 taiwan months g per dayrai2010 uk days g per daymarcia cruz correa2018 richard greil2018 newyorkusa month weeks mg orally twice adaydoses between and mg per minutelynne m howells2019 unitedkingdom days g per dayplasminogen activator genes that are involved in tumorangiogenesis and its invasion curcumin inhibits the expression of this transcription factor curcumin mayinhibit angiogenesis directly by regulating angiogenicgrowth factors growth factors as well as the genes including angiopoietin1 hif1 ho1 and transcription factors such as nfkappab fig [] it isknown that hypoxic stress and activation of betagrowthfactor tgf stimulate vegf expression by activatingap1 and the hypoxiainducible factors hif1 curcumin is an important inhibitor in ap1 activationand it has recently been shown that curcumin is a directinhibitor of hif1 transcription factor activity whichcauses the transcription of many genes associated withcurcumin lowers the concentration of the cea markertumorcurcumin reduces some skin complicationscurcumin increased patient survivalcurcumin reduces some skin problemscurcumin increased the effectiveness of the coloncurcumin reduced glutathione stransferase activitycurcumin reduces prostaglandin e2 productionreduces the number and size of polyps without anysignificant toxicitycurcumin has been shown to improve the overallhealth of patients with colorectal cancercurcumin reduced lipid peroxidation and increasedglutathione content in patientswelltolerated limited absorption and showedactivity in some patientsreduced the serum prostatespecific antigen contentin combination with isoflavonesdecreased para protein load and urinary n telopeptideof type i collagenreduced the urinary excretion of mutagens in smokersreduced the number of micronuclei in mucosal cellsand in circulating lymphocytesimproved the precancerous lesionsincreased vitamins c and e levels decrease dmalondialdehyde and 8hydroxy deoxy guanosine contents inthe serum and salivano difference in polyp size and number betweenplacebo and curcuminno variation in tumor size according to recist criteriacurcumin was a safe and tolerable adjunct to folfoxchemotherapy in patients with metastatic colorectal cancerangiogenesis in tumors [ ] it is also shown thatcurcumin will reduce the expression of membrane surface moleculesadhesionmolecule1 vascular cell adhesion molecule1 and eselectin which play a role in cellular adhesion fig intracellularincludingcurcumin affects a number of adhesive cellular molecules involved in tumor growth and metastasis processes curcumin caused reduction in the expression ofadhesive molecules inside the cells of icam1 vcamvcam or vascular cell adhesion molecule and mmpswhich play an important role in cellular adhesion andmetastasis furthermore curcumin results in increase ofthe expression of various antimetastatic 0cmansouri bmc cancer page of fig the effect of curcumin on angiogenesis and metastasis in cancer cells by coreldraw graphics suite including tissue inhibitor metalloproteinaseproteinstimp the nonmetastatic gene nm23 and ecadherin lack of ecadherin would increase the possibility of metastasis because ecadherin are essential tomaintain cellular adhesion angiogenesis is alsolinked with neoplasia angiogenesis means the formationof new blood vessels which is generally a major step intumor survival and growth curcumin inhibits cancer invarious ans [ ]easyto assess whetherthe antiinflammatory effects of curcumin have beenproven in many studies since oxidative stress leads tochronic inflammatory diseases antioxidant compoundscan be useful in the prevention and treatment of inflammatory disorders [ ] on the other handsince curcumin has a high antioxidant activity it willnot beantiinflammatory activity is also dependent on its antioxidant activity [ ] since many of the antioxidantsthat have been already identified do not have antiinflammatory properties it seems unlikely that the antiinflammatory effects of curcumin are due solely to itsantioxidant properties curcumin as a potent antiinflammatory factor expresses its own effects throughseveral mechanisms first curcumin inhibits the activation of the nfκ factor [ ]curcuminsthe lab based studies have revealed that curcuminneutralizes oxidative stress caused by tumor and restorestnfαnfkappabcurcuminactivityinhibitsproduction thus tcell apoptosis caused by tumor willbe minimised resultsin the initial screening of databases s wereidentified after deleting duplicate s studieswere obtained after deleting unrelated s studies were obtained17 s were also deleted due tolack of access to their fulltext or falling into the lowquality category at the end studies entered the finalphase and analysis as illustrated in fig the specifications and details of the studies considered in this systematic review are summarized in table according to the studies presented in table curcumin has reduced side effects including skin complications depending on the different doses of curcuminprescribed for the patients suffering from cancer theirsurvival rate was increased and their symptoms ofchemotherapy were reduced in studies examining theeffect of curcumin on colorectal cancer curcumin hasincreased efficacy in the large intestine reduced glutathione stransferase activity and reduced prostaglandin e2production curcumin also reduces the number and sizeof polyps without any significant toxicity curcumin inpancreatic cancer reduces lipids peroxidation and increases glutathione content in the patients with this typeof cancer in prostate cancer curcumin reduces theserum levels of prostatespecific antigen in combination 0cmansouri bmc cancer page of with isoflavones and also reduces the severity of urinarysymptoms according to the published studies the useof curcumin during radiation therapy for breast cancerpatients improved treatment outcomes for these patients such as preventing skin symptoms reducing painand suffering of patients improving their quality of lifeduring treatment and reducing delays or unwantedstops during the course of radiation therapy curcumincan regulate multiple signalling pathways and affect different moleculartargets low cost pharmacologicalsafety efficiency and multiple molecular targets makecurcumin a promising product for the prevention andtreatment of various human diseases table after collecting various s from reputable databases and deleting duplicate s and removing unrelated s to the main aim of this paper we finallyconsidered s for further investigation and analysis the main aim of this paper is to review the clinicalstudies about curcumin and its various purposeseffectson cancer the results reported from numerous clinicalstudies that have examined the effects of curcumin onthe patients who are suffering from cancer and undergoing radiotherapy and chemotherapy were very promising here we briefly describe some of these studieswhich are summarised in table garcea evaluated patients in the ukin this study each patient received to mg ofcurcumin per day at the same time that these patientsreceived curcumin they were treated with radiotherapyand chemotherapy the results of this study revealedthat curcumin increased the effectiveness of the treatment plan for colorectal cancer in the patients receivedwith curcumin importantin bayetrobert conducted a study consists offourteen patients with advanced breast cancer who werebeing treated with docetaxel chemotherapy and simultaneously received curcumin at different doses up to amaximum dose of g per day for days per each treatment cycle finally patients participated in this studywere able to complete this treatment plan nutropeniaand leukopenia were the mosttoxicitiescaused by docetaxel administration after days two patients refused to continue treatment because they received curcumintreatment continued by reducing the dosage to a maximum of g per day nine patients were screened fortumor response six weeks after completing the courseof treatment patients partially responded well butthree patients still suffered from the disease in thisstudy the ca tumor marker did not decrease butthe cea tumor marker decreased compared to the initial value prior to the treatment in patients the vegfvascular endothelial growth factor as a tomur markerwhichandcurcumin capsules howeverindicatestumrowth metastasismalignancy was reduced by compared to the baseline before treatment in the effect of curcumin on reducing the sideeffects of radiotherapy and chemotherapy in patientswith ovarian lung colon liver kidney and stomach cancers was investigated eighty patients received mg ofcurcumin simultaneously with radiotherapy the duration of this study was days the incidence of side effects such as nausea diarrhea constipation and weightloss decreased in patients who treated with both radiotherapy and curcumin in the patients who are simultaneously under radiotherapy and received curcumin theprevalence of side effects such as skin lesions mouthand throat ulcers swallowing problems nausea vomitingfatigue weakness and common medications required for treating side effects were statistically lowerthan the control group in a study conducted by hemati in iran patientsreceiving radiation therapy to the breast area due tobreast cancer from days before the start to the end ofradiotherapy mgcapsules containing curcuminwere taken orally times a day yu he evaluated patients in their study andstated that a dose of g of curcumin per day for days improved the general health of patients withcolorectal cancer through increasing the expression ofp53 molecules in tumor cells in a study conductedby cruzcorrea it was stated that a dose of g of curcumin per day will reduce the number and size of polypswithout any significant toxicity discussionin the recent years several studies have been conductedon the biological effects of curcumin in more than studies have been recently published curcumin hasshown to have various effects in cancer treatments curcumin has antioxidant antibacterial antifungal antiviralantiinflammatory antiproliferative proapoptotic effects etc curcumin has tremendous potential for treatment of neurodegenerative diseases arthritis diabetespsoriasis allergies intestinal inflammation kidney poisoning alzheimers depression aids multiple sclerosis cardiovascular disease and especially cancer [] the numerous and multifaceted effects of curcuminin determining the cellular targets and molecular mechanisms involved in curcumin pathways have attractedmuch attention from researchers curcumin is a multifaceted molecule and has many therapeutic effects themultifaceted effects of curcumin are due to its capacityto interact with different molecules and to regulate multiple molecular pathways and their targets one of the compelling properties of curcumin whichmakes it appropriate for therapeutic use is its low toxicity so that even its consumption up to a dose of g 0cmansouri bmc cancer page of per day does not cause any side effects consumption of curcumin in highdose prevents cancer cells frommultiplying although it does not damage healthy cells[ ]minimaltoxicity alongside with possessing manytherapeutic effects have led to the widespread use of natural plantderived compounds in the treatment of cancer the compounds found in nature target various cellular and molecular aspects of cancer cells the researchers have demonstrated that curcumin regulatessignalling pathways in cancer cells reduces the expression of proteins related to drug resistance and increasesthe performance of antitumor drugs at various levelscurcumin reverses drug resistance mechanisms and results in increasing the sensitivity of chemotherapyresistant cells in the research conducted by keyvanighamsari they demonstrated that curcumin is aneffective chemical in cancer treatment in laboratory studies which have been performed onthe cellular categories of colorectal cancer the derivedresults show that curcumin inhibits cell growth and alsostimulates apoptosis by interacting with several molecular targets furthermore curcumin has been used as partof dietary formulations to prevent colon cancer in vitroand in vivo these compounds have been shown to haveanticancer properties for colon cancer and its inflammation the results of this study show that curcuminwould be effective in preventing colorectal cancer in animals this property offers promising expectations inhumans due to the limited number of the human clinical studiesthe corresponding results are somehowcontradictory on the other hand there exist several unanswered questions about dosage bioavailability optimalsigns and potential toxicity which should be investigatedin future studies using sufficiently large samples inaddition curcumin can induce autophagy apoptosisand cell cycle arrest in order to reduce the survival andproliferation oflung cancer cells curcumin has thispromising capability to increase the effectiveness ofradiotherapy in the treatment of lung cancer by targetingdifferent signalling pathways such as epidermal growthfactor receptor and nf κb curcumincontaining nanocarriers increase bioavailability cell uptake and curcumin antitumor activity [ ]in a study conducted by cruzcorrea oral curcumin was prescribed to the patients with adenomatouspolyposis this research was implemented to determinethe safety and efficacy of curcumin in patients with adenomatous polyposis in this study mg of oral curcumin was administered twice per day over monthsto patients with adenomatous polyposis the resultsshowed that there was no significant difference betweenthose who received oral curcumin and those receivingplacebo in another study conducted by grell patients were subjected to receive doses between and mg per minute the main aim of their studywas to evaluate the safety of curcumin locally in patientswith advanced or metastatic cancer the results obtainedfrom their study showed that no change in tumor sizewas observed based on the recist criteria in howells evaluated patients with the age over and with metastatic colorectal cancer using the histological diagnosis quality oflife and neurotoxicity ofthese patients were assessed using questionnaires thederived results showed that curcumin is a safe and tolerable adjunct for folfox chemotherapy in patients withmetastatic colorectal cancer overall the results suggest that curcumin can be usedas an effective combination in inhibiting and controllingcancersimproving clinical symptoms and preventingtumor spread and metastasis this compound wouldaffect various molecular pathways and inhibits vasodilation cell proliferation and metastasiscurcumin is a natural product found in turmeric thathas a unique chemical structure with particular biological and medicinal properties through various cellular and molecular mechanisms curcumin inhibits thecarcinogenesis and their growth due to the fact that nospecific toxic effects of this natural product have beenreported its use has been considered as a drug supplement in therapeutic diets of cancer patients in a number of studies considered in this systematic review haveshown that taking curcumin would increase the expression of antimetastatic proteins in several other studiesit was reported that curcumin has also increased patientsurvival and decreased tumor marker concentrationabbreviationsmmps matrix metalloproteases vcam vascular cell adhesion moleculewho world health anization sid scientific information database prisma preferred reporting items for systematic reviewsacknowledgementsthe authors thank the faculty members of the faculty of nursing andmidwifery kermanshah university of medical sciencesauthorscontributionsshr and ns and km contribute | Colon_Cancer |
" leukotriene receptor antagonists ltras are broadly used for the management of allergic asthmaand have recently been indicated to inhibit carcinogenesis and cancer cell growth in colorectal cancer crcchemoprevention studies the occurrence of adenoma or crc itself is generally set as the trial endpoint althoughthe occurrence rate of crc is the most confident endpoint it is inappropriate for chemoprevention studies becausecrc incidence rate is low in the general population and needed for longterm monitoring aberrant crypt fociacf defined as lesions containing crypts that are larger in diameter and darker in methylene blue staining thannormal crypts are regarded to be a fine surrogate biomarker of crc therefore this prospective study was designedto explore the chemopreventive effect of ltra on colonic acf formation and the safety of the medicine in patientsscheduled for a poly resection as a pilot trial leading the crc chemoprevention trialmethods this study is a nonrandomized openlabel controlled trial in patients with colorectal acf and polypsscheduled for a polypectomy participants meet the inclusion criteria will be recruited and the number of acf inthe rectum will be counted at the baseline colonoscopic examination next the participants will be assigned to theltra or no treatment group participants in the ltra group will continue mg of oral montelukast for weeksand those in the no treatment group will be observed without the administration of any additional drugs at theend of the 8week ltra intervention period a polypectomy will be conducted to evaluate the changes in thenumber of acf and cell proliferation in the normal colorectal epithelium will be analyzeddiscussion this will be the first study to investigate the effect of ltras on colorectal acf formation in humanstrial registration this trial has been registered in the university hospital medical information network uminclinical trials registry as umin000029926 registered november keywords colorectal cancer chemoprevention leukotriene receptor antagonist aberrant crypt foci correspondence takuma_hyokohamacuacjpdepartment of gastroenterology and hepatology yokohama city universityschool of medicine fukuura kanazawa yokohama kanagawa japan the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0chigurashi bmc cancer page of cancer is a one of the major health issues and a leadingcause of death globally the incidence prevalence andmortality rates of colorectal cancer crc continue torise all over the world [ ] the majority of crc casesare derived from adenomatous polyps and their resection has been shown to reduce the risk of the futuredevelopment of advanced adenomas and crc [ ]thereby preventing crcrelated deaths however patients with polyps adenomatous polyps andor hyperplastic polyps represent a highrisk group for theoccurrence of metachronous colorectal polyps andorcrc then a paradigm is shifting from surveillancefor the early detection of advance adenomas or crcsand resection to novel tactics for prevention is requiredto reduce the mortality rate of this disease a number oflargescale epidemiologic andor clinicaltrials haveassessed the prophylactic agentsincluding vitamin dcalcium fiber and nonsteroidal antiinflammatory drugsnsaids such as aspirin and selective cyclooxygenase cox2 inhibitors in preventing against crc development we previously reported that the nsaidsulindac suppressed the development of sporadic colorectal adenomas to this point nsaids particularlycox2 inhibitors have been proven to offer the greatestpossibility for reducing crc risk either alone or in combination with other drugs while it has been reportedan elevated risk of serious cardiovascular events relatedwith the administration of cox2 inhibitors [ ] inview of these adverse cardiovascular effects and the lackof efficacy of other drugs that initially looked promisingthe development of novel agents that meet both safetyand efficacy in preventing crc is essentialleukotriene receptor antagonists ltras such asmontelukast and zafirlukast are commonly used for thetreatment of allergic asthma [ ] and tsai mj reported that ltras reduced cancer risk in a dosedependent manner in asthma patients it was alsoreported that the cancer incidence rate was significantlylower in ltra users than in nonltra users vs per patientsyear this means that apart fromits role in asthma ltra has also been associated withcarcinogenesis and tumormediated immunosuppression for example the overexpression of cysteinyl leukotriene receptor cyslt1r has been observed in crcand montelukast leads the apoptosis of crc cancer cells[ ] previous in vivo studies have shown the chemopreventive effect of ltras [ ] but the chemopreventive effects of ltra have not been studied in clinicalpractice therefore we designed this study to ivestigatethe chemopreventive effect of ltras in clinical practicein crc chemoprevention trials the occurrence of adenomas or crc itself is generally set as the trial endpointthough the occurrence of crc is the most confidentendpoint it is not recommended for chemopreventionstudies because crc incidence rate is low in the generalpopulation and needed for longterm monitoring furthermore there are ethical concerns about conductinglongterm trials to determine whether a test agent is effective or notaberrant crypt foci acf defined as lesions containing crypts that are larger in diameter and darker inmethylene blue staining than normal crypts [] areregarded to be a fine surrogate biomarker of crc our group has previously reported that acf is usefulbiomarker for crc [ ] and study endpoint for achemoprevention study [ ] the advantages of chemoprevention studies with the number of colorectalacf as the trial endpoint are that longterm observationis not needed to investigate the agent efficacy and thenumber of acf can be quantitatively estimated therefore we set the acf count as a good endpoint for thisstudy to the knowledge of us this is the first clinicalstudy investigating the use of ltras as chemopreventive agents against colorectal acf in humansmethodsstudy design and settingthis study was designed as a nonrandomized openlabel controlled trial to be conducted in patients withcolorectal acf it will be performed at the departmentof gastroenterology and hepatology yokohama cityuniversity ycu hospital japan the coordinating office will be at the ycu hospital and patient registrationwill be conducted at the ycu center for novel and exploratory clinical trials ynext and data collectionwill be done using electronic data caputureethical considerations and trial registrationthe trial protocol complies with the declaration ofhelsinki and the ethical guidelines for clinical research of the ministry of health labour and welfarejapan ethical approval of this trial was obtainedfrom the ethics committee of ycu hospital on may the study protocol and informed consent documents were approved by the ycu hospital ethics committee this trial has been approved in the clinical trialact in japan and registered in the japan registry of clinical trials jrct as jrcts031180094 and the universityhospital medical information network umin clinicaltrials registry as umin000029926 all study participants will submit a written study participation informedconsent formparticipation criteriawe will recruit the patients with colorectal acf and resectable polyps for this trial the inclusion criteria are asfollows patients with resectable polyps [adenoma 0chigurashi bmc cancer page of hyperplastic polyp and sessile serrated adenomapolypssap] patients with more than five rectal acfand submit written study participation informed consent formthe exclusion criteria are as follows patients withlesions suitable for early removal a history of ltrause within months before study participation a history of malignant disease within years before studyparticipation a history of heart renal liver failure orliver cirrhosis a history offamilial adenomatouspolyposis hereditary nonpolyposis crc or inflammatory bowel disease pregnancy or the possibility ofpregnancy prohibitions of montelukast allergiesto montelukast regular use of nsaids metforminand pioglitazone and participants considered as unsuitable for the study by the researchersinterventionall eligible participants will be assigned to the ltra orno treatment group because this is an openlabel trialpatients will be assigned to the no treatment group afterthe inclusion of patients in the ltra group participantsin the ltra group will receive mg of oral montelukast for weeks and those in the no treatment groupwill be observed without the administration of any additional drugs at the end of the 8week ltra treatmentperiod a polypectomy will be performed to evaluate thechanges in the number of acf and cell proliferation inthe normal colorectal epithelium will be analyzedoutcome measurementsthe primary endpoint will be the change in the numberof colorectal acf after weeks of treatment a magnifying colonoscope pcfq290azi hz290 olympus cotokyo japan will be used in all cases procedure preparation for the colonoscopy will begin day before theprocedure each participant will be informed to take alowresidue diet and mg oral sodium picosulfate onthe evening before the procedure on the day of the procedure each participant will be given ml polyethylene glycol peg if the stools are not clear enough anadditional ml peg will be given to ensure adequatebowel cleansing in most cases conscious sedation withmidazolam mg and pentazocine mg willbe use at the start of the colonoscopy subcutaneousscopolamine or glucagon will be administered for colonic movements reduction at the time of the first colonoscopy the endoscopists will insert into the cecumand the observe entire colorectum as the endoscope ispulled back one colonic mucosal sample will be collected the number of rectal acf will be counted usinga magnifying endoscope at the end of the 8week ltratreatment period the same endoscopists will performthe polypectomies and countthe number of acfendoscopists will record all procedures on a hard diskdrive and take photograph all acf the number of acfin each participant will first be counted by the endoscopists during the procedure to provide additional validation the number of acf will be recounted by threeblinded expert endoscopists aj ht and ak by observing the recorded hard disk drive cases that these evaluators deem colonoscopy to be inappropriate will beexcluded from the final analysisthe secondary outcomes will be as follows drugsafety adverse events aes will be graded according tothe national cancer institute common toxicity criteriafor adverse events ncictcae version all trialparticipants will be provided with a trial record for thedaily dose of the trial agent and aes participants whodevelop serious ae of grade or higher will be discontinued at that time in the study the effects ofltras on cell proliferation in the rectal mucosa onecolonic mucosal sample will be collected from the samestudy patient by performing a biopsy at the time of thebaseline colonoscopy and polypectomy a biopsy will beobtained from all participants cell proliferation will beevaluated by ki67 staining briefly we will randomly select six crypts and count the number of ki67positivecells per crypt in total cells will be counted at amagnification of à using a brightfield microscopethe results will be presented as the percentage of ki67positive cells all participants will receive laboratory testsand a physical examination at the point of the baselinecolonoscopy and polypectomydrug supplymontelukast capsules will be purchased from kyorincorporation ltd tokyo japan participants will be informed to take one tablet of the study drug every nightbefore bed medication adherence will be monitored bycounting the empty medication sheets returned by theparticipants at the time of their polypectomy the participants will also be interviewed and monitored to confirm thatthey have not used any prohibited drugsaspirin metformin andor other nsaids aes will bemonitored by the investigator and graded according tothe ncictcae version if serious aes or less than drug adherence are confirmed in a participant thisparticipant will be withdrawn from the trialsample size estimation and allocationwe previously reported the administration of mgmetformin per a day for weeks reduced the number ofacf in that trial the mean number of acf per patientdecreased significantly from ± at baseline to ± at weeks p although this study is exploratory research and the accurate chemopreventive efficacy of montelukastis unknown we assume that 0chigurashi bmc cancer page of montelukast may have an effect that is equivalent to of that observed for metformin on the reduction of acfnubmer therefore we estimate that the acf numberwill change by about to on average we determined that a sample size of individuals in theltra group was needed to detect a significant reduction in the number of acfs in the ltra group using apaired ttest with a twosided significance level of and power assuming some dropouts we propose to recruit participants in the ltra group to confirmthat the number of acf does not change during thestudy period we propose to recruit patients in the notreatment group after consecutively accumulating patients in the ltra group therefore we propose to recruit patientsstatistical analysisthe change in the number of acf which is the primaryendpoint will be compared before and after the 8weekstudy period between the ltra and no treatment groupsby the paired ttest drug safety will be assessed by thechisquare test and the remaining results will be compared by the ttest or mannwhitney u test between thetwo groups p will be defined as statistically significant all statistical analyses will be conducted using jmp® software sas institute inc cary nc usatrial steering committee and data monitoring committeethe trial steering committee and data monitoringthe ynext thecommittee will be located atmanagement team will perform central monitoring ofthe trial status and data collection every monthstudy flowa study flow is shown in fig discussionto the knowledge of us this is the first clinical trial proposed to investigate the efficacy of ltras on colorectalacf formation ltras are broadly used for the treatment of allergic asthma and rhinitis [ ] and ltrasare reported to decrease the risk of cancer in asthma patients in a dosedependent manner previous basicresearch has reported that cyslt1r is overexpressed incrc and that montelukast induces the apoptosis ofcrc cells [ ] cyslts have recently been focusedon as significant regulators of gut homeostasis with endogenous cyslt production mediating the proliferationand survival of gut mucosal cells recent evidence focuses on the effect of leukotrienec4 ltc4 in accelerating oxidative dna damage if notadequately repaired can contribute to increase mutationrates and genomic instability dna damage andgenomic instability are major drivers of carcinogenesis cyslts also acts as leukocyte chemoattractants inaddition cyslt1 mediates th17 cell migration the storage of which associates with the progression ofinflammationassociated cancers chronic inflammation is a risk factor for cancer initiation and progression as observed in patients with inflammatory bowelfig study flow chart 0chigurashi bmc cancer page of disease furthermore leukotriene d4 ltd4 antagonists suppress chronic inflammation in a rodent modelof acute enteritis and this may be effective in preventinginflammationassociated crc ltras are leukotriene pathway inhibitors and thus they may have potentialas chemotherapy andor chemoprevention agents to reduce the effect of leukotrienes previous in vivo studieshave elucidated the chemopreventive effect of leukotriene pathway inhibitors [ ] and showed the potentialuse of ltras for chemoprevention therefore we willconduct this trial to explore the chemopreventive effectof ltras in clinical settingthis trial may have some limitations as follow firstacf are believed to be a fine surrogate biomarker ofcrc though its biological significance in humans is stillcontroversial in crc chemoprevention studies typically set the occurrence of adenomas or the crc itselfas endpoint of the study though the occurrence of crcis the most appropriate endpoint it is inappropriate forchemoprevention studies because crc incidence rate islow in the general population and needed for longtermmonitoring our group has previously reported thatacf is useful biomarker for crc and conducted a chemoprevention study for colorectal acf [ ] therefore we designed this study using the number of acf asthe primary endpoint to investigate the chemopreventiveeffect of ltras second an intervention duration of weeks may be too short to reliably detect differences between two groups since our group reported in a previous trial that oral administration of metformin for weeks reduced the number of colorectal acf in humans an intervention period of weeks should beenough to assess the changes in the number of acf ifltras have a chemopreventive effectour group previously conducted a shortterm chemoprevention study of metformin for colorectal acfand reported the preventive effect of the agent on theformation of acf then we conducted a oneyearmetformin chemoprevention studycolorectalpolyps we propose to repeat the same steps as inour metformin study for the chemoprevention studyusing montelukastforif ltras were proved to have efficacy for crc prevention the impact would be significant therefore webelieve it will be very interesting to assess whetherltras inhibit the formation of human colorectal acfabbreviationscrc colorectal cancer nsaids nonsteroidal antiinflammatory drugs cox cyclooxygenase2 ltras leukotriene receptor antagonistscyslt1r cysteinyl leukotriene receptor acf aberrant crypt fociycu yokohama city university umin university hospital medicalinformation network ssap sessile serrated adenomapolyp ncictcae national cancer institute common toxicity criteria for adverseevents peg polyethylene glycol aes adverse events ltc4 leukotriene c4ltd4 leukotriene d4acknowledgmentsthe authors would like to thank the staff for their support in recruitingeligible patients and the patients who participated in this study and theirfamily we thank cathel kerr bsc phd and melissa crawford phd fromedanz group httpsenauthorservicesedanzgroupcom for editing a draftof this manuscriptauthors contributionsth ja and an conceived the study th and ja equally contributed to thismanuscript ja th ka nm ty tm af and ho will perform the baselinecolonoscopies and polypectomies ja th and ka will conduct the secondcount of acf using a hard disk drive recording to ensure validity tt nm tyaf and ho will recruit participants and followup with them at an outpatientclinic the analysis and interpretation of data will be conducted by ja thand ka all authors have read the final manuscript and approved its submission for publicationfundinga grant for this research from the kanagawa institute of industrial scienceand technology kistec was awarded to th we declare that the fundingbody has no role in the design data collection analysis interpretation andwriting of the studyavailability of data and materialsthe datasets used andor analyzed during the current study will be availablefrom the corresponding author on reasonable requestethics approval and consent to participateethical approval of this trial was obtained from the ethics committee of ycuhospital on may the study protocol and informed consentdocuments were approved by the ycu hospital ethics committee this trialhas been registered in the university hospital medical information networkumin clinical trials registry as umin000029926 all study participants willsubmit a written study participation informed consent formconsent for publicationnot applicablecompeting intereststhe authors declare that they have no conflicts of interestsreceived may accepted august referencestorre la bray f siegel rl global cancer statistics ca cancer jclin anderson wf umar a brawley ow colorectal carcinoma in black andwhite race cancer metastasis rev vogelstein b fearon er hamilton sr genetic alterations duringcolorectaltumor development n engl j med winawer sj zauber ag ho mn obrien mj gottlieb ls sternberg ss wayejd schapiro m bond jh panish jf prevention of colorectal cancer bycolonoscopic polypectomy the national polyp study workgroup n engl jmed citarda f tomaselli g capocaccia r barcherini s crespi m italianmulticentre study group efficacy in standard clinical practice ofcolonoscopic polypectomy in reducing colorectal cancer incidence gutzauber ag winawer sj obrien mj lansdorpvogelaar i van ballegooijenm hankey bf shi w bond jh schapiro m panish jf stewart et waye jdcolonoscopic polypectomy and longterm prevention of colorectalcancerdeaths n engl j med maisonneuve p botteri e lowenfels ab fiveyear risk of colorectalneoplasia after negative colonoscopy n engl j med author reply das d arber n jankowski ja chemoprevention of colorectal cancerdigestion epub oct review matsuhashi n nakajima a fukushima y yazaki y oka t effects of sulindacon sporadic colorectal adenomatous polyps gut 0chigurashi bmc cancer page of drazen jm cox2 inhibitorsa lesson in unexpected problems n engl jmed epub feb meyskens fl jr mclaren ce pelot d fujikawabrooks s carpenter pmhawk e kelloff g lawson mj kidao j mccracken j albers cg ahnen djturgeon dk goldschmid s lance p hagedorn ch gillen dl gerner ewdifluoromethylornithine plus sulindac for the prevention of sporadiccolorectal adenoma a randomized placebocontrolled doubleblind trialcancer prev res phila scott jp petersgolden m antileukotriene agents for the treatment of lungdisease am j respir crit care med shen z genomic instability and cancer an introduction j mol cell biolkim hs lee g the cysteinyl leukotriene receptor cysltr1 mediates th17cell migration j immunol bernstein cn blanchard jf kliewer e wajda a cancer risk in patients withinflammatory bowel disease a populationbased study cancer nishikawa m hikasa y hori k tanida n shimoyama t effect of leukotrienec4d4 antagonist on colonic damage induced by intracolonic administrationof trinitrobenzene sulfonic acid in rats j gastroenterol publishers notespringer nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations dahlen se dahlen b drazen jm asthma treatment guidelines meet the realworld n engl j med tsai mj wu ph sheu cc hsu yl chang wa hung jy corrigendumcysteinyl leukotriene receptor antagonists decrease cancer risk in asthmapatients sci rep wang d dubois rn eicosanoids and cancer nat rev cancer nielsen ck the leukotriene receptor cyslt1 and 5lipoxygenase areupregulated in colon cancer adv exp med biol burke l butler ct murphy a moran b gallagher wm osullivan j kennedybn evaluation of cysteinyl leukotriene signaling as a therapeutic target forcolorectal cancer front cell dev biol savari s liu m zhang y sime w sjolander a cyslt1r antagonists inhibittumor growth in a xenograft model of colon cancer plos one e73466 bellamkonda k satapathy sr douglas d chandrashekar n selvanesan bcliu m savari s jonsson g sjolander a montelukast a cyslt1 receptorantagonist reduces colon cancer stemness and tumor burden in a mousexenograft model of human colon cancer cancer lett roncucci l stamp d medline a cullen jb bruce wr identification andquantification of aberrant crypt foci and microadenomas in the humancolon hum pathol roncucci l medline a bruce wr classification of aberrant crypt foci andmicroadenomas in human colon cancer epidemiol biomark prev pretlow tp barrow bj ashton ws aberrant crypts putativepreneoplastic foci in human colonic mucosa cancer res pretlow tp oriordan ma pretlow tg stellato ta aberrant crypts in humancolonic mucosa putative preneoplastic lesions j cell biochem suppl takayama t katsuki s takahashi y ohi m nojiri s sakamaki s kato jkogawa k miyake h niitsu y aberrant crtpt foci of the colon as precursorsof adenoma and cancer n engl j med sakai e takahashi h kato s uchiyama t hosono k endo h maeda syoneda m taguri m nakajima a investigation of the prevalence andnumber of aberrant crypt foci associated with human colorectal neoplasmcancer epidemiol biomarkers prev epub jul ohkubo h takahashi h yamada e sakai e higurashi t uchiyama t hosonok endo h taguri m nakajima a natural history of human aberrant cryptfoci and correlation with risk factors for colorectal cancer oncol rep takahashi h yoneda m tomimoto a endo h fujisawa t iida h mawatarih nozaki y ikeda t akiyama t yoneda m inamori m abe y saito snakajima a nakagama h life stylerelated diseases of the digestive systemcolorectal cancer as a life stylerelated disease from carcinogenesis tomedical treatment j pharmacol sci hosono k endo h takahashi h sugiyama m sakai e uchiyama t suzuki kiida h sakamoto y yoneda k koide t tokoro c abe y inamori mnakagama h nakajima a metformin suppresses colorectal aberrant cryptfoci in a shourtterm clinical trial cancer prev res phila the world medical association wma declaration of helsinki ethicalprinciples for medical research involving human subjects the ministry of health labour and welfare ethics guidelines for clinicalresearch paruchuri s mezhybovska m juhas m sjolander a endogenous productionof leukotriene d4 mediates autocrine survival and proliferation via cyslt1receptor signaling in intestinal epithelial cells oncogene dvash e hartal m barak s meir o rubinstein m leukotriene c4 is themajor trigger of stressinduced oxidative dna damage nat commun 0c" | Colon_Cancer |
ovarian cancer is one of the leading causes of common lethal gynecologic malignancy cortez in worldwide there were an estimated cases and deaths from ovariancancer bray because of the lack of an early diagnosis method and the absence ofspeciï¬c early warning symptoms patients with ovarian cancer are usually diagnosed at an advancedstage and have a poor prognosis scarlett and conejogarcia frontiers in cell and developmental biology wwwfrontiersinaugust volume 0cyang tumor microenvironment in ovarian cancerbased on histological origin ovarian tumors can becategorized into epithelial germ cell sex cord or stromal tumorsjayson around of primary ovarian tumors areof epithelial origin colombo ledermann so we mainly focus on evidence of epithelial ovarian cancer inthis review the world health anization who classiï¬edepithelial ovarian cancer eoc into the following types serousmucinous endometrioid clear celltransitional cell mixedepithelial undiï¬erentiated and unclassiï¬ed ledermann according to architectural features eoc is also classiï¬edinto grades by the international federation of gynecologyand obstetrics figo system colombo in serouseoc figo grade is deï¬ned as lowgrade while figo grade and are combined as highgrade bodurka theclassiï¬cation with histosubtypes and grades are with prognosticsigniï¬cance ledermann plussurgerycytoreductivethe current standardized treatmentoptimalfor ovarian cancerisplatinumbasedchemotherapy with the carboplatinpaclitaxel regimen bolton however with the development of chemotherapyresistant and refractory diseases the sensitivity of chemotherapyhas decreased lim and ledger therefore the longtermsurvival rate for ovarian cancer has decreased and the recurrencerate has increased lim and ledger hennessy reported that despite beneï¬ting from ï¬rstline therapy of patients with advanced ovarian cancer stage iii or ivhave tumor relapse at a median of months from diagnosismoreover for patients with earlystage disease stage i or ii thelongterm survival rates years are in contrastpatients with advanced disease stage iii or iv had a longterm survival rate therefore there is an urgent need toï¬nd new targeted therapies to improve the treatment eï¬cacy ofovarian cancer in recent years the tumor microenvironmenttme has been reported to play a vital role in the tumorigenesisof ovarian cancer and is considered a possible therapeutic targetfor ovarian cancer here we review the interactions betweenthe tme and ovarian cancer and various therapies targeting thetumor environmenttme in ovarian cancerammatory cytokinesthe tme comprises the extracellular matrix ecm whichconsists of chemokinesintegrinsmatrix metalloproteinases mmps and other secreted moleculesand stromal cells including cancer cells cancer stem cellspericytes cancerassociated ï¬broblasts cafs endothelial cellsecs and immune cells figure hanahan and weinberg in this part we reviewed current ï¬ndings on the impactof some key components above on ovarian cancer progressioncancerassociated fibroblastsfibroblasts which diï¬erentiate from mesenchymalderived cellsare part ofthe tme ishii they producevarious mmps tissue inhibitor of metalloproteinases timpsand most ofthe proteins comprising the ecm such ascollagens ï¬bronectin and laminin kalluri and zeisberg erdogan and webb these ï¬broblasts in the tumor milieuare also called cafs additionally cafs can transdiï¬erentiatefrom other cellssuch as pericytes epithelial cells andecs via exposure to plateletderived growth factor pdgftumorderived transforming growth factor tgf vascularendothelial growth factor vegf basic ï¬broblast growth factorbfgf mmps and reactive oxygen species ros cai yu y denton cafs are known to promote tumor progression via variousmechanisms cafs can enhance tumor cell proliferationinvasion and migration sjoberg showed that cafshighly expressed cxcl14 which was an important factor inpromoting cancer growth cafs also express the ï¬broblastactivation protein α fap yangs study indicated that fapαenhanced the migration and invasion ability of ho8910pm cellsa highly metastatic ovarian cancer cell line additionally fapαincreased ho8910pm cell proliferationcafs promote immune inhibition and angiogenesis givel found that cafs increase the ltration offoxp3 regulatory t lymphocytes tregs at the tumor sitewhich exerts immune suppression eï¬ect in the tumor milieuadditionally in orimos research cafs have high expression ofstromal cellderived factor1 sdf1 released sdf1 promotesangiogenesis and tumor proliferation in a paracrine fashionorimo cafs also increase platinum resistance and acceleraterecurrence fauceglias study showed that cafs expressed thefap α by analyzing eoc tissues they found that theoverexpression of fap acted as a hallmark for platinumresistance additionally patients with fap stroma had ashortened recurrence compared to that of patients with fapstroma mhawechfauceglia several studies have indicated that cafs was a biomarker ofpoor prognosis in ovarian cancer yang mhawechfauceglia zhao givel in givels study of cafs in highgrade serous ovarian cancershgsoc the results showed that the expression of cxcl12and the ltration of cafs1 a subtype of cafs implied adismal prognosis givel despite that accumulatingstudies have demonstrated the protumor progression impact ofcafs it is worthy of notifying that there are diï¬erent subtypesof cafs with heterogenous function status recently hussain found caf subsets distinguished by the fapexpression level the faphigh caf subtype instead of thefaplow subtype was found to aggressively enhance tumorprogression and negatively uence patient outcomes whichshed light on therapeutic strategies involving caf modulation toconsider caf status in patient selectioncafscrucialcell population in thetumormicroenvironment cafs promote the proliferation invasionand migration of cancer cells and stimulate angiogenesis bycoordinating with other cells a deeper understanding of cafsis needed to better understand how cafs aï¬ect the tumormicroenvironmentareaendothelial cellsendothelial cells ecs are components of the tme liningfor transporting oxygen andthe vessels ecs are crucialnutrientscloselyassociated with angiogenesisand arefrontiers in cell and developmental biology wwwfrontiersinaugust volume 0cyang tumor microenvironment in ovarian cancerfigure cell components and functions in the tumor microenvironment tme cell components in the tme can be classiï¬ed into cancer cells immune cells andstromal cells these cells actively interact with each other by molecules they secrete [including cytokines chemokines damageassociated molecular patternsdamps etc] and receptors they express such as histocompatibility complex class mhc molecules programmed cell death protein pd1 etc forming anevolving microenvironment on the continuous spectrum from antitumor to protumor effect different cell components can locate at distinct positions and the samegroup of cells may also be repolarized depending on signals in the tme the progression or regression of a single tumor site depends on the overall effect of thecomplex cellular and molecular regulating network in the tmecarmeliet and jain hanahan and weinberg as we all know angiogenesisis a complicated processaccommodated by angiogenesis activators and inhibitorsangiogenesis activators include vegf fgf2 pdgf tgfαand tgf tnfα prostaglandin e2 and interleukin il the angiogenesis inhibitors contain angiopoietin angsthrombospondin tsp1 and endostatin moreoverthesignaltransducing network of endothelial cells is associated withvegf fgf and angs signals cross and claessonwelsh ahmed and bicknell vegf is a protein family consisting of vegfa vegfbvegfc vegfd vegfe and plgf placental growthfactorregulated by the ischaemiahypoxiainducedgenes hifs epidermal growth factor egf and pdgfsemenza there are three receptors for vegf vegfreceptors vegfr1 vegfr2 and vegfr3 vegfr1 andvegfr2 are mainly expressed on ecs and are receptorsfor vegfa hagberg additionally vegfr1is also a receptor for vegfb and plgf vegfr3 is areceptornrp1and nrp2 are coreceptorsthe vegf family withthe binding aï¬nity between vegfathe help of nrp1for vegfc and vegfd neuropilinsforitisferrara and adamisand plgf and vegfr2 increases similarly with the eï¬ect of nrp2 vegfc andvegfd have increased binding aï¬nity with vegfr3 itis well known that the vegf family is implicated in theadjustment of angiogenesis and lymphangiogenesis tammelaand alitalo apte among them vegfafor angiogenesis while vegfc and vegfdis crucialregulatetammela and alitalo apte lymphangiogenesisangs also a protein family consisting of ang1 ang2 ang3and ang4 through combined with the receptors of angs tiesthey perform diï¬erent functions in angiogenesis ang1 and can bind to tie2 and stimulate the tyrosine phosphorylationof tie2 on the contrary ang2 and can competitivecombined tie2 without stimulating tyrosine phosphorylationwhich stopping the signal transduction of angiogenesis sallinen lin sallinen however otherstudy indicated that with ang1 ang2 block the tie2 signalingwhile ang2 induce the tie2 signaling without ang1 yuan additionally ang1 promotes the maturation andstabilization of vessel while ang2 destabilize the stabilized vesseltse frontiers in cell and developmental biology wwwfrontiersinaugust volume 0cyang tumor microenvironment in ovarian cancervegf indicate poor clinical outcomes wimberger shen sopo wimberger foundthat by kaplanmeier analyses vegfr1 expression was closelyrelated to decreased overall survival os and progressionfreesurvival pfs wimberger sopo study discoveredthat vegfr1 vegfa and vegfd were highly expressedin omental metastases compared to expression in primaryovarian epithelial tumors interestingly patients with low vegfa expression were more likely to have a poor prognosis patientswith high vegfc expression were related to a short pfssopo the cd146 expression in the membrane of ecs promotesthe migration of ecs and angiogenesis cd146 is an endothelialbiomarker and the extracellular domain of cd146 directlyinteracts with vegfr2 yans study demonstrated that cd146can promote angiogenesis yan subsequentlyjiangs reportindicates cd146 promotes the migration ofecs and the formation of microvasculature by enhancingvegfr2 phosphorylation and downstream signaling aktp38mapksnfκb activation jiang interestinglyzhous research indicated that the gene and protein levels ofcd146 and vegfra were increased in patients with eoccompared to those of noncancer patients zhou enhanced angs expression increase relapse and decreasesurvival time sallinen lin sallinen observed that patients with ovarian carcinoma hadhigher ang2 levels compared to those of patients with benignovarian tumors furthermore by analyzing the kaplanmeiercurves they found that increased ang2 levels ngml werea biomarker for poor recurrencefree survival sallinen subsequently they discovered that the expression levelsof ang1 and ang2 were and respectively higher inwomen with ovarian cancer than in normal women increasedang2 expression was signiï¬cantly related to advanced stageand grade of cancer and relapse of ovarian cancer additionallyelevated ang2 expression is a predictor of poor os and short pfssallinen as an important part ofthe tumor microenvironmentecs are closely related to angiogenesis vegf and angs arecrucial regulators in angiogenesis both vegf and angs areassociated with poor clinical outcomes which provide possibletargets for treatmentcells myeloidderived suppressorimmune cellsimmune cells include macrophages dendritic cells dcsneutrophils mastcellsmdscs and lymphocytes hanahan and weinberg they play signiï¬cant roles both in tumor progression and tumorsuppression participating in evolving processes of tumorigenesismetastasis and angiogenesis by producing various signalingmolecules such as egf vegf mmp9 ifns ils etcmacrophagesmacrophages are an essential population of immune cells thatparticipate in ammation and tumourigenesis grivennikov among them macrophages residing in tumorsaretamstumorassociated macrophagestermedastams can derive from resident macrophages or ltratingmacrophages from bone marrow monocytes circulating in theblood ghosn depending on stimuli in the tme tams can present twomain phenotypes the antitumor m1 macrophages and protumor m2 macrophages sica grivennikov qian and pollard sica gupta when stimulated with interferongamma ifnγ bacteriallipopolysaccharide lps and granulocytemacrophagecolonystimulating factor gmcsf monocytes diï¬erentiated intom1 macrophages which can secrete il1 il12 tnfα andcxcl12 sica ramanathan and jagannathan m1 macrophages possess cytotoxicity tumor suppression andimmunestimulation functions galdiero when stimulated with cytokinesincluding il4 il10and il13 monocytes diï¬erentiated into m2 macrophagesleyvaillades van dalen in theimmune escape stage the tumor macroenvironment maintainsimmunosuppression due to the secretion of many growthfactors and cytokines such as il4 and il13 by cancer cellsthe immunosuppressive state accelerates monocytes to m2macrophages m2 macrophages in turn can promote tumrowth gordon roy and li in ovarian cancer tams are predominantly m2 macrophagesassociating with tumor invasion angiogenesis metastatic diseaseand early recurrence pollard reinartz yin they produce and secrete cytokines which haveimmunosuppressive eï¬ects such as il1r decoy il10 ccl17and ccl22 gordon via several mechanismstheysuppress adaptive immunity li noy and pollard firstly m2 macrophages can inhibit the proliferationof t cells and accelerate the immunosuppression of treg celltransport to tumors by producing the chemokine ccl22 li secondly m2 macrophages express the ligandreceptors for ctla4 and pd1 the activation of pd1 andctla4 inhibits cytotoxic function and regulates the cell cycleof t cells noy and pollard then m2 macrophagescan also inhibit the activation of t cells through the depletionof larginine which plays an essential role in t cell functiongaldiero arginase i arg1 a hallmark of m2macrophages is an larginine processing enzyme in the tmearg1 decomposes larginine into lornithine and urea thedepletion of larginine suppresses the reexpression of the cd3ζ chain which is internalized by antigen stimulation and t cellreceptor tcr signaling rodriguez aside from immune suppression m2 macrophages alsotake part in tissue repair ecm remodeling and angiogenesiswhich are processes involved in tumor progression as wellmantovani coï¬elt ruï¬ell finkernagel roy and li they canrestructure ecm and regulate ecm components by degradingecm via producing mmps serine proteases and cathepsinsruï¬ell which may facilitate tumor cell migrationinvasion and metastasis additionally they can secrete vegfa which is an angiogenic factor and produce proangiogeniccytokines such as il1 tnfα and upa urokinasetypeplasminogen activator roy and li in m2 macrophagesfrontiers in cell and developmental biology wwwfrontiersinaugust volume 0cyang tumor microenvironment in ovarian cancerthere is a subtype expressing tie2 a tyrosine kinase receptorthe tie2 macrophages are involved in angiogenesis fagianiand christofori these tie2 macrophages recruited byccl3 ccl5 ccl8 and tie2ligand ang are consideredthe most important reason for tumor vascularization becausethe deï¬ciency of this cell type restricts the angiogenic switchngambenjawong tams are plastic the simple dichotomy of m1m2macrophages cannot accountfor the complexity of tamheterogeneity ostuni transcriptome analysisuncovered a spectrum model of tams xue m1 andm2 macrophages can be regarded as two ends of a continuumwith wide ranges of functional states mantovani ostuni the subpopulations of tams in between thetwo ends can share features of both m1 and m2 types qian andpollard for example recently singhal foundthat tams could coexpress m1m2 markers together with tcell coinhibitory and costimulatory receptorsthe dynamic nature of the tme cellular environment gives abasis for the plasticity of tams macrophages present reversiblechanges in their functional phenotypes and distribution inresponse to diï¬erent microenvironmental stimuliincludingvarious cytokines and locally derived molecules which are tissueand tumorspeciï¬c stout okabe and medzhitov ostuni kim and bae therefore indiï¬erent histotypes of tumors zhang cassetta and diï¬erent microregions of the same tumormantovani kim and bae yang m there can be tams with diï¬erent extent of ltration andfunctional statusin ovarian cancer zhang found the densityand the cancerisletstroma ratio of tams vary amongserous mucinous endometrioid clear cell and undiï¬erentiatedhistotypes in the stroma and lumina of a small number of patientovarian tumor samples limited frequencies of inos expressingtams were found which were thought to be cytotoxic klimp in contrast in the malignant ascites of ovariancancer abundant tams can be found which are primarilym2like with protumor capacity gupta as thetumor grows stimuli in the tme alter resulting in changesin tam ltration and polarization in a tumor progressionleveldependent manner in ovarian cancer studies tam andm2 macrophage density were found to increase as cancer stageand ascites volume increased or as lymphatic invasion appearedzhang ke yuan gupta contrarily m1m2 ratio decreased as cancer stageincreased zhang despite expressing similar markers tams may not alwaysimplications in colon cancer studyhave similar functionaltams expressing pd1 presented weakened phagocytic potencyassociating with reduced survival gordon whilein early lung cancer study the pd1 tams did not aï¬ecttumorspeciï¬c t cell attack against tumor singhal this indicates the necessity of future studies focusing on tamfunctional status in the context of tumor tissue types and stagesof the disease this is especially true with ovarian cancer as it hasmany histotypes and high heterogeneityseveral studies revealed the prognostic value of tams inovarian cancer the m1m2 and m2tam ratio have beenreported to be positively associated with pfs and os while theoverall tam density in ovarian tumors indicated no prognosticsigniï¬cance lan zhang yuan m2 density in the ascites or tumor samples is associatedwith reduced relapsefree survival reinartz andpfs lan yuan however there is acontroversy in the relationship between m2 density alone andos lan reported a negative association betweenthe two factors while zhang found no signiï¬cantrelevance this may be due to the diï¬erence ofincludedtumor histotypesdendritic cellsdcs capture endogenous or exogenousdendritic cellsantigens process them and presentthe antigenic peptidesto other immune cells banchereau acting asa bridge connecting the innate and the adaptive immunesystem timmerman and levy riboldi there are two main subtypes of dcs the conventional dccdc thatis specialized in antigen presentation and theplasmacytoid dc pdc that produces ifn upon antigenstimulation aside from activating lymphocytes and other myeloidcells labidigaly vu manh cdcscomprise of myeloid cells in most tumors pdcs arerare in mouse tumors butfound in most human tumorstang the initiating event ofimmune responses againstdcs play key roles in antitumor immunity because it isindispensable for t celltumorscasey dcs are responsible for tumor antigenrecognition which isthe tumorspeciï¬c adaptive immune response in both the animal ovariancancer model and human hgsoc patients dcs can sensedamageassociated molecular patterns damps released fromdead cancer cells such as doublestranded dna dsdnafragments and calreticulin an endoplasmic reticulum erchaperone eliciting th1 polarized immunity ding kasikova after capturing antigens dcs present peptides processedfrom those antigens to cd4 and cd8 t cells via majorhistocompatibility complex class ii mhc ii and mhc imolecules respectively which subsequently initiate a series of tcell activity dudek sabado this processhas been reported to be signiï¬cant for tumor developmentprevention mackie galon besides t cell activation dcs are also crucialfor theaugmentation of cytotoxic t lymphocytes ctls population inthe tme it is reported that intratumoral cdcs are responsiblefor intratumoral ctl proliferation both in vivo and in vitrodiao and they are the only group of phagocytosingtumor myeloid cells that can stimulate cd8 t cell proliferationbroz as the major determinant of success intumor deterrent from the immune aspect budhu is to increase the functional tumorltrated ctl populationthe signiï¬cance of cdcs in the tme for antitumor responsesis selfevidentfrontiers in cell and developmental biology wwwfrontiersinaugust volume 0cyang tumor microenvironment in ovarian cancereï¬ective t cell activation by dcs require dc maturationa process happens after dc exposing to antigen characterizedby increased membrane expression of mhc and costimulatorymolecules cd80 cd86 cd40 bol bhatia alteration of chemokine receptors to favor dc lymph nodeln migration drakes and stiï¬ mature dcs producecytokines that favor th1 antitumor immunity truxova found in cohorts of hgsoc patients thattumorltrated mature lamp dcs is robustly associated with th1immune responses clinically favorable cytotoxic activities in thetme and favorable osthe process of dc maturation can be hampered by multiplefactors leaving dc immatured potentially developing into atolerogenic status and promote immune tolerance dhodapkar immature dcs express low levels of costimulatorymolecules and cytokines and mount limited immune activitiesdrakes and stiï¬ factors that lead to dc dysfunctionincluding the inhibition of dc maturation involve the immunemodulating molecules in the tme such as il6 il10 andvegftheactivation of oncogene stat3 in dcs the er stress responseand the abnormal intracellular lipid accumulation cubillosruiz tang devito thesefactors suppress dc functions by reducing the expression ofcostimulatory molecules and the secretion of proammatorycytokines inhibiting dc lymph node chemotaxis dampeningdc diï¬erentiation inducing tolerogenic phenotypes on dcs andshortening the lifespan of dcs tang tumorderived soluble mediators and exosomestolerogenic dcs suppresses antitumor immunity via severalmechanisms first they produce less proammatory cytokinesand induce immune suppressive cytokines labidigaly found in a cohort of ovarian cancer patients that intratumoural tolerogenic pdcs secreted fewer ifnα tnfα il6macrophage ammatory protein1 and ccl5 while inducedil10 from cd4 t cells promoting immune tolerance in thesepatients second they harbor enzymes negatively regulatingt eï¬ector cell functions such as nitric oxide synthase nosand indoleamine 23dioxygenase ido casey ido is an enzyme catalyzing tryptophan degradation capableofsuppressing tumorltrated lymphocyte proliferationpromoting treg diï¬erentiationinducing t cell anergy andpromoting tumor angiogenesis as well as metastasis munn tanizaki munn and mellor in eocpatients there was signiï¬cantly increased frequency of idodcs in tumor draining ln compared to the normal donorln besides in vitro study revealed ido signiï¬cantly inhibitedproliferation oftumorassociated lymphocytes derived fromeoc patients qian many factors are aï¬ecting the actual dc functions andbehaviors which are with high plasticity contributing to eitherprotumor or antitumor eï¬ect tumor expressing moleculesare associated with mature dc ltration recently macgregor 2019a found higher surface expression of b7h4 ab7 family molecule was correlated with higher mature dccd11chladrhigh ltration in eoc patient sampleswhich may be associated with increased expression of cxcl17a monocyte and dc chemoattractant in those tumors thisgroup have also found that tumourtostroma ratio tsr whichrepresents the percentage of malignant cell component relativeto the stroma in the tumor tissue have an impact on ltrateddc phenotype high tsr was associated with elevated pdl1expression on mature dcs cd11chladrhigh ltrating inovarian tumor tissue macgregor 2019bdc functions can be regulated by their interactions withthe proximal milieu so diï¬erent locations of dcs may resultin diï¬erentfunction labidigaly discoveredthat in ovarian cancer patients tumor pdcs produced lessproammatory cytokinesfrom ascites orperipheral bloodthan pdcscan vary by diï¬erentalso dc performancetumordevelopment stage in an ovarian cancer mouse model atthe early stage tumor growth was prevented by ltrating dcsand dc depletion at this stage accelerated tumor expansion atthe advanced stage however dcs become immunosuppressivein the tme abrogating enduring activity of antitumor t cellsand dc depletion at this stage signiï¬cantly delayed diseaseprogression scarlett similarly in a mouse model ofovarian cancer krempski also found progressivelygained immunosuppressive phenotype of ltrating dcs as thetumor progressed over time represented by gradually increasedpd1 expressionmore studies are favored in the future to reveal facts onhow dcs functions are regulated thereby providing clues fortherapeutic strategies in maintaining their antitumor potentialmyeloidderived suppressor cellsmyeloidderived suppressor cells mdscs are a heterogeneouspopulation of myeloid cells that coexpress the myeloid surfacemarkers gr1 and cd11b atretkhany and drutskaya mdscs consist of three phenotypes pmnmdsc mmdsc anda small group of cells that have myeloid colonyforming activityincluding myeloid progenitors and precursors gabrilovich pmnmdscs are similar to neutrophils in phenotypeand morphology and represent over of mdscs whilemmdscs are similar to monocyte gabrilovich studieshave conï¬rmed that mdscs promote tumor progression byvarious mechanisms first mdscs are implicated in immunesuppression ostrandrosenberg and fenselau despitetheir involvementin the inhibition of many cells in theimmune system mdscs mainly target t cells we summarizedthe mechanisms involved in immune suppression mdscsaccelerate lymphocyte nutrient depletion rodriguez srivastava both larginine and lcysteine areessential amino acids that are important for t cell activationand function mdscs produce arg1 and depletion of largininethrough an arg1dependent manner rodriguez mdscs also sequester lcysteine srivastava thereforethe amount of ζchain in the tcr complex isdownregulated and the proliferation of antigenactivated tcells is suppressed mdscs disturb lymphocyte traï¬ckingand viability hanson sakuishi galectin which is expressed in mdscs binds to tim3 onlymphocytes which induces the apoptosis of t cells sakuishi similarly mdscs express adam17 which canfrontiers in cell and developmental biology wwwfrontiersinaugust volume 0cyang tumor microenvironment in ovarian cancerdecrease the lselectin level on t cells and limit t cell recruitmentin lymph nodes hanson mdscs promotetreg cell activation and expansion gabrilovich mdscs stimulate cd4 t cells to translate into induced tregitreg cells and expand natural treg ntreg cells theseprocesses are associated with cd40cd40l interactions ifnγ il10 and tgf mdscs stimulate the generation ofoxidative stress oxidative stress is linked to ros and rnsreactive nitrogen species gabrilovich superoxide reactswith no and generates pnt peroxynitrite which nitratestcell receptors and limits the response of antigenmhccomplexes thus suppressing t cells directly pnt also nitratestcellspeciï¬c chemokines which decreases the combination ofantigenic peptides to mhc and limits the migration of t cellsmolon moreover mdscsfacilitate neovascularization throughdiï¬erent mechanisms hypoxia in tumors induces mdscs toproduce vegf fgf2 and mmp9 interestingly the activation ofstat3 in mdscs also stimulates neovascularization through il1 ccl2 and cxcl2 release bruno additionallythese factors stimulate invasion and metastasis by producingmmps ostrandrosenberg and fenselau mdsc is an important part of the tumor microenvironmentmdsc promote tumor progression by regulating immunesuppression and facilitating neovascularization moreover therelated to diï¬erentdiï¬erentfunctions and diï¬erentiation of mdsc neverthelessthemechanism is still not cleartumor microenvironmentisthe tmelymphocyteslymphocytes a major component ofinclude blymphocytes and t lymphocytes and mediate innate and adaptiveimmunity respectively sadelain b lymphocytesaccelerate tumor progression by producing protumorigeniccytokines and regulating the th1 th2 ratio quail and joyce t lymphocytes a major component of the tme are crucialfor adaptive immunity sadelain t cells developin the thymus before encountering the initial antigen t cellsare regarded as naïve tn cells after antigen encounter naïvetn cells are activated and start diï¬erentiation smithgarvin they proliferate rapidly and release ammatorycytotoxic granules and cytokines which activate the immuneresponse according to the cytokine environment t cellsdiï¬erentiate into various subsets wang m due to the exclusive expression of cd4 or cd8 markersmature t cells are categorized into cd3cd4 cd3cd8t cells and cd4 treg cells kishton cd3cd4t cells are also called helper t cells th cells and regulateimmune responses by releasing cytokines that promote orinhibit ammation joyce and pollard cd3cd4t cells can be divided into th1 and th2 cells among themth1 cells produce and release proammatory cytokines andassist cd3cd8 t cells in tumor rejection therefore th1cells are antitumorigenic however th2 cells release antiammatory cytokines and promote tumor progression joyceand pollard quail and joyce cd3cd8 t cellscalled cytotoxic t lymphocytes ctls produce ammatorycytokines and cell lytic molecules such as perforin and granzymewhich speciï¬cally recognize and destroy pathogeninfected ormalignant cells joyce and pollard zhang and bevan treg cells cd4cd25foxp3 also play a crucial rolein the immune response patsoukis duringdevelopmentin the thymus treg cells universally expressfoxp3 representing of cd4 t cells when respondingshow suppression tregto tcr and tgf treg cellscells protect hosts against autoimmune diseasesthroughinhibiting self and autoreactive cells de aquino additionally treg cells play a tumourigenic role mainly throughimmunosuppression monitoring lindau tregcells regulate the immune response through four mechanismsvignali facciabene secretingimmunosuppressive molecules treg cells suppress eï¬ector t cellfunctions by secreting cytokines such as il10 il35 and tgfadditionally il10 and tgf are reported as key mediatorsthat limit antitumor immunity and promote tumor progressionfacciabene interestingly these cytokines not onlyinhibit the function of eï¬ector cells but also promote dcpolarization to tolerogenic phenotypes additionally treg cellssecrete vegf which is also an immunosuppressive moleculevignali through vegf treg cells exert inhibitionand regulate the diï¬erentiation of dcs cytolysis treg cellsinduce the apoptosis of eï¬ector cells by secreting granzyme b andperforin vignali metabolic disruption severalmechanisms have been reported for the metabolic disruptionregulated by treg cells however it is still controversial tregcells deplete the local level of il2 which causes eï¬ector cellsto starve and results in the apoptosis of eï¬ector cells moreoverwith the expression of cd73 and cd39 treg cells catalyze atp toadenosine which inhibits the function of eï¬ector t cells deaglio vignali modulation of dc maturationand function ctla4 cytotoxic tlymphocyte antigen isexpressed on treg cells and cd80 and cd86 are expressedon dcs treg cells induce dcs through ctla4cd80cd86interactions which induces the release of ido indoleamine23dioxygenase ido expression depletes essential tryptophanand inhibits the function | Colon_Cancer |
introduction postoperative ileus poi a common complication after surgery severely affects postoperative recovery it is unclear whether pretreatment with transcutaneous electrical acupoint stimulation teas can improve recovery from poi this trial will evaluate the effects of pretreatment with teas on poimethods and analysis this will be a prospective randomised controlled trial american society of anesthesiologists asa physical status classification iiii level patients aged years and scheduled for laparoscopic colon surgery will be included in the study it is planned that subjects will be randomised to the teas and sham teas steas groups the groups will undergo two sessions of teassteas daily for days before surgery with a final teassteas treatment min before anaesthesia the primary endpoint of the study will be time to first defaecation secondary endpoints will include time to first flatus time to tolerance of oral diet gi2 composite outcome of time to first defaecation and time to tolerance of oral diet time to independent walking length of hospital stay postoperative pain visual analogue scale score on the first days after surgery analgesic requirements complications and plasma concentrations of interferon ifn ifnÎ interleukin6 il6 and il1 multiple linear regression will be used to identify independent predictors of outcome measuresethics and dissemination this study has been approved by the chinese registered clinical trial ethics review committee no chiecrct20170084 the results of the trial will be published in an international peer reviewed trial registration number this study has been registered with the chinese clinical trial registry no chictr inr17013184trial status the study was in the recruitment phase at the time of manuscript submissionintroductionpostoperative dysfunction ileus poi of gastrointestinal is a transient gi strengths and limitations of this study º this study aims to evaluate whether pretreatment with transcutaneous electrical acupoint stimulation teas can prevent postoperative ileus poi º teas is a safe non invasive and easily accepted adjunctive intervention º this study will provide deeper insights into the mechanism by which teas pretreatment reduces the inflammatory response º this is a single centre study which is a potential limitationpropulsion that often occurs after abdominal surgery and may also occur after surgery at other sites1 the main symptoms of poi include abdominal pain and distention nausea vomiting difficult defaecation and intolerance to solid food poi is usually temporary but if prolonged may lead to surgical incision dehiscence intestinal anastomotic fistula abdominal cavity infection intestinal ischaemia aspiration pneumonia and other serious complications2 a retrospective cohort study involving nearly hospitals in the usa showed that poi is a key reason for prolonged hospitalisation and increased medical costs for patients undergoing abdominal surgery1 the usa spends more than billion treating poi every year5 at present the most common methods used to treat poi include rational perioperative use of narcotic drugs and opioids eating as soon as possible after surgery avoidance of nasogastric tubes after the operation early ambulation postoperative epidural analgesia restriction of fluid intake the use wang a0j et a0al bmj open 202010e030694 101136bmjopen2019030694 0copen access of minimally invasive surgery such as laparoscopic drug therapy and the use of chewing gum despite the numerous treatment strategies poi remains a difficult clinical challenge that compromises the rapid recovery of postoperative patients it is therefore necessary to find more effective convenient and economical treatment methods6the main mechanism underlying poi may be activation of macrophages in the external muscular layer during the surgical procedure11 intestinal manipulation during surgery can activate macrophages in the outer muscle layer of the small intestine leading to release of inflammatory factors interleukin6 il6 il1 and the chemokine mip1α together with increased expression of the adhesion molecule icam1 on endothelial cells and induction of neutrophils and monocytes in the circulation into the small intestine muscle layer these cells and activated macrophages can release a large amount of inducible nitric oxide synthase and prostaglandin which inhibit the movement and contraction of the gi tract12 transport of these inflammatory mediators in the bloodstream causes activation of macrophages in the distal gi tract leading to poi over the entire intestinal tract14 it has been confirmed by a large number of animal experiments that reducing the inflammatory response is an effective way to treat poi15there is a long history in traditional chinese medicine tcm of using acupuncture to treat functional gi diseases and in recent years there has been significant global interest in the beneficial effects of acupuncture on poi the positive effect of electroacupuncture ea on poi has been clearly demonstrated ng used ea to treat poi in patients undergoing laparoscopic colon surgery18 defaecation time and length of hospital stay were significantly shortened in patients who received ea compared with those who did not receive the treatment in patients undergoing hepatic resection you found a significant reduction in the incidence of poi in patients treated with a combination of acupuncture and chinese herbal medicine the length of hospitalisation was also significantly shortened in the treated group ± days vs ± days p001419in the previous studies we proved that pretreatment with acupuncture could reduce excessive activation of the innate immune system and inhibit the inflammatory response this effect may be achieved by activation of the vagal nervous system20 other studies have shown that transcutaneous electrical acupoint stimulation teas and ea have similar effects in the treatment of pain and alleviating the inflammatory response22 tcm holds that the best treatment for disease is prevention based on all of the above studies we hypothesise that the use of teas as a preoperative treatment may reduce the incidence of poi there have so far not been any studies that address this questionwe have therefore designed a randomised controlled trial to investigate whether pretreatment with teas can reduce the incidence of poi in patients undergoing laparoscopic colon resection the study is also designed to verify that the anti inflammatory effect is associated with the immunomodulatory function of teasmethods and analysisstudy objectivethe primary objective is to assess the effect of teas on clinical recovery of bowel function after laparoscopic colon surgery the secondary objective is to verify that suppression of overactivation of the innate immune system and reduction of the inflammatory response are the mechanisms underlying the ability of pretreatment of percutaneous acupuncture to prevent poistudy locationa prospective single centre double blinded randomised controlled trial will be conducted at shuguang hospital which is affiliated to the shanghai university of traditional chinese medicine chinastudy populationparticipants will be recruited according to the inclusion and exclusion criteriainclusion criteria male and female patients aged years patients undergoing elective laparoscopic colonic surgery and upper rectal resection such as left collect right colectomy and anterior resection of the upper part of the rectum and lower part of the sigmoid body mass index kgm2 asa classification iiii patients provide signed informed consent the consent form can be viewed in online supplementary appendix exclusion criteria middle and lower rectal resection totalproctocolectomy or the need for complex endoscopic surgery need for abdominal wall fistula gi fistula fistula surgery or stoma creation history of abdominalpelvic operations or complications patients receiving epidural anaesthesia or epidural analgesia patients with skin infections surgical incision or scar at the point of application of acupuncture patients have a history of limb surgery spinal surgery or nerve injury patients who participated in other clinical trials or received other acupuncture therapy in the previous weeks patients with cardiac pacemakers patients have one of the following conditions before surgery chronic pain drug addiction or alcohol dependence patients with preoperative combination of severe central nervous system disease and severe mental illnesswang a0j et a0al bmj open 202010e030694 101136bmjopen2019030694 0cendpointsprimary endpointfirst defaecation time h that is time to first anal defaecation after laparoscopic surgerysecondary endpointstime to first flatus h time to tolerance of solid oral diet h gi2 composite outcome of time to first defaecation and time to tolerance of oral diet time to walk independently h length of hospital stay defined as the number of days from operation to discharge d criteria for hospital discharge include stability of vital signs with no fever achievement of flatus or defaecation ability to tolerate solid food without vomiting control of postoperative pain absence of other postoperative complications and ability to function at home independently or with home care provided pain will be assessed using the visual analogue scale vas on postoperative days and scale of to where represents complete absence of pain and represents the worst pain intensity postoperative requirements for analgesia will also be assessed inflammatory mediators interferon ifn ifnÎ interleukin6 il6 and il1 in blood will be measured before teassteas intervention and on days and after the operation postoperative complications will be recorded using the clavien dindo classification for complication assessment24 the follow up period will be at least monthswe add gi2 as a secondary outcome to the original protocol after recruitment of the study had already begun gi2 is a time indicator which will be calculated from two existing outcomes time to first defaecation and time to tolerance of oral diet there will be no harm to subjects no additional cost and no more workopen accessrandomisation and blindingpatients will be randomised to receive either teas or steas by stratified randomisation according to sex in a ratio figure using a computer generated random sequence a sealed envelope will be opened to determine to which group the patient has been assigned the acupuncturist will be aware of the treatment group patients as well as the outcome investigator nurse anaesthetist will be blinded to the treatment allocationcurrent sample size justificationaccording to wang jian and song jiangangs preliminary study of teas pretreatment for prevention of poi in patients undergoing laparoscopic colon surgery in shuguang hospital the mean time to first defaecation following laparoscopic colon surgery was ± hours m±sd working on the assumption that a clinically meaningful difference in mean time to first defaecation between the teas and steas groups is day or hours patients would be needed in each group to reach a power of and a type i error rate if the dropout rate is a total sample size of patients for the two groups is needed for this studystatistical analysisdata for continuous variables ie first defaecation time first passage of flatus time to tolerance of oral diet time to walking independently length of hospital stay will be reported using the mean and sd m±sd for normally distributed data or median range for skewed data data for categorical variables will be expressed as a number percentage intergroup differences will be assessed using the students t test or mann whitney u test intergroup differences in inflammatory mediators at time points of pre teassteas treatment and on figure flowchart of the study protocolwang a0j et a0al bmj open 202010e030694 101136bmjopen2019030694 0copen access figure acupoints selected in this trial a hegu il4 and neiguan p6 b zusanli st36 and shangjuxu st37 c hans acupoint nerve stimulatorpostoperative days and were assessed by two way repeated measures analysis of variance with bonferroni post hoc test the significance level will be set at all data will be analysed using spss v170 software or other appropriate statistical software packagespretreatmentpatients randomised to the teas and steas groups will undergo two treatment sessions daily for three consecutive days before surgery the patients will then be treated for a final time min before anaesthesiafor patients in the teas group the zusanli st36 shangjuxu st37 hegu li4 and neiguan p6 acupoints will be identified before electrical stimulation with surface electrodes figure selection of these acupoints is based on a consensus between the acupuncturists carrying out the study the acupuncturist will stimulate these acupoints using a hans acupoint nerve stimulator hans200a nanjing jisheng medical technology nanjing china at a frequency of hz the intensity will be adjusted for each individual to maintain a slight twitching of the regional muscle and achieve de qi sensations such as soreness numbness distention and heaviness the steas group will receive a strong but comfortable current for s and the current will then gradually vanish over the next s25 the participants of both groups will be told that they are receiving current stimulation each session of acupoints treatment will last for min during the application of teas patients will be required not to change the current settings themselves a prompt beep at the end of teas will indicate the end of treatmentall surgery will be carried out under general anaesthesia using standardised anaesthetic procedures patients will be fasted for hours before surgery right upper extremity venous access will be established before the patients entering the operating theatre ringers lactate solution mlkg will be administered by intravenous infusion for compensatory expansion before induction of anaesthesia patients will then receive midazolam mgkg fentanyl µgkg vecuronium bromide mgkg and propofol mgkg intravenously for induction of anaesthesia anaesthesia will be maintained using a cp600 anaesthesia delivery system slgo medical technology beijing china the dose wang a0j et a0al bmj open 202010e030694 101136bmjopen2019030694 0cof propofol will be adjusted to maintain the bispectral index in the range of after surgery all patients will remain in the post anaesthesia care unit and then return to the ward for recovery until dischargethe perioperative management of all patients will be standardised early ambulation will be encouraged and oral feeding will be resumed as early as possible all patients will be followed up for at least months after discharge from the hospitaladverse eventsall adverse reactions will be closely monitored through spontaneous reports by patients or direct observation by clinicians or by asking the patients about adverse events using open questions all adverse reactions will be recorded and appropriate treatment will be provided if necessary serious adverse events will be reported to the ethics committeedata collection and managementdemographic variables and clinical data will be collected from all patients during the study blood pressure heart rate and oxygen saturation will also be monitored any adverse events will be recorded data will be collected throughout the study and will be securely managed under conditions of confidentiality data collection will be performed by a nurse anaesthetist the participants will be referred to by their participant number rather than by their name throughout the study unless otherwise specified all relevant documents and files will be archived for years the data will be accessible only by investigators who sign the confidential disclosure agreement and by institutional or governmental auditors during the study data without patient identifiers will be publicly accessible after the study data collection and management will be monitored by the institutional ethics committee for clinical research of shuguang hospitalpatient and public involvementthis study is currently in the recruitment phase patients andor the public were not involved in study design or conduct of the study the participants will be able to access the study results through social mediadiscussionpoi continues to represent an important cause of morbidity after colon surgery the prevention of poi is thus of great importance in reducing perioperative complications and reducing hospitalisation costs although it has been shown that ea can shorten the duration of poi18 the effectiveness of teas which is a similar technique in preventing poi has not been investigated it is therefore important to assess the effectiveness of teas in preventing poi through a clinical studythis study has several strengths first the intervention strategy of the protocol will be pretreatment with teas previous studies have shown that pretreatment open accesshas a prophylactic effect for example pretreatment with teas has been shown to improve pain treatment26 and to improve resuscitation after anaesthesia with reduction of postoperative nausea and vomiting28 it is however unclear whether preoperative teas can prevent poi studies suggest that early preoperative intervention may be more beneficial in regulating physiological functions and preventing poi29 in an extension to these findings the present study will help to determine whether teas pretreatment could improvement poisecond the effectiveness of teas will be evaluated by assessing clinical function and by serological examination in this randomised controlled trial of patients undergoing laparoscopic colorectal surgery our aim is to assess the effects of preoperative teas on poi using relevant clinical parameters associated with bowel function these include time to first defaecation time to first flatus time to tolerance of oral diet and gi2 importantly we will also measure serum concentrations of inflammatory mediators associated with poi such as ifn ifnÎ il6 and il1 our findings may thus provide deeper insights into the mechanisms by which teas improves poithere are also limitations to this protocol various clinical indicators have been used in studies for the diagnosis of poi but there is no consensus on which clinical parameter is the best for assessment of gi transit9 two indicators that are widely used to assess bowel movement will be used in this study time to first defaecation will be the primary outcome and time to first flatus will be one of the secondary outcomes there is a possibility that we may observe conflicting results ie significant improvement in time to flatus but not defaecation because flatus can vary considerably between patients clinical trials support the time to tolerance of oral diet and gi2 defined as the later of the following two events time to first tolerance of solid food and time to first bowel movement as supplementary secondary outcomes to measure the recovery time of gi function and these will be used in this study32 other limitations of these indicators are that they require objective measurement of motility and are time consuming to measure34 recently this situation has been improved by the use of in vivo monitoring techniques to assess the function of gi movements innovative devices such as sitz markers have been used to evaluate postoperative recovery of small bowel movement by counting the number of sitz markers that did not pass through the ileocecal valve but remained in the small intestine using radiography36 the smartpill is a swallowable device that record parameters within the gi tract indicators such as ph temperature and intracavitary pressure can be collected to analyse gi transit times in vivo37 these devices acquire objective parameters to evaluate bowel movement and could save time research into the satisfaction of both doctors and patients with these device needs to be carried out furthermore this study is a single centre trial and because the therapeutic effect of teas may be affected by ethnicity and region it will wang a0j et a0al bmj open 202010e030694 101136bmjopen2019030694 0copen access be necessary to conduct multicentre and large sample studies in the futurenotwithstanding its limitations this study can clearly indicate the overall effects of teas on postoperative recovery we hypothesise that pretreatment with teas could improve recovery of gi function in patients undergoing laparoscopic surgery if this study provides positive results it will be possible to recommend this pretreatment strategy for patients undergoing abdominal surgery relevant cost effectiveness studies are also worthy of considerationauthor affiliations1anesthesiology shuguang hospital affiliated to shanghai university of traditional chinese medicine shanghai china2anesthesiology wenzhou medical university the sixth affiliated hospital lishui china3research institute of acupuncture anesthesia shuguang hospital affiliated to shanghai university of traditional chinese medicine shanghai chinaacknowledgements we thank dr stanley tao from shanghai ruihui biotech for his valuable assistance in the statistical design of this studycontributors jw conceived the study dl wt jg and gf participated in its design and coordination wc yy ws and jg collected references and developed the protocol gy and ly will perform statistical analyses rf will follow up with patients and record data jw lf and js drafted the manuscript all authors have read and approved the final manuscriptfunding the present study is supported by the project of the national natural science foundation of china nos and and the commercial sponsorship of sinch pharmaceuticals techcompeting interests none declaredpatient consent for publication obtainedprovenance and peer review not commissioned externally peer reviewedopen access this is an open access article distributed in accordance with the creative commons attribution non commercial cc by nc license which permits others to distribute remix adapt build upon this work non commercially and license their derivative works on different terms provided the original work is properly cited appropriate credit is given any changes made indicated and the use is non commercial see a0http creativecommons org licenses by nc orcid idlihua a0fan http orcid org references iyer s saunders wb stemkowski s economic burden of postoperative ileus associated with colectomy in the united states j manag care pharm boelens pg heesakkers ffbm luyer mdp et a0al reduction of postoperative ileus by early enteral nutrition in patients undergoing major rectal surgery prospective randomized controlled trial ann surg melis m fichera a ferguson mk bowel necrosis associated with early jejunal tube feeding a complication of postoperative enteral nutrition arch surg moghadamyeghaneh z hwang gs hanna mh et a0al risk factors for prolonged ileus following colon surgery surg endosc goldstein jl matuszewski ka delaney cp et a0al inpatient economic burden of postoperative ileus associated with abdominal surgery in the united states p and t bragg d el sharkawy am psaltis e et a0al postoperative ileus recent developments in pathophysiology and management clin nutr wolthuis am bislenghi g fieuws s et a0al incidence of prolonged postoperative ileus after colorectal surgery a systematic review and meta analysis colorectal dis 201618o1 nguyen dl maithel s nguyen et et a0al does alvimopan enhance return of bowel function in laparoscopic gastrointestinal surgery a meta analysis ann gastroenterol studies and clinical aspects of gadolinium salts and chelates cardiovasc drug rev koscielny a kalff jc t helper cell type memory cells and postoperative ileus in the entire gut curr opin gastroenterol mikkelsen hb thuneberg l opop mice defective in production of functional colony stimulating factor1 lack macrophages in muscularis externa of the small intestine cell tissue res van bree shw nemethova a cailotto c et a0al new therapeutic strategies for postoperative ileus nat rev gastroenterol hepatol hilton wm lotan y parekh dj et a0al alvimopan for prevention of postoperative paralytic ileus in radical cystectomy patients a cost effectiveness analysis bju int wehner s behrendt ff lyutenski bn et a0al inhibition of macrophage function prevents intestinal inflammation and postoperative ileus in rodents gut wehner s straesser s vilz to et a0al inhibition of p38 mitogen activated protein kinase pathway as prophylaxis of postoperative ileus in mice gastroenterology schwarz nt kalff jc türler a et a0al prostanoid production via cox2 as a causative mechanism of rodent postoperative ileus gastroenterology engel dr koscielny a wehner s et a0al t helper type memory cells disseminate postoperative ileus over the entire intestinal tract nat med adding lc bannenberg gl gustafsson le basic experimental ng ssm leung ww mak twc et a0al electroacupuncture reduces duration of postoperative ileus after laparoscopic surgery for colorectal cancer gastroenterology you x m mo x s ma l et a0al randomized clinical trial comparing efficacy of simo decoction and acupuncture or chewing gum alone on postoperative ileus in patients with hepatocellular carcinoma after hepatectomy medicine 201594e1968 song jg li hh cao yf et a0al electroacupuncture improves survival in rats with lethal endotoxemia via the autonomic nervous system anesthesiology zhang j yong y li x et a0al vagal modulation of high mobility group box1 protein mediates electroacupuncture induced cardioprotection in ischemia reperfusion injury sci rep balogun ja biasci s han l the effects of acupuncture electroneedling and transcutaneous electrical stimulation therapies on peripheral haemodynamic functioning disabil rehabil jiang y wang h liu z et a0al manipulation of and sustained effects on the human brain induced by different modalities of acupuncture an fmri study plos one 20138e66815 dindo d demartines n clavien p a classification of surgical complications a new proposal with evaluation in a cohort of patients and results of a survey ann surg rakel b cooper n adams hj et a0al a new transient sham tens device allows for investigator blinding while delivering a true placebo treatment j pain huang l pan y chen s et a0al prevention of propofol injection related pain using pretreatment transcutaneous electrical acupoint stimulation turk j med sci zhang q gao z wang h et a0al the effect of pre treatment with transcutaneous electrical acupoint stimulation on the quality of recovery after ambulatory breast surgery a prospective randomised controlled trial anaesthesia zheng lh sun h wang gn et a0al effect of transcutaneous electrical acupoint stimulation on nausea and vomiting induced by patient controlled intravenous analgesia with tramadol chin j integr med stakenborg n labeeuw e gomez pinilla pj et a0al preoperative administration of the ht4 receptor agonist prucalopride reduces intestinal inflammation and shortens postoperative ileus via cholinergic enteric neurons gut vather r trivedi s bissett i defining postoperative ileus results of a systematic review and global survey j gastrointest surg wu z boersema gsa dereci a et a0al clinical endpoint early detection and differential diagnosis of postoperative ileus a systematic review of the literature eur surg res deng g wong wd guillem j et a0al a phase ii randomized controlled trial of acupuncture for reduction of postcolectomy ileus ann surg oncol van bree shw bemelman wa hollmann mw et a0al identification of clinical outcome measures for recovery of gastrointestinal motility in postoperative ileus ann surg wang a0j et a0al bmj open 202010e030694 101136bmjopen2019030694 0c maffezzini m campodonico f canepa g et a0al current perioperative management of radical cystectomy with intestinal urinary reconstruction for muscle invasive bladder cancer and reduction of the incidence of postoperative ileus surg oncol bungard tj kale pradhan pb prokinetic agents for the treatment of postoperative ileus in adults a review of the literature pharmacotherapy open access chae h d kwak m a kim i h effect of acupuncture on reducing duration of postoperative ileus after gastrectomy in patients with gastric cancer a pilot study using sitz marker j altern complement med vilz to pantelis d lingohr p et a0al smartpill® as an objective parameter for determination of severity and duration of postoperative ileus study protocol of a prospective two arm open label trial the pidusa study bmj open 20166e011014wang a0j et a0al bmj open 202010e030694 101136bmjopen2019030694 0c' | Colon_Cancer |
" recently copy number alteration cna of 9p241 were demonstrated in of diffuse large bcelllymphoma dlbcl with gene expression and mutation profiles that were similar to those of primary mediastinallarge bcell lymphoma pmbcl however their cnabased profile and clinical impact still remain unclearmethods multiplex ligationdependent probe amplification were employed to investigate the prevalence of jak2pdl2 amplification in dlbcl and their cnabased pattern of driver genes the clinical outcome and characteristicswere also analyzedresults using unsupervised hierarchical clustering a small group of dlbcl was clustered togetherwith pmbcl as cluster_2 demonstrating amplification of jak2 and pdl2 this subgroups ofdlbcl demonstrated significant higher expression of pdl1 than those with myd88 l265p mutationp andthey exhibited dismal os and pfs as compared with dlbcl_othersp and respectively which issimilar to dlbcl with myd88 l265p mutations dlbcl with amplification of jak2pdl2 exhibits cna pattern that is similar to pmbcl anddemonstrates unfavorable clinical outcome that resembles those with myd88 l265p mutation it is essential toidentify this subgroup of dlbcl who may acquire more benefits from the jak2 and pdl1 signaling inhibitionkeywords diffuse large bcell lymphoma jak2 pdl2 amplification prognosis diffuse large bcell lymphoma dlbcl is a highly heterogeneous disease recently several distinctive geneticsubtypes were identified including schmitz r studymcd bn2 n1 and ezb subtypes and chapuy b study c0 c5 clusters [ ] godfrey j study also correspondence jmyingcicamsaccn lvningcicamsaccn xuemin xue and wenting huang are cofirst authors jianming ying andning lv are cosenior authors1department of pathology national cancer centernational clinical researchcenter for cancercancer hospital chinese academy of medical sciencesand peking union medical college beijing chinafull list of author information is available at the end of the identified an unique biological subset of dlbcl withpdl1 gene alterations having high risk features thus the genetics of dlbcl relating to potential therapeutic targets for immune checkpoint inhibitors shouldbe paid much more attention tojanus kinase jak2 programmed cell death ligand pdl1cd274pdcd1lg1 and programmed celldeath ligand pdl2cd273pdcd1lg2 are adjacent to each other on chromosome 9p241 playing keyroles in host immune surveillance amplification of9p241 were frequently seen in celllines of classical and primaryhodgkin lymphoma chl the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cxue bmc cancer page of mediastinal large bcell lymphoma pmbcl but much less in dlbcl cell lines ] correspondingly pd1 ligands pdl1 and pdl2transcripts and proteins were more abundant in chl andpmbcl cell lines than that in dlbcl cell lines recently y wang study demonstrated that ofdlbcl had copy number alteration cna of 9p241with a gene expression and mutation profile similar tothose of pmbcl however their cnabased profileand clinical impact still remain unclearin thisthereforestudy we employed multiplexligationdependent probe amplification mlpa to investigate the prevalence of jak2pdl2 amplification indlbcl and their cnabased pattern of driver genesincluding bcl2 cdkn2a and tp53 and we analyzed their longterm survival outcome after treatmentof rchoplike regimemethodscase selectionwe collected consecutive cases of dlbcl and pmbclin our clinical ffpe archives of excisional biopsy database between jan and oct and cases ofdlbcl and cases of pmbcl were found after confirmation one case of dlbcl was diagnosed as pmbclthus cases of dlbcl and cases of pmbcl wereacquired finally see additional file all patients werediagnosed at national cancer centernational clinicalresearch center for cancercancer hospital chineseacademy of medical sciences and peking union medicalcollege according to the revised 4th edition ofthewho classification of tumours of haematopoietic andlymphoid tissues the data regarding treatment andprognosis were acquired by means of medical recordconsultation and telephone conversationmultiplex ligationdependent probe amplification mlpagenomic dna were extracted from formalinfixedparaffinembedded ffpe blocks using qiaamp dnaffpe tissue kit qiagen valencia ca then dnacopy number quantification and myd88 l265p mutation were detected using mlpa kitmrchollandnetherlands the pcr products were detected on anabi genetic analyzer applied biosystems usaand the final result were analyzed using coffalyser software the relative peak ratio prr of probe largerthan was defined as amplification and less than was defined as deletion see additional file geneswhich had two or more probes covering two differentexomes were put into final analysis including jak2 pdl2 mdm2 rel pus10 bcl2 nfatc1 spib foxp1nfkbiz bcl6 prdm1 tnfaip3 cdkn2a ptening1 and tp53 the details of mlpa probes of drivergenes in dlbcl are shown in the online supportingmaterial see additional file true amplification of onegene was regarded only when all probes of this gene exhibited amplification and vice versa see additional file myd88 l265p mutation was identified when theprobe had a high peak myd88 wildtype didnt show anypeak see additional file immunohistochemistry ihc staining of pdl122c3ihc staining was performed on dako autostainer link asl48 platform each ffpe block were cut at athickness of 4μm and then deparaffinized antigen retrieval were performed using the envision¢ flex targetretrieval solution at low ph monoclonal pdl1clone 22c3 dako were used as primary antibodyfollowed by incubation with envision¢ flex mouselinker and then envision¢ flex hrp reagent finally the ihc was visualized by envision¢ flex dabeach ihc slide contained a positive controllungcarcinomaihc score of pdl1 were calculated by multiplyingthe percentage of positive cells with mean intensity no staining weak staining moderate staining strong staining which was reported in previous study the results were evaluated by an experienced hematopathologist xueminstatistical analysisthe differences of clinicopathological characteristicsamong different groups were analyzed using chisquaretest fisher exact test or kruskalwallis rank sum testpdl1 ihc score between different groups was analyzedusing wilcoxon test overall survival os and progressfree survival pfs times were defined from the date ofpathologic diagnosis to the date of the event or the lastfollowup the hazard ratio hr of each parameter wascalculated by univariate cox proportional regressionanalysis firstly in which parameters with p wereevaluated together using multivariate cox proportionalregression analysis the survival curve were made according to kaplanmeier procedure the day oflastfollowup was march 1st all statistical analysiswere two sided and p was defined as significanceunsupervised hierarchical clustering was carried outusing euclidean distance and complete method heatmap was plot using pheatmap packageall above statistical analyses were run in r statistic softwareresultsunsupervised hierarchical clustering of cnas of drivergenes and its survival analysis in dlbcl and pmbclpatientsbased on array cgh lenz g study previouslyidentified specific cnas in pmbcl which were different 0cxue bmc cancer page of from abc and gcb of dlbcl abc dlbcls oftenhave cnas in foxp1 nfkbiz cdkn2a cdkn2binf4a bcl2 nfatc1 and spib while gcb dlbclsfrequently harbor cnas in rel pten mdm2 mihg1and ing1 pmbcl often demonstrate cnas of jak2 andpdl2 using unsupervised hierarchical clustering we explored the cnabased pattern of these genes in dlbcl andpmbcl the result showed that a small group of dlbcl was clustered together with pmbcl as cluster_2 with amplification of jak2 and pdl275068fig 1a this subgroup of dlbcl occurred atthe site of cervical lymph node cases gastrointestinal tract cases nasal cavity case and spleen cases fig 1atable 1additional file the frequency of jak2 and pdl2 amplification in the whole cohort of dlbcl were and while both of them were inpmbcl fig 1a see additional file meanwhile all casesin cluster_3 harbored amplification of nfkbiz which is essential for nfκb activation in abc dlbcl but noamplification of nfkbiz was found in cluster_1as to survival dlbcl in cluster_2 demonstrated significant worse os p and pfs p as compared with dlbcl in cluster_1fig 1b howevercluster_1 and cluster_3 didnt reveal significant differencein survival fig 1b we also analyzed the os and pfs between dlbcl with and without jak2pdl2 amplification and got statistical significance see additional file of note we found that dlbcl in cluster_2 enrichedfor jak2pdl2 amplification had less frequency ofmyd88_l265p mutation fig 1a table fig heatmap and survival analysis based on unsupervised hierarchical clustering and status of jak2pdl1 amplification and myd88 mutationin tcga dataset a heatmap of cnabased profiles of driver genes in dlbcl and pmbcl by using unsupervised hierarchical clustering b survivalcurves and coxregression analysis of os and pfs among three cnabased clusters after rchoplike treatment c status of amplifications of jak2pdl1cd274 and pdl2pdcd1lg2 and mutation of myd88 in dlbcl tcga pancancer atlas from cbioportal [ ] 0ccervical lymphnodefemale high_intermediatehigh_intermediatedlbclnasal cavitymalegcbbreak_apartdlbcldlbcldlbcldlbcldlbcldlbclcervical lymphnodestomachstomachcolondlbclpmbclpmbclpmbclpmbclcervical lymphnodecervical lymphnodemediastinummediastinummediastinummalelow_intermediate non_gcbnormalfemale low_intermediate non_gcbmalemalelowhighnon_gcbgcbnon_gcbnormalnormalnormalnormalfemale low_intermediate non_gcbnormalmalelowfemale female female lowlowhigh_intermediatenanananananormalnormalnormalnormalnormaljak2_amppdl2_ampxue bmc cancer page of table the clinicopathological characteristics of dlbcl with jak2pdl2 amplification and pmbclmyd88_no diagnosis sitel265pmyc_ breakapartnormalhansalgorithmnon_gcbage ipi _riskspleenfemale lowsexpmbclna not applicablemediastinummalelowwhich was supported by the cancer genome atlas datatcga pancancer atlas from cbioportal [ ] fig 1ccategory and myc breakapart didnt show any significant differences table jak2pdl2 amplification identify a distinctive cnabasedpattern of dlbcl similar to that of pmbclsince dlbcl with jak2pdl2 amplification had less frequency of myd88 l265p mutation our study separateddlbcl patients into three subgroups dlbcl with jak2pdl2 amplification dlbcl_jak2pdl2_amp dlbclwith myd88 l265p mutation dlbcl_myd88_l265pjak2pdl2 amplification norand dlbcl withoutmyd88_l265p mutation dlbcl_others fig 2a basedon the unsupervised cluster result fig 1a one patientwho had both jak2pdl2 amplification and myd88l265p mutation was clustered into cluster_2 thereforethis patient was put into dlbcl_jak2pdl2_amp subgroup accordingly we also analyzed the data when thiscase was included in dlbcl_myd88_l265p subgroupand got the similar result see additional file unlike dlbcl_myd88_l265p and dlbcl_othersdlbcl_jak2pdl2_amp showed a distinctive pattern similar to that of pmbcl with high frequencyof rel and nfkbiz amplifications but no amplification of bcl2 and nfatc1 and no deletion ofprdm1 was found fig 2awith respectto clinicopathologicdlbcl_jak2pdl2_amp tend to be youngerdlbcl_myd88_l265p p hans modelcharacteristicsthantable whileinternational prognostic index ipi riskpdl1 expression in dlbcl with jak2pdl2 amplificationwas significantly higher than that in dlbcl with myd88l265p mutationtotally cases were performed pdl1 22c3 ihc detection including dlbcl_myd88_l265p cases dlbcl_jak2pdl2_amp cases dlbcl_others cases andpmbcl cases the result showed that pdl1 expressionin dlbcl_jak2pdl2_amp was significantly higher thanthat in dlbcl_myd88_l265p p and dlbcl_others p fig 2b and d while no significant difference was found between dlbcl_jak2pdl2_amp andpmbcl p fig 2bjak2pdl2 amplification identify a subgroup of dlbclwith unfavorable survival outcome similar to that ofmyd88 l265p mutationtrying to explore the survival indication of jak2pdl2 amplification and myd88 l265p mutation cases of dlbcls who received rchoplike regimentwith or without surgical resection were enrolled toperformed cox proportional regression analysis of osand pfs the median followup time was monthsrange monthsin the univariatecompared withdlbcl_others dlbcls with myd88 l265p mutationhad significantly worse os and pfs p andanalysisas 0cxue bmc cancer page of fig comparison of cnabased pattern pdl1 expression and survival analysis among pmbcl and three subgroups of dlbcl a comparison ofcnabased patterns of driver genes among pmbcl and three subgroups of dlbcl according to the status of jak2pdl2 amplification andmyd88 l265p mutation b comparison of pdl1 expression ihc score among pmbcl and three subgroups of dlbcl c survival curves and coxregression analysis of os and pfs among three subgroups of dlbcl after rchoplike treatment d representative images of heà and pdl1à ihc in dlbcl_jak2pdl2_amp and dlbcl_ myd88_l265p respectively and the same to dlbcls withjak2pdl2 amplification p and respectively meanwhile ipi risk category were significantly associated with os and pfs fig 2c tables and in the multivariate analysis ipi risk category andthree subgroups of dlbcl were put into analysis ascompared with dlbcl_others dlbcl with myd88l265p mutation still showed poor os and pfs p and respectively and the same todlbcl with jak2pdl2 amplification for pfs andos p and respectively meanwhile ipirisk category was still an independent risk predictorsfor os and pfs fig 2c tables and either jak2pdl2 amplification or myd88 l265pmutation are frequently seen in relapserefractory dlbclwith pfs less than yearsdlbcl with pfs less than years was defined as primaryrelapserefractory cases among these cases who treated byrchoplike regime the frequency of jak2 and pdl2amplification were and meanwhilethe frequency of myd88 l265p mutation were dlbcl with either jak2pdl2 amplification ormyd88 l265p accounted for discussiondlbcl presents with a wide spectrum of genetic aberration recently shi study exhibited pdl2 amplification in pmbcl and of dlbcl chapuy demonstrated of 9p241 amplification indlbcl meanwhile dlbcl with pdl1 gene alterations was identified as a unique biological subgrouphaving high risk features y wang study demonstrated that of dlbcl had cna of 9p241 withgene expression and mutation profiles that were similarto those of pmbcl in our study by using unsupervised hierarchical clustering cases ofdlbcl were clustered together with pmbcl as cluster_2 indicating that they shared recurrent cnas theywere enriched for jak2 amplification and pdl2 amplification fig 1a 0cxue bmc cancer page of table comparison of characteristics among pmbl and three subgroups of dlbcldlbclothersmyd88_l265pjak2pdl2_amppmbclpatientsage median range bmnegativepositiveihc hans algorithmgcbnongcbipilowrisklow_intermediatehigh_intermediatehighmyc breakapartnegativepositive p_valueÏ2test kruskalwallis rank sum testusing hans model most of dlbcl in cluster_2 werenongcb and tend to be younger than othergroups of dlbcl table which was consistent withprior study therefore coupled with y wang study we confirmed that dlbcl with jak2pdl2 amplification is a unique subgroup resembling the pmbclwith respect to cna patternwith regard to survival increasingly data exhibited thatthe suppression of immune surveillance in dlbcl was associated with poor survival godfrey j study hasdemonstrated that dlbcl with pdl1 gene alterationsshowed high risk features metaanalysis also showedthat pdl1 expression was associated with poor os andadverse clinicopathologic features in dlbcl in y wang study of dlbcl harbored cnaof 9p241 of which were gains and were amplifications and as compared with those who have nogain of 9p241 dlbcl with 9p24 amplification had atrend of better efs while patients with only gain tend tothey didnthave worse prognosis unfortunatelyshow any statistical significance in our study of dlbcl were found that had cna of jak2when jak2 cna was separated into gain mlpa valuebetween and amplification mlpa value as described cases in dlbcl_jak2pdl2_amp group were found that had jak2 gain whichwas slightly lower than that in wang j study asshown in additional file and both dlbcl withjak2 gain and with amplification demonstrated significant poor prognosis as compared with rest of dlbclas shown in additional file more interesting unlikey wang study cases of pmbcl were included in our study as control all of which demonstrating jak2 gains rather than amplifications as shown inadditional file of note we found that dlbcl in cluster_2 enrichedfor jak2pdl2 amplification had less frequency ofmyd88_l265p mutation fig 1a table which was supported by the cancer genome atlas datatcga pancancer atlas from cbioportal [ ] fig 1cmyd88 l265p is a poor indicator of survival for dlbcl which may lead to primary refractoryrelapsed diseasethis is a gainoffunction driver mutation occurring in of dlbcl but absent in pmbcl [] inour study the frequency of myd88 l265p in dlbcl andpmbcl were and which were in linewith prior studies [] of greatinterest myd88l265p mutation occurred less frequently in cluster_2 which was supported by the data tcga pancancer atlas from cbioportal [ ] thus when we divided dlbcl patients into three subgroups dlbcl_jak2pdl2_amp dlbcl_myd88_l265p and dlbcl_others both dlbcl_jak2pdl2_amp and dlbcl_myd88_l265p demonstrated dismal os and pfs with amedian followup of years as compared with dlbcl_others therefore dlbcl with jak2pdl2 amplification 0cxue bmc cancer page of table os in dlbcl treated by rchoplike regimeage ¥ bmnegativepositivesiteextranodalnodalihc hans algorithmgcbnongcbmyc fish breakapartnegativepositiveipi risk categorylowlow_intermediatehigh_intermediatehightherapyrchopresection rchopcna_based_clustercluster_1cluster_2cluster_3three subgroups of dlbcldlbcl_othersdlbcl_jak2pdl2_ampdlbcl_myd88_l265poshr_u95ci p_valueoshr_m95cip_value hr_u hazard ratio by univariate analysis hr_m hazard ratio by multivariate analysis because age was contained in the ipi thus it wasnt put into multivariate analysiswas identified as a poor survival subgroup that is similar todlbcl with myd88 l265p mutationmeanwhile we also compared the cna patterns ofdriver genes among dlbcl_jak2pdl2_amp dlbcl_myd88_l265p dlbcl_others and pmbcl dlbcl_jak2pdl2_amp showed a distinctive pattern similarto pmbcl with high frequency of rel and nfkbizamplifications but no amplification of bcl2 and nfatc1 and no deletion of prdm1 was found the profile ofdlbcl_myd88_l265p was closed to dlbcl_othersshowing relatively high frequency of cdkn2a deletionnfatc1 amplification and bcl2 amplificationin our study of dlbcl_jak2pdl2_ampharbored both jak2 and pdl2 amplifications simultaneouslyindicating that they may also have the pdl1amplification because pdl1 located in the middle ofjak2 and pdl2 at 9p241 thus we hypothesized thatpdl1 expression would be upregulated in this subgroup as what we expected using pdl1 22c3 ihcdetection pdl1 expression in dlbcl_jak2pdl2_in dlbcl_amp was significantly higher than thatmyd88_l265p p and dlbcl_othersp fig 2b and d but not in pmbcl p fig 2b meanwhile pdl1 expression could be 0cxue bmc cancer page of table pfs in dlbcl treated by rchoplike regimeage ¥ bmnegativepositivesiteextranodalnodalihc hans algorithmgcbnongcbmyc breakapartnegativepositiveipilowlow_intermediatehigh_intermediatehightherapyrchopresection rchopcna_based_clusterscluster_1cluster_2cluster_3three subgroups of dlbcldlbcl_othersdlbcl_ jak2pdl2_ampdlbcl_ myd88_l265ppfshr_u95ci p_value pfshr_m95cip_value hr_u hazard ratio by univariate analysis hr_m hazard ratio by multivariate analysis because age was contained in the ipi thus it wasnt put into multivariate analysisenhanced not only by pdl1 amplification but also byjak2 activation [ ] therefore dlbcl with jak2pdl2 amplification was confirmed as an unique subtype that is different from dlbcl with myd88 l265pand othersobjective response rates orr of pd1 blockade therapy was in unselected patients with relapsedrefractory dlbcl [ ] the wide spectrum of orrmay be due to high heterogeneity of this subgroupansell sm study demonstrated patients with9p241 alteration in relapsedrefractory dlbcl inour cohort the frequency of jak2 and pdl2 amplification in relapsedrefractory dlbcl were and which were within the range of orr in the prior studies[ ] while patients were found thathad myd88 l265p mutation who may not be suitablefor antipd1 therapy thus the genetic analysis in refractoryrelapsed dlbcl is required for future therapyselection to increase the orr of immune checkpointinhibitorsjak2 amplification could augment the expression of itself and pd1 ligands pdl1 and pdl2 enhancing the 0cxue bmc cancer page of sensitivity to jak2 kinase inhibitor chemical jak2inhibition could reduce the rna transcription and protein expression of pdl1 thus selective inhibitionof jak2 would be a valuable complementary therapy forpdl1 blockadeauthors contributionsxx contributed to pdl1 ihc staining clinical followup data analysis andmanuscript writing wh contributed to ffpe tissues collection mlpa detection and clinical followup tq and lg provided experiment guidance anddata interpretation jy and nl contributed to study design coordination discussion and manuscript editing all authors read and approved the finalmanuscriptsjak2pdl2 exhibitsdlbcl with amplification ofpmbcllike cnas pattern and demonstrates unfavorable outcome resembling those with myd88l265p mutation thusit is essential to identify thissubgroup of dlbcl who may acquire more benefitsfrom the jak2 and pdl1 signaling inhibition andjak2 amplification detection by mlpa would be feasible in routine practice meanwhile the difference ofsurvival outcome between our study and wang j study indicated that pmbcllike dlbcl suggested by 9p241 cna could be an intermixed subgroup which required further explorationsupplementary informationsupplementary information accompanies this paper at httpsdoi101186s12885020072933additional file mlpa results and clinical followup data the clinicopathological characteristics clinical followup data and mlpa results areshowed in this fileadditional file figure s1 representative results of mlparepresentative results of mlpa are showed in this figureadditional file table s1 the details of mlpa probes of genes indlbcl the locations and lengths of mlpa probes of genes are showedin this tableadditional file the detailed information of dlbcl with jak2pdl2amplification the detailed data about clinicopathological characteristicsmorphology immunohistochemistry and treatments of dlbcl with jak2pdl2 amplification are showed in this fileadditional file figure s2 the os and pfs of dlbcl with or withoutjak2pdl2_amp the os and pfs of dlbcl with or without jak2pdl2_ampadditional file figure s3 comparison of cnabased pattern andtheir survival outcome among pmbcl and three subgroups of dlbclone case of dlbcl with jak2pdl2 amplification and myd88 l265p mutation were included in dlbcl_myd88_l265p group a comparison ofcnabased patterns of driver genes among pmbcl and three subgroupsof dlbcl according to the status of jak2pdl2 amplification and myd88l265p mutation b survival curves and coxregression analysis of os andpfs among three subgroups of dlbcl after rchoplike treatmentadditional file figure s4 the frequencies of jak2 gain andamplification and their survival analysis a the frequencies of jak2 gainand amplification in dlbcl_jak2pdl2_amp and pmbcl b the os andpfs of dlbcl with jak2 gain or with jak2 amplificationabbreviationsdlbcl diffuse large bcell lymphoma pmbcl primary mediastinal large bcell lymphoma mlpa multiplex ligationdependent probe amplificationtcga the cancer genome atlas ipi international prognostic indexffpe formalinfixed paraffinembedded os overall survival pfs progressfree survival hr hazard ratioacknowledgementsnot applicablefundingthis study was partly supported by the beijing municipal science technology commission grant number z151100004015121 the cancerfoundation of china grant number lc2014l13 and cams innovation fundfor medical sciences grant number 2016i2m1001 to perform ffpe tissuescollection and mlpa detection and was partly supported by the cancerfoundation of china grant number lc2018b10 and pumc youth fundand the fundamental research funds for the central universities grantnumber to conduct pdl1 ihc staining and clinical followupand collect fish data of cmycavailability of data and materialsall data generated or analyzed during this study are included in thispublished and its supplementary information filesethics approval and consent to participatethis is a retrospective study that was launched in november the casesenrolled in this project were diagnosed between jan and oct whose ffpe samples were used the data regarding treatment andprognosis were acquired by means of medical record consultation andtelephone conversation thus the need for consent was waived by theindependent ethics committee of cancer hospital chinese academy ofmedical sciences national gcp center for anticancer drugs ncc2015st05consent for publicationnot applicablecompeting intereststhe authors declare that they have no competing interestsauthor details1department of pathology national cancer centernational clinical researchcenter for cancercancer hospital chinese academy of medical sciencesand peking union medical college beijing china 2department ofpathology national cancer centernational clinical research center forcancercancer hospital shenzhen hospital chinese academy of medicalsciences and peking union medical college shenzhen chinareceived march accepted august referencesschmitz r wright gw huang dw johnson ca phelan jd wang jqroulland s kasbekar m young rm shaffer al genetics andpathogenesis of diffuse large bcell lymphoma n engl j med chapuy b stewart c dunford aj kim j kamburov a redd ra lawrencems roemer mgm li aj ziepert m molecular subtypes of diffuse largeb cell lymphoma are associated with distinct pathogenic mechanisms andoutcomes nat med godfrey j tumuluru s bao r leukam m venkataraman g phillip jfitzpatrick c mcelherne j macnabb bw orlowski r pdl1 genealterations identify a subset of diffuse large bcell lymphoma harboring a tcellinflamed phenotype blood green mr monti s rodig sj juszczynski p currie t o'donnell e chapuy btakeyama k neuberg d golub tr integrative analysis reveals selective9p241 amplification increased pd1 ligand expression and furtherinduction via jak2 in nodular sclerosing hodgkin lymphoma and primarymediastinal large bcell lymphoma blood wang y wenzl k manske mk asmann yw sarangi v greipp pt krull jehartert k he r feldman al amplification of 9p241 in diffuse large bcell lymphoma identifies a unique subset of cases that resemble primarymediastinal large bcell lymphoma blood cancer j 0cxue bmc cancer page of lenz g wright gw emre nc kohlhammer h dave ss davis re carty slam lt shaffer al xiao w molecular subtypes of diffuse large bcelllymphoma arise by distinct genetic pathways proc natl acad sci u s aswerdlow sh campo e harris nl jaffe es pileri sa stein h thiele j whoclassification of tumours of haematopoietic and lymphoid tissues revised4th edn lyon iarc cerami e gao j dogrusoz u gross be sumer so aksoy ba jacobsen abyrne cj heuer ml larsson e the cbio cancer genomics portal anopen platform for exploring multidimensional cancer genomics datacancer discov gao j aksoy ba dogrusoz u dresdner g gross b sumer so sun yjacobsen a sinha r larsson e integrative analysis of complex cancergenomics and clinical profiles using the cbioportal sci signal pl1 nogai h wenzel ss hailfinger s grau m kaergel e seitz v wollertwulf bpfeifer m wolf a frick m ikappabzeta controls the constitutive nfkappab target gene network and survival of abc dlbcl blood shi m roemer mg chapuy b liao x sun h pinkus gs shipp ma freemangj rodig sj expression of programmed cell death ligand pdl2 is adistinguishing feature of primary mediastinal thymic large bcelllymphoma and associated with pdcd1lg2 copy gain am j surg pathol qiu l zheng h zhao x the prognostic and clinicopathological significanceof pdl1 expression in patients with diffuse large bcell lymphoma a metaanalysis bmc cancer moelans cb monsuur hn de pinth jh radersma rd de weger ra vandiest pj esr1 amplification is rare in breast cancer and is associated withhigh grade and high proliferation a multiplex ligationdependent probeamplification study anal cell pathol amst fernandezrodriguez c bellosillo b garciagarcia m sanchezgonzalez bgimeno e vela mc serrano s besses c salar a myd88 l265p mutation isan independent prognostic factor for outcome in patients with diffuse largebcell lymphoma leukemia ngo vn young rm schmitz r jhavar s xiao w lim kh kohlhammer h xuw yang y zhao h oncogenically active myd88 mutations in humanlymphoma nature dubois s viailly pj bohers e bertrand p ruminy p marchand vmaingonnat c mareschal s picquenot jm penther d biological andclinical relevance of associated genomic alterations in myd88 l265p andnonl265pmutated diffuse large bcell lymphoma analysis of casesclin cancer res gupta s cheville jc jungbluth aa zhang y zhang l chen yb tickoo skfine sw gopalan a alahmadie ha jak2pdl1pdl2 9p241amplifications in renal cell carcinomas with sarcomatoid transformationimplications for clinical management mod pathol ansell sm minnema mc johnson p timmerman jm armand p shipp marodig sj ligon ah roemer mgm reddy n nivolumab for relapsedrefractory diffuse large bcell lymphoma in patients ineligible for or havingfailed autologous transplantation a singlearm phase ii study j clin oncollesokhin am ansell sm armand p scott ec halwani a gutierrez mmillenson mm cohen ad schuster sj lebovic d nivolumab inpatients with relapsed or refractory hematologic malignancy preliminaryresults of a phase ib study j clin oncol hao y chapuy b monti s sun hh rodig sj shipp ma selective jak2inhibition specifically decreases hodgkin lymphoma and mediastinal largebcell lymphoma growth in vitro and in vivo clin cancer res publishers notespringer nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations 0c" | Colon_Cancer |
continuously increasing with development of the economy and the environment [] the prognosis for hcc patients remains extremely poor although significant progress has been achieved strategies for early diagnosis are urgently needed because the majority of patients with hcc are diagnosed in very late stages however the molecular mechanism of hcc has not been clearly defined circular rnas circrnas are a new class of rna molecules that have functions as regulators of parental gene transcription in alternative splicing and as mirna sponges through use of rna deep sequencing gtechnology numerous circrnas have been identified as the predominant regulatory elements in diseases moreover accumulating evidence shows that circrnas play pivotal roles in many diseases in particular abnormally expressed circrnas are involved in tumor progression including cell proliferation migration and invasion [] in addition some research indicates that circrnas level are closely correlated wit specific phenotypes and tumorigenesis in hcc [] nevertheless the research concerning circrnas is frankly in its infancy which greatly hinders the application of circrnas as biomarkers for diagnosis of hcc in clinicsrelated research shows that circrnas possess great potential to be used for diagnosis of hcc recent studies have found that hsa_circ_0067934 plays oncogenic roles by accelerating cell proliferation and metastasis in glioblastoma gbm circsmarca5 was significantly elevated and thereby suppressed cell apoptosis and arrested cell cycle in prostate cancer in addition previous studies have shown that downregulation of hsa_circ_0005986 facilitated cell proliferation by promoting the g0g1 to s phase transition in hcc similarly alteration in expression of circrnas correlated with development and metastasis of malignant tumors these data suggest that circrnas may be of greater benefit in clinical diagnosis of hcc however reliable circrna biomarkers for hcc are still lacking therefore this review synthetically integrates available data on the role of circrna in hcc progression and attempts to provide crucial clues for investigating the molecular mechanism regarding hccoverview of circrnacircrnas are a category of singlestranded closedcircle molecules which take part in multifaceted biological regulation recently research has verified that the majority of circrnas are synthesized by backspliced exons and that others are formed from intron intergenic and untranslated regions utr therefore biogenesis of circrnas can be divided into eicirnas exonintron circrnas ecircrnas circular exonic rnas and cirnas circular intronic rnas meanwhile over circrnas have been identified and this type of transcript has been considered a new form of gene expression generally the structure of the transcription is inverted and the order of genomic exons is altered and these exons are spliced over time the biological functions of circrnas gradually have been recognized including roles in embryonic development maintainenance of homeostasis and promotion of tumor progression figure properties of circrnascircrnas recently have attracted great attention related to their pathological role in disease development compared with linear rnas circrnas have special properties including biological roles and clinical use circrnas are mainly enriched in certain body fluids comprising blood saliva and urine they are covalently closed loop structures degradation of most rna is highly dependent on rna exonuclease or rnase hence circrnas remain highly stable based on their high resistance to enzyme degradation moreover studies have shown that expression of circrnas is tissuespecific and correlated with different phases of development and they exhibit different expression patterns at different developmental stages roles of circrnasaccumulating evidence shows that circrnas play a crucial role in the pathogenesis of diseases as a result of their complex biological functions generally the molecular functions of circrnas mainly include being sponges of mirna acting as rnabinding proteins performing alternative splicing of premrnas regulating transcription and translation and potentially encoding proteins these properties are described in detail belowsponges of mirnathe different types of circrnas have different mirna binding sites some circrnas negatively regulate mirnas by absorbing and specifically binding to mirnas then decreasing mirna activity and elevating expression of mirnarelated target genes researchers have shown that cirs7 inhibits mir7 function and positively mediates mir7 target genes acting as a molecular sponge in addition functional analyses have indicated that circrnas constitute an entire molecular regulatory network which specifically regulates degradation of mirnas as mirna sponges this work is licensed under creative common attributionnoncommercialnoderivatives international cc byncnd e9238322indexed in [current contentsclinical medicine] [sci expanded] [isi alerting system] [isi s master list] [index medicusmedline] [embaseexcerpta medica] [chemical scas]review s 0czou rc research progress on circrnas in hcc med sci monit e923832premrnae1e2abcbasepairinge3dguriche4ecrichpretrnarna bindingproteinsrbpgnicilpskcablariat splicingaecircrnaelcirnacirnatrnatricrnafigure1 biogenesis of circular rnas a lariatdriven circularization the hydroxyl of the upstream exon reacts with the phosphate of the downstream exon to form a covalent linkage then producing a lariat including exons and introns the hydroxyl of the intron interacts with the phosphate of the intron to form an ecircrna following an interaction between the hydroxyl of the exon and the phosphate of the exon b rnabinding protein rbpdriven circularization rbps accelerate interaction of the downstream intron and upstream intron thereby promoting formation of ecircrna c basepairingdriven circularization the downstream introns and upstream introns are paired depends on inverserepeatingcomplementary sequences formation of ecircrnaeicirna was derived from the introns are removedretained d biosynthesis of cirna formation of cirnas mainly based on a 7nt gurich element and an 11nt crich element to escape debranching and exonucleolytic degradation e formation of tricrna trna splicing enzymes divide pretrna into two parts tricrnas are generated by a phosphodiester bond and the other part generates trnascircrnasbinding proteinsrna binding proteins rbps are a broad class of proteins involved in gene transcription translation and interaction studies suggest that distribution of rbps is widespread in many tissue types furthermore rbps participate in development of disorders by regulating posttranscriptional regulation of rnas rbps assemble ribonucleoprotein complexes to bind rna sequences thereby affecting the function of the target rnas previous research has shown that circrnas serve as protein decoys to harbor binding sites of specific proteins and block protein activity circfoxo3 induces cell cycle arrest resulting in defective cdk2 gene function by binding to p21 and cdk2 moreover circrna ciacgas binds to cgas protein and suppresses enzymatic activity of cgas thereby preventing cgas from recognizing selfdna e9238323indexed in [current contentsclinical medicine] [sci expanded] [isi alerting system] [isi s master list] [index medicusmedline] [embaseexcerpta medica] [chemical scas]review sthis work is licensed under creative common attributionnoncommercialnoderivatives international cc byncnd 0czou rc research progress on circrnas in hcc med sci monit e923832circrnas regulate alternative splicing transcription and translationcellular localization of most circrnas is cytoplasmic which is the basis for the biological function of mirna and protein decoys several studies have suggested that circrnas participate in rna splicing assembly and biosynthesis recently research has shown that circrnas may play pivotal roles in regulating alternative splicing transcription and translation in addition the exon of the splicing factor may form a circrna by affecting formation of linear rna eicirnas interact with the u1 small nuclear ribonucleoproteinsnrnp thereby regulating parental gene transcription by binding to rna polymerase ii interestingly translation of circrnas is mediated by ires and n6methyladenosine m6a and translation efficiency of circrna is regulated by the level of m6a modification moreover circfbxw7 effectively inhibits glioma proliferation and cell cycle progression by antagonizing usp28induced cmyc stabilization potential to encode proteinscircrnas are implicated in numerous physiological processes and pathogenesis of diseases strong evidence indicates that circrnas can encode proteins by mimicking dna rolling circle amplification related studies indicate that circrna circppp1r12a plays a key molecular role by encoding a functional protein circppp1r12a73aa which promotes proliferation migration and invasion of colon cancer circanril interacts with pescadillo zebrafish homolog pes1 to mediate ribosome biogenesis and prerrna processing in vascular macrophages and smooth muscle cells these studies have significantly increased the knowledge base about the biological functions of circrnascircrnas in diseasescircrnas are involved in processes that lead to development of various disorders such as neuronal and cardiovascular diseases and cancers circrnas participate in regulating gene transcription and protein expression and are indirectly and directly associated with time and regionspecific variations as mentioned previously abnormal expression of circrnas is implicated in neurological disorders atherosclerosis and ribosomal rna maturation reportedly are regulated by circanril simultaneously some studies have suggested that circrnas upregulation significantly affects sprouting and proliferation of vascular endothelial cells and elicits vascular dysfunction recently several experiments have implicated circrnas in pathogenesis of cancer via activation of a series of cascade reactions however the underlying mechanism for the effect of circrnas in initiation and progression of tumors has not been fully clarified to date related studies have revealed that certain circrnas are highly expressed in tumor tissues and overexpression of circrnas promotes tumor proliferation and deterioration an investigation revealed that hsa_circ_002059 was downregulated in gastric cancer while hsa_circ_0004018 was upregulated in hcc meanwhile tumorspecific circrnas candidates were screened in lung adenocarcinoma tissue by microarrays and circrnas were identified downregulated and upregulated of the circrnas hsa_circ_0013958 clearly was positive correlated with lymph node metastasis and tnm stage these findings indicate that circrnas have important roles in tumor progression and may have potential for broad applicatoins in medicine scienceoverview of hcchcc is one of the most prevalent tumors worldwide with diagnoses and approximately deaths annually epidemiological survey data indicate that morbidity and mortality from hcc are gradually increasing risk factors for hcc include diabetes mellitus obesity smoking alcohol consumption older age male sex chronic hbv liver cirrhosis and chronic hepatitis c virus hcv the primary risk factors include liver cirrhosis viral hepatitis alcohol intake and obesity worldwide approximately hcc patients are infected with hepatitis b virus hbv or hcv in addition alcohol abuse is a crucial factor for onset of hcc [] obesity hypertension and diabetes are closely linked with development of hcc but specific correlations remain unknown moreover regular screening has been widely applied for early detection and to ensure effective treatment of hcc most commonly good results have been achieved with regular screening with ultrasonography blood alphafetoprotein content testing mri and ct generally surgical resection and chemotherapy are mainstays of therapy in patients with hcc yet some tumors cannot be fully removed which results in tumor growth invasion and metastasis local and systemic metastases are the main reasons for the unsatisfactory prognosis in patients with hcc therefore more effective therapeutic approaches need to be developedroles of circrnas in hccnumerous studies have documented the important role that circrnas play in tumorigenesis metastasis and invasion research has shown that circrnas are localized in the nucleus and interfere with transcription and promote alternative this work is licensed under creative common attributionnoncommercialnoderivatives international cc byncnd e9238324indexed in [current contentsclinical medicine] [sci expanded] [isi alerting system] [isi s master list] [index medicusmedline] [embaseexcerpta medica] [chemical scas]review s 0czou rc research progress on circrnas in hcc med sci monit e923832hsa_circ_0001649circzkscan1circitchwntbetacatenincircmto1mir9p21hsa_circ_00059836mir1295phmgb1 ragenfκbmir7hsa_circ_101368hsa_circ_001569cdr1ashsa_circ_0000673figure the function of circrnas in hcc carcinogenesis this graph demonstrates the role of circrnas in hcc carcinogenesis including positive and negative effects respectivelytable brief summary of circrnas as biomarkers for hccnamediseaseconclusiondoicirs7hsa_circ_0003570hsa_circ_0005075hepatocellular carcinomahepatocellular carcinomahepatocellular carcinomacirs7 was one of the independent factors and may be a promising biomarker for hepatic mvi and a novel therapy target for restraining mvi101007s0043201622567hsa_circ_0003570 expression levels were associated with hcc clinicopathological characteristics101002jcla22239hsa_circ_0005075 promotes proliferation migration and invasiveness of hcc via mir431 regulation101016jbiopha201801150splicing circpvt1 is overexpressed in gastric cancer tissues compared with nontumor tissues and circpvt1 acts as an oncogene to mediate expression of mir4975p however studies concerning the role of circrnas in development and progression of hcc remain in their infancytumor inhibitioncurrently circrnas are considered promising diagnostic biomarkers and ideal therapeutic targets for hcc studies have revealed that circitch inhibits tumor proliferation by suppressing the wntbetacatenin pathway expression of circitch has been positively correlated with good survival outcome in patients with hcc analysis of the circrnas expression profile in human hcc tissues showed that circmto1 was markedly decreased in hcc tissues and that expression of circmto1 was positively correlated with survival rate circmto1 reportedly inhibits hcc progress by sponging mir9 and thereby increasing p21 expression meanwhile overexpression of hsa_circ_0001649 negatively affects invasion and proliferation and promotes apoptosis of hcc cells downregulation of zkscan1 and circzkscan1 enhances cell proliferation and promotes progression of hcc tumor promotionin patients with hcc cdr1was more abundant in tumor specimens than in adjacent normal tissues cdr1as effectively suppresses the invasion and proliferation of hcc cells by targeting mir7 some reports have shown that hsa_circ_0000673 is significantly upregulated in hcc tissues and hsa_circ_0000673 downregulation markedly inhibits proliferation and invasion of hcc cells in vitro meanwhile a positive correlation was found between circ_001569 expression level and tumor size advanced tnm stages and unfavorable prognosis in patients with hcc circrna101368 was abundantly expressed in hcc tissue which correlated with poorer prognosis in addition circrna101368 inhibited cell migration by reducing protein levels in nfkb rage and hmgb1 figure e9238325indexed in [current contentsclinical medicine] [sci expanded] [isi alerting system] [isi s master list] [index medicusmedline] [embaseexcerpta medica] [chemical scas]review sthis work is licensed under creative common attributionnoncommercialnoderivatives international cc byncnd 0czou rc research progress on circrnas in hcc med sci monit e923832biomarkerconclusionsprevious studies have shown that circrnas are closely related to development of tumors clinicopathological features in patients with hcc are correlated to with levels of expression of cirs7 and its targeted mrnas global circrna expression profile analysis showed that hsa_circ_0005075 exhibited significant differences in tumor tissue versus adjacent tissues in patients with hcc expression of hsa_circ_0005075 also was related to tumor proliferation and metastasis therefore an increasing number of circrnas have been identified as diagnostic markers as summarized in table given the high incidence and mortality fo hcc worldwide it is one of the most serious diseases threatening human health increasing attention is being paid due to this serious situation evidence is increasing to support the close association between circrnas progression of hcc circrnas may play an important role in the occurrence and development of tumors however the molecular mechanism underlying the relationship between circrnas and hcc has not been fully elucidated therefore indepth research is needed on the potential regulatory relationships and to uncover regulatory patterns between circrnas and hcc so that new diagnostic markers for hcc can be developedreferences bray f ferlay j soerjomataram i global cancer statistics globocan estimates of incidence and mortality worldwide for cancers in countries cancer j clin feng rm zong yn cao sm xu rh current cancer situation in china good or bad news from the global cancer statistics cancer commun lond jemal a bray f center mm global cancer statistics cancer j clin li r jiang j shi h circrna a rising star in gastric cancer cell mol life sci salzman j gawad c wang pl circular rnas are the predominant transcript isoform from hundreds of human genes in diverse cell types plos one e30733 lukiw wj circular rna circrna in alzheimers disease ad front genet liu y yang y wang z insights into the regulatory role of circrna in angiogenesis and clinical implications atherosclerosis zhao y alexandrov pn jaber v lukiw wj deficiency in the ubiquitin conjugating enzyme ube2a in alzheimers disease ad is linked to deficits in a natural circular mirna7 sponge circrna cirs7 genes basel shen f liu p xu z circrna_001569 promotes cell proliferation through absorbing mir in gastric cancer j biochem song t xu a zhang z circrna hsa_circrna_101996 increases cervical cancer proliferation and invasion through activating tpx2 expression by restraining mir8075 j cell physiol min l wang h zeng y circrna_104916 regulates migration apoptosis and epithelialmesenchymal transition in colon cancer cells front biosci landmark ed verduci l strano s yarden y blandino g the circrnamicrorna code emerging implications for cancer diagnosis and treatment mol oncol wei j wei w xu h circular rna hsa_circrna_102958 may serve as a diagnostic marker for gastric cancer cancer biomark li p chen s chen h using circular rna as a novel type of biomarker in the screening of gastric cancer clin chim acta xin j zhang xy sun dk upregulated circular rna hsa_circ_0067934 contributes to glioblastoma progression through activating pi3kakt pathway eur rev med pharmacol sci kong z wan x zhang y androgenresponsive circular rna circsmarca5 is upregulated and promotes cell proliferation in prostate cancer biochem biophys res commun fu l chen q yao t hsa_circ_0005986 inhibits carcinogenesis by acting as a mir1295p sponge and is used as a novel biomarker for hepatocellular carcinoma oncotarget zhu x wang x wei s hsa_circ_0013958 a circular rna and potential novel biomarker for lung adenocarcinoma febs j zhang q wang w zhou q roles of circrnas in the tumour microenvironment mol cancer qu z jiang c wu j ding y exosomes as potent regulators of hcc malignancy and potential biotools in clinical application int j clin exp med memczak s jens m elefsinioti a circular rnas are a large class of animal rnas with regulatory potency nature cocquerelle c mascrez b hetuin d bailleul b missplicing yields circular rna molecules faseb j zhao x cai y xu j circular rnas biogenesis mechanism and function in human cancers int j mol sci qu s yang x li x circular rna a new star of noncoding rnas cancer lett bahn jh zhang q li f the landscape of microrna piwiinteracting rna and circular rna in human saliva clin chem hsu mt cocaprados m electron microscopic evidence for the circular form of rna in the cytoplasm of eukaryotic cells nature yu x odenthal m fries jw exosomes as mirna carriers formationfunctionfuture int j mol sci hanan m soreq h kadener s circrnas in the brain rna biol constantin l circular rnas and neuronal development adv exp med biol van rossum d verheijen bm pasterkamp rj circular rnas novel regulators of neuronal development front mol neurosci ebert ms sharp pa microrna sponges progress and possibilities rna ebert ms neilson jr sharp pa microrna sponges competitive inhibitors of small rnas in mammalian cells nat methods hansen tb jensen ti clausen bh natural rna circles function as efficient microrna sponges nature hsiao ky lin yc gupta sk noncoding effects of circular rna ccdc66 promote colon cancer growth and metastasis cancer res janga sc mittal n construction structure and dynamics of posttranscriptional regulatory network directed by rnabinding proteins adv exp med biol du ww yang w liu e foxo3 circular rna retards cell cycle progression via forming ternary complexes with p21 and cdk2 nucleic acids res xia p wang s ye b a circular rna protects dormant hematopoietic stem cells from dna sensor cgasmediated exhaustion immunity 701e7 li z huang c bao c exonintron circular rnas regulate transcription in the nucleus nat struct mol biol this work is licensed under creative common attributionnoncommercialnoderivatives international cc byncnd e9238326indexed in [current contentsclinical medicine] [sci expanded] [isi alerting system] [isi s master list] [index medicusmedline] [embaseexcerpta medica] [chemical scas]review s 0czou rc research progress on circrnas in hcc med sci monit e923832 yang y fan x mao m extensive translation of circular rnas driven by n6methyladenosine cell res yang y gao x zhang m novel role of fbxw7 circular rna in repressing glioma tumorigenesis j natl cancer inst abe n hiroshima m maruyama h rolling circle amplification in a prokaryotic translation system using small circular rna angew chem int ed engl zheng x chen l zhou y a novel protein encoded by a circular rna circppp1r12a promotes tumor pathogenesis and metastasis of colon cancer via hippoyap signaling mol cancer holdt lm stahringer a sass k circular noncoding rna anril modulates ribosomal rna maturation and atherosclerosis in humans nat commun gokul s rajanikant gk circular rnas in brain physiology and disease adv exp med biol idda ml munk r abdelmohsen k gorospe m noncoding rnas in alzheimers disease wiley interdiscip rev rna luo q chen y long noncoding rnas and alzheimers disease clin interv aging li cy ma l yu b circular rna hsa_circ_0003575 regulates oxldl induced vascular endothelial cells proliferation and angiogenesis biomed pharmacother chen j cui l yuan j circular rna wdr77 target fgf2 to regulate vascular smooth muscle cells proliferation and migration by sponging mir biochem biophys res commun kristensen ls hansen tb veno mt kjems j circular rnas in cancer opportunities and challenges in the field oncogene fu l yao t chen q screening differential circular rna expression profiles reveals hsa_circ_0004018 is associated with hepatocellular carcinoma oncotarget massarweh nn elserag hb epidemiology of hepatocellular carcinoma and intrahepatic cholangiocarcinoma cancer control salem r gilbertsen m butt z increased quality of life among hepatocellular carcinoma patients treated with radioembolization compared with chemoembolization clin gastroenterol hepatol 65e1 ozer ed suna n boyacioglu as management of hepatocellular carcinoma prevention surveillance diagnosis and staging exp clin transplant 15suppl lou w liu j ding b identification of potential mirnamrna regulatory network contributing to pathogenesis of hbvrelated hcc j transl med yang t hu ly li zl liver resection for hepatocellular carcinoma in nonalcoholic fatty liver disease a multicenter propensity matching analysis with hbvhcc j gastrointest surg nishibatake km minami t tateishi r impact of directacting antivirals on early recurrence of hcvrelated hcc comparison with interferonbased therapy j hepatol toyoda h kumada t tada t the impact of hcv eradication by directacting antivirals on the transition of precancerous hepatic nodules to hcc a prospective observational study liver int zhao j oneil m vittal a prmt1dependent macrophage il6 production is required for alcoholinduced hcc progression gene expr vandenbulcke h moreno c colle i alcohol intake increases the risk of hcc in hepatitis c virusrelated compensated cirrhosis a prospective study j hepatol fabris c toniutto p falleti e mthfr c677t polymorphism and risk of hcc in patients with liver cirrhosis role of male gender and alcohol consumption alcohol clin exp res vernon g baranova a younossi zm systematic review the epidemiology and natural history of nonalcoholic fatty liver disease and nonalcoholic steatohepatitis in adults aliment pharmacol ther bruix j reig m sherman m evidencebased diagnosis staging and treatment of patients with hepatocellular carcinoma gastroenterology zhang bh yang bh tang zy randomized controlled trial of screening for hepatocellular carcinoma j cancer res clin oncol verduci l ferraiuolo m sacconi a the oncogenic role of circpvt1 in head and neck squamous cell carcinoma is mediated through the mutant p53yaptead transcriptioncompetent complex genome biol yu j xu qg wang zg circular rna csmarca5 inhibits growth and metastasis in hepatocellular carcinoma j hepatol wang m yu f li p circular rnas characteristics function and clinical significance in hepatocellular carcinoma cancers basel guo w zhang j zhang d et al polymorphisms and expression pattern of circular rna circitch contributes to the carcinogenesis of hepatocellular carcinoma oncotarget han d li j wang h circular rna circmto1 acts as the sponge of microrna9 to suppress hepatocellular carcinoma progression hepatology qin m liu g huo x hsa_circ_0001649 a circular rna and potential novel biomarker for hepatocellular carcinoma cancer biomark yao z luo j hu k zkscan1 gene and its related circular rna circzkscan1 both inhibit hepatocellular carcinoma cell growth migration and invasion but through different signaling pathways mol oncol xu l zhang m zheng x the circular rna cirs7 cdr1as acts as a risk factor of hepatic microvascular invasion in hepatocellular carcinoma j cancer res clin oncol yu l gong x sun l the circular rna cdr1as act as an oncogene in hepatocellular carcinoma through targeting mir7 expression plos one e0158347 jiang w wen d gong l circular rna hsa_circ_0000673 promotes hepatocellular carcinoma malignance by decreasing mir7673p targeting set biochem biophys res commun liu h xue l song c overexpression of circular rna circ_001569 indicates poor prognosis in hepatocellular carcinoma and promotes cell growth and metastasis by sponging mir4115p and mir4325p biochem biophys res commun li s gu h huang y circular rna 101368mir200a axis modulates the migration of hepatocellular carcinoma through hmgb1rage signaling cell cycle shang x li g liu h comprehensive circular rna profiling reveals that hsa_circ_0005075 a new circular rna biomarker is involved in hepatocellular carcinoma development medicine baltimore e3811 yao t chen q shao z circular rna as a new biomarker for hepatocellular carcinoma metastasis j clin lab anal e22572e9238327indexed in [current contentsclinical medicine] [sci expanded] [isi alerting system] [isi s master list] [index medicusmedline] [embaseexcerpta medica] [chemical scas]review sthis work is licensed under creative common attributionnoncommercialnoderivatives international cc byncnd 0c' | Colon_Cancer |
" breast cancer bc is the most commonly diagnosed malignant cancer in women bc is the main cause of cancerrelated death in women and seriously threatens the life and health of women worldwide micrornas mirnasmirs have been reported to regulate the development and progression of different types of cancer however the regulatory functions of mir1885p in bc have not been thoroughly demonstrated in this present research we identified that mir5p was downregulated in bc tissues and several bc cell lines downregulation of mir5p was significantly associated with advanced tnm stage moreover we identified that mir5p mimics significantly inhibited proliferation using cck8 assay colony formation and xenograft animal model suppressed invasion and migration detected by transwell invasion assay and increased the cellular apoptosis of bc cells as determined by cell apoptosis assay moreover mir1885p mimics also reduced the expression of nfκb p65rel to further investigate its regulatory mechanism transcription factor zinc finger protein zfp91 was predicted as the targeted protein of mir1885p by bioinformatic method we confirmed their specific binding by dual luciferase dlr assay we demonstrated that the overexpression of mir1885p significantly inhibited the expression of zfp91 in bc cell lines and reduced the expression of nfκb p65rel an inverse correlation was found between the expression of mir1885p and zfp91 in bc tissues importantly we demonstrated that the restoration of zfp91 was able to block the effect of mir1885p on the progression of mdamb231 cells therefore our study showed that mir1885p may be one of the important indicators and could inhibit the progression of human bc via targeting the zfp91nfκb p65rel signaling pathway suggesting that mir1885p may be a promising future target for bc treatmentcorrespondence to dr zhaoyu liu department of radiology shengjing hospital of china medical university sanhao street shenyang liaoning pr chinaemail liuzy1226126comkey words breast cancer mir1885p zinc finger protein zfp91 proliferation apoptosisintroductionbreast cancer bc is one of the most commonly diagnosed malignancy in the world the mortality rate for bc ranks first among all female malignant tumors globally the number of newly diagnosed bc cases reached approximately million in accounting for almost of cancer cases among women bc exhibits a complex pathogenesis and is a clinically heterogeneous disease with a wide range of clinical behaviors and treatment responses although many dysregulated molecular pathways have been discovered in bc the development of effective therapeutic methods has been limited it is urgent to discover novel molecules to suppress bc proliferation induce apoptosis and inhibit invasion and provide potential therapeutic strategies to improve the survival and quality of life of bc patients micrornas mirnasmirs are a class of endogenous noncoding rnas of approximately nucleotides in length which are generally located in unstable regions of the human genome and are usually dysregulated in malignant tumors to regulate gene functions mirnas regulate target genes through binding to the 'untranslated regions 'utrs of the target mrnas subsequently inhibiting gene expression through regulation of the targeted proteins mirnas play an important role in many tumor cellular processes such as proliferation cell cycle apoptosis invasion and metastasis and participate in almost all signaling pathways mir1885p has been reported to be an inhibitor of tumor growth and metastasis in prostate cancer and hepatocellular carcinoma however to the best of our knowledge the functions of mir1885p in bc remain elusivein the present study we detected the expression of mir1885p in tumor tissues of bc patient tissues and several bc cell lines furthermore we investigated its regulatory role in bc proliferation apoptosis and invasion we also predicted and confirmed the targeted protein of mir5p transcription factor zinc finger protein zfp91 elucidating the regulatory mechanisms of mir1885p in bcmaterials and methodspatients and tissues one hundred paired bc tissue specimens including malignant and normal tissues used in this study were obtained from shengjing hospital of china medical university shenyang liaoning china during the period 0cyang mirna1885p inhibits proliferation and induces apoptosis in bc via zfp91from january to december with the informed consent of patients the age range of the patients was from to years with a mean age of years all experiments were approved by the ethics committee of shengjing hospital of china medical university no 2016ps18j the samples were snapfrozen in liquid nitrogen and stored at Ëc tnm staging system was performed for tumor grading of bc and for evaluating and staging of patients respectively which was carried out according to the 7th edition of the american joint committee on cancer ajcc tnm classification system cell cultivation the bc cell lines mdamb231 atcc crmhtb26 bt549 atcc htb122 and mcf7 atcc crl3435 were cultured in rpmi1640 medium sigmaaldrich merck kgaa containing heatinactivated fetal bovine serum fbs mp biomedicals penicillinstreptomycin invitrogen thermo fisher scientific inc no the nonmalignant mammary epithelial cell line mcf 10a atcc crl10317 was cultivated in dmemf12 ham's mixture supplemented with equine serum hyclone ge healthcare egf ngml insulin µgml hydrocortisone mgml and cholera toxin ngml all from sigmaaldrich merck kgaa all cells were incubated at Ëc in a humidified co2 atmosphererna isolation and quantitative qpcr total rnas from tissues or cells were isolated using rnx¢plus reagent cinnagen and cdna was synthesized using the primescript¢ rt reagent kit takara according to the manufacturer's instructions qpcr was performed using a sybr premix extaq¢ kit takara with the following primer sets on the abi qpcr system applied biosystems actin was used to normalize the relative expression of the target genes mirnas were detected through a miscript ii rt kit qiagen in a fluorescence thermal cycler biorad laboratories inc the primers for mir1885p and the reference gene u6 were purchased from novland biopharm the thermocycling condition were Ëc for min followed by cycles of Ëc for sec and Ëc for 1min followed by a hold at Ëc the relative expression ratio of mir5p was quantified using the 2δδcq method the relative expression of mir1885p was normalized to u6 the primer sequences are listed in table iplasmid preparation the coding region of human zfp91 was amplified from human breast cancer cell mdamb cdna library by pcr then we cloned the prepared zfp91 fragment into pcmvtag2b stratagene to obtain pcmvtag2bzfp91 the primer sequences are listed in table icell transfection the mir1885p mimics and mirnc were purchased from thermo fisher scientific inc firstly lipofectamine transfection reagent thermo fisher scientific inc was used to transfect mir1885p mimics mirnc into mdamb231 cells in accordance with the manufacturer's instructionsafter the whole detection the pcmvtag2b vector pc was transfected into mdamb231 cells with mirnc ncpc or mir1885p mimics pcmir1885p pcmvtag2bzfp91 was transfected into mdamb cells with mir5p mimics mir5pzfp91 as the cells were grown to mir mimics or vector was transfected into cells according to the manufacturer's instructions then cells were cultivated for up to h finally the total rna and protein were extracted and properly stored for further researchcell proliferation assay cell counting kit8 cck8 kit dojindo was performed to detect cell proliferation based on the reduction of wst to wst formazan briefly the mdamb231 cells were seeded in a 96well plate at a density of 5x103 cellswell on the day of the experiment the cells were transfected with empty vector and mir1885p cck8 reagent was added into the culture medium at the indicated time and incubated for min the absorbance at nm was measured by a microplate readercolony formation assay mdamb231 cells from the different treated groups were seeded in a 60mm dish containing cells followed by a 14day cultivation at Ëc with co2 the supernatant was discarded and cells were washed twice with pbs the colonies were fixed in paraformaldehyde for min and then stained with giemsa staining solution solarbio science technology co ltd beijing china for min colonies were counted and images were captured under an inverted microscope nikon tokyo japan this assay was repeated timescell apoptosis assay mdamb231 cells were stained by annexin valexa fluor488propidium iodide pi staining to identify the apoptotic mdamb231 cells after transfection with mir1885p for h mdamb231 cells were stained with annexin valexa fluor488 for min on ice followed by the addition of pi solution for the secondary staining process all experimental procedures were strictly protected from lights the data were calculated by flowjo software v87 tree star after facs calibur bd biosciences analysiscell migration and invasion assays after the counting mdamb231 cells in the different groups were inoculated equally at a density of 5x105 cellsml in the upper compartments of polycarbonate membrane filters cell migration and invasion assays were performed uncoated for the migration assay and coated with matrigel bd biosciences for the invasion assay after h the migrated and invaded cells in the membrane were fixed with methanol and then stained with crystal violet for min at room temperature cells were observed under a light microscope with magnification x100western blotting protein samples extracted from tissues or cultivated cells were lysed in ripa buffer containing protease and phosphatase inhibitor cocktail and incubated at Ëc followed by the quantified measurement of protein using bca kit fujifilm wako pure chemical corp after protein samples µgeach sample were loaded and separated on sdspage gels for electrophoresis the proteins were then transferred onto a polyvinylidene difluoride pvdf membranes millipore usa the membranes were blocked in wv skim milk for h at room temperature and incubated at Ëc overnight with primary antibodies antizfp91 dilution 0concology reports bidirectional primer sequencetable i primer informationgene name f 'ccctctctcacatcccttgcat3'mir1885p r 'atcctgcaaaccctgcatgtg3' f 'tgagacctacaaaccccactt'zfp91 r 'ccttttgggtaaacgtggacttt3'homoactin f 'ttcctccgcaaggatgacacgc3'r 'ccttttgggtaaacgtggacttt3' f 'cgggtttgttttgcatttct3'u6 snrna f 'agtcccag catgaacagctt3'zfp91 zinc finger protein homolog f forward r reverse abcam ab30970 and antivimentin dilution cell signaling technology inc antiecadherin dilution cell signaling technology inc ncadherin dilution cell signaling technology inc matrix metalloproteinase mmp2 dilution cell signaling technology inc mmp9 dilution cell signaling technology inc nfκb p65 dilution cell signaling technology inc relb dilution cell signaling technology inc and gapdh dilution cell signaling technology inc as internal control on the following day all membranes were incubated with antirabbit igg hrplabeled secondary antibodies dilution cell signaling technology inc finally the signals were detected and analyzed with the application of luminata forte western hrp substrate millipore in the biorad chemidox xrs imaging system biorad laboratoriesluciferase reporter assay to further investigate the specific correlation between mir5p and zfp91 targetscan wwwtargetscanorgmamm_31 and miranda wwwmicrornaorgmicrornahomedo were performed the zfp91 was selected to be the predicted targeting of mir1885p the fragments of the 'utr of zfp91 containing mir5p binding sites and its mutants were amplified by pcr and then the pcr products were inserted into pmirglo dualluciferase mirna target expression vector promega corp the reporter and control vector were transfected into 293t cells using lipofectamine thermo fisher scientific inc after cultivation for h the relative luciferase activity was examined by the dualluciferase reporter assay kit thermo fisher scientific inc according to the manufacturer's instructionspreparation of tumor xenograft animal model and treatment with mir5p mimics thirtysix nude mice female weighing ± g were purchased from huafukang biotech beijing china the experiments were performed in the animal facility at the department of laboratory animal science of china medical university and approved by the animal ethics committee of shengjing hospital approval no nude mice were randomly divided into a control group n12 mir1885p group n12 and nc group n12 a density of 5x106 cells in logarithmic phase were transfected with 1x pbs control group nc or mir1885p then the different groups of cells were resuspended in 1x pbs and injected into the nude mice respectively then tumor size was measured every days using a slide caliper and the tumor volume v was calculated using the formula vlength x width22 after days the mice were euthanasia by cervical dislocation and the tumors were excised imaged weighed and stored properly for further investigationsstatistical analysis graphpad prism graphpad software inc was used to perform statistical analysis the results are represented as mean ± sd of at least independent experiments the comparisons between groups were evaluated by student' ttest oneway anova followed by tukey test was used to evaluate the differences for multiple comparisons the statistical significance of correlations between mir5p and zfp91 expression in bc tissue were analyzed by pearson's correlation coefficient p005 was considered to indicate a statistically significant differenceresultsmir5p is signif\ufeff\ufefficantly decreased in bc tissue and cell lines firstly we analyzed the expression level of mir1885p in cases of bc tissues and adjacent counterparts by rtqpcr the results showed that the level of mir1885p in bc tissues was significantly lower than that in the normal adjacent counterparts fig 1a p005 we also found that mir1885p was correlated with bc tnm stage fig 1b p005 the expression level of mir5p in advanced bc tumors was lower than that in early stage tumors suggesting that mir1885p is inversely correlated with the malignancy of bcwe also compared the expression level of mir1885p in the nonmalignant mammary epithelial cell line mcf10a and bc cell lines mdamb231 bt549 and mcf7 our data showed that the levels of mir1885p in the mdamb231 bt549 and mcf7 cells were lower than that in the mcf10a cells fig 1c p005 meanwhile the lowest mir1885p expression was detected in mdamb231 therefore mdamb231 cells were selected for further experimentsmir5p inhibits proliferation induces cell apoptosis and suppresses migration and invasion of bc cells as the expression of mir1885p in both bc cell lines and tumor tissues of bc patients were clearly downregulated we sought to investigate the effects of mir1885p on bc development by using both in vitro bc cell line cultivation and in vivo mouse tumor xenografts as shown in fig 2a transfection of mdamb cells with mir5p mimics significantly elevated the expression level of mir1885p when compared to the control and mirnc groups p005 importantly the increased level of mir5p in mdamb cells significantly inhibited the cell proliferation when compared to the control and mirnc groups fig 2b and c p005 it was also observed that the apoptotic mdamb231 cell numbers were significantly increased by the upregulation of mir5p when compared to the control and mirnc groups fig 2d 0cyang mirna1885p inhibits proliferation and induces apoptosis in bc via zfp91figure mir1885p is downregulated in bc tissue and bc cell lines a rtqpcr results showed that the level of mir1885p in bc tissues was downregulated compared with counterpart bc tissues the comparisons between groups were evaluated by student's ttest b relative expression level of mir1885p in patients at different clinical stages c relative expression level of mir1885p in bc cell lines mdamb231 bt549 and mcf7 relative to the normal human breast epithelial cell line mcf10a oneway anova followed by tukey's test was used to evaluate the difference for multiple comparisons in b and c all data are presented as means ± sd n3 bc breast cancerp005 importantly mir5p mimics significantly inhibited the invasion and migration abilities of the mdamb23 cells under transwell assay detection when compared to the control and mirnc groups fig 2e p005 moreover mir5p mimics significantly enhanced the expression of vimentin and ncadherin and reduced the level of ecadherin when compared to the control and mirnc groups fig 2f p005 the matrix metalloproteinases mmp2 and mmp9 mmp29 possess the ability to hydrolyze components of the basement membrane and stimulate tumor growth metastasis and epithelialmesenchymal transition emt mir5p mimics were demonstrated to significantly inhibit the expression of mmp2 and mmp9 fig 2f p005 these data provide robust evidence that mir1885p inhibits the tumor proliferation induces apoptosis reduces tumor invasion and migration and inhibits emt of bc which may be through the regulation of mmp29 expressionzfp91 is the downstream target of mir5p to further investigate the specific correlation between mir1885p and zfp91 targetscan wwwtargetscanorgmamm_31 and miranda wwwmicrornaorgmicrornahomedo were performed the results predicted that mir1885p possesses the binding sites of zfp91 fig 3a hence we sought to discover the regulatory mechanisms of mir1885p on bc development through targeting on zfp91 as hypothesized the upregulation of mir5p in mdamb cells decreased zfp91 mrna and protein levels when compared to the mirnc and control groups fig 3b p005 moreover the luciferase assay confirmed that mir1885p specifically binds to the 'utr of zfp91 fig 3c p005 it was also discovered that the injection of mdamb231 transfected with mir1885p mimics in tumor xenograft mice inhibited zfp91 expression fig 6b p005 these results suggested that zfp91 is the downstream target gene of mir1885p in bcmir5p regulates bc cell progression through targeting zfp91 to further investigate the biological functions of mir1885p in bc development we established a zfp91overexpressing mdamb cell line the expression of zfp91 was confirmed by rtqpcr fig s1 p005 with this system inhibition of zfp91 by mir5p mimics was reversed fig 4a p005 then it was found that the cotransfection of mdamb cells mir5pzfp91 group significantly enhanced the cell proliferation compared to that in pcmir5p group fig 4b and c p005 significantly suppressed cell apoptosis fig 4d p005 and significantly promoted invasion migration fig 4e p005 and emt fig 4f p005 in contrast with the monotransfection of mir1885p mimics in mdamb231 cells moreover the regulatory role of mir1885p on mmp2 and mmp9 was also reversed by overexpression of zfp91mir5p and zfp91 are correlated in tumor tissues of bc patients furthermore we examined the levels of zfp91 in the tumor tissues and adjacent normal tissues of bc patients the aberrantly high level of zfp91 was observed in the tumor tissues of the bc patients fig 5a p005 spearman's correlation analysis showed a significantly inverse correlation between mir5p and zfp91 in the bc patient tissues fig 5b p005 taken together these results further confirmed that the proliferation and apoptosis of bc is regulated by mir5pzfp91mir5p inhibits the proliferation of mdamb cells and reduces the expression of zpf91 in a bc xenograft mouse model moreover to evaluate the regulatory role of mir1885p in a bc xenograft mouse model we injected the mdamb231 cells transfected with mir1885p mimics or mirnc into nude mice the results showed that mir1885p mimics inhibited the tumor volume and weight compared to the mirnc group fig 6a p005 protein expression and the mrna level of zpf91 were also suppressed by mir1885p mimics in the tumor tissues of the xenograft mouse model when compared with the mirnc and control groups fig 6b p005mir5pzfp91 axis regulates nfkbp65 and relb expression numerous studies have reported that zinc finger protein zfp91 promotes proliferation and tumorigenesis 0concology reports figure effect of mir1885p on bc cell line mdamb231 mdamb231 cells were transfected with mir1885p mimics and mirnc a the mrna levels of mir1885p in mdamb231 cells were determined by rtqpcr p005 the proliferation of mdamb cells was examined by b cck kit post transfection and c colony formation p005 compared to control group p005 compared to mirnc group d the apoptotic mdamb cells were analyzed using annexin vpi staining and facs p005 e the invasion and migration capability of mdamb cells were detected by transwell assay p005 f expression of ecadherin ncadherin vimentin mmp2 mmp9 and nfκb p65rel were detected by western blotting the data are represented as the mean ± sd n3 p005 oneway anova followed by tukey's test was used to evaluate the difference for multiple comparisons bc breast cancer mmp matrix metalloproteinase nc negative controlof different cancer types via regulation of the nfκb p65 pathway therefore to further investigate the regulatory mechanism of the mir5pzfp91 axis we detected the expression of nfκbp65 and relb in bc cells the results showed that mir5p mimics significantly reduced the expression of nfκbp65 and relb together fig s2a p005 moreover the cotransfection of mir5p mimics and zfp91 also upregulated the expression levels of 0cyang mirna1885p inhibits proliferation and induces apoptosis in bc via zfp91figure zfp91 is a target of mir5p a zfp91 provides binding sites with mir5p b rtqpcr and western blotting were used for analysis of transcription and translation of zfp91 in vitro and in vivo p005 c the binding between mir5p and zfp91 was confirmed by luciferase assay p005 relative gene expression was normalized by gapdh expression the data are represented as the mean ± sd n3 oneway anova followed by tukey's test was used to evaluate the difference for multiple comparisons in b the comparisons between two groups were evaluated by ttest in c zfp91 zinc finger protein nfκbp65 and relb compared to the monotransfection of mir1885p mimics fig s2b p005 in summary these results illustrated that the mir5pzfp91 axis regulates the progression of bc via the noncanonical nfκb signaling pathwaydiscussionbreast cancer bc is one of the most common types of tumors diagnosed in women worldwide bc is the second leading cause of cancerrelated mortality worldwide in more than women were diagnosed with bc in china and almost percent of all newly diagnosed cancer cases were in women however the molecular mechanisms of bc still await elucidation and effective molecular targets for the diagnosis and treatment of bc are urgently required recently research has reported that mirnas are small noncoding rna molecules which regulate target protein expression to play critical roles as tumorpromotors or suppressors several studies have demonstrated that mir1885p promotes cell proliferation migration and metastasis in gastric cancer and hepatocellular carcinoma moreover iwakawa detected higher expression of mir1885p in stage iii breast cancer and tnbc wang reported that circulating mir1885p was upregulated in bc patients and associated with tnm of bc interestingly mir1885p was downregulated in bc mdamb231 and mcf7 cells moreover using gainof and lossoffunction analyses of mir1885p in breast cancer cells the authors demonstrated that mir1885p inhibited the proliferation and invasion of bc mdamb231 cells via targeting il6st however we demonstrated that the expression of mir1885p was drastically downregulated in bc tissue specimens which was also decreased in bc cell lines mdamb231 bt549 and mcf7 compared to normal breast epithelial cell line mcf10a moreover the downregulation of mir5p was significantly 0concology reports figure mdamb cell proliferation and apoptosis are regulated by mir5pzfp91 pcmvtag2b vector pc was transfected into bc mdamb cells with mirnc ncpc or mir5p mimics pcmir5p pcmvtag2bzfp91 was transfected into mdamb cells with mir5p mimics mir5pzfp91 a the mrna levels of mir5p in the cotransfected overexpressing zfp91mdamb cells were quantified by rtqpcr p005 cell proliferation was measured by b the cck kit and c colony formation assay p005 compared to control group p005 compared to mirnc group d cell apoptosis was detected by annexin vpi staining and facs p005 e the invasion and migration capability of mdamb231 cells were detected by transwell assay p005 f expression of ecadherin ncadherin vimentin mmp2 mmp9 and nfκb p65rel were detected by western blotting relative genes expression was normalized by gapdh expression the data are represented as the mean ± sd n3 p005 oneway anova followed by tukey's test was used to evaluate the difference for multiple comparisons bc breast cancer zfp91 zinc finger protein mmp matrix metalloproteinase nc negative control 0cyang mirna1885p inhibits proliferation and induces apoptosis in bc via zfp91figure mrna and protein levels of zpf91 in bc tissues a the mrna and protein levels of zpf91 in bc tissue and corresponding nontumor normal tissue were quantified using rtqpcr and western blot analysis the comparisons between two groups were evaluated by ttest n noncancer tissue c cancer tissue p005 b correlation between zpf91 mrna and mir5p was analyzed using pearson's correlation coefficient the data are represented as the mean ± sd n100 bc breast cancer zfp91 zinc finger protein figure the regulatory role of mir1885p in a bc xenograft mouse model bc mdamb231 cells were transfected with mir1885p mimics and mirnc and then injected into nude mice a the tumor volume and weight in the tumor xenograft mouse were compared among the mir1885p mimics group mirnc group and control group p005 compared to control group p005 compared to mirnc group b the protein expression and mrna level of zpf91 were detected by c western blotting and rtqpcr the data are represented as the mean ± sd n3 p005 oneway anova followed by tukey's test was used to evaluate the difference for multiple comparisons bc breast cancer zfp91 zinc finger protein nc negative controlassociated with advanced tnm stage however to investigate the relationship of mir1885p and bc patient prognosis we found that kaplanmeier analysis of mir1885p was limited due to the small sample size in tcga these results illustrated that the downregulation of mir1885p may be related with bc progression in the clinic suggesting that mir1885p may be a valuable bc diagnostic indicator moreover we confirmed that mir1885p mimics considerably inhibited the proliferation induced the apoptosis and inhibited the invasion of bc cells suggesting that mir1885p plays an inhibitory role in bc cellstranscription factor zinc finger protein zfp91 was firstly identified in the mouse in which was found to be overexpressed in colon liver prostate stomach and breast cancer zfp91 has a molecular mass of kda with amino acids containing five zincfinger motives a leucine zipper a coiledcoil structure and nuclear localization sequences zfp91 was confirmed to be a transcription factor located in the cellular nucleus ma reported that zfp91 functions as an oncogene in cancer development by activating hif1α transcription the overexpression of zfp91 0concology reports was also found to result in the promotion of nkκb signaling pathway activation through increasing nkκb inducing kinase nik whose activity and overexpression are related to cancer progression in melanoma pancreatic breast and lung cancer the inhibition of zfp91 was demonstrated to promote apoptosis in bc stomach cancer cells colon cancer and endometrial cancer in addition the overexpression of zfp91 was found to increase the cancer cell growth rate and metastatic capability zfp91 was also reported to interact with cyclindependent kinase inhibitor 2a cdkn2a which is an alternative reading frame arf tumor suppressor inhibiting the induction of p53dependent cell death to illuminate the molecular mechanisms of mir188 we predicted that il6st foxn2 zfp91 may be the targets of mir5p using targetscan and miranda furthermore peng reported that zfp91 is the target protein of mir5p in gastric cancer in addition overexpression of mir1885p was confirmed to inhibit the progression of breast cancer thus we chose the reported oncogene zfp91 for further investigation in the present study we confirmed that the 'untranslated region 'utr of zfp91 was bound by mir5p through dual luciferase assay moreover transfection of mir1885p mimics in mdamb cells reduced the zfp91 mrna and protein levels together mirnas usually bind to the 'utrs of target mrnas and do not reduce the level of mrnas however mirnas also were reported to decay the target mrnas and decease mrna level restoration of zfp91 largely reversed the decreased proliferation and induced apoptosis which were both regulated by mir1885p overexpression moreover in the tumor xenograft mouse model we observed that the expression of zfp91 was downregulated by an increased level of mir1885p furthermore the expression of mir5p and zfp91 were negatively correlated in bc patient tissues therefore our studies confirmed that mir5p can inhibit the progression of human bc via targeting zfp91zfp91 has been reported to promote proliferation in colon cancer prostate cancer and gastric cancer ma reported that zfp91 activates nfkappabp65 to promote proliferation and tumorigenesis of colon cancer paschke identified that zfp91 is a noncanonical nfκb signaling pathway regulator with oncogenic properties in prostate cancer in the present study we also confirmed that a decrease in zfp91 could significantly inhibit nfκbp65 and relb expression in bc cells therefore mir1885p overexpression reduced zfp91 via the noncanonical nfκb signaling pathway to inhibit the progression of bcin conclusion our data showed that mir1885p is downregulated in bc cell lines and tissues and the downregulated expression of mir1885p is associated with the poor prognosis of patients with bc we further investigated that overexpression of mir1885p could inhibit proliferation and induce the apoptosis of mdamb cells furthermore zfp91 was predicted and confirmed as a target gene of mirna5p and the effects of mir1885p on bc cells were dependent on the inhibition of zfp91 additionally a decrease in zfp91 significantly inhibited the nfκbp65 and relb expression in bc cells moreover the expression levels of mir5p and zfp91 were highly correlated with bc progression therefore we suggest that mir1885p can inhibit breast cancer progression via the zfp91nfκbp65 axis and may be a potential diagnostic indicator for bcacknowledgementsnot applicablefundingthis study was supported by the national natural science foundation of china grant nos and and talent projectavailability of data and materialsthe datasets used and analyzed during the current study are available from the corresponding author upon reasonable requestauthors' contributionszy and zl conceived and designed the study zy zc gy performed the experiments zy wrote the paper zy zl zc and gy reviewed the results and data and edited the manuscript all authors read and approved the manuscript and agree to be accountable for all aspects of the research in ensuring that the accuracy or integrity of any part of the work are appropriately investigated and resolvedethics approval and consent to participatetissues used in this study were obtained from shengjing hospital of china medical university shenyang liaoning china with the informed consent of patients and all experiments were approved by the ethics committee of shengjing hospital of china medical university no 2016ps18jpatient consent for publicationnot applicablecompeting intereststhe authors declare that they have no competing interestsreferences davidson ne armstrong sa coussens lm cruzcorrea mr deberardinis rj doroshow jh foti m hwu p k | Colon_Cancer |
purposeconjunctival squamous cell carcinoma scc is primarily treated with surgical resectionscc has various stages and local recurrence is common the purpose of this study was todetermine molecular localization of epidermal growth factor receptor egfr and the possibility of egfr as a biomarker for the management of conjunctival sccmethodsin this retrospective study we performed immunohistochemistry to evaluate egfr expression and localization in tumor cells egfr mutationspecific expression e746a750del andl858r and human papillomavirus expression in a series of conjunctival sccsresultsall tumors in our cohort were egfr positive twentyone of tumors showed focal egfr staining and seven showed diffuse egfr staining in additionwe calculated the percentages of the two most important mutations in egfr exon a750del exon l858r mutant in conjunctival sccs weobserved that the translocation of egfr from the membrane into the cytoplasm was relatedto clinical prognosis as we detected correlations between egfr cytoplasmic staining andfinal orbital exenteration and between decreased egfr membrane staining and progressionfree survivala1111111111a1111111111a1111111111a1111111111a1111111111open accesscitation sakai a tagami m kakehashi akatsuyamayoshikawa a misawa n wanibuchi h expression intracellular localizationand mutation of egfr in conjunctival squamouscell carcinoma and the association with prognosisand treatment one e0238120 101371 pone0238120editor sanjoy bhattacharya bascom palmer eyeinstitute united statesreceived april accepted august published august peer review history recognizes thebenefits of transparency in the peer reviewprocess therefore we enable the publication ofall of the content of peer review and authorresponses alongside final published s theeditorial history of this is available here101371 pone0238120copyright sakai this is an openaccess distributed under the terms of thecreative commons attribution license whichpermits unrestricted use distribution andreproduction in any medium provided the originalauthor and source are crediteddata availability statement all relevant data arewithin the manuscript and supporting informationfiles one 101371 pone0238120 august one 0cfunding none of the authors have any proprietaryor financial interests to declarecompeting interests none of the authors haveany proprietary or financial interests to declaresegfr is important in the pathology of ocular surface squamous neoplasia including sccand is a prognostic factor increased understanding of egfr mutations may have importantimplications for future treatment optionsegfr in conjunctival squamous cell carcinomaintroductionocular surface squamous neoplasia ossn includes several diseases such as conjunctival premalignant dysplasia carcinoma in situ and invasive conjunctival squamous cell carcinomascc the annual incidence of ossn was casesmillionyear conjunctival intraepithelial neoplasia casesmillionyear scc casesmillionyear in the united kingdom [ ] in the united states the rate of scc is 5fold higher among males and whites other previous research revealed that the risk increases with exposure to direct daylightand in outdoor workers metaanalysis demonstrated an association with human immunodeficiency virus odds ratio and human papillomavirus hpv odds ratio howeverno large epidemiological studies have been performed on people living in the far eastscholz examined clinicopathological factors and biomarkers and identified promotermutations in telomerase reverse transcriptase in of samples of conjunctival ossn associated with ultraviolet light induction recent research demonstrated that pdl1 isexpressed in almost half of conjunctival scc cases and noted the potential application ofimmune checkpoint blockade as a treatment strategy for conjunctival scc molecular targeted therapy is now used to treat most carcinomas and its use is continuingto increase uveal melanoma also has recently been reported in the ocular oncology area gefitinib is a relatively old tyrosine kinase inhibiter tki that is used as a molecular targeted therapy and its effects have been reported in various carcinomas on the other hand nobasic clinical studies on ocular tumors have been reported [] in our current study weinvestigated epidermal growth factor receptor egfr expression in our cases to assess thepossible effect of gefitinib we also examined the molecular expression and intracellular localization of egfr in conjunctival scc in east asian patientsmaterials and methodsselection of cases and collation of clinicopathologic datathis study was approved by the institutional review boards of osaka city university andkobe kaisei hospital and adhered to the tenets of the declaration of helsinki writteninformed consent was obtained from all patients before enrollment we identified patientstreated by ophthalmologists aa mt between november and july from whom wewere able to procure tissue blocks with residual tumor for each patient we collected demographic features age at initial diagnosis and at presentation to our institution and sex andprimary tumor features disease status at presentation [primary or recurrent] and in situ versusinvasive disease the american joint committee on cancer ajcc stage local recurrenceanatomic site and date metastases regional or distant and date vital status at last followup cause of death types of surgery and adjuvant therapy were also recordedimmunohistochemistry ihcimmunohistochemical studies for egfr and hpv were performed on 6μmthick tissue sections using the following antibodies antihuman egfr rabbit monoclonal antibody clone one 101371 pone0238120 august one 0cegfr in conjunctival squamous cell carcinomasp84 414r14 cell marque rocklin ca usa antihpv mouse monoclonal antibodyclone k1h8 ab75574 abcam cambridge uk horseradish peroxidaseconjugated antirabbit igg hl goat polyclonal antibody histofine nichirei corporation tokyojapan and horseradish peroxidaseconjugated antimouse igg hl goat polyclonal antibody histofine nichirei corporationegfr mutationspecific immunohistochemical staining was performed on cases as primary antibodies we used egfr e746a750del cell signaling technologies danversma usa and egfr l858r cell signaling technologies which were manuallyapplied to the slides stained sections were viewed with an olympus bx53dp74as controls for staining benign conjunctival lesions were also stained for egfr and coloncancer samples were stained as a positive controlimage analysisslides immunostained for egfr egfr mutations and hpv were evaluated in a blinded manner by two specialists mt and ak egfr expression was visually estimated as the percentageof tumor cells with complete or partial membranous staining tumors with egfr staining in� of tumor cells were considered the diffuse staining type diffuse type and those with of tumor cells were considered the focal staining type focal type the presence orabsence and intensity of cell membrane staining were semiquantitatively divided into groupswith a score of none weak strong very strong the presence or absence andintensity of cell cytoplasmic staining were also divided into groups with a score of andsemiquantitatively analyzed none weak strong very strong egfr mutationspecific immunostaining was divided into two groups those with immunostaining that wasclearly present and those without immunostainingslides immunostained for hpv were assessed with visual evaluation for the presence ofpunctate nuclear signals within tumor nuclei at magnification and were scored as positive or negativeegfr expression in tumorsegfr expression in the tumor was analyzed with nanostring analysis archival formalinfixedparaffinembedded tumor tissue was retrieved and manually macrodissected total mrnawas isolated from the macrodissected tumor tissues using a qiagen mirneasy kit qiagenvalencia ca usa according to the manufacturers instructions the rna sample was quantified with nanodrop thermo scientific wilmington de usa and regarded as adequate ifit contained ng at minimum the sample was subsequently analyzed with the ncounterpancancer progression panel nanostring seattle wa usa according to the manufacturers instructions nanostring data processing was done with the r statistical programmingenvironment v342 considering the counts obtained for positive control probe sets rawnanostring counts for each gene were subjected to technical factorial normalization whichwas carried out by subtracting the mean counts plus two times the standard deviation from thecodeset inherent negative controls subsequently biological normalization using the includedmrna reference genes was performed additionally all counts with p after a onesidedttest versus negative controls plus two times the standard deviation were interpreted as notexpressed over basal noisestatistical analysisthe clinical and histopathologic characteristics were summarized using descriptive statisticscorrelations between immunohistochemical demographic and clinicopathologic factors were one 101371 pone0238120 august one 0cegfr in conjunctival squamous cell carcinomaassessed using the wilcoxon rank sum and fishers exact tests progressionfree survival pfswas defined as the time from surgery to disease recurrence or death from any cause coxregression modeling was used to evaluate correlations between clinicopathologic and immunohistochemical features and survival outcomes statistical analyses were performed usingspss statistics version software ibm japan tokyo japan values of p were considered statistically significantresultsclinicopathologic findings of our cohort are summarized in table all patients in ourcohort were east asian and included men and women with a mean age at presentation of years fourteen patients had invasive scc and had an in situtumor primary orbital exenteration was necessary for local disease control in two patients and two patients underwent additional orbital exenteration nine patients table clinicopathologic findings of cases of conjunctival squamous cell carcinomaage years mean rangesexmalefemalefollowup after primary surgery months rangetstage ajccall n n tist1t2t3t4primary surgery typelocal excisionorbital exenterationadjuvant therapynoyesadditional excisiontopical chemotherapyradiation therapyimmunohistochemical markershpv status in tumor cellsnegativepositiveegfr expression in tumorsdiffuse stainingfocal stainingnegativecell membrane egfr expression in tumorsvery strongstrongweak one 101371 pone0238120 august continued one 0ctable continuednegativecell cytoplasm egfr expression in tumorsvery strongstrongweaknegativeoutcomeorbital exenterationyesnolocal recurrence after curative therapyyesnometastasisdistantregional distantregionalnonevital status at last followupdeadalivecause of deathconjunctival scc metastasisother101371 pone0238120t001egfr in conjunctival squamous cell carcinomaall n n underwent adjuvant therapy most commonly additional local surgery topical chemotherapyand radiation therapy were performed in one patient in the adjuvant therapy group of thisgroup one patient died with disease months after diagnosis of regional and lung metastasesthe other patient was alive without disease at months after diagnosis of regional metastasestwo patients died one of which was due to conjunctival scc described above ninepatients experienced local recurrence after curative surgeryall tumors were egfr positive in our cohort twentyone of tumors showed focal egfr staining and seven showed diffuse egfr staining fig analysisof egfr intracellular staining patterns showed scores of for membrane staining and for cytoplasmic staining no significant difference was found between carcinoma in situ tisand invasive carcinoma tadv table no significant difference was found in the scoredepending on the stage egfr expression in colon cancer was used as a positive control fig2aon the other hand seven benign conjunctival lesions three pinguecula three pterygiumone dermoid cyst showed partial weak positive staining in conjunctival squamous epithelialcells especially on the cell membrane fig 2b in addition cytoplasmic staining was seen inonly one case benign cases showed scores of for membrane staining and for cytoplasmic staining cytoplasmic staining patterns were significantly different in benign compared to scc cases p table the correlation between egfr staining focal ordiffuse and egfr localization cytoplasmic staining group was not significantly different one 101371 pone0238120 august one 0cegfr in conjunctival squamous cell carcinomafig egfr expression in conjunctival scc focal egfr staining a and diffuse egfr staining b scale bar μm inset corresponding field in ahematoxylineosinstained section membrane staining very strong c and cytoplasm staining very strong d scale bar μm101371 pone0238120g001but the diffuse egfr group tended to have a higher score p and respectivelytable egfr e746a750 del and egfr l858r expression were assessed with immunohistochemistry in all patients fig the mutation at exon egfr e7446a750 del was confirmedin cases and that at exon egfr l858r point mutation was confirmed in cases with ihc table the relationship between egfr mutation and egfr stainingtable staining patterns of egfrcell membranetis in situtadv invasiven n benign tumorn cell cytoplasmtis in situtadv invasiven n benign tumorn �p value based on the nonpaired ttest101371 pone0238120t002staining patterns n totaltotalaverageaveragepp� one 101371 pone0238120 august one 0cegfr in conjunctival squamous cell carcinomafig a egfr expression in colon cancer as a positive control scale bar μm b egfr expression in a control benign lesion pinguecula scale bar μminset corresponding field in a hematoxylineosinstained section101371 pone0238120g002focal or diffuse was determined using univariate linear regression analysis with correctionfor age p regarding egfr expression in tumors we compared the tis and tadv groups according toajcc t grading n4 no significant difference was found p fig the majority of patients in our cohort were hpv negative n table the positive rate of hpv immunoreactivity increased with increases in ajcc t grading but the correlation was not statistically significantthe cox regression model was used to examine and analyze the relationship between longterm prognosis including orbital exenteration and pfs and the clinicopathological statusegfr staining pattern and egfr mutation univariate cox regression analyses revealed significant correlations between egfr cytoplasmic staining and final orbital exenteration hazard ratio hr p table additionally a significant correlation was seenbetween the t stage ajcc and pfs and between egfr membrane staining and pfs hr p p respectively table local recurrence distant metastasisrate and overall survival rate were not statistically significant in addition the egfr mutationwas not significantly correlated with final orbital exenteration or pfs tables and discussionto the best of our knowledge this is one of the first studies to survey the prevalence of egfrmutations and intracellular localization in conjunctival scc and to evaluate the prognostic significance of tumor cells that express egfr in conjunctival sccin this study we found that the tumor tissue of all conjunctival sccs expressedegfr in addition we determined the percentages of the two most important mutations intable egfr staining and localizationcell membranecell cytoplasmicegfr focaln ±±egfr diffusen ±±p101371 pone0238120t003 one 101371 pone0238120 august one 0cegfr in conjunctival squamous cell carcinomafig egfr mutationspecific expression in conjunctival scc a basement membrane staining in a tumor with egfr e746a750 del bwhole tumor staining in an egfr e746a750 del mutant c conjunctival scc layer cells with strong staining in an egfr l858r mutant scalebar μm101371 pone0238120g003egfr exon 746a750del exon l858r mutant in conjunctival sccs we also showed that the translocation of egfr from the membrane into the cytoplasm was related to clinical activation of cancer as correlations between egfr cytoplasmicstaining and final orbital exenteration and between decreased egfr membrane staining andpfs were noted although the number of cases examined was small the expression of cytoplasmic staining of egfr was weak but significantly different from membrane staining in thebenign disease group our hypothesis is that as egfr transitions from the membrane into thecytoplasm malignant changes progress in addition a correlation between egfr stainingfocal or diffuse and egfr cytoplasmic staining was seen and a higher score tended to bepresent in the diffuse egfr staining grouptable summary of egfr e746a750 del and egfr l858r point mutationsmutationn age y sex mt stage egfr staining patterns diffuseegfr localization score membraneexon egfr e746a750 del n fexon egfr l858r point mutationn t3 t2 tis t3 tis focalcytoplasmicm male f female101371 pone0238120t004 one 101371 pone0238120 august one 0cegfr in conjunctival squamous cell carcinomafig for egfr expression in tumors we compared carcinoma in situ tis and invasive carcinoma tadv groups according toajcc t grading n4 ns not significant101371 pone0238120g004intracellular transfer of egfr in the group with diffuse staining may indicate progressionand although no statistical differences were observed in this study significant findings mayemerge by increasing the number of cases in the futurein the past especially in african countries several studies on conjunctival sccs and egfrexpression have been reported they suggested a potential association with hpv [ ]other previous studies reported that posttranslational modification can promote egfrtable relationship between orbital exenteration and clinicopathologic and molecular factorsunivariate analysisvariablesagesextstage ajccegfr stainingn mean yearsmale female tis t1 t2 21t3 �focal 21diffuse egfr membrane stainingvery strong 1strong 21weak 7negative egfr cytoplasmic stainingegfr mutationhpv positivevery strong 4strong 6weak 19negative exon e746a750 del 8exon l858r point mutation positive 7negative hr ci p�ci indicates confidence interval hr hazard ratestatistically significant differences are underlined�p value based on the cox proportional hazard model101371 pone0238120t005 one 101371 pone0238120 august one 0ctable relationship between pfs and clinicopathologic and molecular factorsunivariate analysisvariablesagesextstage ajccegfr stainingn mean yearsmale 15female tis t1 t2 21t3 �focal 21diffuse egfr membrane stainingvery strong 1strong 21weak 7negative egfr cytoplasmic stainingegfr mutationhpv positivevery strong 4strong 6weak 19negative exon e746a750del 8exon l858r point mutation positive 7negative ci indicates confidence interval hr hazard ratestatistically significant differences are underlined�p value based on the cox proportional hazard model101371 pone0238120t006egfr in conjunctival squamous cell carcinomahr ci p��endocytosis and lysosomal degradation of egfr thereby ensuring termination of receptor signaling [ ]in our cohort expression and localization of egfr and its association with prognosis werefirst reported in the asian race additionally intracellular translocation of egfr from membrane staining to cytoplasm staining likely by endocytosis was associated with the percent offinal orbital exenteration cytoplasmic staining hr p and pfs membranestaining hr p in our cohort regarding the difference in local changes inegfr immunoreactivity in patients without egfr expression in the tumor we compared thetis and tadv groups according to ajcc t grading a recent study showed that feedback regulatory loops can modulate growth factors and receptor tyrosine kinases such as egfr to regulate cellular functions including abnormal states such as cancer our study examined thisphenomenon clinically and confirmed a pathological difference without changes in geneexpressionegfr mutations in ossn including invasive sccs have not been examined in asianpatients since approximately egfr mutations in lung cancer had been registeredin the cosmic the catalog of somatic mutations in cancer database most are concentrated in the exon region of the intracellular tyrosine kinase domain the most frequentone is at codon of exon a deletion mutation is present at a site centered on five aminoacids elrea near amino acid and a point mutation changes leucine to argininel858r at codon of exon shigematsu in and mitsudomi in reported that egfr mutations are common in asians females nonsmokers and adenocarcinomas in lung cancer [ ] generally when egfr mutation occurs the tyrosine kinaseactivity of egfr at the atp binding site is constantly active even without growth factor cancer cell growth and survival depend on this pathway oncogene addiction egfr tkis competitively inhibit atp binding in the kinase domain and suppress autophosphorylation ofegfr blockade of signal transmission has antitumor effects previous reports of egfractivating mutations common mutations described the frequency of exon deletion mutations as and for l858r mutations in lung cancer [ ]egfr mutations were examined to verify the effect of gefitinib on positive nonsmall celllung carcinoma in two phase iii clinical trials from japan in the nej002 trial and thewjtog3405 trial gefitinib was the test treatment group the standard treatment in the former one 101371 pone0238120 august one 0cegfr in conjunctival squamous cell carcinomafig schematic of movement of egfr into the cytoplasm by endocytosis to avoid excessive signaling and for recycling101371 pone0238120g005was carboplatin paclitaxel and in the latter was cisplatin in all studies the gefitinib groupshowed superior pfs [ ] in view of these findings in lung cancer in asians our findingsregarding egfr expression and mutations will provide further options for potential treatmentof ossn for pre and postsurgical treatmentthe association of scc with hpv was not confirmed because the number of cases wassmall in addition our results may not be accurate because we did not use multiplex pcrwhich is currently the most suitable genotyping method ours is the first report to show that differences in the expression form and mutations inegfr in ossn are associated with prognosis and treatmentin an animal model egfr inhibition affected epithelial cell proliferation and stratificationduring corneal epithelial wound healing and may play a role in maintaining normal cornealepithelial thickness gefitinib is an egfr inhibitor and is the first approved molecular targeted therapy for cancer treatment in japan thus understanding the pathological role of egfr in ossn andapplying it to treatment are of great significance for seeking new treatment indications inossn including conjunctival sccs in this study egfr may translocate from the cell membrane into the cytoplasm tumor cells may transfer egfr into the cytoplasm by endocytosisto avoid excessive signaling by the feedback system fig furthermore in this study theegfr mutation was present in many patients with ossn this finding may suggest a courseof treatment in the future in addition the method we used for identification of egfr mutations was not general genotyping but was a judgment of immunohistochemically stained sections although the sensitivity and specificity were high in a previous report this is still alimitation one 101371 pone0238120 august one 0cegfr in conjunctival squamous cell carcinomathis study has important limitations first regarding egfr expression on the ocular surface changes in benign diseases and agerelated changes in normal tissues may not have beensufficiently investigated our study found that egfr mutations were also present in conjunctival scc in east asians however we did not obtain results that correlated with the final prognosis further studies including further multiinstitutional studies and an increase in thenumber of cases will be needed in the future another limitation is that double testing of formalinfixed paraffinembedded specimens and plasma with realtime pcr for detection ofegfr mutations is more common than ihc in actual clinical practice according to the literature both the sensitivity and specificity were satisfactory for these two types of mutations in addition the size of our study cohort was small n and the length of followup lessthan year in some patients may not have been sufficient for longterm outcome analysestherefore additional studies will be needed to corroborate our findingsin the results of this study indicate that egfr is an active molecular target inthe pathology of ossn including scc and is a prognostic factor the finding also suggests thatdiscovery of mutations may have important implications for future treatment optionssupporting informations1 filexlsxacknowledgmentswe gratefully acknowledge the technical assistance of the research support platform osakacity university graduate school of medicine and the clinical laboratory department of kobekaisei hospitalauthor contributionsconceptualization mizuki tagami atsushi azumidata curation atsushi sakai mizuki tagami atsuko katsuyamayoshikawa norihiko misawa atsushi azumiformal analysis mizuki tagami anna kakehashi norihiko misawafunding acquisition mizuki tagamiinvestigation mizuki tagami atsuko katsuyamayoshikawa atsushi azumimethodology mizuki tagami anna kakehashi atsuko katsuyamayoshikawa atsushiazumiproject administration mizuki tagamisupervision anna kakehashi hideki wanibuchi atsushi azumi shigeru hondavisualization atsushi sakai mizuki tagami anna kakehashiwriting original draft atsushi sakai mizuki tagamiwriting review editing mizuki tagami shigeru hondareferenceslee ga hirst lw ocular surface squamous neoplasia surv ophthalmol 101016s0039625705800542 pmid one 101371 pone0238120 august one 0cegfr in conjunctival squamous cell carcinoma kiire ca stewart rmk srinivasan s heimann h kaye sb dhillon b a prospective study of the incidence associations and outcomes of ocular surface squamous neoplasia in the united kingdom eyelond mcclellan aj mcclellan al pezon cf karp cl feuer w galor a epidemiology of ocular surfacesquamous neoplasia in a veterans affairs population cornea 101097ico0b013e31829e3c80 pmid sun ec fears tr goedert jj epidemiology of squamous cell conjunctival cancer cancer epidemiolbiomarkers prev pmid scholz sl thomasen h reis h frequent tert promoter mutations in ocular surface squamousneoplasia invest ophthalmol vis sci 101167iovs1517469pmid nagarajan p elhadad c gruschkus sk ning j hudgens cw sagiv o pdl1pd1 expressioncomposition of tumorassociated immune infiltrate and hpv status in conjunctival squamous cellcarcinoma invest ophthalmol vis sci 101167iovs1926894pmid le tourneau c delord jp gonc¸alves a gavoille c dubot c isambert n molecularly targetedtherapy based on tumour molecular profiling versus conventional therapy for advanced cancershiva a multicentre openlabel proofofconcept randomised controlled phase trial lancetoncol 101016s1470204515001886 pmid el zaoui i bucher m rimoldi d nicolas m kaya g pescini gobert r conjunctival melanomatargeted therapy mapk and pi3kmtor pathways inhibition invest ophthalmol vis sci 101167iovs1826508 pmid ciardiello f tortora g a novel approach in the treatment of cancer targeting the epidermal growth factor receptor clin cancer res pmid lynch tj bell dw sordella r gurubhagavatula s okimoto ra brannigan bw activating mutations in the epidermal growth factor receptor underlying responsiveness of nonsmallcell lung cancer togefitinib n engl j med 101056nejmoa040938 pmid paez jg ja¨nne pa lee jc tracy s greulich h gabriel s egfr mutations in lung cancer correlation with clinical response to gefitinib therapy science 101126science1099314 pmid cesano a ncounter® pancancer immune profiling panel nanostring technologies inc seattlewa j immunother cancer 101186s4042501500887 pmid yu jj fu p pink jj dawson d wasman j orem j hpv infection and egfr activationalteration in hivinfected east african patients with conjunctival carcinoma one e10477101371 pone0010477 pmid mwololo a nyagol j rogena e ochuk w kimani m onyango n correlation of egfr pegfrand p16ink4 expressions and high risk hpv infection in hivaidsrelated squamous cell carcinoma ofconjunctiva infect agent cancer 1011861750937897 pmid haglund k dikic i the role of ubiquitylation in receptor endocytosis and endosomal sorting j cell sci 101242jcs091280 pmid zhang x gureasko j shen k cole pa kuriyan j an allosteric mechanism for activation of the kinasedomain of epidermal growth factor receptor cell 101016jcell pmid avraham r yarden y feedback regulation of egfr signalling decision making by early and delayedloops nat rev mol cell biol 101038nrm3048 pmid kobayashi y mitsudomi t not all epidermal growth factor receptor mutations in lung cancer are created equal perspectives for individualized treatment strategy cancer sci 101111cas12996 pmid shigematsu h lin l takahashi t nomura m suzuki m wistuba ii clinical and biological features associated with epidermal growth factor receptor gene mutations in lung cancers j natl cancerinst 101093jncidji055 pmid mitsudomi t yatabe y mutations of the epidermal growth factor receptor gene and related genes asdeterminants of epidermal growth factor receptor tyrosine kinase inhibitors sensitivity in lung cancercancer sci 101111j13497006200700607x pmid yun ch mengwasser ke toms av woo ms greulich h wong kk the t790m mutation inegfr kinase causes drug resistance by increasing the affinity for atp proc natl acad sci u s a 101073pnas0709662105 pmid one 101371 pone0238120 august one 0cegfr in conjunctival squamous cell carcinoma kobayashi y togashi y yatabe y mizuuchi h jangchul p kondo c egfr exon mutationsin lung cancer molecular predictors of augmented sensitivity to afatinib or neratinib as comparedwith first or thirdgeneration tkis clin cancer res 10115810780432ccr151046 pmid wu jy yu cj chang yc yang ch shih jy yang pc effectiveness of tyrosine kinase inhibitors onuncommon epidermal growth factor receptor mutations of unknown clinical significance in nonsmallcell lung cancer clin cancer res 10115810780432ccr10 pmid maemondo m inoue a kobayashi k sugawara s oizumi s isobe h gefitinib or chemotherapyfor nonsmallcell lung cancer with mutated egfr n engl j med 101056nejmoa0909530 pmid mitsudomi t morita s yatabe y negoro s okamoto i tsurutani j gefitinib versus cisplatin plusdocetaxel in patients with nonsmallcell lung cancer harbouring mutations of the epidermal growth factor receptor wjtog3405 an open label randomised phase trial lancet oncol 101016s147020450970364x pmid nishiwaki m yamamoto t tone s murai t ohkawara t matsunami t genotyping of humanpapillomaviruses by a novel onestep typing method with multiplex pcr and clinical applications j clinmicrobiol 101128jcm0079307 pmid nakamura y sotozono c kinoshita s the epidermal growth factor receptor egfr role in cornealwound healing and homeostasis exp eye res 101006exer2000 pmid fukuoka m yano s giaccone g tamura t nakagawa k douillard jy multiinstitutional randomized phase ii trial of gefitinib for previously treated patients with advanced nonsmallcell lung cancer the ideal trial [corrected] j clin oncol 101200jco pmid oldrini b hsieh wy erdjumentbromage h codega p carro ms curielgarcı´a a egfr feedbackinh | Colon_Cancer |
" levels of physical activity change throughout the year however little is known to what extentactivity levels can vary based on accelerometer determined sedentary and physicallyactive time the aim of thislongitudinal study was to examine older adults activity changes from a nonsnowfall season to a subsequentsnowfall season with consideration of the codependence of domains of time usemethods participants were older japanese adults women aged years living in a rural area ofheavy snowfall who had valid accelerometer active style pro hja750c data during nonsnowfall and snowfallseasons activity was classified as sedentary behavior sb lightintensity pa lpa and moderatetovigorous pamvpa compositional changes from the nonsnowfall to the snowfall season were analyzed using aitchisonsperturbation method the ratios of each component in the composition such as [sbsnowsbnonsnow lpasnowlpanonsnow mvpasnowmvpanonsnow] for seasonal changes were calculated and were then divided by thesum of these ratiosresults in men the percentages of time spent in each activity during the nonsnowfallsnowfall seasons were for sb for lpa and for mvpa these corresponded to mean seasonal compositionalchanges δsb δlpa δmvpa of and respectively in women the percentages of time spent ineach activity during the nonsnowfallsnowfall seasons were for sb for lpa and formvpa these corresponded to mean seasonal compositional changes δsb δlpa δmvpa of and respectively the degree of seasonal change was greatest in mens in older adults activity behaviors were changed unfavorably during snowfall season particularly so formen the degree of seasonal change was greatest for sb development of strategies to keep rural older adultsactive during the snowfall season may be needed for maintaining a consistentlyactive lifestyle for their healthkeywords accelerometry aging environment exercise sedentary lifestyle correspondence inouetokyomedacjp1department of preventive medicine and public health tokyo medicaluniversity shinjuku shinjukuku tokyo japanfull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0camagasa bmc public health page of global physical activity guidelines state that older adultsleast min per week of moderateshould do atintensity physical activity atleast min vigorousintensity physical activity or an equivalent combinationof both in bouts lasting min or more physical inactivity increases the risk of many adverse health outcomes including mortality cardiovascular disease type diabetes several types of cancer and cognitive decline[] in recent years there has been significant growthin research investigating the detrimental health effects ofsedentary behavior sb put simply too much sitting reducing sb and increasing physical activity are keyingredients of initiatives to reduce the global burden ofnoncommunicable diseases [ ]there is a growing body of evidence identifying the importance of natural environments the attributes of whichcan vary greatly by season as well as built environments asinfluences on physical activity [ ] one systematic review of studies from eight different countries showed thatlevels of physical activity tended to be lowest during winter to date however most of these studies have relied onselfreport physical activity assessments and have not focusedon older adults objective assessment via accelerometers can now overcome problems of recall and reportingbias and allow more accurate and finergrained assessmentsof activity behavior patterns sb lightintensity physical activityactivitymvpa there is limited evidence on how season affects devicebased activity behaviors [] compositionaldata analysis coda allows consideration ofthe codependence of time spent in all behaviors arising within aday or other fixed period [ ] to date no previous studyhas investigated seasonal changes of activity behaviors takingtime spent in each activity behavior into accountlpa and moderatetovigorous physicalwe compared times spent in accelerometermeasuredactivity behaviors during a nonsnowfall and a snowfallseason in a sample of communitydwelling older japanese adults using the coda approach we also exploredwhich activity behaviors were most affected by seasonmethodsstudy sample and data collectionwe used longitudinal data from the neuron to environmental impact across generations study neige study themethods of this study are described in previous study participants were older adults without longterm care livingin tokamachi city japan tokamachi is a rural city officiallyregistered as a heavy snowfall region during winter locatedin the southernmost region of niigata prefecture participants were from two areas matsunoyama mountain areaor tokamachi area downtown the mountain area mmaximum snow depth had more snow than the downtownarea maximum snow depth during winter theaverage temperature at tokamachi city in february was °c lowest °c highest °c brieflyin a total of residents were recruited from a resident registry using stratified randomsampling in the nonsnowfall season autumn of we conducted a questionnaire survey and health examination of older adults who agreed to enrollment inthe neige study at the same time they were asked towear an accelerometer of these participants agreedto also wear an accelerometer during the snowfall seasonwinter of accelerometermeasured activity behaviorshabitual time spent in activity behaviors were evaluatedby active style pro hja750c omron healthcarekyoto japan active style pro is a validated accelerometer [] and comparable to the devices most commonly used in studies conducted in western countries[ ] its measurement algorithm has been explainedin detail elsewhere [ ] participants were instructedto wear an accelerometer over the waist on an elasticated belt for seven consecutive days except duringsleep and waterbased activities during snowfall andnonsnowfall season respectively in the survey duringsnowfall season participants were mailed an accelerometer no acceleration signal detected for longer than consecutive minutes was defined as nonwear participants with a wear time corresponding to at least hduring waking time per day collected over four ormore valid days were included in the analysis thedata were collected in 60s epochs activity behaviorswere classified into three intensity categories based onmetabolic equivalents mets sb mets lpa mets and mvpa ¥ mets [ ]sociodemographic biological and psychological factorsparticipants reported their age gender living arrangement with others alone and selfrated health verygood good fair poor in autumn we classified participants between the ages of and years as youngoldand those between ages and years as oldold bodymass index bmi was calculated from height and weightkgm2 measured by body composition analyzer mc780a tanita corporation tokyo japanstatistical analysesall analyses were performed using r version r foundation for statistical computing vienna austria we usedr package compositions for coda approach for all analyses pvalues were considered statistically significantanalyses were applied stratified by gender since activity behavior patterns were significantly different between men andwomen 0camagasa bmc public health page of the chisquare test or t test was performed to compare participant characteristics mcnemars test wasused to compare nonsnowfall and snowfall seasonsproportions of those adhering to physical activity guidelines ie ¥ minweek of mvpa in bouts of at least min a minbout mvpa was defined as ormore consecutive minutes above the moderate intensitythreshold with the allowance of min interruptionintervals we described activity behaviors during the nonsnowfalland snowfall season using coda approach as all participantsspent some time in every behavior there was no need for amethod to deal with zeros compositional changes [δsbδlpa δmvpa] were then determined by aitchisons perturbation method [] the ratios of each component inthe composition such as [sbsnowsbnonsnow lpasnowlpanonsnow mvpasnowmvpanonsnow] for seasonalchanges were calculated and were then divided by the sumof these ratios an equal composition of these three activitiesin the nonsnowfall and snowfall seasons would result in acompositional change of[ ] compositionalchanges were plotted as ternary diagrams with some significant guide values illustratedresultsparticipant enrollment and characteristicsof the older adults who agreed to wear an accelerometer during both the nonsnowfall and snowfallseasons response rate were excluded for notmeeting wearing time criteria n bone fracturen unreturned accelerometer n and malfunction n the analytic sample was who had validaccelerometer datawhen compared to participant characteristics betweenthose who had valid accelerometer data in snowfall season and those who did not a significant difference wasfound in age groups youngold oldold chisquare test participants who participated in thesurvey in the snowfall season were significantly morephysically active approx more minutes of mvpaper day than those who did not ttesttable presents the characteristics of the participantsthe mean sd age was years womenand most of the participants were living with others kgm2 bmi and good perceived health comparedto women men were more likely to be from the mountain area be living with others and to have attained¥ minweek of mvpa there were no significant seasonal differences in proportion of those adhering to global physical activity guidelines nonsnowfall seasonoverall men women snowfall season overall men women activity behaviors in snowfall and nonsnowfall seasonmean accelerometer wear time was minday innonsnowfallseason and minday in snowfalltable participants characteristics at baseline nonsnowfall seasonoverall n mean ± sd n ± men n mean ± sd n ± women n mean ± sd n ± ± ± pvalueage yrsage categoriesyoungold yrsoldold ¥ yrsarea of residencemountain areadowntownliving arrangementliving with othersliving alonebmi kgm2bmi categoriesnormal kgm2obese ¥ kgm2perceived healthgood very good good ± poor fair poorpvalue was calculated using t test or chisquare test as appropriate 0camagasa bmc public health page of season older adults spent most of their time on sb andlpa table presents the descriptive statistics of timespent in each activity behavior in men time spent ineach activity sb lpa and mvpa was and respectively during the nonsnowfall season and and respectively during the snowfall season corresponding to mean seasonal change of forsb for lpa and for mvpa fig sincelarger distance from indicates greater change in timespent in activity the largest seasonal change was observed in sb compared to lpa and mvpa in womentime spent in each activity was and respectively during nonsnowfall season while that was and respectively during snowfall seasoncorresponding to mean seasonal change of for sb for lpa and for mvpa significant unfavorable seasonal changes in activity behaviors were observed in both men and women but the degree ofchange was larger in men in every behavior if we contrast the changes the ratios between men and womensimilar findings were observed after stratification byresidential area but the degree of change in activity behaviors was larger in mountain area than in downtownregardless of genderdiscussionwe identified seasonal differences of accelerometer determined activity behaviors in communitydwelling olderadults in a rural snowfall area using the coda approacholder adults had more unfavorable activity behavior patterns during the snowfall season compared to nonsnowfall season with the magnitude of these differencesgreater in men no significant seasonal differences werefound in the proportions adhering to global physical activity guidelinesour findings are consistent with previous studies usingselfreport [ ] and pedometer accelerometer [] data where levels of adults physical activity duringwinter was less than the other seasons a study iniceland found that both older men and women weremore active during summer than winter with less timespent in accelerometer determined sb in men in women a study in the uk found that onlytime spent in lpa was significantly higher during springthan that during winter among older adults winter ± hday spring ± hday summer ± hday and autumn ± hday we found thatthe magnitude of decline during winter seems to belarge compared to these icelandic and uk studies thismay result from winter conditions including amount ofsnowfall furthermore our study participants were relatively more active and less sedentary during autumn thenonsnowfall season more highly active participantsmay experience activity decline to a greater degree compared to low activity participants in this sample therewere no significant seasonal changes of adherence toglobal physical activity guidelines this may be due tovery little time spent in mvpa in bouts of more than mingender differences of activity patterns were in linewith our previous findings [ ] that japanese olderwomen were more physically active than older men provided that all activity behaviors were assessed in thissample men experienced greater seasonal activity declines than did women in japan women are more responsible for household chores and thus engage in acertain amount of activities throughout the year theremay be needed to develop a strategy to combat this seasonal decrease in physical activity for menin the current study activity behavior patterns wereunfavorably changed with approximately increasein sb and decrease in mvpa the degree of the changein activity behaviors may be significant for health a previous study found decline of physical activity during winter unfavorably affected the physical performance levelafter one year in communitydwelling older women particularly its effect on maximum walking speed given that the rate of muscle mass decline is higher inolder adults compared to middleaged adults maintaining physical activity may be required to keep physicaltable compositional geometric means of time spent in activity behaviors during the nonsnowfall and snowfall seasonnonsnowfall season minday wear timewear timesb lpa snowfall season minday wear timewear timesb lpa mvpa mvpa overallmenmountain areadowntownwomenmountain areadowntownabbreviation sb sedentary behavior lpa lightintensity physical activity mvpa moderatetovigorous physical activity 0camagasa bmc public health page of fig changes in sedentary behavior sb light intensity physical activity lpa and in moderatetovigorous physical activity mvpa from thenonsnowfall to snowfall season in ruraldwelling older japanese adults a stratified by gender blue men red women b stratified by genderand residential area blue mountain area green downtown abbreviation sb sedentary behavior lpa lightintensity physical activity mvpamoderatetovigorous physical activityability and prevent a negative cycle of frailty another study showed shortterm decreased physical activity with increased sb caused impairment of metabolism which increases risk of cardiovascular disease therefore promoting physical activity during wintermay be one way to tackle agerelated diseases and lossof physical functioningstrengths and limitationsstrengths of this study included objective assessment ofactivity behaviors and a novel statistical approach eventhough there has been increasing research using objective methods [ ] no study has treated with consideration to the codependence of time spent in activitybehaviors moreover we provided resultsfrom aseldomstudied elderly population in a rural snowfallarea however several limitations should be consideredto interpret the findings we may underoverestimate ofsb and lpa since active style pro cannot provide posturalinformation also some activities eg snow removal and skiing may be not captured accuratelysecond we may underestimate the decline of activitylevel since most of the days that participants wore an accelerometer were relatively good weather in spite ofheavy snowfall area it has previously been observed thatlonger day length is associated with increased devicebased physical activity in the older population [ ]third as tokamachi city is a rural area where heavysnowfall is common during winter it is not necessarilyrepresentative ofjapanese rural areas with different 0camagasa bmc public health page of climates people in areas with less snowfall may experience a smaller decline of physical activity during snowfall season more research is needed in the differentclimate zones from different geographic areas finallyselection bias may have occurred in general accelerometry respondents have been more active and healthierthan nonparticipating older adults in this studythose had valid accelerometer data in snowfall seasonwere more physically active than those who did notimplications for future research policy and practiceour findings suggest several implications in terms of bothdevelopment of interventions to protect against seasonalphysical activity decline and physical activity surveillancemonitoring further research regarding how to stay activeduring winter may be required for health promotion particularly in regions that experience long winters and withsevere weather eg heavy snow given that approximately of the land in japan has snow and a cold winter and that a quarter of the population lives in thoseareas leading an active lifestyle during winter potentially has a significant public health impactsince sb is the most affected by season it may be better tofocus on developing strategies to reduce time spent in sittingolder adults might be particularly influenced by seasonaloutside conditions due to reduced physical functioning andmobility and spend much of their time indoors it thus maybe effective to provide supplementary resources for indooractivities eg gymnastic exercise programs sharing ofhousehold chores and making educational instrumentalsupports for safe snow removal further approach includes replacing mentallypassive with mentallyactive sbmay be effective for health particularly for preventing cognitive decline [ ] and depression [ ]as for physical activity surveillance monitoring investigators should be aware of the potential for under oroverestimation of levels of activity especially when theinterest is in its betweenindividual variation includingcommunitylevel and countrylevel comparisons seasonality also should be considered when interventionstudies are performedsaccelerometer determined activity behaviors were greatlyaffected by season in communitydwelling older adults ina rural snowfall area resulting in unfavorable changesparticularly in sb time during snowfall season development of strategies to keep rural older adults active duringthe snowfall season may be needed for maintaining aconsistentlyactive lifestyle for their healthabbreviationssb sedentary behavior lpa lightintensity physical activitymvpa moderatetovigorous physical activity coda compositional dataanalysis mets metabolic equivalentsacknowledgementswe thank the city workers and the participants for their time and effort inthe neige studyauthors contributionsys hm si and tf developed the neige study ys hm si tf hk nf mmand sa collected the data sa performed the analysis and prepared themanuscript sc and no gave critical feedback and revised the manuscript allauthors advised on the data analysis and interpretation and reviewed themanuscriptfundingthis study was funded by grant from the policy research institute ministryof agriculture forestry and fisheries in japan and the pfizer healthresearch foundation and by jsps kakenhi grant number 16h03249 k19794 k10829 and 19h03910 the funding bodies were not involved inany portion of the study design data collection and analysis interpretationof the results and preparation of the manuscriptshiho amagasa is supported by jsps research fellowships for youngscientists neville owen is supported by a national health and medicalresearch council of australia nhmrc centre of research excellence grant nhmrc senior principal research fellowship and thevictorian governments operational infrastructure support programavailability of data and materialsthe datasets used and analyzed during the current study are available fromthe corresponding author on reasonable requestethics approval and consent to participatethe university ethics committees niigata university and tokyo medicaluniversity granted ethical approval written informed consent was obtainedfrom all participantsconsent for publicationnacompeting intereststhe authors declare no conflict of interestauthor details1department of preventive medicine and public health tokyo medicaluniversity shinjuku shinjukuku tokyo japan 2institute ofgerontology the university of tokyo hongo bunkyoku tokyo japan 3department of global health promotion tokyo medical anddental university yushima bunkyoku tokyo japan 4school ofhealth and life science institute of applied health research glasgowcaledonian university cowcaddens road glasgow uk 5department ofsport and movement science ghent university ghent belgium6behavioral epidemiology laboratory baker heart diabetes institute level commercial road melbourne vic australia 7centre for urbantransitions swinburne university of technology po box hawthornmelbourne australia 8division of international health niigata universitygraduate school of medical and dental sciences asahimachidoriniigata city japan 9department of active ageing niigatauniversity graduate school of medical and dental sciences asahimachidori niigata city japanreceived february accepted august references world health anization global recommendations on physical activity forhealth httpappswhointirisbitstream106654439919789241599979_engpdf accessed jan lee im shiroma ej lobelo f puska p blair sn katzmarzyk pt effect ofphysical inactivity on major noncommunicable diseases worldwide ananalysis of burden of disease and life expectancy lancet kyu hh bachman vf alexander lt mumford je afshin a estep k veermanjl delwiche k iannarone ml moyer ml physical activity and risk ofbreast cancer colon cancer diabetes ischemic heart disease and ischemic 0camagasa bmc public health page of stroke events systematic review and doseresponse metaanalysis for theglobal burden of disease study bmj 2016354i3857aune d norat t leitzmann m tonstad s vatten lj physical activity andthe risk of type diabetes a systematic review and doseresponse metaanalysis eur j epidemiol arem h moore sc patel a hartge p berrington de gonzalez avisvanathan k campbell pt freedman m weiderpass e adami ho leisure time physical activity and mortality a detailed pooled analysis of thedoseresponse relationship jama intern med sofi f valecchi d bacci d abbate r gensini gf casini a macchi c physicalactivity and risk of cognitive decline a metaanalysis of prospective studiesj intern med young dr hivert mf alhassan s camhi sm ferguson jf katzmarzyk ptlewis ce owen n perry ck siddique j yong cm sedentary behavior andcardiovascular morbidity and mortality a science advisory from theamerican heart association circulation biswas a oh pi faulkner ge bajaj rr silver ma mitchell ms alter dasedentary time and its association with risk for disease incidence mortalityand hospitalization in adults a systematic review and metaanalysis annintern med ding d lawson kd kolbealexander tl finkelstein ea katzmarzyk pt vanmechelen w pratt m the economic burden of physical inactivity a globalanalysis of major noncommunicable diseases lancet tucker p gilliland j the effect of season and weather on physical activity asystematic review public health barnett dw barnett a nathan a van cauwenberg j cerin e builtenvironmental correlates of older adults' total physical activity and walking asystematic review and metaanalysis int j behav nutr phys act shephard rj tudorlocke c the objective monitoring of physical activitycontributions of accelerometry to epidemiology exercise science andrehabilitation new york springer international publishing davis mg fox kr hillsdon m sharp dj coulson jc thompson jlobjectively measured physical activity in a diverse sample of older urbanuk adults med sci sports exerc arnardottir ny oskarsdottir nd brychta rj koster a van domelen drcaserotti p eiriksdottir g sverrisdottir je johannsson e launer lj comparison of summer and winter objectively measured physical activityand sedentary behavior in older adults age geneenvironmentsusceptibility reykjavik study int j environ res public health yasunaga a togo f watanabe e park h park s shephard rj aoyagi y sexage season and habitual physical activity of older japanese the nakanojostudy j aging phys act chastin sf palareaalbaladejo j dontje ml skelton da combined effectsof time spent in physical activity sedentary behaviors and sleep on obesityand cardiometabolic health markers a novel compositional data analysisapproach plos one 201510e0139984 dumuid d stanford te martinfernandez ja pedisic z maher ca lewis lkhron k katzmarzyk pt chaput jp fogelholm m compositional dataanalysis for physical activity sedentary time and sleep research statmethods med res shobugawa y murayama h fujiwara t inoue s cohort profile of the neigestudy in tokamachi city japan j epidemiol japan meteorological agency historical weather data search httpswwwjmagojpjmaindexehtml accessed jul ohkawara k oshima y hikihara y ishikawatakata k tabata i tanaka s realtime estimation of daily physical activity intensity by a triaxial accelerometerand a gravityremoval classification algorithm br j nutr oshima y kawaguchi k tanaka s ohkawara k hikihara y ishikawatakata ktabata i classifying household and locomotive activities using a triaxialaccelerometer gait posture park j ishikawatakata k tanaka s bessyo k tanaka s kimura t accuracyof estimating step counts and intensity using accelerometers in olderpeople with or without assistive devices j aging phys act nagayoshi s oshima y ando t aoyama t nakae s usui c kumagai stanaka s validity of estimating physical activity intensity using a triaxialaccelerometer in healthy adults and older adults bmj open sport exercmed 20195e000592kurita s yano s ishii k shibata a sasai h nakata y fukushima n inoue stanaka s sugiyama t comparability of activity monitors used in asianand westerncountry studies for assessing freeliving sedentary behaviourplos one 201712e0186523 murakami h kawakami r nakae s nakata y ishikawatakata k tanaka smiyachi m accuracy of wearable devices for estimating total energyexpenditure comparison with metabolic chamber and doubly labeledwater method jama intern med tudorlocke c camhi sm troiano rp a catalog of rules variables anddefinitions applied to accelerometer data in the national health andnutrition examination survey prev chronic dis 20129e113troiano rp berrigan d dodd kw masse lc tilert t mcdowell m physicalactivity in the united states measured by accelerometer med sci sportsexerc haskell wl lee im pate rr powell ke blair sn franklin ba macera ca heathgw thompson pd bauman a physical activity and public health updatedrecommendation for adults from the american college of sports medicineand the american heart association circulation pate rr o'neill jr lobelo f the evolving definition of sedentary exercsport sci rev aitchison j ng kw the role of perturbation in compositional data analysisstat model boogaart kg tolosanadelgado r analyzing compositional data with rberlin springerverlag berlin heidelberg winkler eah chastin s eakin eg owen n lamontagne ad moodie m dempsey pckingwell ba dunstan dw healy gn cardiometabolic impact of changing sittingstanding and stepping in the workplace med sci sports exerc uitenbroek dg seasonal variation in leisure time physical activity med scisports exerc matthews ce freedson ps hebert jr stanek ej 3rd merriam pa rosal mcebbeling cb ockene is seasonal variation in household occupational andleisure time physical activity longitudinal analyses from the seasonalvariation of blood cholesterol study am j epidemiol amagasa s fukushima n kikuchi h takamiya t oka k inoue s light andsporadic physical activity overlooked by current guidelines makes olderwomen more active than older men int j behav nutr phys act amagasa s inoue s ukawa s sasaki s nakamura k yoshimura a tanaka akimura t nakagawa t imae a ding d kikuchi h tamakoshi a are japanesewomen less physically active than men findings from the dosanco healthstudy j epidemiol httpsdoi102188jeaje20200185 mizumoto a ihira h makino k saitoh s ohnishi h furuna t physical activityduring winter in oldold women associated with physical performance afterone year a prospective study curr gerontol geriatr res volpi e nazemi r fujita s muscle tissue changes with aging curr opin clinnutr metab care fried lp tangen cm walston j newman ab hirsch c gottdiener jseeman t tracy r kop wj burke g mcburnie ma frailty in older adultsevidence for a phenotype j gerontol series a 200156m146 bowden davies ka sprung vs norman ja thompson a mitchell klhalford jcg harrold ja wilding jph kemp gj cuthbertson dj shorttermdecreased physical activity with increased sedentary behaviour causesmetabolic derangements and altered body composition effects inindividuals with and without a firstdegree relative with type diabetesdiabetologia ormazabal v nair s elfeky o aguayo c salomon c zuñiga fa associationbetween insulin resistance and the development of cardiovascular diseasecardiovasc diabetol wu yt luben r wareham n griffin s jones ap weather day length andphysical activity in older adults crosssectional results from the europeanprospective investigation into cancer and nutrition epic norfolk cohortplos one 201712e0177767schepps ma shiroma ej kamada m harris tb lee im day length isassociated with physical activity and sedentary behavior among olderwomen sci rep inoue s ohya y odagiri y takamiya t kamada m okada s tudorlocke cshimomitsu t characteristics of accelerometry respondents to a mailbasedsurveillance study j epidemiol ministry of land infrastructure transport and tourism snow disaster preventionhttpwwwmlitgojproadbosaifuyumichiprojecthtml accessed jan chastin sf de craemer m lien n bernaards c buck c oppert jm nazareja lakerveld j o'donoghue g holdsworth m the sosframeworksystems of sedentary behaviours an international transdisciplinaryconsensus framework for the study of determinants research priorities andpolicy on sedentary behaviour across the life course a dedipacstudy int jbehav nutr phys act 0camagasa bmc public health page of kesseguyot e charreire h andreeva va touvier m hercberg s galan poppert jm crosssectional and longitudinal associations of differentsedentary behaviors with cognitive performance in older adults plos one20127e47831 bakrania k edwardson cl khunti k bandelow s davies mj yates tassociations between sedentary behaviors and cognitive function crosssectional and prospective findings from the uk biobank am j epidemiol hallgren m owen n stubbs b zeebari z vancampfort d schuch fbellocco r dunstan d trolle lagerros y passive and mentallyactivesedentary behaviors and incident major depressive disorder a 13yearcohort study j affect disord hallgren m nguyen tt owen n stubbs b vancampfort d lundin adunstan d bellocco r lagerros yt crosssectional and | Colon_Cancer |
thyroid carcinoma is presently the malignancy with the most rapidly increasing incidence in the worldand is the most widely recognized endocrine carcinoma in the western world thyroid cancers derivedfrom follicular thyroid cells can be sorted into papillary thyroid carcinoma ptc follicular thyroid carcinoma ftc and anaplastic thyroid carcinoma atc according to the histological subtype clinicalresults vary across these subtypesthe annual rate of thyroid cancer has more than doubled within the past two decades with the vast majority of this increase being ascribed to ptc which accounts for of all thyroid carcinomas inaddition patients with ptc suffer from cervical lymph nodes metastasis or remote metastasis which leadsto unfavorable results and approximately of cases may progress to a potentially fatal recurrentailment due to these reasons uncovering the causes of ptc and its fundamental mechanisms andfinding molecular biomarkers for early diagnosis and customized treatment are significant and important the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commons attributionlicense cc by 0cbioscience reports bsr20201555101042bsr20201555taskswith the advancement and continuous improvement of gene sequencing and geneediting technology it is nowconvenient to recognize the hub biomarkers related to neoplasm metastasis and survival status using a large amountof information available by applying bioinformatics currently there are no effective sensitive biomarkers for earlydiagnosis treatment and prevention of lymph node metastasis of ptc an examination of differentially expressedgenes degs between tumor and paracarcinoma tissue may help identify critical biomarkers of papillary thyroidcarcinoma as a form of molecular marker mrna containing the most abundant genetic information is necessaryfor protein translation and it is separate from the pathological process of cancer at various stages some studiesutilized public databases such as the cancer genome atlas tcga and the gene expression omnibus geo toidentify significant biomarkers of papillary thyroid carcinoma however these investigations were only founded onsingle datasets with constrained sample sizes or just based on online databases used to screen out the degsin the present study we analyzed the degs in ptc tissues versus the matched adjacent tissues by rnaseq andbioinformatics methods to obtain the degs then we screened out the key modules and extracted the key genes inthose modules by constructing degs interaction network then the possible role of differentially expressed geneswas analyzed using go annotation and kegg pathway enrichment analysis the expression validation survivalanalysis and functional enrichment analysis of key genes were conducted by using relevant databases finally wefound that the three genes adora1 apoe and lpar5 were highly expressed in ptc and were associated withptc methylation tnm staging and immune infiltrationmethodstissue samplesthirty pairs of ptc and adjacent tissues were collected from january to july at the first affiliated hospitalof hebei north university this experiment was approved by the ethical committee of the first affiliated hospitaland all patients provided informed consent all tissues were frozen in liquid nitrogen after surgical resectionrna library construction and sequencingtotal rna was isolated from four adjacent normal and cancerous thyroid samples utilizing trizol reagent qiagenvalencia ca usa as indicated by the manufacturers guidelines rnas of ptc tissues and paracancerous tissuessample numbers 1c 1p 2c 2p 3c 3p 4c 4p the number represents different samples the c indicates a cancersample and the p represents a matched paracancerous tissue sample were used six libraries were built utilizingan illumina standard kit as indicated by the manufacturers protocol all sequencing was carried out on an illuminahiseq lc bio chinadifferentially expressed genes screeningthe level of expression of mrnas was evaluated using stringtie by calculating fpkm the degs between ptcand paracancerous tissue were screened with log2 fold change1 and p005 was regarded as statistically significant the analyses were conducted using the r package ballgown functional enrichment analysis and pathway analysisto reveal the functional roles of the degs the annotation visualization and integrated discovery function annotation tool david httpdavidabccncifcrfgovhomejsp was used to perform gene ontology go enrichmentanalysis and kyoto encyclopedia of genes and genomes kegg pathway enrichment analysis p values less than were considered as cutoff criteriappi network construction and identiï¬cation of hub genesppi networks were constructed successively using string database tringdb the interactions ofdegs with a combined score were set as significant and cytoscape software version was utilized tovisualize and analyze the results of the string database to find key hub genes in this ppi network the significantmodule was analyzed by using the plugin mcode of cytoscape software the criteria for selection were set to thedefault the key genes were chosen with degrees ¥ subsequently genes in that module were used to analyse theirfunctional roles with funrich software the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commons attributionlicense cc by 0cbioscience reports bsr20201555101042bsr20201555table pcr primersgene symboladora1actinapoelpar5bp base pair f forward primer r reverse primerprimer sequencef5cid3ccacagacctacttccacacc3cid3r5cid3taccggagagggatcttgacc3cid3f5cid3cactcttccagccttccttcc3cid3r5cid3aggtctttgcggatgtccac3cid3f5cid3 gttgctggtcacattcctgg 3cid3r5cid3 gcaggtaatcccaaaagcgaccid3f5cid3 cacttggtggtctacagcttg3cid3r5cid3 gcgtagtaggagagacgaacg3cid3data validation and analysisto verify the accuracy of our rnaseq results we used the gene expression profiling interactive analysis databaseto verify the expression of key genes in ptc and adjacent tissues the overall survival and diseasefree survivalanalyses were performed by kaplanmeier plots for these ptcrelated hub genes genetic alterations of hub genesin ptc and their correlations with other genes were analyzed utilizing the cbioportal for cancer genomics hub genesrelated to clinicopathological features were analyzed using the online database ualcan httpualcanpathuabedu the correlation of adora1 apoe and lpar5 expression with the immune infiltration level in ptc and theexpression of these three genes in different kinds of cancers was performed using the tumor immune estimationresource database for qrtpcr analysis total rna was isolated from normal and cancerous papillary thyroid samples utilizingtrizol reagent qiagen valencia ca usa cdna was synthesized with rna reverse transcription kit tiangenbiotech beijing china qrtpcr was performed with an abi realtime pcr system applied biosystems life technologies usa the expression of the genes of interest was normalized to actin the primers foradora1 apoe lpar5 and actin are shown in table for western blot ripa buffer was used to extract protein from four pairs of tissue from ptc patients and theprotein concentrations were measured via bca methods briefly the sdspage gel was used for electrophoresis andpdvf membrane was used for transmembrane transfer pdvf membrane was blocked and then incubated with primary antiadora1 antibodies dilution bioss bs6649r apoe dilution bioss bs4892r lpar5antibodies dilution bioss bs15366r and actin dilution bioss bs0061r at ¦c overnight followed by incubation with secondary antibodies zhongshanjinqiao dilution at ¦c for h the signal wasdetected using ecl methodstatistical analysisall the data were analyzed by r and spss spss inc usa kaplanmeier method was used to estimate thesignificant difference in survival between the overexpression group and the lowexpression group of key genes inpapillary thyroid carcinoma the statistical difference was set at p resultsdifferentially expressed genes screening based on rnaseqto screen out the genes or modules that may play a role in promoting cancer in papillary thyroid carcinoma weperformed rnaseq experiments on four pairs of thyroid cancer tissues and their matched paracancerous tissues toobtain differentially expressed genes after rnaseq we acquire million reads for each sample the fold changesbetween ptc cancer tissues and matched paracancerous samples were calculated setting the cutoff criterion as pvalue and a fold change there were upregulated and downregulated genes these degswere considered to be candidate genes for subsequent study figure 1a showed the expression of the top genes inptc versus matched paracancerous tissuesfunctional enrichment analysis and pathway analysisconsidering that there were many falsepositive genes among these degs we verified our results one by onethrough the tcga database we found that only genes in our data were consistent with the gene expression of the the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commons attributionlicense cc by 0cbioscience reports bsr20201555101042bsr20201555figure identiï¬cation of degs by rnaseqthe heat map a and ppi network of the degs b c the volcano plots of the degs d the most significant module was selectedby mcode in cytoscape red represents the upregulated genes and blue represents the downregulated genesfigure go and kegg pathway enrichment analysis of degs through rnaseqa bubble plot of gene ontology enrichment analysis of degs b bubble plot of kyoto encyclopaedia of genes and genomespathway enrichment analysis of degstcga database to investigate the potential function of these degs in ptc genes functional enrichment was conducted by using go and kegg pathway analyses for the biological process category the degs were significantly involved in the regulation of axonogenesis regulation of cell morphogenesis extracellular structure anization extracellular matrix anization synapse anization cellsubstrate adhesion and urogenital system development thecellular component category results showed ptcrelated degs were enriched in collagencontaining extracellularmatrix synaptic membrane cellcell junction glutamatergic synapse neurontoneuron synapse postsynaptic membrane basolateral plasma membrane degs in molecular function were mainly involved in cell adhesion moleculebinding passive transmembrane transporter activity extracellular matrix structural constituent glycosaminoglycanbinding growth factor binding transmembrane receptor protein kinase activity and transmembrane receptor proteintyrosine kinase activity figure 2aas figure 2b showed the kegg pathway results showed degs were enriched in cytokinecytokine receptorinteraction mapk signaling pathway proteoglycans in cancer rap1 signaling pathway axon guidance cushing the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commons attributionlicense cc by 0cbioscience reports bsr20201555101042bsr20201555figure go enrichment analysis and kegg analysis for the key genesa top elements involved in biological processes b top elements involved in molecular function c top elementsinvolved in cellular components d top pathways related to the key genes through kegg analysissyndrome parathyroid hormone synthesis secretion and action agerage signaling pathway in diabetic complications growth hormone synthesis secretion and action salivary secretion circadian entrainment cholinergic synapse p53 signaling pathway ecmreceptor interaction arrhythmogenic right ventricular cardiomyopathyarvc endocrine resistance renin secretion type ii diabetes mellitus bladder cancer nicotinate and nicotinamidemetabolism and apoptosismultiple speciesppi network construction and module analysisppi networks were constructed successively by the database by loading the ptc related dges into the stringdatabase figure 1bc using cytoscape we analyzed the most significant module in the ppi network figure 1dthe ppi network consisted of nodes and edges following the use of mcode in cytoscape the significantmodule was selected the top hub genes adcy8 adora1 adra2c apoe c5ar1 ccl13 ccl20 cdh2chgb cxcl12 eva1a fam20a fn1 gnai1 gpc3 grm4 lpar5 meltf or mfi2 mfge8 nmu oprm1serpina1 sstr3 timp1 and tnc were evaluated by degree in the ppi network figure 1d the resultsshowed that the functions of the key genes were mainly concentrated in signal transduction cell communicationgprotein coupled receptor activity cell adhesion molecule activity and gpcr ligand binding figure data analysis and validationafter the key genes were selected the expression of key genes in ptc and its adjacent tissues were verified by thegepia database figure adora1 apoe eva1a lpar5 mfge8 oprm1 serpina1 sstr3 and timp1were positively related to the overall survival analysis of ptc patients while c5ar1 and gnai1 were negativelyrelated figure adcy8 adora1 chgb fn1 lpar5 nmu and tnc showed positive associations withdiseasefree survival analysis of ptc patients but not apoe figure next we analyzed the alterations of the key genes by using the cbioportal database figure the key geneswere changed in of queried samples figure 7b figure 7a showed the frequency of alterations of eachptc related key gene sstr3 fn1 and adora1 were altered the most and respectively figure 7dshowed the network of the genes and their altered neighbouring genes in ptc patients out of a total of among these genes only adora1 apoe and lpar5 genes simultaneously showed statistical significance foroverall survival analysis and diseasefree survival analysis of ptc patients the qpcr experiments and western blotdata verified that these three survivalrelated genes were all overexpressed in ptc figure then based on ual the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commons attributionlicense cc by 0cbioscience reports bsr20201555101042bsr20201555figure validation of the key degs in the gepia databaseadora1 apoe ccl13 cdh2 cxcl12 eva1a fam20a fn1 gnai1 lpar5 mfge8 nmu serpina1 timp1 and tnc areoverexpressed in ptc tissues compared with paracancerous tissue while gnai and gpc3 are downregulated the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commons attributionlicense cc by 0cbioscience reports bsr20201555101042bsr20201555figure overall survival analysis of key genes in ptc using kaplanmeier plotsexpression levels of adora1 apoe c5ar1 eva1a fam20a gnai1 lpar5 mfge8 oprm1 serpina1 sstr3 and timp1are related to the overall survival of patients with ptccan the clinical features and degree of methylation of these three genes were analyzed the transcription levels ofadora1 apoe and lpar5 were significantly higher in ptc patients than normal tissues according to subgroupsof sample types individual stages and nodal metastasis status figure in addition ador1 and lpar5 exhibiteda hypomethylation state in the cancer group but apoe showed a hypermethylation state in ptc samples figure10ato further clarify the role of these genes we conducted an analysis of immune infiltration the ador1 expression level was positively corelated with infiltrating levels of b cells r0111 p151e2 cd8 t cells r0246p396e neutrophils r0162 p331e and dcs r0232 p232e the expression of apoe was the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commons attributionlicense cc by 0cbioscience reports bsr20201555101042bsr20201555figure diseasefree survival analysis of key genes in ptc using kaplanmeier plotsexpression levels of adcy8 adora1 apoe chgb fn1 lpar5 nmu and tnc are significantly related to the diseasefreesurvival of patients with ptcpositively associated with b cells r0228 p439e cd8 t cells partialcor p930e neutrophils r0197 p114e and dcs partialcor p358e lpar5 expression level was positively related to b cells r0259 p815e cd4 t cells r0238 p103e macrophages r0175p105e neutrophils r027 p142e and dcs r0256 p104e and negatively related to purity r p294e and cd8 t cells r p618e figure 10b these findings stronglysuggested that lpar5 ador1 and apoe may play specific roles in immune infiltration in ptc especially those ofdcs finally we examined the expression of these three genes in common cancer tissues and adjacent tissues and wefound that these three genes were highly expressed in most cancer tissues figure the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commons attributionlicense cc by 0cbioscience reports bsr20201555101042bsr20201555figure the key genes expression and mutation analysis in ptc by the cbioportal for cancer genomicsa the genetic alterations of key genes of ptc samples queried genes are altered in of queried patientssamplesb the expression heatmap of key genes c the alteration frequency of key genes in ptc d network of key genesmutations and their frequently altered neighboring genes in ptc the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commons attributionlicense cc by 0cbioscience reports bsr20201555101042bsr20201555figure the mrna and protein expressions of adora1 apoe and lpar5 in ptc tissuesac validation of expression levels of adora1 apoe and lpar5 by rtqpcr in cases of ptc and matched adjacent tissuesd adora1 apoe and lpar5 protein levels are increased in four cases of ptc and matched adjacent tissues as measured bywestern blot p0001discussionptc is a common cancer with great heterogeneity in morphological features and prognosis although most papillary thyroid carcinomas exhibit low biological activity there are still a few patients with higher invasive and metastaticclinical features activation of oncogene expression and loss of function of tumor suppressor genes may lead tothe development or progression of ptc to better clarify the molecular mechanism of ptc occurrence development and metastasis we identified key genes related to ptc progression through comprehensive bioinformaticsmethods and we screened three of the ptc prognosisrelated genes for a comprehensive analysisin the present study we identified differentially expressed genes by rnaseq with go enrichment analysis showing that the degs were enriched in the regulation of the axonogenesis regulation of cell morphogenesis extracellular structure anization extracellular matrix anization synapse anization cellsubstrate adhesion urogenital system development collagencontaining extracellular matrix synaptic membrane cellcell junction glutamatergic synapse neuron to neuron synapse postsynaptic membrane basolateral plasma membranecell adhesion molecule binding passive transmembrane transporter activity extracellular matrix structural constituent glycosaminoglycan binding growth factor binding transmembrane receptor protein kinase activity andtransmembrane receptor protein tyrosine kinase activity and kegg pathway results showed degs were enrichedin cytokinecytokine receptor interaction mapk signaling pathway proteoglycans in cancer rap1 signaling pathway axon guidance cushing syndrome parathyroid hormone synthesis secretion and action agerage signalingpathway in diabetic complications growth hormone synthesis secretion and action salivary secretion circadian entrainment cholinergic synapse p53 signaling pathway ecmreceptor interaction arrhythmogenic right ventricularcardiomyopathy arvc endocrine resistance renin secretion type ii diabetes mellitus bladder cancer nicotinateand nicotinamide metabolism and apoptosismultiple speciesto further explore the interrelationship of differentially expressed genes in papillary thyroid carcinoma we constructed a ppi regulatory network a total of degs with nodes greater than were screened out in the networkthe key genes were adcy8 adora1 adra2c apoe c5ar1 ccl13 ccl20 cdh2 chgb cxcl12 eva1afam20a fn1 gnai1 gpc3 grm4 lpar5 meltf mfge8 nmu oprm1 serpina1 sstr3 timp1 andtnc biological process and molecular function analyses of these key degs indicated that they were significantly the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commons attributionlicense cc by 0cbioscience reports bsr20201555101042bsr20201555figure relative expression of adora1 apoe and lpar5 in normal thyroid tissues and ptc tissues individual cancerstages and nodal metastasis status respectively ualcanp0001involved in cancer regulation processes such as adjustment of cell growth or maintenance cell immune response celladhesion molecular activity and extracellular matrix structural constituentto verify the credibility of the experiments and data the degs screened were verified by the gepia databaseamong the selected genes genes showed expression differences consistent with our rnaseq data among the the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commons attributionlicense cc by 0cbioscience reports bsr20201555101042bsr20201555figure methylation level and immune inï¬ltration level of adora1 apoe and lpar5a relative methylation level of adora1 apoe and lpar5 based on normal thyroid tissues and ptc tissues individual cancerstages and nodal metastasis status respectively ualcan b the correlation between the three genes and tiics timer tiicstumor infiltrating immune cells genes adora1 apoe ccl13 cdh2 eva1a fam20a fn1 lpar5 mfge8 nmu serpina1 timp1and tnc levels were overexpressed in ptc tissues while gpc3 and gnai1 were downregulatedadora1 belongs to the gprotein coupled receptor family and protects human tissues and cells under physiological conditions lin et al suggested that adora1 may promote the proliferation of breast cancer cellsby positively regulating oestrogen receptoralpha in breast cancer cells similarly jayakar indicated that knockdown of apoe expression can reduce the level of mmps by regulating the ap1 signaling pathway and thus reducethe invasion and metastasis of oral squamous cell carcinoma bioinformatics predictions were that apoe mrnashows a significant increase in ptc ccl13 is a coding gene involved in immune regulation and inflammatoryresponses and it has been reported that ccl13 has a role in promoting the proliferation of tumorforming volumein nude mice cdh2 is overexpressed in various cancers some research results indicate that overexpression ofcdh2 can increase the invasive ability and induce emt in lung cancer cells qiu et al confirmed cdh2 actsas an oncogene in papillary thyroid carcinoma which is consistent with our findings eva1a acts as a regulatorof programmed cell death and shen et al indicates that eva1a can inhibit the proliferation of gbm cells by inducing autophagy and apoptosis via inactivating the mtorrps6kb1 signaling pathway fam20a may play a keyrole in haematopoiesis there are few reports on the relationship between fam20a and cancer and our experimentfound that fam20a is more highly expressed in papillary thyroid carcinoma than in other cancers fn1 is involvedin regulating cell adhesion cell movement wound healing and maintaining cell morphology some researchersindicated that fn1 participates in regulating many types of cancer progression such as gastric cancer skin squamous cell carcinoma and papillary thyroid carcinoma it has been shown that lpar5 is related tothe pathogenesis of pancreatic cancer consistent with our study zhang et al believes that lpar5 may be involved in the development of papillary thyroid carcinoma according to previous reports mfge8 is involvedin the progression of various malignancies such as breast cancer melanoma bladder tumors and ovarian cancer the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commons attributionlicense cc by 0cbioscience reports bsr20201555101042bsr20201555figure the expression of adora1 apoe and lpar5 in thyroid cancer tissues and normal thyroid tissuesthe three genes expression were analyzed in different kind of cancer tissues and normal tissues via the timer database p005p001 p001 the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commons attributionlicense cc by 0cbioscience reports bsr20201555101042bsr20201555[] mfge8 is considered to be a potential therapeutic target for ovarian cancer owing to its carcinogenic effect consistent with our data zhang et al indicate that nmu is one of the degs of papillary thyroid carcinoma recently a researcher has shown that abnormal expression of nmu is associated with a variety of cancers for serpina1 there are currently six s pointing out that serpina1 may be a key gene for ptc consistentwith our results [] clinical studies have shown that high expression of timp1 is positively correlated witha poor prognosis of colon brain prostate breast lung and several other cancers tnc is a component of theextracellular matrix ecm and is closely related to the malignant biological behavior of cancer in particular tncoverexpression is positively associated with liver cancer oral squamous cell carcinoma and lymph node metastasisof breast cancer gpc3 belongs to the glypicans family it has been reported that overexpression of gpc3can promote the metastasis of hepatocellular carcinoma but we found that it is expressed at low levels in ptcsimilar to gpc3 some scholars believe that gnai1 is a tumorpromoting gene and reported upregulated gnai1mrna in human glioma which is inconsistent with our data only the adora1 apoe and lpar5 genes simultaneously showed statistical significance for overall survivaland diseasefree survival of ptc patients considering that the occurrence and metastasis of cancer is a complexand multiregulated process we further analyzed the regulatory mechanisms of these three genes we found thatthe mrna and methylation levels of these three genes were significantly correlated with tnm staging in additionadora1 apoe and lpar5 were all related to immune infiltration especially to dendritic cells finally we foundthat these three genes were more highly expressed in cancer tissues than matched adjacent tissueshowever our research has certain limitations first only four pairs of cancer and adjacent tissues were analyzedusing rnaseq in this experiment so further research requires a larger sample size second further experiments areneeded to validate the specific mechanisms of these key genesdata availabilitythe data used to support the findings of this study are available from the corresponding author upon requestcompeting intereststhe authors declare that there are no competing interests associated with the manuscriptfundingthis study was supported by grants from the hebei provincial department of finance specialist capacity building and specialistleadership program [grant number ] the hebei provincial natural science foundation project [grant number h201840505]and the hebei north university basic research business expenses project [grant number jyt2019015]author contributionxu lin conducted the bioinformatics analysis xu lin and gang xue contributed as first authors xu lin wrote the manuscriptjingfang wu and gang xue critically revised the gang xue and da pei obtained clinical specimens and the others contributed to verification of the rnaseq resultsabbreviationsatc anaplastic thyroid carcinoma ecm extracellular matrix ftc follicular thyroid carcinoma ptc papillary thyroid carcinomareferences kitahara cm and sosa ja the changing incidence of thyroid cancer nat rev endocrinol 101038nrendo2016110 aschebrookkilfoy b ward mh sabra mm and devesa ss thyroid cancer incidence patterns in the united states by histologic type thyroid 101089thy20100021 pourseiraï¬ s shishehgar m ashraf mj and faramarzi m papillary carcinoma of thyroid with nasal cavity metastases a case report iranj med sci ullmann tm gray kd moore md zarnegar r and fahey iii tj current controversies and future directions in the diagnosis andmanagement of differentiated thyroid cancers gland surg 1021037gs20170908 jin x deng b ye k et al comprehensive expression proï¬les and bioinformatics analysis reveal special circular rna expression and potentialpredictability in the peripheral blood of humans with idiopathic membranous nephropathy mol med rep 103892mmr201910671 rapisuwon s vietsch ee and wellstein a circulating biomarkers to monitor cancer progression and treatment comput struct biotechnolj 101016jcsbj201605004 the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commonsattribution license cc by 0cbioscience reports bsr20201555101042bsr20201555 pertea m pertea gm antonescu cm et al stringtie enables improved reconstruction of a transcriptome from rnaseq reads natbiotechnol 101038nbt3122 frazee ac pertea g jaffe ae et al ballgown bridges the gap between transcriptome assembly and expression analysis nat biotechnol 101038nbt3172 von mering c huynen m jaeggi d et al string a database of predicted functional associations between proteins nucleic acids res 101093nargkg034 chandrashekar ds bashel b balasubramanya sah et al ualcan a portal for facilitating tumor subgroup gene expression and survivalanalyses neoplasia 101016jneo201705002 li t fan j wang b et al timer a web server for comprehensive analysis of tumorinï¬ltrating immune cells cancer res e108e11010115800085472can170307 nikiforov ye and nikiforova mn molecular genetics and diagnosis of thyroid cancer nat rev endocrinol 101038nrendo2011142 pusztaszeri m and auger m update on the cytologic features of papillary thyroid carcinoma variants diagn cytopathol 101002dc23703 borea pa gessi s merighi s and varani k adenosine as a multisignalling guardian angel in human diseases when where and howdoes it exert its protective effects trends pharmacol sci 101016jtips201602006 lin z yin p reierstad s et al adenosine a1 receptor a target and regulator of estrogen receptoralpha action mediates the proliferativeeffects of estradiol in breast cancer oncogene 101038onc2009409 jayakar sk loudig o brandweingensler m et al apolipoprotein e promotes invasion in oral squamous cell carcinoma am j pathol 101016jajpath201706016 tan j qian x song b et al integrated bioinformatics analysis reveals that the expression of cathepsin s is associated with lymph nodemetastasis and poor prognosis in papillary thyroid cancer oncol rep kuo cy wang jc hsu sl and hwang gy functional characterization of hepatitis b virus x protein based on the inhibition oftumorigenesis in nude mice injected with ccl13hbx cells intervirology 101159000158522 yamauchi m yoshino i yamaguchi r et al ncadherin expression is a potential survival mechanism of geï¬tinibresistant lung cancer cellsam j cancer res qiu j zhang w zang c et al identiï¬cation of key genes and mirnas markers of papillary thyroid cancer biol res 101186s4065901801881 shen x kan s liu z et al eva1a inhibits gbm cell proliferation by inducing autophagy and apoptosis exp cell res 101016jyexcr201702003 liao yx zhang zp zhao j and liu jp effects of fibronectin on cell proliferation senescence and apoptosis of human glioma cellsthrough the pi3kakt signaling pathway cell physiol biochem 10115 | Colon_Cancer |
glioma initiates from glial cells and contains several types such as astrocytoma and oligodendroglioma1 over a quarter of brain tumors are glioma which causes a large number of cancerrelated deaths every year around the world1 the current clinically therapeutic strategies are surgery combined with chemotherapy and radiotherapy2 however the prognosis of glioma patients remains not well post therapy3 hence it is urgently required to discover new molecular mechanism for glioma therapyboth long noncoding rna lncrna and microrna mirna belong to noncoding rnas which have no proteincoding ability lncrna is characterized with more than nucleotides while mirna is about nucleotides in length4 lncrna and mirna are involved in various cellular processes including cell division invasion and survival5 dysregulation of lncrna or mirna usually causes tumor initiation and progression67 for example lncrna linc00152 upregulation promotes gastric cancer growth and metastasis8 lncrna snhg6 overexpression facilitates lung cancer cell proliferation and metastasis9 mir3405p dysregulation promotes tumorigenesis of esophageal squamous cell carcinoma10 in addition mir126 cancer management and research du this work is published and licensed by dove medical press limited the full terms of this license are available at wwwdovepresscomtermsphp and incorporate the creative commons attribution non commercial unported v30 license httpcreativecommonslicensesbync30 by accessing the work you hereby accept the terms noncommercial uses of the work are permitted without any further permission from dove medical press limited provided the work is properly attributed for permission for commercial use of this work please see paragraphs and of our terms wwwdovepresscomtermsphpcorrespondence jun wu weiwen qiu email wwwwjjjj924163com weiwenqhotmailcomsubmit your manuscript wwwdovepresscomdovepresshttp102147cmars262279 0cdu dovepresssuppresses colon cancer cell survival and induces apoptosis11 besides lncrna has been identified as potential competing endogenous rna cerna for mirna to function in cancer12 the potential roles underlying lncrna and mirna still require much investigation and the relationship between lncrna and mirna also needs to be definedlinc00173 is an oncogene in lung cancer and breast cancer1314 the function of linc00173 in glioma is unclear in the current study we found that linc00173 was upregulated in glioma tissues linc00173 high expression was associated with a low survival rate linc00173 depletion suppressed proliferation migration and invasion of glioma cells linc00173 was discovered to sponge mir765 to elevate nutf2 expression taken together our findings supported that linc00173 plays essential oncogenic roles in glioma through activating mir765nutf2 pathwaymaterials and methodsclinical samplesthirtyseven glioma tissues and normal tissues were collected from lishui city peoples hospital patients received no radiotherapy or chemotherapy before surgery all tissues were stored in liquid nitrogen association between linc00173 expression and clinical characteristics in glioma tissues was analyzed in table written informed consent was obtained from every patient this study was approved by the ethics committee of lishui city peoples hospital no and the table association between linc00173 expression and clinical characteristics in glioma tissuesfeaturesage yearsgendermalefemalegradeiiiiiiivtumor size cmlow n19high n18pvalueexperiments were conducted in accordance with the declaration of helsinkicell culture and treatmentthe normal human astrocyte nha and glioma cell lines were purchased from institute of biochemistry and cell biology of the chinese academy of sciences shanghai china cells were cultured using pmi1640 medium invitrogen carlsbad ca usa supplemented with fetal bovine serum fbs invitrogen shrnas against linc00160 mir6293p mimics mir6293p inhibitors and negative controls were obtained from genepharma and transfected into glioma cells using lipofectamine invitrogen according to the manufacturers instructions efficiency was validated using qrtpcr after hqrtpcrtotal rna was extracted from tissues and cell lines using trizol invitrogen carlsbad ca primescript rt reagent kit rr047a takara holdings inc tokyo japan was used to generate cdna from rna template qpcr was completed through sybr premix ex taq¢ ii takara japan gapdh was the normalized control relative expression was calculated through the δδct methodluciferase reporter assaythe fragment of linc00173 or nutf2 containing indicated mir765 binding site was constructed into pmir report vector for luciferase reporter assay glioma cells were transfected with report vector and mir765 mimics after h the luciferase reporter activity was measured through the dualluciferase reporter assay system promega madison wiwestern blot assaycells were lyzed using radioimmunoprecipitation buffer beyotime shanghai china protein concentration was determined by a bca protein assay kit thermo fisher scientific ma then proteins were separated using sdspage and transferred onto pvdf membranes after blockage using bsa for h the membrane was incubated the primary antibodies at °c overnight after washed times using pbst the membranes were incubated with horseradish peroxidaselabeled second antibody followed by detection the enhanced chemiluminescence reagent emd millipore usathrough submit your manuscript wwwdovepresscom dovepress cancer management and research 0cdovepress du cck8 and colony forming assaysproliferation was measured using cck8 and colony formation assay cck8 assay was performed using the cck reagent dojindo kumamoto japan according to the manufacturers instructions and absorbance was determined at nm using a microplate reader biotek winooski vt for colony formation assay cells were seeded into 6well plates and cultured for days then the cells were fixed with methanol and stained with crystal violet for minutesedu assaycells were plated into 96well plates and incubated with edu μl at °c for h followed by detection using facstranswell migration and invasion assaystranswell plates corning ny were used to measure migration and invasion according to the manufacturers instructions in brief cells were suspended into μl serumfree medium and seeded into the upper chamber while the lower chamber was filled with µl of complete medium after incubation for cells in the lower chamber were fixed with methanol and stained with crystal violet for minutes migrated and invaded cells were counted through a light microscopestatistical analysisgraphpad prism graphpad ca usa was used to analyze results data were presented as means±standard deviation sd significant differences were analyzed using students ttest or oneway anova survival rate was analyzed by the kaplanmeier method and log rank test p005 was considered to be significantresultslinc00173 expression is elevated in gliomathe expression of linc00173 was firstly analyzed through qrtpcr we found that linc00173 level was elevated in glioma tissues compared with normal tissues figure 1a besides we found that linc00173 was also upregulated in glioma cell lines compared to nha cells figure 1b then according to the median value of linc00173 glioma tissues were classified into two groups after analysis we found that linc00173 high expression correlated with poor prognosis figure 1ctransfection of linc00173 enhanced glioma cell proliferation migration and invasionto explore the function of linc00173 u87 and u251 cells were chosen after shlinc00173 linc00173 expression was significantly downregulated figure 2a through cck8 assay we observed that linc00173 knockdown suppressed the proliferation capacity of glioma cells figure 2b and c which was validated by edu and colony formation assays figure 2d and e afterwards transwell assay was performed results indicated that linc00173 loss inhibited migration and invasion of glioma cells figure 2f and g thus linc00173 exerted oncogenic roles by affecting proliferation migration and invasionlinc00173 worked as the sponge for mir765linc00173 has been found to serve as cerna for several mirnas such as mir490 and mir2181314 to determine the mechanism of linc00173 in glioma we also figure linc00173 expression is elevated in glioma a the level of linc00173 in glioma tissues was measured b the expression of linc00173 in glioma cell lines and nhas c association between overall survival and linc00173 expression in glioma patients p005cancer management and research submit your manuscript wwwdovepresscom dovepress 0cdu dovepressfigure linc00173 enhanced glioma cell proliferation migration and invasion a qrtpcr analysis of linc00173 expression in u87 and u251 cells be proliferation ability was measured using cck8 edu and colony formation assays f and g migration and invasion capacity was evaluated after linc00173 knockdown in glioma cells p005suppressed the supporting their direct performed bioinformatics analysis using mirdb we identified that mir765 was the most potential candidate because it scored the highest to validate it we constructed luciferase reporters figure 3a followed by luciferase reporter assay results showed that mir765 activity of linc00173wt only figure 3b interaction pulldown assay further demonstrated their interaction figure 3c qrtpcr found that linc00173 overexpression suppressed the level of mir765 figure 3d next bioinformatics analysis using mirdb and targetsan implied that nutf2 is the most potential target of mir765 the corresponding luciferase reporters were further constructed figure 3e luciferase reporter assay also demonstrated the interaction between nutf2 and mir765 figure 3f besides nutf2 expression was suppressed by mir765 mimics figure 3g moreover nutf2 level was decreased after linc00173 knockdown while mir765 inhibitors reversed it figure 3h finally we found that mir765 level was negatively correlated with linc00173 or nutf2 in glioma tissues figure 3i and jlinc00173 promoted glioma progression through mir765nutf2 pathwaywe noticed that nutf2 expression was upregulated in glioma tissues figure 4a and b suggesting an oncogenic role to investigate whether linc00173 regulates glioma progression through mir765nutf2 we restored the expression of nutf2 in shlinc00173 transfected cells cck8 and transwell assays demonstrated that nutf2 restoration successfully rescued the capacities of proliferation migration and invasion in glioma cells transfected with shlinc00173 figure 4cf therefore linc00173 submit your manuscript wwwdovepresscom dovepress cancer management and research 0cdovepress du figure linc00173 worked as the sponge for mir765 a bioinformatics analysis indicated the binding sites between linc00173 and mir765 b u87 cells were transfected with mir765 mimics or negative controls mirnc and luciferase reporter linc00173wt or linc00173mut then relative luciferase activity was determined c rna pulldown assay using biotinlabeled mirnas d relative expression of mir765 after linc00173 knockdown e bioinformatics analysis indicated the binding sites between mir765 and nutf2 f u87 cells were transfected with mir765 mimics or negative controls mirnc and luciferase reporter nutf2wt or nutf2mut then relative luciferase activity was determined g qrtpcr analysis for nutf2 expression h western blotting analysis for nutf2 protein level i and j correlation analyses of linc00173 mir765 and nutf2 in glioma tissues using pearsons correlation coefficient p005contributes to glioma progression through mir765nutf2 pathwaydiscussionas the most malignant brain tumor glioma leads to a huge number of deaths patients with glioma display a poor prognosis therefore it is of great significance to reveal the mechanism underlying glioma progression in this study we found that linc00173 was upregulated in glioma tissues and cells linc00173 overexpression predicted a poor prognosis moreover linc00173 knockdown the proliferation migration and invasion of glioma cells linc00173 was also found to inhibit mir765 and promote nutf2 expression summarily our research discovered that linc00173 is an important oncogenic lncrna in gliomasuppressed the potential roles of lncrna in glioma have been researched for a long time many lncrnas have been identified to participate in glioma development for example lncrna nck1as1 enhances growth and metastasis of glioma through targeting mir13823p to activate β catenin signaling2 lncrna ccat2 contributes to glioma progression by activating vegfa pathway15 lncrna linc00467 upregulation promotes glioma development through repressing p53 level16 previous study showed that linc00173 downregulation promotes nonsmall cell lung cancer cell growth and survival17 however another study showed that linc00173 enhances chemoresistance and facilitates tumor progression in small cell lung cancer13 besides linc00173 contributes to breast cancer development14 yet how linc00173 works in glioma remains undermined in our study we found that linc00173 was upregulated in glioma tissues linc00173 knockdown inhibited the proliferation migration and invasion of glioma cells therefore our data discovered that linc00173 is a new oncogene in glioma for the first timecancer management and research submit your manuscript wwwdovepresscom dovepress 0cdu dovepressfigure linc00173 promoted glioma progression through mir765nutf2 pathway a and b nutf2 expression in glioma tissues and normal tissues according to tcga data using gepia tool and qrtpcr c and d proliferation was measured by cck8 assay e and f migration and invasion potential was determined by transwell assay p005lncrna has been found to serve as mirna sponge in tumor cells for instance lncrna ttnas1 sponges to promote breast cancer metastasis18 mir1405p lncrna cdkn2bas1 sponges mir3245p to regulate cellcycle progression in laryngeal squamous cell cancer19 previous studies also revealed linc00173 was a sponge for some mirnas such as mir4903p and mir2181314 in our study we did not observe linc00173 sponges above mirnas however through bioinformatics we identified linc00173 targeted mir765 in glioma we demonstrated their direct interaction and found that linc00173 overexpression inhibited mir765 expression mir765 has important roles in cancers mir765 was found to suppress tongue squamous cell carcinoma development20 mir765 also promotes myeloma and osteosarcoma progression2122 besides mir765 plays oncogenic or anticancer roles in gastric cancer and breast cancer2324 its role in glioma remains unclear our results suggested that mir765 was a tumor suppressor in gliomalncrnamirnamrna regulatory axis is widely observed in cancer for example linc00703mir181a klf6 axis suppresses the development of gastric cancer25 linc00312mir9cdh1 axis was found to promote breast cancer progression26 through bioinformatics we found that mir765 targeted nutf2 in glioma moreover we showed that nutf2 expression was regulated by linc00173mir axis the function of nutf2 in cancer is nearly unknown in our work we found that nutf2 expression was upregulated in glioma tissues compared to normal tissues moreover we found that nutf2 overexpression promoted the proliferation migration and invasion of glioma cells indicating nutf2 was an oncogene in gliomain conclusion our study showed that linc00173 acted as a sponge for mir765 to promote nutf2 expression and linc00173mir765nutf2 axis plays a critical function in promoting glioma progressionfunding this work was supported by zhejiang province analytical testing and experimental animal program lgd19h and zhejiang province welfare technology applied research project 2017c37111 disclosureall authors declare no conflicts of interest in this workreferences ostrom qt cioffi g gittleman h cbtrus statistical report primary brain and other central nervous system tumors diagnosed in the united states in neuro oncol 201921suppl 5v1 v100 101093neuoncnoz150the of glioma huang l li x ye h et al long noncoding rna nck1as1 promotes sponging microrna13823p and activating the trim24wntbetacatenin axis j exp clin cancer res 101186s13046 tumorigenesis through chen w lei c liu p et al progress and prospects of recurrent glioma a recent scientometric analysis of the web of science in world neurosurg 2020134e387e399 101016jwneu20 submit your manuscript wwwdovepresscom dovepress cancer management and research 0cdovepress du sun b meng m wei j wang s long noncoding rna pvt1 contributes to vascular endothelial cell proliferation via inhibition of mir190a5p in diagnostic biomarker evaluation of chronic heart failure exp ther med 103892etm20208599 feng s yao j chen y functional role of reprogrammingrelated long noncoding rna lincrnaror in glioma j mol neurosci 101007s120310140488z zhang d zhou h liu j mao j long noncoding rna asb16as1 promotes proliferation migration and invasion in glioma cells biomed res int sun l zhao m wang y neuroprotective effects of mir27a against traumatic brain injury via suppressing foxo3amediated neuronal autophagy biochem biophys res commun 101016jbbrc201612001 shi y sun h downregulation of lncrna linc00152 suppresses gastric cancer cell migration and invasion through inhibition of the erkmapk signaling pathway onco targets ther 102147otts217452 li k jiang y xiang x et al long noncoding rna snhg6 promotes the growth and invasion of nonsmall cell lung cancer by downregulating mir1013p thorac cancer wang x gu m ju y zhou j pik3c3 acts as a tumor suppressor in esophageal squamous cell carcinoma and was regulated by mir340 5p med sci monit 202026e920642 1012659msm923909 wei l chen z cheng n microrna126 inhibit viability of colorectal cancer cell by repressing mtor induced apoptosis and autophagy onco targets ther 102147 otts238348 chen y shen z zhi y long noncoding rna ror promotes radioresistance in hepatocellular carcinoma cells by acting as a cerna for microrna145 to regulate rad18 expression arch biochem biophys 101016jabb201803018 zeng f wang q wang s et al linc00173 promotes chemoresistance and progression of small cell lung cancer by sponging mir218 regulate etk expression oncogene to 101038s4138801909842 fan h yuan j li x et al lncrna linc00173 enhances triplenegative breast cancer progression by suppressing mir490 3p expression biomed pharmacother 1010 16jbiopha2020109987 sun sl shu yg tao my lncrna ccat2 promotes angiogenesis in glioma through activation of vegfa signalling by sponging mir424 mol cell biochem 101007 s1101002003712y zhang y jiang x wu z et al long noncoding rna linc00467 promotes glioma progression through inhibiting p53 expression via binding to dnmt1 j cancer 107150 jca41942 yang q tang y tang c diminished linc00173 expression induced mir1825p accumulation promotes cell proliferation migration and apoptosis inhibition via agernfkappab pathway lung cancer am j transl res in nonsmallcell xue j zhang z li x ren q wang q long noncoding rna ttnas1 promotes breast cancer cell migration and invasion via sponging mir1405p oncol lett 1038 92ol201911222 liu f xiao y ma l wang j regulating of cell cycle progression by the lncrna cdkn2bas1mir3245prock1 axis in laryngeal squamous cell cancer int j biol markers 1011771724600819898489 ding j yang c yang s linc00511 interacts with mir765 and modulates tongue squamous cell carcinoma progression by targeting lamc2 j oral pathol med 101111 jop12677 long s long s he h chen g microrna765 is preregulated in multiple myeloma and serves an oncogenic role by directly targeting sox6 exp ther med 103892 etm20197473 lv db zhang jy gao k microrna765 targets mtus1 to promote the progression of osteosarcoma via mediating erkemt pathway eur rev med pharmacol sci 1026355eurrev_201906_18040 jiao y yuan c wu h li x yu j oncogenic microrna765 promotes the growth and metastasis of breast carcinoma by directly targeting ing4 j cell biochem yuan l ma t liu w et al linc00994 promoted invasion and proliferation of gastric cancer cell via regulating mir7653p am j transl res yang h peng m li y zhu r li x qian z linc00703 acts as a tumor suppressor via regulating mir181aklf6 axis in gastric cancer j gastric cancer 105230jgc2019 19e43 chen y qiu f huang l et al long noncoding rna linc00312 regulates breast cancer progression through the mir9cdh1 axis mol med rep 103892mmr201910895cancer management and research publish your work in this journal cancer management and research is an international peerreviewed open access journal focusing on cancer research and the optimal use of preventative and integrated treatment interventions to achieve improved outcomes enhanced survival and quality of life for the cancer patient the manuscript management system is completely online and includes a very quick and fair peerreview system which is all easy to use visit httpwwwdovepresscomtestimonialsphp to read real quotes from published authors dovepress submit your manuscript here wwwdovepresscomcancermanagementandresearchjournalcancer management and research submit your manuscript wwwdovepresscom dovepress 0c' | Colon_Cancer |
chronic rhinosinusitis crs characterized by dysfunctionalmucociliary clearancemcc with subsequent microbialcolonization is known as a multifactorial disease process wherebacterial infection may play a role in the commencement orprogression of the ammatory response ramakrishnan crs patients have complex sinus microbial communitiesthat incite persistent ammation and airway damage lee andlane despite the high density of bacteria that colonizethe airway nutrient sources that sustain bacterial growth in vivoand the derivation of those nutrients are not wellcharacterizedrecently our laboratory successfully created a rabbit model ofcrs by blocking the sinus drainage pathway cho 2017akey data generated from this model indicate a clear sequenceof events that augment our understanding of recalcitrant crspathophysiology blockage of sinus ostia generates a shiftin microbiota to a predominance of anaerobes and theshift from acute to chronic sinus ammation is subsequentlyassociated with a robust increase in pathogenic bacteria egpseudomonas how the two events are mechanistically related isunknown but critical to understanding disease pathogenesis andthe potential for interventionabundant nutrient sources are provided by airway mucus toliving anisms in the microenvironment and mucins are themajor macromolecular constituents of mucus that provide a largecarbon reservoir [eg short chain fatty acids scfa] flynn mucins are the main nutrient source for nichespeciï¬c microbiota of the gut and oral cavity flynn therefore mucin degrading microbes mdm primary mucindegrader are thought to modify the nutritional landscape ofthe microenvironment and stimulate the growth of secondarycolonizers kolenbrander it is wellknown that similarinteractions exist between the commensal gut microbiota and themucus layer of the human intestine cameron and sperandio although the common sinus pathogen pseudomonasaeruginosa cannot derive scfas endogenously flynn little is known regarding the degradation of airway mucinsas a source of scfas by these or other opportunistic pathogens inthe upper airwaydata from observational studies tunney andthe rabbit model cho 2017a indicate a shift in themicrobiota to predominately anaerobic bacteria with impairedmcc in rabbits production of bioavailable nutrients scfafor pathogenic bacteria follows and there was a subsequent shiftfrom acute to chronic ammation with robust generation ofpathogenic microbes eg p aeruginosa since p aeruginosacannot derive scfas eg acetate and propionate from the hostmucus through fermentation on their own we hypothesized thatanaerobic bacteria may ferment mucins into scfa forms usableby p aeruginosa this would provide a novel mechanistic basisfor recalcitrant crs pathogenesis following mcc disruptionthat occurs with acute respiratory infectionsammation andsubsequent compromise of the sinus ostia by edema crabbe thereforethis study is toevaluate the concentrations of scfa within the sinonasal mucusfrom rabbit and human and its contribution to the growthof p aeruginosathe objective ofmethodspao1 stock preparationpseudomonas aeruginosa pao1 strain was expanded fromglycerol frozen stock by inoculating ml of lysogeny brothlb followed by overnight growth at ¦c on a shaker at rpm cultures were streaked on lb agar plates accordingto the quadrant method and grown in a static incubatorat ¦c overnight at least twice to conï¬rm conformity ofcultures from the plate an isolated colony was grown in ml of lbmiller broth at ¦c on a shaker at rpmovernight cultures were diluted with fresh lbmiller broth toan inoculation concentration of to make a pao1 stockfor further experimentsanimal modelthis study was approved by the institutional animal careand use committee iacuc approval number at theuniversity of alabama at birmingham uab pasteurellafreefemale new zealand white rabbits kg were used forthe study before initiation rabbits were acclimatized to theanimal facility for at least week for any procedure rabbitswere anesthetized with [ketamine mgkg mwi boiseid dextomitor mgkg zoetis inc kalamazoo mibuprenorphine mgkg reckitt benckiser pharmaceuticalsinc richmond va and carprofen mgkg zoetis incfrontiers in cellular and infection microbiology wwwfrontiersinaugust volume 0ccho anaerobes in recalcitrant crskalamazoo mi] in a warm room for comfort rabbits did notreceive any antibiotics before or during this studymucus collection from rabbit model ofsinusitisbased on our previous experiments mdms dominated on week after blocking the sinus drainage pathway in the rabbit andtherefore mucus samples were collected at week cho 2017a a total of four rabbits were used to create a rabbitmodel of acute sinusitis without providing exogenous bacteriaor pathogens as described previously cho 2017a asterile synthetic sponge merocel rcid13 medtronic minneapolismn was inserted in the left unilateral middle meatus naturaloutï¬ow tract of maxillary sinus of new zealand white rabbitsfor weeks and the sponge was removed on week toconï¬rm acute sinusitis rabbits were examined with microcomputed tomography microct scanning using spectctxspect system gamma medica northridge ca and nasalendoscopy [ mm 30degree endoscope karl storz tuttlingengermany] on week and mucus samples were collectedon week control and week sinusitis using a specialsuction catheter created by our laboratory to evaluate whethertargeting fermentative anaerobes halts the sinusitis progressionfrom acute to chronic metronidazole mgkg twice a dayfor days was administered to the acute sinusitis rabbits week microct scans were repeated at week between acute andchronic to assess for ct evidence of sinusitis opaciï¬cationsinus opaciï¬cation grading was performed using kerschnersrabbit sinus ct grading system [scoring each imaging studybased on estimated percent opaciï¬cation of the maxillarysinus for for for for for for for for for and for ] kerschner opaciï¬cation was measured using the imagej version 150i national institutesof health bethesda md by two blinded judgesin vitro coculturingto test whether mdms at week are able to generate metabolitesfrom mucin that could simultaneously stimulate p aeruginosagrowth mucus samples collected at week were cocultured withpao1 in a polystyrene culture tube fisher scientiï¬c companypittsburg pa a bottom agar plug was made by adding µl of agar inoculated with µl of mucus collected from rabbitsday or week or agar negative control n afterallowing this to solidify a top plug was made with µl of minimal media agar inoculated with the dilution of anovernight culture of p aeruginosa pao1 after solidiï¬cationcocultures were placed at ¦c for h agar plugs were thenremoved from the upper phase and homogenized by pipettingin ml of phosphate buï¬ered saline colony forming unitscfu per tube were determined by serial dilution and platingon lb agarmucus collection and culture from humanchronic sinusitisto understand the concentration of scfas in human crs withp aeruginosa mucus samples were collected from the middlemeatus this study was approved by the institutional reviewboard irb approval number at the universityof alabama at birmingham and all patients provided writteninformed consent subjects ¥ years of age visiting theuniversity of alabama at birmingham sinus clinic were recruitedfor the study patient eligibility criteria were designed to limitenrollment to healthy individuals and patients who clearly havecrs based on sinus and allergy health partnership criteriabenninger orlandi control patientswere enrolled based on endoscopic procedures for unilateralbenign tumors where the opposite side could be tested andother disease entities where sinus ammation was not presenteg csf leaks nasal septal deviation benign nasal tumor andturbinate hypertrophy demographic and clinical data wereprospectively collected deidentiï¬ed and stored in a securelyencrypted electronic database for cultures specimens wereobtained in the clinic or operating room or with eswabscopan diagnostics inc murrieta ca for hospital laboratoryculture culture swabs were endoscopically guided to the areaof interest with care taken to avoid contamination from thenares the mucosal surface and overlying mucus of the middlemeatus from frontal ethmoid andor maxillary sinuses wasaggressively swabbed for at least ï¬ve full rotations until fullysaturated culture swabs were sent to the hospital clinicalmicrobiology laboratory for aerobic and anaerobic culture forbacterial growth and isolation for metabolite quantiï¬cationmucus samples from the area of interest were collected in theclinic or or using a modiï¬ed catheter suction created by ourlaboratory cho 2017ab durmowicz metabolite quantiï¬cationtargeted quantiï¬cation of scfas was performed via highperformance liquid chromatography hplc the systemconsisted of a shimadzu scl10a system controller lc10at liquid chromatograph sil10af autoinjector spd10auvvis detector and cto10a column oven separationof compounds was performed with an aminex hpx87hguard column and an hpx87h cationexchange column biorad hercules ca the mobile phase consisted of nh2so4 set at a ï¬ow rate of ml min the column wasmaintained at ¦c and the injection volume was µl aminoacid and metabolite acetate and propionate quantiï¬cationfrom enrichmentsupernatants were performed by millisscientiï¬c inc baltimore md using liquid chromatographymass spectrometry and gas chromatographymass spectrometrygcms samples for amino acid quantiï¬cation were spikedwith µl of uniformly labeled amino acids cambridge isotopelabs and derivatized using accqtag reagent waters corp for min at ¦c a waters micromass quatro lcms interfacedwith a waters atlantis dc18 µm mm columnwas used reversephase lc was used for separation mobilephases a mm ammonium formate in formic acid bmethanol with a constant ï¬ow rate ml min and acolumn temperature of ¦c electrospray ionization was usedto generate ions in the positive mode and multiple reactionmonitoring was used to quantify amino acids samples ¼µl for acetate and propionate quantiï¬cation were ï¬rst dilutedfrontiers in cellular and infection microbiology wwwfrontiersinaugust volume 0ccho anaerobes in recalcitrant crs µl water spiked with internal standards µl of ppm acetate [13c2] and ppm propionate [13c1] andacidiï¬ed using µl of 12n hcl after equilibration at ¦c for h carboxenpolydimethylsiloxane solid phase microextractionspme ï¬ber was used to adsorb the headspace at ¦c for min acids were then desorbed into the gas chromatographinlet for min a m mm id db624 column attached toa thermo electron trace gas chromatograph with helium carriergas ml min was used for separation of analytes a watersmicromass quatro gc mass spectrometer was used for detectionand quantiï¬cation of target ionseffect of scfa on pao1 growthto test the eï¬ects of scfa on p aeruginosa growth we incubatedpao1 strains with scfa at varying concentrations each scfamedium was prepared by adding individual scfa to m9 minimalsalts media at diï¬erent concentrations obtained from sigma stlouis mo pao1 seeding solution was prepared by adding µl pao1 stock to ml lbmiller broth µl of pao1 seedingsolution was inoculated into µl scfas media solutionson a 48well plate and incubated at ¦c for h the ï¬nalconcentration of each scfa was or mm based onin vivo hplc data optical density od of planktonic pao1growth was measured at nm using a microplate readeradditionally to assess the growth of pao1 strains in the presenceof all scfas the colony counts of pao1 were comparedbetween the single the most dominant scfa vs scfas afterincubating h in m9 minimal salts media repeated statistical analysisstatistical analyses were conducted using excel andgraphpad prism software la jolla ca with signiï¬cance setat p statistical evaluation utilized unpaired student ttestsor analysis of variance anova based on the characteristics ofanalysis data is expressed ± standard error of the meanresultsmucus samples from acute sinusitisrabbits support the growth of pao1in vitroto test whether the mucus samples from acute sinusitis inthe rabbit model enriched with mdms and scfas couldsimultaneously stimulate pao1 growth the mucus samples werecocultured with pao1 in a minimal mucin medium once thelower agar phase containing the mucus had solidiï¬ed pao1 wassuspended in buï¬ered agar medium without mucin ie nocarbon source and was placed in the upper portion of the tubethis experimental setup establishes an oxygen gradient allowinganaerobes to grow and restricts the movement of microbes butallows metabolites to freely diï¬use flynn underthese conditions p aeruginosa would be expected to achieve ahigher cell density if provided with diï¬usible growth substratesfrom the mucus lower phase coculture growth was monitoredover a 72h period after h a diï¬usible bluegreen pigmentpyocyanin characteristic of p aeruginosa growth was observedthroughout the coculture tubes contained with sinusitis mucusweek figure 1a by contrast no observable pigment wasproduced in the tubes contained with control mucus day or without any mucus colony counts of the upper phase weresigniï¬cantly higher in those tubes containing mucus samplesfrom week sinusitis cfutube ± n compared to tubes containing control mucus ± n or no mucus ± n with a magnitude 5fold increase p figure 1bmucus samples from acute sinusitisrabbits contain short chain fatty acidsscfato provide evidence of fermentative activity in vivo scfasacetate propionate and butyrate were quantiï¬ed using gcms within the mucus samples from rabbits on day controland week sinusitis scfas were found at millimolar or lessconcentrations in all mucus samples and we were able to detect allthree scfas figure acetate concentrations were signiï¬cantlyhigher in the mucus samples collected on week sinusitisrelative to day control ± vs ± mm p n propionate and butyrate concentrations werenot signiï¬cantly elevated in the mucus samples from sinusitiscompared to those from control [propionate ± vs ± mm p n butyrate ± vs ± mm p n ] even though there wasa trendhuman mucus samples from crs withp aeruginosa contain signiï¬cantly higherscfasto provide the evidence of fermentative activity in human crswith p aeruginosa we analyzed the presence of scfas in humanmucus samples from controls and crs with p aeruginosa ofthose human mucus samples collected in the clinic or or eightsamples were collected from controls and six from crs withp aeruginosa all crs patients with p aeruginosa presentedwith purulence in the sinus cavity without any recent use oforal or intravenous antibiotics for at least weeks of those crs with p aeruginosa mucus samples mucusdegradingmicrobes were present in ï¬ve samples from our hospitalclinical microbiology laboratory report table using gcms scfas acetate propionate and butyrate were quantiï¬edin human mucus samples collected from eight controls and crs patients with p aeruginosa with active purulent drainagescfas were found at mm concentrations in all mucus samplesfigure scfas from crs patients with p aeruginosa were alsosigniï¬cantly higher than those from controls acetate ± vs ± mm p propionate ± vs ± p butyrate ± vs ± p figure collectively datapresented here demonstrate the presence of signiï¬cantly higherquantities of fermentation metabolites in crs with p aeruginosascfas increase pao1 growth in vitroto understand the role of scfas in the growth of pao1 invitro the growth of pao1 with multiple concentrations offrontiers in cellular and infection microbiology wwwfrontiersinaugust volume 0ccho anaerobes in recalcitrant crsfigure mucus samples from rabbit sinusitis support the growth of pao1 in vitro a the mucus samples were cocultured with pao1 in an anaerobic minimalmucin medium after h a diffusible bluegreen pigment pyocyanin characteristic of p aeruginosa growth was observed throughout the coculture tubescontaining sinusitis mucus week b colony counts were signiï¬cantly higher in those tubes contained with the mucus samples from week sinusitis cfutube ± compared to tubes containing control mucus ± or no mucus ± with a magnitude increasep represents statistical signiï¬cance p frontiers in cellular and infection microbiology wwwfrontiersinaugust volume 0ccho anaerobes in recalcitrant crsfigure concentrations of short chain fatty acids scfa in mucus samples from rabbits acetate concentrations were signiï¬cantly higher in the mucus samplescollected on week sinusitis relative to day control ± vs ± mm p n propionate and butyrate concentrations were higher in themucus samples from sinusitis compared to those from control propionate ± vs ± mm p butyrate ± vs ± mm p but statistical signiï¬cance was lacking n all groups consisted of at least four experiments represents statistical signiï¬cance p table presence of mucin degrading microbes in p aeruginosa culturepositive patientsno age comorbiditieslocation ofculturemucin degradingmicrobes dmfrontal sinusstreptococcusintermedius asthma mannosebindingethmoid sinusenterobacter cloacaelectin protein deï¬ciency cf 01f508 01f508frontal sinuscutibacterium acnesenterobacter cloacae immunocompromisedmaxillary sinusnonepostkt dm prostate cancermiddle meatusklebsiella oxytoca dm copdethmoid sinusrothia mucilaginosastreptococcus salivariusdm diabetes mellitus cf cystic ï¬brosis kt kidney transplantation copd chronicobstructive pulmonary diseasescfas was monitored concentrations of scfas were chosenbased on the previous hplc ï¬ndings above figure asacetate concentrations were above mm in the rabbit mucussamples we utilized four diï¬erent concentrations of acetate and mm because propionate and butyratewere mm two diï¬erent concentrations and mmbelow mm were used normalized od values were comparedto the average od from control without adding scfas foracetate propionate and butyrate figure 4a in the presence ofacetate pao1 exhibited increased growth from to mmand statistical signiï¬cance was noted between and mmcompared to control [normalized od values at nm mm ± n mm ± n mm ± n and mm ± n p anova with posthoc tukeykramer] in thepresence of propionate statistical signiï¬cance was noted in bothconcentrations [normalized od values at nm mm ± n mm ± n p anova] however the growth increases of pao1 observedwith butyrate were not statistically signiï¬cant in the analysis ofthese two concentrations [ mm ± n mm ± n p anova] additionallyas acetate is the major scfa at least or 200fold higherthan propionate or butyrate in figure the growth of pao1strains was compared in the presence of acetate alone mmvs scfas acetate mm propionate mm butyrate mm colony counts were signiï¬cantly higher when pao1strains were grown with acetate alone or scfas compared tocontrols control ± cfuml n acetate ± cfuml n threescfas ± cfuml n anovap figure 4b even though there was a trend towardhigher colony counts when pao1s were treated with scfasstatistical signiï¬cance was lacking between the acetate alone and scfas tukeys multiple comparison test p targeting anaerobes halts theradiographic evidence of sinusitisprogressionto evaluate whether targeting fermentative anaerobes haltssinusitis progression from acute to chronic stages acute sinusitisrabbits week were treated with metronidazole for days atweek midpoint between acute and chronic the metronidazoletreatment group n had marked improvement ofopaciï¬cation compared to the control group without treatmentn figure ct scores were signiï¬cantly higher in rabbitswithout metronidazole treatment kerschner scale n ± compared to those rabbits treated with metronidazole n ± p at week radiographically theprogression to sinusitis was halted by metronidazole treatmentdiscussionin this study we investigated the role of scfas produced bymucin fermenting anaerobes in the growth of p aeruginosafrontiers in cellular and infection microbiology wwwfrontiersinaugust volume 0ccho anaerobes in recalcitrant crsfigure concentrations of short chain fatty acids scfa in human mucus samples scfas from crs patients with p aeruginosa pa n were compared tothose from controls n acetate ± vs ± mm p propionate ± vs ± p butyrate ± vs ± p [represents statistical signiï¬cance p p and p ]in rhinosinusitis to our knowledge this is the ï¬rst study toquantitatively assess the presence of scfas in the human mucusincluding patients with crs while pseudomonas ineï¬ectuallyutilizes mucins as a carbon source on its own flynn we determined that mucin fermentation by mdms canstimulate the growth of p aeruginosa moreover we revealed thatscfas were also abundant and available in crs patients withand without p aeruginosa together these results suggest thatthe high levels of utilizable metabolites present in sinus mucusmay be derived from bacterial mucin degradation by anaerobesin the sinus cavity which may contribute to the establishmentand progression of recalcitrant crs cho under normal oxygen conditions most bacteria preferentiallyoxidize glucose and other saccharides to pyruvate and shuttlepyruvate through the citric acid cycle both processes requireoxygen as the ï¬nal electron acceptor in anaerobic conditionsbacteria must choose an alternative route by using the energyof another biochemical reaction and thus bypassing oxidativerespiration zumft ragsdale and pierce the processof fermentation involves the release of energy from compoundswithout utilizing exogenous oxygen eg muscle cells duringexercise through the formation of lactic acid ghorbani cystic ï¬brosis cf airway disease is one conditionwhich gives rise to persistent bacterial colonization coupledwith an anaerobic microenvironment the cf airway includesa thick mucus layer rendered hypoxic through the metabolismof host immune cells and bacteria under these circumstancesbacteria can produce scfas through the fermentation processfermentation of carbohydrates within the mucus results in theformation of scfas eg propionate butyrate and acetatewhich can thus uence the progression and resolution ofinfection and ammation in the airways ghorbani scfas have been shown to downregulate immune cellammatory responses promote neutrophil chemotaxis induceammatory protein expression in epithelial cellsinhibitproliferation and strengthen epithelial tight junctions in thegastrointestinal tract ferreira vieira inthe large intestine scfas are found at concentrations rangingfrom to mm which is signiï¬cantly higher than theconcentrations found in the airway mortensen and clausen smith while high mm concentrations impairgrowth low mm concentrations mm boost the growthof potential pathogens eg pseudomonas and upregulate il8in cf primary airway epithelium ghorbani thesemolecules easily penetrate the airway tissue because of their lowmolecular weight and subsequently interrupt host cell activityand host defense systems by inducing apoptosis in ï¬broblastsand lymphocytes tonetti kuritaochiai sato it is also possible that scfas may uencep aeruginosa bioï¬lm formation which will be investigated infuture experimentsit is interesting to note that scfa levels were higher inrabbits than human samples in both controls and noncontrolsevery species appear to have diï¬erent microbial fermentationpatterns even if they have similar diets kroliczewska this is the ï¬rst report to our knowledge to evaluatescfa levels in rabbit sinonasal mucus and we will requiremore numbers to strictly deï¬ne normal scfa levels in rabbitsinuses however the high ï¬ber intake in the diet encouragesthe growth of species that ferment ï¬ber into metabolites asscfas and thus rabbits baseline could be higher than humansas previously shown in the gut cummings tomova furthermore for experimental conditions weintentionally created an anaerobic environment in the sinuscavity and the scfa levels were measured during the acutesinusitis phase in the rabbit model by contrast the humansamples were collected from the postoperative sinuses ormiddle meatus in crs patients therefore the scfas fromrabbits may have been generated at higher levels than observedin human samplesbased on our experiments all scfas were signiï¬cantlyhigher in crs with p aeruginosa our clinical results are alsoconsistent with cf bronchoalveolar lavage ï¬uid ï¬ndings in otherstudies ghorbani mirkovic flynn the ratio of propionate to acetate in our humansinusitis samples was comparable to flynn s experimentsin human cf sputumsaliva samples flynn asexplained by flynn only a small amount of propionate isgenerated in vivo or it may be used by microanisms in a crossfeeding relationship when we compared the cfus betweenfrontiers in cellular and infection microbiology wwwfrontiersinaugust volume 0ccho anaerobes in recalcitrant crsfigure growth of pao1 with scfas a pao1 growths were compared to controls in the presence of different concentrations of acetate between and mm normalizzed od values at nm mm ± n mm ± n mm ± n and mm ± n in the presence of propionate statistical signiï¬cance was noted in both concentrations compared to controls normalized od values at nm mm ± n mm ± n however no statistically signiï¬cant growth of pao1 was seen with butyrate at these two concentrations [ mm ± n mm ± n p ] oneway anova ns no signiï¬cance b colony counts were signiï¬cantly higher when pao1 strainswere grown with acetate alone or three scfas compared to controls control ± cfuml n acetate ± cfuml n scfas ± cfuml n anova p p p and p frontiers in cellular and infection microbiology wwwfrontiersinaugust volume 0ccho anaerobes in recalcitrant crsfigure radiographic progression of sinusitis when targeting anaerobes a at week midpoint between acute and chronic the metronidazole treatment groupn had marked improvement of opaciï¬cation compared to the control group without treatment n b scores were signiï¬cantly higher in rabbits withoutmetronidazole treatment kerschner scale n ± compared to those rabbits treated with metronidazole n ± p at week p frontiers in cellular and infection microbiology wwwfrontiersinaugust volume 0ccho anaerobes in recalcitrant crsfigure model for the development of recalcitrant chronic rhinosinusitis this model demonstrates a sequence of events that augment our understanding ofrecalcitrant crs pathophysiology blockage of sinus ostia generates a shift in microbiota to a predominance of anaerobes mucin metabolites [short chain fattyacids scfas] produced by these anaerobes are abundant within the mucus during an acute anaerobic stage and the shift from acute to chronic sinusammation is associated with a robust increase in pathogenic bacteria eg p aeruginosathe acetate alone and three scfas an additional growth ofp aeruginosa could be clinically presumable in the presenceof multiple nutrients even though statistical signiï¬cance waslacking in this experiment when comparing the total scfaconcentrations before and after antibiotic treatment in patientspresenting with a cf pulmonary exacerbation the mean scfaconcentrations were signiï¬cantly lower after the treatmentghorbani our results suggest the role of hypoxia in recalcitrant crspathophysiology figure hypoxia due to sinus closure at theostiomeatal complex at the junction of the ethmoid and maxillarysinuses is widely considered a major pathogenic mechanismleading to the development of crs and is strongly supportedby proof that hypoxia is present at the surface epithelium indiseased sinuses of closed ostia aanaes targetingmucinfermenting anaerobes and their metabolites could be anovel therapeutic strategy for the treatment of crs by restoringthe microbial community in diseased sinuses to the originalnoninfected pristine statusthere are several limitations to this study our hypothesisis from the animal model not human subjects and this maynot reï¬ectthe human in vivo environment even thoughsome wellcontrolled microbiota studies using animal modelshave also shown interstudy variations due to confoundingfactors eg origin maternal eï¬ects environmental conditionsnguyen the advantages of the rabbit modelare numerous in that it enables us to control for a numberof variables that are inherent in human samples includinggenetics medical history environmental allergiespollutants andmedication use cho as this is a clinical pilotstudy to assess the concentrations of scfas in crs patientsthe size and homogeneity of the clinical samples limit thestudys generalizability in addition we did not include anymicrobiomerelated analyses or molecular based quantiï¬cationofthe microanisms that colonize the mucus layers ofhuman samples a much larger group of patients will berecruited for future studies additionally bacteroides one ofthe most common anaerobes in the rabbit model is not oneof the common anaerobes in human sinusitis and there couldbe other nutrients other than scfas which are derived byfermentation that sustain bacterial growth in humans thereforewe are planning to testthe capability of patientderivedanaerobic microbiota taxa streptococcus peptostreptococcuspropionibacterium fusobacterium and prevotella to support thegrowth of p aeruginosa isolates using an in vitro and in vivocrossfeeding coculture modelconclusiongiven that scfas are solely derived from bacterial fermentationour experiments propose a critical role for mucinfermentingbacteria in generating carbonsource nutrients for pathogenicfrontiers in cellular and infection microbiology wwwfrontiersinaugust volume 0ccho anaerobes in recalcitrant crsbacteria in the airway mucin fermenting anaerobes maycontribute to the development of recalcitrant crs by degradingmucinsfor potential pathogenslike p aeruginosathus providing nutrientsdata availability statementexperimentsfrom rh sr ws and bw concept ofand editing ds dl ht crw and sz carried out therabbit and in vitro experiments and contributed to samplepreparationjg performed statistical analysis all authorsprovided critical feedback helped shape the research analysisand manuscriptthe datasets generated for this study are available on request tothe corresponding authorfundingethics statementthe studies involving human participants were reviewed andapproved by institutional review board irb approval number at the university of alabama at birmingham thepatientsparticipants provided their written informed consentto participate in this study the animal study was reviewedand approved by institutional animal care and use committeeiacuc approval number at the university of alabamaat birmingham uabthis work was supported by national institutes of healthnihnational heart lung and blood institute r01hl13300605 to bw nationalinstitute of diabetes anddigestive and kidney diseases 5p30dk07248202 to srand nihnational institutes of allergy and infectious diseasek08ai146220john w kirklin research and educationfoundation fellowship award uab faculty developmentresearch award american rhinologic society new investigatoraward triological society career development award andcystic fibrosis foundation k08 boost award cho20a0kbto dycauthor contributionsacknowledgmentsdyc designed the study carried outthe experimentsand took the lead in writing the manuscript with supporta part of this manuscript was presented at north americancystic fibrosis conference in denver coloradoreferencesaanaes k rickelt l fjohansen h k von buchwald c presslert hoiby n decreased mucosal oxygen tension in themaxillary sinuses in patients with cystic ï¬brosis j cyst fibros doi 101016jjcf201012002benninger m s fergusonm b j hadley j a hamilos d l jacobs mkennedy d w adult chronic rhinosinusitis deï¬nitionsdiagnosis epidemiology and pathophysiology otolaryngol head neck surg129suppl3 s1 doi 101016s0194599803013974cameron e a and sperandio v frenemies signaling and nutritionalintegration in pathogenmicrobiotahost interactions cell host microbe doi 1 | Colon_Cancer |
" the activation of the nfκb pathway plays a crucial role in the progression of breast cancer bca andalso involved in endocrine therapy resistance on the contrary to the canonical nfκb pathway the effect of thenoncanonical nfκb pathway in bca progression remains elusivemethods bca tumor tissues and the corresponding cell lines were examined to determine the correlation betweenrelb and the aggressiveness of bca relb was manipulated in bca cells to examine whether relb promotes cellproliferation and motility by quantitation of apoptosis cell cycle migration and invasion rnaseq was performedto identify the critical relbregulated genes involved in bca metastasis particularly relbregulated mmp1transcription was verified using luciferase reporter and chip assay subsequently the effect of relb on bcaprogression was further validated using bca mice xenograft modelsresults relb uniquely expresses at a high level in aggressive bca tissues particularly in triplenegative breastcancer tnbc relb promotes bca cell proliferation through increasing g1s transition andor decreasing apoptosisby upregulation of cyclin d1 and bcl2 additionally relb enhances cell mobility by activating emt importantlyrelb upregulates bone metastatic protein mmp1 expression through binding to an nfκb enhancer elementlocated at the ²flanking region accordingly in vivo functional validation confirmed that relb deficiency impairstumor growth in nude mice and inhibits lung metastasis in scid micekeywords relb emt mmp1 cancer progression and breast cancer according to the latest statistic data the numbers ofnew cases and deaths of breast cancer bca accountedfor and of total cancers worldwide sincethe past decade bca incidence has been consistently increasing in developing countries such as south america correspondence yxu4696njmueducn tangjhnjmueducn2jiangsu cancer hospital jiangsu institute of cancer research theaffiliated cancer hospital of nanjing medical university baiziting nanjing p r china1department of general surgery the first affiliated hospital with nanjingmedical university guangzhou road nanjing p r chinafull list of author information is available at the end of the asia and africa owing to improved early diagnosisand advanced therapy strategies the current death ratesof bca have appreciatively decreased in the western developed countries including the united states howeverdistantan metastasis associated with endocrine therapy resistance still remains as a large obstacle to successful control of advanced bca [ ] to data bca patientswith distant metastasis at the time of diagnosis appearedto be worse prognosis with a 5year survival rate of since metastasis is the main cause of death ofbca patients the key for improving the bca survival rateis to accurately evaluate the metastatic potential for the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cwang cell communication and signaling page of guidance of clinical implementation of the individualizedtreatment plan current therapeutic strategies for bcamainly consisted of surgical reduction and radiotherapybased locally therapy with anticancer drugs providedsystemic therapy including chemotherapy endocrinetherapy and targeted therapy however the comprehensive treatment strategy is still limited and a large numberof patients eventually developed to more aggressive malignant forms that are resistant to the most of treatments[ ] thus the discovery of new biomarkers for surveillance of the disease progression and novel molecular targets for improving therapeutic efficiency are urgentlyneededthe reoccurrence ofnuclear factorκb nfκb a family of transcriptionfactors regulates immune responses and inflammation it has been welldocumented that nfκb plays a crucial role in bca endocrine therapy resistance in particular nfκb activation is thought to promote bcaprogression toward erindependent phenotypes especially fortriplenegative bcatnbc after hormone treatment [ ] mammals express five nfκb membersincluding nfκb1 p50p105 nfκb2 p52p100 rela p65 relb and crel[ ] based on the components of the signaling cascade the nfκb signaling can be activated via either canonical pathway p50rela or noncanonical pathwayp52relb [ ] the canonical nfκb signaling pathway is regarded as the central regulator of the inflammatory response but it has been extensively studied in thecontext of hormone disorders autoimmunity obesityand cancer [ ] nevertheless the molecular basisfor the activation of the noncanonical nfκb pathway awell as its role in cancer progression have been recentlyreceiving increased attention tissuematerials and methodsbca patient tissue samples and immunohistochemistryihcthe first affiliated hospital with nanjing medical university nanjing china has collected fresh tumor tissuesfrom bca patients prior to treatment and noncancerousbreast tissues the ethics committee of nanjing medicaluniversity has approved the study protocol with writteninformed consents obtained from all the patients participated in this study a total of bca cases including cases with lymphatic metastasis were selected to examine the correlation between the level of relb and bcapathological grade or stage the tissues were fixedparaffinembedded slides theslides weredewaxed by xylene and then ethanol washing off excessliquid the slides were further rehydrated by rinsingwith dh2o for ihc the slides were soaked in bsabuffer for h and then incubated with 400x diluted primary antibodies within bsa buffer at °c overnightthe slides were washed and then incubated with biotinylated secondary antibody at room temperature for min after washing the slides with 1x bps a dabsubstrate kit cell signaling tech usa was used toobserve immunostaining images under a microscopethe intensity of ihc staining was scored as negativescore weak medium and strong totalcell positivity was scored as the percentage of positivecells including no positive cell and respectively the hscore was calculated using pi i for all slides inwhich pi represented the percentage of positive and irepresented the staining intensityrelb has been originally identified in bcells and therelbbased noncanonical nfκb signaling has beenrecognized to impair antigen presentation by dcs whichis associated with skin inflammation and excessive immune cell infiltration to various ans in the past decade [] recently relb has been implicated incancer progression particularly in sex hormonerelatedcancers including bca prostate cancer pca and cervical cancer [] we have reported that relb ishighly expressed in advanced pca and enhances radioresistance speculating that the activation of noncanonicalnfκb pathway contributes to pca malignancy from androgen receptorto arindependentphenotypes [ ] the present study further demonstrated that relb highly expresses in advanced bcawhich is necessary for sustaining bca malignant progression mechanistically relb promoted bca cell proliferation emt and metastasis through transcriptionalupregulation of several oncogenic proteinsincludingbcl2 cycline d1 and mmp1ardependentlinesthe selected human breast cellcell culture and gene manipulationalllines were purchased from the american type culture collectionatcc usa and grown in the recommended mediaincluding normal breast epithelial cellline mcflines mcf7 and t47d10a erpositive bca celland tnbc cellmdamb231 and bt549relb was ectopically expressed in erpositive cellswith low constitutive relb conversely relb wasknocked out from the tnbc cells using crisprcas9approach briefly several grnas were designed usingthe optimized crispr design tool doublestrand oligo dna for each grna was cloned into theecorisite in pgl3u6 spsgrna vector the constructwas cotransfected with 331sp cas9 plasmid intomdamb231 and bt549 cells singlecell colonieswere selected using puromycin and neomycin andfurther validated by western blots dna was extractedfrom the candidate clones and confirmed by dna sequencing prior to t7en1 digestion 0cwang cell communication and signaling page of cell proliferationcell proliferation was determined using cell countingand colony formation assay respectively bca cells wereseeded into 96well plates at cellswell and treatedwith cck8 reagent dojindo mol tech japan andcell viability was measured as the optical density at nm for colony formation assay bca cells were seededinto 6well plates at cellswell and cultured for weeks the cells were fixed in paraformaldehyde for min stained with crystal violet beyotime biotech china for h after rinsing three times with1xpbscolonies were photographed andcountedcelltheflow cytometryflow cytometry was used to analyze apoptosis and cellcycle for apoptotic cell qualification à cells wereseeded in 6well plates and cultured for h the cellswere collected and washed three times with cold pbsand then stained with μm annexin vfitc and μmpi dojindo mol tech in a supplied binding buffer for min at room temperature and dark condition apoptotic cells were counted using a bd facscalibur flowcytometer bd biosciences usa and the percentage ofapoptotic cells was calculated by the number of preapoptotic and apoptotic cells divided by the amount oftotal cells for cell cycle analysis the cultured cells weredetached washed and fixed with ethanol overnightat °c the cells were then washed times with coldpbs and treated with μl propidium iodide bd biosciences usa for min the cell cycling phases weredetermined by flow cytometrycell motilitycell movability was analyzed using wound healing assayand transwell assay respectively bca cells were platedinto 6well plates at à cellswell with serumfreedmem for h after rinsing with à pbs three timesthe cultured cells were separated using pipette tips tocreate three parallel wounds the wells were thenwashed several times with pbs to remove floating cellsthe cells were continuatively cultured in the completedmedia the wound closure was monitored at and h using a microscope and the wound surface area wasquantified in addition transwell assay corning usawas used to quantify cell migration and invasion abilitiesfor migration assay the cells were plated into the upperchambers without coated membrane at à cellswellfor invasion assay the chamber inserts were coated with μl serumfree medium mixed with matrigels bdbiosci usa and solidified at °c for h the cellswere placed into the upper chamber at cellswell inboth assays the cells were plated in μl serumfreemedium in the upper chambers and μl mediumsupplemented with fbs was placed into the lowerchambers after culturing the cells for h the invadedcells on the lower surface were fixed in paraformaldehyde stained with crystal violet and counted using amicroscoperna sequencingtotal rna was isolated from bca cells using a totalrna isolation kit invitrogen mrna was converted tocdna libraries and added adapters for sequencing theprocedure of ngs sequencing on illumina hiseq platform was conducted by vazyme biotech co ltd nanjing chinachromatin immunoprecipitation chipa chipit system active motif usa was used toquantify relb binding to the nfκb enhancer elementlocated at the ²flanking region of the human mmp1gene briefly chromatin isolated from bca cells werepulled down using a relb antibody cell signalingusa unprecipitated chromatin was used as input control and chromatin pulled down by an igg antibodyserved as a negative antibody control the pulled downthe enhancer fragment was quantified using a quantitative pcr with the genespecific primerswestern blottingcytosolic and nuclear proteins were extracted from cellsand tumor tissues using a ripa lysis buffer containingpmsf and then quantified using a bca assay kit keygen biotech china the extracted proteins μg were separated on sdspage gels and thentransferred to pvdf membranes the membranes weresubsequently incubated overnight at °c with theprimary antibodies against relb bcl2 cyclin d1 and βactin santa cruz biotech usa against er ecadherin vimentin snail slug twist cell signalingtech usa thereafter the membranes were washedthree times with tbst buffer and incubated at roomtemperature for h with hrpconjugated secondaryantibody santa cruze biotech the immunoblots werevisualized using an enhanced chemiluminescence detection system biorad usa the intensities of blots werequantified using quantity one software and protein expression was normalized by loading controls such as βactin and gapdhanimal experimentthe effects of relb on tumorigenesis and metastasiswere validated using bca cells bearing mouse xenografttumor experimental models all animal studies wereconducted according to the institutional animal careand use approved by the research committee ofnanjing medical university no iacuc1711030 five 0cwang cell communication and signaling page of weekold female balbc athymic nude mice beijingvital river lab animal tech co ltd china wereused for studying tumor growth and fiveweekold female scid mice nanjing medical university chinawere used for studying tumor metastasis respectivelyfor the tumor growth experiment à bca cellswere subcutaneously implanted into the right axilla ofmice tumor volume was measured using digital calipersevery other day and calculated using a standard formulav à ab2 a and b represent the diagonal tumorlengths the mice were executed when tumor volumereached to mm3 and tumor tissues were removedfor tumor metastasis study bca cells were injectedinto mice through tail vein and assessed for lung metastasis the mice were sacrificed at weeks and the number of metastatic lung nodules was countedusage of tcga databasethe tcga bca dataset was analyzed to assess the association of rela or relb expression with bca occurrenceand the correlation between the mrna level of rela orrelb and ernegative bca patient survival ratestatistical analysisdata were presented as the mean ± standard deviationsd from at least three replicates significant differencesbetween the experimental groups were analyzed by unpaired students ttest oneway analysis of varianceanova followed by dunnetts or bonferronis multiple comparison test was performed using prismgraphpad san diego usa statistical significance wasaccepted at p additional materials and methods are in additional fileresultsrelb is correlated with bca aggressivenesswe analyzed the bca cohort in tcga database toexamine whether the expression level of relb is correlated to bca progression and patient survival the statistical analytic data indicated that the mrna levels ofrela and relb in tumor tissues were higher than theirlevels in normal breast tissues particularly the differencein relb levels appeared to be highly associated with bcaprogression fig 1a consistently the expression ofrela and relb was also associated with the overall survival of ernegative bca patients notable compared torela the high level of relb led to lower survival ratesfig 1b to assess the correlation between nfκb andbca normal breast tissues and tumor tissues from theselected bca patients with different stages were analyzedby ihc additional file table s1 compared to thenormal breast tissue control the levels of four membersof the nfκb family increase in bca tumor tissuesespecially in the ernegative phenotype notably the expression of relb is apparently correlated to the aggressiveness of bca fig 1c subsequently we furtherexamined the levels of relb in tnbc tissues vs erpositive bca tissues by western blots as expected thehigh levels of relb were detected in all the tnbc tissues suggesting the relb is inversely related to er inbca fig 1dfurthermore several bca cell lines correspondent tothe different stages of bca progression vs a normalbreast epithelial cell line mcf10a were examined consistently the high constitutive levels of the nfκb members were detected in bca cell lines particularly relbuniquely expressed at high levels in tnbc celllinesfig 2a accordingly the nfκb activity was consistently elevated in bca cells especially in tnbc cellsfig 2b furthermore erpositive mcf7 cells vstriplenegative mdamb231 cells were used to measure cytoplasmic and nuclear levels of the nfκb members as expected all the members highly increased inmdamb231 cells compared to mcf7 cells importantly the nfκb upstream ikkα and its phosphorylatedlevels also heighten in tnbc cells fig 2c the distribution of the nfκb members was further confirmed byconfocal microscope fig 2d taken together these results indicated that the high level of relb is associatedwith aggressiveness of bca suggesting that relb maycontribute to the advanced bcarelb promotes bca cell proliferationto investigate the potential mechanism by which relbregulates downstream gene expression involved in bcaprogression relb was ectopically expressed into mcf7and t47d cells with low levels of constitutive relbfig 3a in parallel relb was knocked out from tnbccells mdamb231 and bt549 using a crisprcas9gene edition system additional file figure s1 fig 3bthe nfκb activity was slightly elevated in the relboverexpressed ernegative cells but significantly reduced in relbknocked out tnbc cells fig 3c and dconsistently the elevated relb in erpositive cells led toincreased cell colony number and the cell proliferationrate fig 3e g and i conversely cell colony formationand the cell proliferation rate significantly decreased inrelbknocked out tnbc cells fig 3f h and jrelb promotes g1s transition and inhibits apoptosisto elucidate the role of relb in bca cell proliferationthe cell cycling process and the cell apoptotic rate wereanalyzed by flow cytometry the enforced expression ofrelb in mcf7 cells resulted in decreasing g1 phase butincreasing s and g2m phases which enhances the acceleration of cell division fig 4a in contrast knockout of relb in mdamb231 cells led to an increasing 0cwang cell communication and signaling page of fig see legend on next page 0cwang cell communication and signaling page of see figure on previous pagefig the correlation between relb and aggressiveness of bca a b tcga database geo accession gse11121 was analyzed to examine thecorrelation between mrna expression profiles of rela and relb and bca occurrence and over survival of patients c bca tumor tissues withdifferent pathological grades and normal breast tissues were screened by ihc using nfκb antibodies the luminal group was characterized aserpositive and her2negative lowgrade genotype her2 group was assessed as a middlegrade genotype with high tumor growth tnbcgroup was determined as a high grade d the levels of relb in tnbc tumors vs erpositive bca tumors were quantified βactinnormalizedimages were plottedfig the nfκb activation in bca cell lines a several corresponding cell lines were selected to confirm the relationship between nfκb proteinsand the aggressiveness of bca cells b the relative nfκb activity was quantified in the bca cell lines c cytoplasmic and nuclear levels of nfκbproteins in erpositive mcf7 and tnbc mdamb231 cells were quantified accordingly the nfκb upstream phosphorylated ikkα levels in thetwo cell lines were measured d the cellular distribution of the nfκb proteins in the two cell lines were further examined using aconfocal microscope 0cwang cell communication and signaling page of fig see legend on next page 0cwang cell communication and signaling page of see figure on previous pagefig determination of relbenhanced bca cell survival and growth a relb was ectopically expressed in erpositive bca cells by stablytransfected a human relb cdna construct b relb was knocked out in tnbc cells using a crisprcas9 gene edition system c d the relative nfκb activity was quantified in relbmanipulated bca cells e f the survival rates in the relbmanipulated bca cells were examined by colonyformation assay gj the proliferation rates in the relbmanipulated bca cells were quantified by cck8 assay mean ± sd was representative ofthree independent experiments carried out in duplication p p show the significances between two groups as indicatedg1 phase but decreasing s phase fig 4b furthermoreapoptosis was examined in the relbmanipulated bcacells although the elevated relb in mcf7 cells unlikelychanged the cell apoptotic rate the depletion of relb inmdamb231 cells obviously increased apoptotic cellrates fig 4c and d consistently the overexpression ofrelb in mcf7 cells resulted in upregulation of cyclind1 but no effect on bcl2 expression fig 4e additionally knock out of relb in mdamb231 cells led to suppression of cyclin d1 and bcl2 suggesting that relbsustains tnbc cell growth via activation of prosurvivaland antiapoptotic pathways fig 4f moreover relb depletion led to increases in p21 and p27 but decreases incmyc cyclin e1 and bclxl additional file table s2additional file figure s2relb enhances bca cell mobility by enhancing emtit has been widely recognized that bca progression isgenerally through the erdependent phenotype to erindependent malignancyimportantly emerging evidence demonstrated the inverse relationship between erand nfκb in bca cells consistently the expression of relb appeared to be inversely correlated to erlevels in the tested cell lines additional file figures3a furthermore er elementsdriven luciferase reporter gene expression construct was constructed toexamine whether relb is able to regulate the er response expectedly the erreporter response was remarkably reduced due to the increased relb in mcf7cells suggesting that relb negatively regulates er signaling additional file figure s3bfurthermore to testify whether relb contributes toestrogendeprived bca progression the effect of relb onthe metastatic ability of bca cells was examined sincethe emt process has been well documented in bca metastasis several emt markers were measured in ourpanel of bca cell lines the elevated relb in erpositivecells led to decreasing ecadherin but increasing snail islug i twist i and vimentin fig 5a in parallel knockout of relb in tnbc cells resulted in decreasing thoseemt markers while ecadherin was not detectable intnbc cells fig 5b accordingly the effect of relb onthe cell motility was analyzed by quantifying cell capacities of wound healing migration and invasion as expected the enforced expression of relb in mcf7 cellssignificantly enhanced the cell capacity for wound recovery compared to a vehicle control fig 5c consistentlythe elevated relb in mcf7 cells also resulted in enhancing cell migration and invasion fig 5e subsequentlyknock out of relb in mdamb231 cells led to decreases in those capacities fig 5d and f these resultssuggest that relb promotes erindependent bca progression mainly through activating emt processrelb upregulates bone metastasis associated proteinmmp1to identify the important relbregulated genes involvedin bca metastasis we applied rnaseq analysis usingtotal rna isolated from relbknocked out mdamb cells vs the parent cells the results indicated thatthe mrna expression profiles of numerous genes werealtered in the relbknocked out cells fig 6a and b importantly multiple genes relating to metastatic signalingpathways were downregulated fig 6c and table among of them mmp1 an activator in bca bone metastasis was highly related to the level of relb toassess the effect of relb on mmp1 expression we verified the expression level of mmp1 increased in relboverexpressed mcf7 cells but decreased in relbknocked out mdamb231 cells suggesting that relbmay directly regulate the mmp1 gene fig 6dto elucidate how relb regulates mmp1 expressionwe identified a putative nfκb element located in the²finking region of the human mmp1 gene for verifying this nfκb element functional response to transcriptional activation by relb a ²flanking region containingthe nfκb enhancer element and the core promoter inthe human mmp1 gene was cloned into a pgl4 vectorto drive the luciferase reporter gene expression compared to the basic vector control the reporter gene response driven by the mmp1 enhancer highly increasedin relboverexpressed mcf7 cells but significantly decreased in relbknocked out mdamb231 cellsfig 6e moreover to confirm that relbmediated transcriptional activation through binding to the nfκb enhancer element located at the ²flanking region of themmp1 gene a 240bp fragment containing the nfκbenhancer element was pulled down using a specific relbantibody and amplified using pcr the amount ofpulleddown dna fragment from mcf7 cell chromatinwas less than the level from mdamb231 cell chromatin fig 6ffurthermore to validate whether the increased mmp1can further promote emt mmp1 was manipulated by 0cwang cell communication and signaling page of fig the effect of relb on cell cycle and apoptosis a b the cell cycling process in relbmanipulated bca cells was analyzed using flowcytometry c d preapoptotic and apoptotic cells in the relbmanipulated bca cells were quantified by flow cytometry e f the levels of bcl2 andcyclin d1 proteins were measured by western blots statistical significance between two groups as described in fig 0cwang cell communication and signaling page of fig the effect of relb on emt a b the emtassociated markers in relbmanipulated bca cells were measured by western blots c d the cellwound healing abilities were analyzed e f the cell a migration and invasion capacities were examined using a transwell assay statisticalsignificance between two groups as described in fig 0cwang cell communication and signaling page of fig see legend on next page 0cwang cell communication and signaling page of see figure on previous pagefig relbmediated transcriptional regulation mrna expression profiles in the relbknocked out mdamb231 cell vs the parent cell wereexamined using a rnaseq platform a the distribution of mrna profiles in the relbdeprived cell was illustrated b cluster analysis of rnaseqdata indicates updownregulated mrna profiles heatmap c kegg pathway enrichment analysis was performed to assess emtmetastasisrelating mrna profiles d the protein level of mmp1 was quantified in the relbmanipulated bca cells e a ²flanking region of the human mmp1gene mmp1 containing the nfκb element underline and the core promoter region was cloned in pgl4 vector to drive the luciferasereporter gene expression as illustrated the enhancer activity modulated by relb in bca cells was estimated by normalized reporter responses f asmall dna fragment containing the nfκb element was pulled down from chromatins using a relb antibody and then quantified by pcr iggserves as a negative antibody control statistical significance between two groups as described in fig overexpressing in mcf7 cells but silencing in mdamb231 cells the elevated mmp1 in mcf7 cells led toa slight decrease in ecadherin but increases in snail twist1 and vimentin in contrast the silence of mmp1in mdamb231 cells resulted in decreasing snail twist slug and vimentin regardless of the undetectable of ecadherin in the cells additional file figures4a in addition to determine the functional contribution of mmp1 to emt under the relb regulationmmp1 was either ectopically expressed in relbknockedout mbamb231 cells or wassilenced in relboverexpressed mcf7 cells intriguingly the elevatedmmp1 in relbknocked out mbamb231cells resultedin partially restored the emt proteins while the silenceof mmp1 in relboverexpressed mcf7 cells was ableto abrogate the elevated emt proteins except for no effect on twist expression additionalfile figures4brelbmediated bca progression was validated in micetwo mouse xenograft tumor experimental models wereused to validate the effects of relb on tumor growth andmetastasis in vivo relbknocked out mdamb231 cellsand the parent cells were subcutaneously injected intothe front leg area of nude mice one week after injection tumors were formed and continuously grew toreach the maximum volume mm3 compared toinjection of the control cells tumor formation by therelbknocked out cells was approximately week late month after injectionthe tumor volumes in thecontrol group reached the maximal volume howeverthe tumor growing speed and tumor weight wereremarkably reduced in the group injected with the relbknocked out cellsfig 7ac according to relbknocked out bcl2 and cycline d1 in the tumor tissueswere significantly reduced fig 7d which just reflectedto in vitro results as shown in fig furthermore the reduction in the metastatic capacityof the relbknocked out mdamb231 cells was validated by injecting the cells through tail vents of scidmice six weeks after the injection the tumor occurredon the lung of mice injected with the control cells however the incidence of metastatic lung tumors was dramatically reduced in the relbknocked out groupfig 7e the excised tumor tissues were analyzed byihc analysis using the relative antibodies compared tothe control group tumor aggressiveness stain ki67 wassignificantly reduced in the relbknocked out groupconsistently the levels of mmp1 and emt markerswere also reduced in relbknocked out group fig 7fadditionally tumor images by hematoxylin and eosinhe staining confirmed that the metastasized tumorstable mrna expression profiles in mdamb231 cellsgene namevimrelbkoctrlcdh11zeb1snai1mmp1mmp9mmp28junhsp70dmbt1smad2log2relbkoctrlupordowndowndowndowndowndowndowndownupupupunchangedp value500e05500e05500e05500e05vim vimentin cdh11 cadherin zeb1 zinc finger eboxbinding homeobox snai1 snail1 mmp1 matrix metallopeptidase mmp9 matrix metallopeptidase mmp28 matrix metallopeptidase jun jun protooncogene ap1 transcription factor hsp70 heat shock protein dmbt1 deleted in malignant brain tumor tumor suppressor smad2 a member in tgfbsmad signaling family 0cwang cell communication and signaling page of fig see legend on next page 0cwang cell communication and signaling page of see figure on previous pagefig validation of relbenhanced tumor growth and metastasis in mice a relbknocked out mdamb231 and the parent cells weresubcutaneously injected into the right axilla of female nude mice and allowed to form tumors thirty days after bca cell implantation tumortissues were removed out prior to the execution the excised tumor tissues were photographed two out of five mice injected with relbknockedout cells didnt form the tumor b c tumor growth rates and the excised tumor weights were measured and plotted d total proteins wereextracted from the tumor tissues and immunoblotted with the relating antibodies βactin served as an internal control e female scid mice wereinjected with relbknocked out mdamb231 and the parent cells through the tail vein after weeks the mice were sacrificed and lungmetastasis was examined the excised lungmetastasized tumors indicated by arrows were photographed f the metastatic lung tissues werescreened by ihc with relative antibodies incorporated with ki67 staining the relative images were plotted g he staining was applied toconform the lung metastatic tissues and the numbers of micrometastasis in the two groups were plotted statistical significance between twogroups as described in fig were obviously reduced in the relbknocked out groupcompared to the control group fig 7g it should benoted that the detection of low levels of relb in therelbknocked out group reflected the constitutive mouserelb additionallythe enforced expression relb inmcf7 cells was still insufficient to tumor metastasizeto the lung altogether these results suggest the noncanonical nfκb pathway may serve as a major contributor to the progression of malignant bcadiscussionit has been widely recognized that bca is | Colon_Cancer |
chronic rhinosinusitis crs characterized by dysfunctionalmucociliary clearancemcc with subsequent microbialcolonization is known as a multifactorial disease process wherebacterial infection may play a role in the commencement orprogression of the ammatory response ramakrishnan crs patients have complex sinus microbial communitiesthat incite persistent ammation and airway damage lee andlane despite the high density of bacteria that colonizethe airway nutrient sources that sustain bacterial growth in vivoand the derivation of those nutrients are not wellcharacterizedrecently our laboratory successfully created a rabbit model ofcrs by blocking the sinus drainage pathway cho 2017akey data generated from this model indicate a clear sequenceof events that augment our understanding of recalcitrant crspathophysiology blockage of sinus ostia generates a shiftin microbiota to a predominance of anaerobes and theshift from acute to chronic sinus ammation is subsequentlyassociated with a robust increase in pathogenic bacteria egpseudomonas how the two events are mechanistically related isunknown but critical to understanding disease pathogenesis andthe potential for interventionabundant nutrient sources are provided by airway mucus toliving anisms in the microenvironment and mucins are themajor macromolecular constituents of mucus that provide a largecarbon reservoir [eg short chain fatty acids scfa] flynn mucins are the main nutrient source for nichespeciï¬c microbiota of the gut and oral cavity flynn therefore mucin degrading microbes mdm primary mucindegrader are thought to modify the nutritional landscape ofthe microenvironment and stimulate the growth of secondarycolonizers kolenbrander it is wellknown that similarinteractions exist between the commensal gut microbiota and themucus layer of the human intestine cameron and sperandio although the common sinus pathogen pseudomonasaeruginosa cannot derive scfas endogenously flynn little is known regarding the degradation of airway mucinsas a source of scfas by these or other opportunistic pathogens inthe upper airwaydata from observational studies tunney andthe rabbit model cho 2017a indicate a shift in themicrobiota to predominately anaerobic bacteria with impairedmcc in rabbits production of bioavailable nutrients scfafor pathogenic bacteria follows and there was a subsequent shiftfrom acute to chronic ammation with robust generation ofpathogenic microbes eg p aeruginosa since p aeruginosacannot derive scfas eg acetate and propionate from the hostmucus through fermentation on their own we hypothesized thatanaerobic bacteria may ferment mucins into scfa forms usableby p aeruginosa this would provide a novel mechanistic basisfor recalcitrant crs pathogenesis following mcc disruptionthat occurs with acute respiratory infectionsammation andsubsequent compromise of the sinus ostia by edema crabbe thereforethis study is toevaluate the concentrations of scfa within the sinonasal mucusfrom rabbit and human and its contribution to the growthof p aeruginosathe objective ofmethodspao1 stock preparationpseudomonas aeruginosa pao1 strain was expanded fromglycerol frozen stock by inoculating ml of lysogeny brothlb followed by overnight growth at ¦c on a shaker at rpm cultures were streaked on lb agar plates accordingto the quadrant method and grown in a static incubatorat ¦c overnight at least twice to conï¬rm conformity ofcultures from the plate an isolated colony was grown in ml of lbmiller broth at ¦c on a shaker at rpmovernight cultures were diluted with fresh lbmiller broth toan inoculation concentration of to make a pao1 stockfor further experimentsanimal modelthis study was approved by the institutional animal careand use committee iacuc approval number at theuniversity of alabama at birmingham uab pasteurellafreefemale new zealand white rabbits kg were used forthe study before initiation rabbits were acclimatized to theanimal facility for at least week for any procedure rabbitswere anesthetized with [ketamine mgkg mwi boiseid dextomitor mgkg zoetis inc kalamazoo mibuprenorphine mgkg reckitt benckiser pharmaceuticalsinc richmond va and carprofen mgkg zoetis incfrontiers in cellular and infection microbiology wwwfrontiersinaugust volume 0ccho anaerobes in recalcitrant crskalamazoo mi] in a warm room for comfort rabbits did notreceive any antibiotics before or during this studymucus collection from rabbit model ofsinusitisbased on our previous experiments mdms dominated on week after blocking the sinus drainage pathway in the rabbit andtherefore mucus samples were collected at week cho 2017a a total of four rabbits were used to create a rabbitmodel of acute sinusitis without providing exogenous bacteriaor pathogens as described previously cho 2017a asterile synthetic sponge merocel rcid13 medtronic minneapolismn was inserted in the left unilateral middle meatus naturaloutï¬ow tract of maxillary sinus of new zealand white rabbitsfor weeks and the sponge was removed on week toconï¬rm acute sinusitis rabbits were examined with microcomputed tomography microct scanning using spectctxspect system gamma medica northridge ca and nasalendoscopy [ mm 30degree endoscope karl storz tuttlingengermany] on week and mucus samples were collectedon week control and week sinusitis using a specialsuction catheter created by our laboratory to evaluate whethertargeting fermentative anaerobes halts the sinusitis progressionfrom acute to chronic metronidazole mgkg twice a dayfor days was administered to the acute sinusitis rabbits week microct scans were repeated at week between acute andchronic to assess for ct evidence of sinusitis opaciï¬cationsinus opaciï¬cation grading was performed using kerschnersrabbit sinus ct grading system [scoring each imaging studybased on estimated percent opaciï¬cation of the maxillarysinus for for for for for for for for for and for ] kerschner opaciï¬cation was measured using the imagej version 150i national institutesof health bethesda md by two blinded judgesin vitro coculturingto test whether mdms at week are able to generate metabolitesfrom mucin that could simultaneously stimulate p aeruginosagrowth mucus samples collected at week were cocultured withpao1 in a polystyrene culture tube fisher scientiï¬c companypittsburg pa a bottom agar plug was made by adding µl of agar inoculated with µl of mucus collected from rabbitsday or week or agar negative control n afterallowing this to solidify a top plug was made with µl of minimal media agar inoculated with the dilution of anovernight culture of p aeruginosa pao1 after solidiï¬cationcocultures were placed at ¦c for h agar plugs were thenremoved from the upper phase and homogenized by pipettingin ml of phosphate buï¬ered saline colony forming unitscfu per tube were determined by serial dilution and platingon lb agarmucus collection and culture from humanchronic sinusitisto understand the concentration of scfas in human crs withp aeruginosa mucus samples were collected from the middlemeatus this study was approved by the institutional reviewboard irb approval number at the universityof alabama at birmingham and all patients provided writteninformed consent subjects ¥ years of age visiting theuniversity of alabama at birmingham sinus clinic were recruitedfor the study patient eligibility criteria were designed to limitenrollment to healthy individuals and patients who clearly havecrs based on sinus and allergy health partnership criteriabenninger orlandi control patientswere enrolled based on endoscopic procedures for unilateralbenign tumors where the opposite side could be tested andother disease entities where sinus ammation was not presenteg csf leaks nasal septal deviation benign nasal tumor andturbinate hypertrophy demographic and clinical data wereprospectively collected deidentiï¬ed and stored in a securelyencrypted electronic database for cultures specimens wereobtained in the clinic or operating room or with eswabscopan diagnostics inc murrieta ca for hospital laboratoryculture culture swabs were endoscopically guided to the areaof interest with care taken to avoid contamination from thenares the mucosal surface and overlying mucus of the middlemeatus from frontal ethmoid andor maxillary sinuses wasaggressively swabbed for at least ï¬ve full rotations until fullysaturated culture swabs were sent to the hospital clinicalmicrobiology laboratory for aerobic and anaerobic culture forbacterial growth and isolation for metabolite quantiï¬cationmucus samples from the area of interest were collected in theclinic or or using a modiï¬ed catheter suction created by ourlaboratory cho 2017ab durmowicz metabolite quantiï¬cationtargeted quantiï¬cation of scfas was performed via highperformance liquid chromatography hplc the systemconsisted of a shimadzu scl10a system controller lc10at liquid chromatograph sil10af autoinjector spd10auvvis detector and cto10a column oven separationof compounds was performed with an aminex hpx87hguard column and an hpx87h cationexchange column biorad hercules ca the mobile phase consisted of nh2so4 set at a ï¬ow rate of ml min the column wasmaintained at ¦c and the injection volume was µl aminoacid and metabolite acetate and propionate quantiï¬cationfrom enrichmentsupernatants were performed by millisscientiï¬c inc baltimore md using liquid chromatographymass spectrometry and gas chromatographymass spectrometrygcms samples for amino acid quantiï¬cation were spikedwith µl of uniformly labeled amino acids cambridge isotopelabs and derivatized using accqtag reagent waters corp for min at ¦c a waters micromass quatro lcms interfacedwith a waters atlantis dc18 µm mm columnwas used reversephase lc was used for separation mobilephases a mm ammonium formate in formic acid bmethanol with a constant ï¬ow rate ml min and acolumn temperature of ¦c electrospray ionization was usedto generate ions in the positive mode and multiple reactionmonitoring was used to quantify amino acids samples ¼µl for acetate and propionate quantiï¬cation were ï¬rst dilutedfrontiers in cellular and infection microbiology wwwfrontiersinaugust volume 0ccho anaerobes in recalcitrant crs µl water spiked with internal standards µl of ppm acetate [13c2] and ppm propionate [13c1] andacidiï¬ed using µl of 12n hcl after equilibration at ¦c for h carboxenpolydimethylsiloxane solid phase microextractionspme ï¬ber was used to adsorb the headspace at ¦c for min acids were then desorbed into the gas chromatographinlet for min a m mm id db624 column attached toa thermo electron trace gas chromatograph with helium carriergas ml min was used for separation of analytes a watersmicromass quatro gc mass spectrometer was used for detectionand quantiï¬cation of target ionseffect of scfa on pao1 growthto test the eï¬ects of scfa on p aeruginosa growth we incubatedpao1 strains with scfa at varying concentrations each scfamedium was prepared by adding individual scfa to m9 minimalsalts media at diï¬erent concentrations obtained from sigma stlouis mo pao1 seeding solution was prepared by adding µl pao1 stock to ml lbmiller broth µl of pao1 seedingsolution was inoculated into µl scfas media solutionson a 48well plate and incubated at ¦c for h the ï¬nalconcentration of each scfa was or mm based onin vivo hplc data optical density od of planktonic pao1growth was measured at nm using a microplate readeradditionally to assess the growth of pao1 strains in the presenceof all scfas the colony counts of pao1 were comparedbetween the single the most dominant scfa vs scfas afterincubating h in m9 minimal salts media repeated statistical analysisstatistical analyses were conducted using excel andgraphpad prism software la jolla ca with signiï¬cance setat p statistical evaluation utilized unpaired student ttestsor analysis of variance anova based on the characteristics ofanalysis data is expressed ± standard error of the meanresultsmucus samples from acute sinusitisrabbits support the growth of pao1in vitroto test whether the mucus samples from acute sinusitis inthe rabbit model enriched with mdms and scfas couldsimultaneously stimulate pao1 growth the mucus samples werecocultured with pao1 in a minimal mucin medium once thelower agar phase containing the mucus had solidiï¬ed pao1 wassuspended in buï¬ered agar medium without mucin ie nocarbon source and was placed in the upper portion of the tubethis experimental setup establishes an oxygen gradient allowinganaerobes to grow and restricts the movement of microbes butallows metabolites to freely diï¬use flynn underthese conditions p aeruginosa would be expected to achieve ahigher cell density if provided with diï¬usible growth substratesfrom the mucus lower phase coculture growth was monitoredover a 72h period after h a diï¬usible bluegreen pigmentpyocyanin characteristic of p aeruginosa growth was observedthroughout the coculture tubes contained with sinusitis mucusweek figure 1a by contrast no observable pigment wasproduced in the tubes contained with control mucus day or without any mucus colony counts of the upper phase weresigniï¬cantly higher in those tubes containing mucus samplesfrom week sinusitis cfutube ± n compared to tubes containing control mucus ± n or no mucus ± n with a magnitude 5fold increase p figure 1bmucus samples from acute sinusitisrabbits contain short chain fatty acidsscfato provide evidence of fermentative activity in vivo scfasacetate propionate and butyrate were quantiï¬ed using gcms within the mucus samples from rabbits on day controland week sinusitis scfas were found at millimolar or lessconcentrations in all mucus samples and we were able to detect allthree scfas figure acetate concentrations were signiï¬cantlyhigher in the mucus samples collected on week sinusitisrelative to day control ± vs ± mm p n propionate and butyrate concentrations werenot signiï¬cantly elevated in the mucus samples from sinusitiscompared to those from control [propionate ± vs ± mm p n butyrate ± vs ± mm p n ] even though there wasa trendhuman mucus samples from crs withp aeruginosa contain signiï¬cantly higherscfasto provide the evidence of fermentative activity in human crswith p aeruginosa we analyzed the presence of scfas in humanmucus samples from controls and crs with p aeruginosa ofthose human mucus samples collected in the clinic or or eightsamples were collected from controls and six from crs withp aeruginosa all crs patients with p aeruginosa presentedwith purulence in the sinus cavity without any recent use oforal or intravenous antibiotics for at least weeks of those crs with p aeruginosa mucus samples mucusdegradingmicrobes were present in ï¬ve samples from our hospitalclinical microbiology laboratory report table using gcms scfas acetate propionate and butyrate were quantiï¬edin human mucus samples collected from eight controls and crs patients with p aeruginosa with active purulent drainagescfas were found at mm concentrations in all mucus samplesfigure scfas from crs patients with p aeruginosa were alsosigniï¬cantly higher than those from controls acetate ± vs ± mm p propionate ± vs ± p butyrate ± vs ± p figure collectively datapresented here demonstrate the presence of signiï¬cantly higherquantities of fermentation metabolites in crs with p aeruginosascfas increase pao1 growth in vitroto understand the role of scfas in the growth of pao1 invitro the growth of pao1 with multiple concentrations offrontiers in cellular and infection microbiology wwwfrontiersinaugust volume 0ccho anaerobes in recalcitrant crsfigure mucus samples from rabbit sinusitis support the growth of pao1 in vitro a the mucus samples were cocultured with pao1 in an anaerobic minimalmucin medium after h a diffusible bluegreen pigment pyocyanin characteristic of p aeruginosa growth was observed throughout the coculture tubescontaining sinusitis mucus week b colony counts were signiï¬cantly higher in those tubes contained with the mucus samples from week sinusitis cfutube ± compared to tubes containing control mucus ± or no mucus ± with a magnitude increasep represents statistical signiï¬cance p frontiers in cellular and infection microbiology wwwfrontiersinaugust volume 0ccho anaerobes in recalcitrant crsfigure concentrations of short chain fatty acids scfa in mucus samples from rabbits acetate concentrations were signiï¬cantly higher in the mucus samplescollected on week sinusitis relative to day control ± vs ± mm p n propionate and butyrate concentrations were higher in themucus samples from sinusitis compared to those from control propionate ± vs ± mm p butyrate ± vs ± mm p but statistical signiï¬cance was lacking n all groups consisted of at least four experiments represents statistical signiï¬cance p table presence of mucin degrading microbes in p aeruginosa culturepositive patientsno age comorbiditieslocation ofculturemucin degradingmicrobes dmfrontal sinusstreptococcusintermedius asthma mannosebindingethmoid sinusenterobacter cloacaelectin protein deï¬ciency cf 01f508 01f508frontal sinuscutibacterium acnesenterobacter cloacae immunocompromisedmaxillary sinusnonepostkt dm prostate cancermiddle meatusklebsiella oxytoca dm copdethmoid sinusrothia mucilaginosastreptococcus salivariusdm diabetes mellitus cf cystic ï¬brosis kt kidney transplantation copd chronicobstructive pulmonary diseasescfas was monitored concentrations of scfas were chosenbased on the previous hplc ï¬ndings above figure asacetate concentrations were above mm in the rabbit mucussamples we utilized four diï¬erent concentrations of acetate and mm because propionate and butyratewere mm two diï¬erent concentrations and mmbelow mm were used normalized od values were comparedto the average od from control without adding scfas foracetate propionate and butyrate figure 4a in the presence ofacetate pao1 exhibited increased growth from to mmand statistical signiï¬cance was noted between and mmcompared to control [normalized od values at nm mm ± n mm ± n mm ± n and mm ± n p anova with posthoc tukeykramer] in thepresence of propionate statistical signiï¬cance was noted in bothconcentrations [normalized od values at nm mm ± n mm ± n p anova] however the growth increases of pao1 observedwith butyrate were not statistically signiï¬cant in the analysis ofthese two concentrations [ mm ± n mm ± n p anova] additionallyas acetate is the major scfa at least or 200fold higherthan propionate or butyrate in figure the growth of pao1strains was compared in the presence of acetate alone mmvs scfas acetate mm propionate mm butyrate mm colony counts were signiï¬cantly higher when pao1strains were grown with acetate alone or scfas compared tocontrols control ± cfuml n acetate ± cfuml n threescfas ± cfuml n anovap figure 4b even though there was a trend towardhigher colony counts when pao1s were treated with scfasstatistical signiï¬cance was lacking between the acetate alone and scfas tukeys multiple comparison test p targeting anaerobes halts theradiographic evidence of sinusitisprogressionto evaluate whether targeting fermentative anaerobes haltssinusitis progression from acute to chronic stages acute sinusitisrabbits week were treated with metronidazole for days atweek midpoint between acute and chronic the metronidazoletreatment group n had marked improvement ofopaciï¬cation compared to the control group without treatmentn figure ct scores were signiï¬cantly higher in rabbitswithout metronidazole treatment kerschner scale n ± compared to those rabbits treated with metronidazole n ± p at week radiographically theprogression to sinusitis was halted by metronidazole treatmentdiscussionin this study we investigated the role of scfas produced bymucin fermenting anaerobes in the growth of p aeruginosafrontiers in cellular and infection microbiology wwwfrontiersinaugust volume 0ccho anaerobes in recalcitrant crsfigure concentrations of short chain fatty acids scfa in human mucus samples scfas from crs patients with p aeruginosa pa n were compared tothose from controls n acetate ± vs ± mm p propionate ± vs ± p butyrate ± vs ± p [represents statistical signiï¬cance p p and p ]in rhinosinusitis to our knowledge this is the ï¬rst study toquantitatively assess the presence of scfas in the human mucusincluding patients with crs while pseudomonas ineï¬ectuallyutilizes mucins as a carbon source on its own flynn we determined that mucin fermentation by mdms canstimulate the growth of p aeruginosa moreover we revealed thatscfas were also abundant and available in crs patients withand without p aeruginosa together these results suggest thatthe high levels of utilizable metabolites present in sinus mucusmay be derived from bacterial mucin degradation by anaerobesin the sinus cavity which may contribute to the establishmentand progression of recalcitrant crs cho under normal oxygen conditions most bacteria preferentiallyoxidize glucose and other saccharides to pyruvate and shuttlepyruvate through the citric acid cycle both processes requireoxygen as the ï¬nal electron acceptor in anaerobic conditionsbacteria must choose an alternative route by using the energyof another biochemical reaction and thus bypassing oxidativerespiration zumft ragsdale and pierce the processof fermentation involves the release of energy from compoundswithout utilizing exogenous oxygen eg muscle cells duringexercise through the formation of lactic acid ghorbani cystic ï¬brosis cf airway disease is one conditionwhich gives rise to persistent bacterial colonization coupledwith an anaerobic microenvironment the cf airway includesa thick mucus layer rendered hypoxic through the metabolismof host immune cells and bacteria under these circumstancesbacteria can produce scfas through the fermentation processfermentation of carbohydrates within the mucus results in theformation of scfas eg propionate butyrate and acetatewhich can thus uence the progression and resolution ofinfection and ammation in the airways ghorbani scfas have been shown to downregulate immune cellammatory responses promote neutrophil chemotaxis induceammatory protein expression in epithelial cellsinhibitproliferation and strengthen epithelial tight junctions in thegastrointestinal tract ferreira vieira inthe large intestine scfas are found at concentrations rangingfrom to mm which is signiï¬cantly higher than theconcentrations found in the airway mortensen and clausen smith while high mm concentrations impairgrowth low mm concentrations mm boost the growthof potential pathogens eg pseudomonas and upregulate il8in cf primary airway epithelium ghorbani thesemolecules easily penetrate the airway tissue because of their lowmolecular weight and subsequently interrupt host cell activityand host defense systems by inducing apoptosis in ï¬broblastsand lymphocytes tonetti kuritaochiai sato it is also possible that scfas may uencep aeruginosa bioï¬lm formation which will be investigated infuture experimentsit is interesting to note that scfa levels were higher inrabbits than human samples in both controls and noncontrolsevery species appear to have diï¬erent microbial fermentationpatterns even if they have similar diets kroliczewska this is the ï¬rst report to our knowledge to evaluatescfa levels in rabbit sinonasal mucus and we will requiremore numbers to strictly deï¬ne normal scfa levels in rabbitsinuses however the high ï¬ber intake in the diet encouragesthe growth of species that ferment ï¬ber into metabolites asscfas and thus rabbits baseline could be higher than humansas previously shown in the gut cummings tomova furthermore for experimental conditions weintentionally created an anaerobic environment in the sinuscavity and the scfa levels were measured during the acutesinusitis phase in the rabbit model by contrast the humansamples were collected from the postoperative sinuses ormiddle meatus in crs patients therefore the scfas fromrabbits may have been generated at higher levels than observedin human samplesbased on our experiments all scfas were signiï¬cantlyhigher in crs with p aeruginosa our clinical results are alsoconsistent with cf bronchoalveolar lavage ï¬uid ï¬ndings in otherstudies ghorbani mirkovic flynn the ratio of propionate to acetate in our humansinusitis samples was comparable to flynn s experimentsin human cf sputumsaliva samples flynn asexplained by flynn only a small amount of propionate isgenerated in vivo or it may be used by microanisms in a crossfeeding relationship when we compared the cfus betweenfrontiers in cellular and infection microbiology wwwfrontiersinaugust volume 0ccho anaerobes in recalcitrant crsfigure growth of pao1 with scfas a pao1 growths were compared to controls in the presence of different concentrations of acetate between and mm normalizzed od values at nm mm ± n mm ± n mm ± n and mm ± n in the presence of propionate statistical signiï¬cance was noted in both concentrations compared to controls normalized od values at nm mm ± n mm ± n however no statistically signiï¬cant growth of pao1 was seen with butyrate at these two concentrations [ mm ± n mm ± n p ] oneway anova ns no signiï¬cance b colony counts were signiï¬cantly higher when pao1 strainswere grown with acetate alone or three scfas compared to controls control ± cfuml n acetate ± cfuml n scfas ± cfuml n anova p p p and p frontiers in cellular and infection microbiology wwwfrontiersinaugust volume 0ccho anaerobes in recalcitrant crsfigure radiographic progression of sinusitis when targeting anaerobes a at week midpoint between acute and chronic the metronidazole treatment groupn had marked improvement of opaciï¬cation compared to the control group without treatment n b scores were signiï¬cantly higher in rabbits withoutmetronidazole treatment kerschner scale n ± compared to those rabbits treated with metronidazole n ± p at week p frontiers in cellular and infection microbiology wwwfrontiersinaugust volume 0ccho anaerobes in recalcitrant crsfigure model for the development of recalcitrant chronic rhinosinusitis this model demonstrates a sequence of events that augment our understanding ofrecalcitrant crs pathophysiology blockage of sinus ostia generates a shift in microbiota to a predominance of anaerobes mucin metabolites [short chain fattyacids scfas] produced by these anaerobes are abundant within the mucus during an acute anaerobic stage and the shift from acute to chronic sinusammation is associated with a robust increase in pathogenic bacteria eg p aeruginosathe acetate alone and three scfas an additional growth ofp aeruginosa could be clinically presumable in the presenceof multiple nutrients even though statistical signiï¬cance waslacking in this experiment when comparing the total scfaconcentrations before and after antibiotic treatment in patientspresenting with a cf pulmonary exacerbation the mean scfaconcentrations were signiï¬cantly lower after the treatmentghorbani our results suggest the role of hypoxia in recalcitrant crspathophysiology figure hypoxia due to sinus closure at theostiomeatal complex at the junction of the ethmoid and maxillarysinuses is widely considered a major pathogenic mechanismleading to the development of crs and is strongly supportedby proof that hypoxia is present at the surface epithelium indiseased sinuses of closed ostia aanaes targetingmucinfermenting anaerobes and their metabolites could be anovel therapeutic strategy for the treatment of crs by restoringthe microbial community in diseased sinuses to the originalnoninfected pristine statusthere are several limitations to this study our hypothesisis from the animal model not human subjects and this maynot reï¬ectthe human in vivo environment even thoughsome wellcontrolled microbiota studies using animal modelshave also shown interstudy variations due to confoundingfactors eg origin maternal eï¬ects environmental conditionsnguyen the advantages of the rabbit modelare numerous in that it enables us to control for a numberof variables that are inherent in human samples includinggenetics medical history environmental allergiespollutants andmedication use cho as this is a clinical pilotstudy to assess the concentrations of scfas in crs patientsthe size and homogeneity of the clinical samples limit thestudys generalizability in addition we did not include anymicrobiomerelated analyses or molecular based quantiï¬cationofthe microanisms that colonize the mucus layers ofhuman samples a much larger group of patients will berecruited for future studies additionally bacteroides one ofthe most common anaerobes in the rabbit model is not oneof the common anaerobes in human sinusitis and there couldbe other nutrients other than scfas which are derived byfermentation that sustain bacterial growth in humans thereforewe are planning to testthe capability of patientderivedanaerobic microbiota taxa streptococcus peptostreptococcuspropionibacterium fusobacterium and prevotella to support thegrowth of p aeruginosa isolates using an in vitro and in vivocrossfeeding coculture modelconclusiongiven that scfas are solely derived from bacterial fermentationour experiments propose a critical role for mucinfermentingbacteria in generating carbonsource nutrients for pathogenicfrontiers in cellular and infection microbiology wwwfrontiersinaugust volume 0ccho anaerobes in recalcitrant crsbacteria in the airway mucin fermenting anaerobes maycontribute to the development of recalcitrant crs by degradingmucinsfor potential pathogenslike p aeruginosathus providing nutrientsdata availability statementexperimentsfrom rh sr ws and bw concept ofand editing ds dl ht crw and sz carried out therabbit and in vitro experiments and contributed to samplepreparationjg performed statistical analysis all authorsprovided critical feedback helped shape the research analysisand manuscriptthe datasets generated for this study are available on request tothe corresponding authorfundingethics statementthe studies involving human participants were reviewed andapproved by institutional review board irb approval number at the university of alabama at birmingham thepatientsparticipants provided their written informed consentto participate in this study the animal study was reviewedand approved by institutional animal care and use committeeiacuc approval number at the university of alabamaat birmingham uabthis work was supported by national institutes of healthnihnational heart lung and blood institute r01hl13300605 to bw nationalinstitute of diabetes anddigestive and kidney diseases 5p30dk07248202 to srand nihnational institutes of allergy and infectious diseasek08ai146220john w kirklin research and educationfoundation fellowship award uab faculty developmentresearch award american rhinologic society new investigatoraward triological society career development award andcystic fibrosis foundation k08 boost award cho20a0kbto dycauthor contributionsacknowledgmentsdyc designed the study carried outthe experimentsand took the lead in writing the manuscript with supporta part of this manuscript was presented at north americancystic fibrosis conference in denver coloradoreferencesaanaes k rickelt l fjohansen h k von buchwald c presslert hoiby n decreased mucosal oxygen tension in themaxillary sinuses in patients with cystic ï¬brosis j cyst fibros doi 101016jjcf201012002benninger m s fergusonm b j hadley j a hamilos d l jacobs mkennedy d w adult chronic rhinosinusitis deï¬nitionsdiagnosis epidemiology and pathophysiology otolaryngol head neck surg129suppl3 s1 doi 101016s0194599803013974cameron e a and sperandio v frenemies signaling and nutritionalintegration in pathogenmicrobiotahost interactions cell host microbe doi 1 | Colon_Cancer |
craigpattersontelecaretoj rajani r perry m the reality of medical work the case for a new perspectiveon telemedicine virtual real bf01421807 hoek pd schers hj bronkhorst em vissers kcp hasselaar jgj the eï¬ectof weekly specialist palliative care teleconsultations in patients with advancedcancer a randomized clinical trial bmc med s1291601708669 ministero della salute telemedicina linee diavailabledocumentazionep6_2_2_1jsplinguaitalianoid2129 onlineatindirizzo nazionalihttpwwwsalutegovitportaleaccessed april aiom indicazioni aiom cipomo su covid19 per loncologia wwwaiomitwpcontentuploads202003accessedavailable20200313_covid19_indicazioni_aiomcipomocomupdfapril onlineat cox a lucas g marcu a piano m grosvenor w mold f cancer survivors experience with telehealth a systematic review andthematic synthesis j med internet res 19e11 102196jmir rogante m giacomozzi c grigioni m kairy d telemedicine in palliativecare a review of systematic reviews ann ist super sanita 104415ann_16_03_16 kruse cs krowski n rodriguez b tran l vela j brooks mtelehealth and patient satisfaction a systematic review and narrativeanalysis bmj open 7e016242 101136bmjopen2017 holzner b giesinger jm pinggera j zugal s sch¶pf f oberguggenbergeras the computerbased health evaluation software ches aelectronic patientreported outcome monitoring bmcsoftwaremedinform decis makfor gutteling jj busschbach jj de man ra darlington as logistic feasibilityin clinical practiceof health related quality ofresults of a prospective study in a large population of chronic liverpatients health qual life outcomes life measurement taenzer p bultz bd carlson le speca m degagne t olson k impact of computerized quality of life screening on physician behaviourand patient satisfaction in lung cancer outpatients psychooncology wright ep selby pj crawford m gillibrand a johnston c perren tj feasibility and compliance of automated measurement of quality of life inoncology practice j clin oncol 101200jco200311 jazieh ar alenazi th alhejazi a alofoncoloncologypatientssaï¬f al olayaninfected with coronavirus101200go20a outcomejcoglobtelemed flodgren ginteractivehealthsandrevrachas afarmer ajtelemedicinecareoutcomes2015cd002098eï¬ectsoninzitari mprofessionalshepperdpracticesyst10100214651858cd0databasecochrane ye z hong y wu x hong d zhang y dong x[management of a colon cancer patientdisease ] zhejiang da xue xue bao yi xue banalinfected with corona viruset ogorman ld hogenbirkin northern ontario astelemedicineunitstohealthcare telemed j e health 101089tmj20a measure of potentialjc driving distanceaccessto perri fc ottaiano acancertranslionna f longo f della vittoria scarpati g de angelisagainst head and necktherapy101016jtranon2019immunebiological mechanismsoncolresponseandimplicationonaletfrontiers in oncology wwwfrontiersinaugust volume 0ccrispo covid19 emergency and postemergency hisada y mackman n cancerassociated pathways and biomarkers of venousthrombosis blood 101182blood20170374 xu x lai y hua zc apoptosis and apoptotic body disease message andtherapeutic target potentials biosci rep 39bsr20180992 bsr20180992 zhao z bai h duan j wang j recommendations ofandlungtreatmentcancermedicalforepidemic thorac cancerpatientsindividualizedevents managementof covid19 commonduringadversetheoutbreakconï¬ict of interest the authors declare that the research was conducted in theabsence of any commercial or ï¬nancial relationships that could be construed as apotential conï¬ict of interestcopyright crispo montagnese perri grimaldi bimonte augustin amorecelentano di napoli cascella and pignata this is an openaccess distributedunder the terms of the creative commons attribution license cc by the usedistribution or reproduction in other forums is permitted provided the originalauthors and the copyright owners are credited and that the original publicationin this is cited in accordance with accepted academic practice no usedistribution or reproduction is permitted which does not comply with these termsfrontiers in oncology wwwfrontiersinaugust volume 0c' | Colon_Cancer |
high throughput methods in biological and biomedical fields acquire alarge number of molecular parameters or omics data by a single experimentcombining these omics data can significantly increase the capability for recoveringfinetuned structures or reducing the effects of experimental and biological noise indataresultsfhclust for identifying patient subgroups from different omics information eg geneexpression mirna expression methylation in particular hierarchical structures of patientdata are obtained in each omic or view and finally their topologies are merged byconsensus matrix one of the main aspects of this methodology is the use of ameasure of dissimilarity between sets of observations by using an appropriate metricfor each view a dendrogram is obtained by using a hierarchical clustering based on afuzzy equivalence relation with Åukasiewicz valued fuzzy similarity finally a consensusmatrix that is a representative information of all dendrograms is formed by combiningmultiple hierarchical agglomerations by an approach based on transitive consensusmatrix construction several experiments and comparisons are made on real data egglioblastoma prostate cancer to assess the proposed approachs fuzzy logic allows us to introduce more flexible data agglomerationtechniques from the analysis of scientific literature it appears to be the first time that amodel based on fuzzy logic is used for the agglomeration of multiomic data theresults suggest that fhclust provides better prognostic value and clinical significancecompared to the analysis of singleomic data alone and it is very competitive withrespect to other techniques from literaturekeywords multiomics data data integration hierarchical clustering fuzzy similarityfuzzy aggregation the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriatecredit to the original authors and the source provide a link to the creative commons licence and indicate if changes weremade the images or other third party material in this are included in the s creative commons licence unlessindicated otherwise in a credit line to the material if material is not included in the s creative commons licence and yourintended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtain permission directlyfrom the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40 the creativecommons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to the data madeavailable in this unless otherwise stated in a credit line to the data 0cciaramella bmc bioinformatics 21suppl page of nowadays high throughput methods in biological and biomedical fields acquire a largenumber of molecular parameters by a single experiment in particular such measuredparameters are collected in omics datasets eg genomics transcriptomics methylomics among multiple measured parameters dna genome sequence rna expressionand dna methylation are representative instances for individually analysing suchdata several methodologies have been introduced in literature even though recentlya number of studies pointed out the best performance coming from the integration ofmultiomics data for instance analysing each omic or view in the machine learningjargon set separately fundamental patterns can be detected from data however somefinetuned structures such as cancer subtypes can be highlighted by both gene expression and dna methylation information so that multiomics analysis can reduce theeffects of experimental and biological noise in data from literature three kinds ofintegration methodologies emerge¢ early integration builds a single featurebased matrix by concatenating each omic¢ intermediate integration builds a joint representation of data given the views¢ late integration each omic is analysed separately and the solutions are integratedin general late integration methods and in particular clustering are preferred whenthe analysis combines continuous and discrete data together for a review of integrationapproaches and their comparisons the reader may refer to in this work a multiviewclustering methodology named fhclust is introduced see fig for its general schemafor identifying patient subgroups from different omics information or datasets eg geneexpression mirna expression methylation specifically for each omic dataset a fuzzybased hierarchical clustering approach is adopted and finally the results are mergedby consensus matrix the idea behind the proposed approach comes from observingthat a hierarchical clustering dendrogram can be associated with a Åukasiewicz fuzzydataset ie view and applies a singleomic analysisfig proposed approach a data preparation b data normalization and feature selection c multiomicshierarchical agglomerations d data integration e clustering and visualization 0cciaramella bmc bioinformatics 21suppl page of similaritybased equivalence relation so that a consensus matrix that is the representative information of all dendrograms is derived by combining multiple hierarchicalagglomerations following an approach based on transitive consensus matrix constructionmethodscluster analysis or clustering is an unsupervised technique that aims at agglomerating aset of patterns in homogeneous groups or clusters [ ] hierarchical clustering hc isone of several different available techniques for clustering which seeks to build a hierarchyof clusters and it can be of two types namely agglomerative where each sample starts inits own cluster and pairs of clusters are merged as one moves up the hierarchy or divisivewhere all samples start in one cluster and splits are performed recursively as one movesdown the hierarchy thus hc aims at grouping similar objects into a cluster and werethe endpoint is a set of clusters where each cluster is distinct from each other and theobjects within each cluster are broadly similar to each other hc can be performed eitheron a distance matrix or raw data agglomerative hc starts by treating each observationas a separate cluster and it repeatedly executes the following two steps identifies thetwo clusters that are closest together and merges the two most similar clusters thisprocess continues until all the clusters are merged togetherthe main output of hc is a dendrogram which shows the hierarchical relationshipbetween the clusters distances many distance metrics have been developed and thechoice should be made based on theoretical concerns from the domain of studylater on it is necessary to determine how the distance is computed eg singlelinkagecompletelinkage averagelinkage as with distance metrics the choice of linkage criteria should be based on theoretical considerations from the application domainin nonfuzzy clustering or hard clustering data is divided into distinct clusters andeach data point can only belong to exactly one cluster in fuzzy clustering data pointscan potentially belong to multiple clusters for example in hard clustering given someparameters a symptom can be in a mutually exclusive way present or absent red orblue whereas in fuzzy clustering that symptom could simultaneously be of somegrade red and some other grade blue in fig a comparison between hard and fuzzycategorisation is shown the reader can refer to for a recent comparison betweenhard and fuzzy clustering in this work we introduce a data integration methodologybased on fuzzy concepts in particular we associate a dendrogram to a fuzzy equivalencerelation ie Åukasiewicz valued fuzzy similarity so that a consensus matrix in a multiview clustering that is the representative information of all dendrograms can be obtainedfrom multiple hierarchical agglomerations [ ] the main steps of fuzzy agglomerationcan be summarised as follows characterisation of membership functions computation of a fuzzy similarity matrix or dendrogram for all models at a giventime construction of a consensus matrix for all hierarchical agglomerationsmembership functionswhen dealing with clustering tasks fuzzy logic fl permits to obtain a soft clusteringinstead of an hard clustering of data specifically data points can belong to more 0cciaramella bmc bioinformatics 21suppl page of fig hard vs fuzzy in symptom risk example a hard categorization b fuzzy categorizationthan one cluster simultaneously the fundamental concept in fl upon which all thesubsequent theory is constructed is the notion of fuzzy set a generalisation of a crisp setfrom classical set theorya fuzzy set generalises a crisp set by allowing its characteristic function ie itsmembership function assuming values in the interval [ ] rather than in the set in this way a given item belongs to the fuzzy set with a degree of truthranging from do not belong at all ie its membership function assumes value to completely belong ie the membership function assumes value in fl applications fuzzy sets make it possible to represent qualitative nonnumeric values ielinguistic variables such as high medium low for approximate reasoninginference or fuzzy control systems linguistic variables can be represented by fuzzy setsthrough a transformation step called fuzzification and it is achieved by using different types of membership functions representing the degree of truth to whicha given input sample belongs to a fuzzy set see membership functions sectionin supplementary material 0cciaramella bmc bioinformatics 21suppl page of fuzzy similarity matrixa measure of similarity or dissimilarity defines the resemblance between two samples orobjects similarity measure is a significant means for measuring uncertain informationfuzzy similarity measure is a measure that depicts the closeness among fuzzy sets and hasbeen used for dealing issues of pattern recognition and clustering analysisa binary fuzzy relation that is reflexive symmetric and transitive is known as a similarity relation fuzzy similarity relations are the generalisation of equivalence relationsin binary crisp relations to binary fuzzy relations in details a fuzzy similarity relationcan be considered to effectively group elements into crisp sets whose members are similar to each other to some specified grade and it is a generalization of classical equivalencerelation as described in fuzzy similarity section in supplementary material in orderto introduce the fuzzy similarity in the following we focus on the properties of theÅukasiewicz tnorm tl and the biresiduum in this way we obtain a fuzzy equivalencerelation that can be used for building dendrogram for more details in the derivation ofthese results see fuzzy similarity section in supplementary materialdendrogram and consensus matrixif a similarity relation is mintransitive ie t min then it implies the existence ofthe dendrogram see dendrogram and consensus matrix section in supplementarymaterial for details the mintransitive closure of a relation matrix r can be easilycomputed and the overall process is described in algorithm the last ingredient to accomplish an agglomerative clustering is a dissimilarity relationhere we considered the following result lemma letting r be a similarity relation with the elements rcid2x ycid3 [ ] and lettingd be a dissimilarity relation which is obtained from r bydx y rcid2x ycid3then d is ultrametric iif r is mintransitivein other words we have a onetoone correspondence between mintransitive similaritymatrices and dendrogram and between ultrametric dissimilarity matrices and dendrograms finally after the dendrograms have been obtained each time a consensus matrixie the representative information of all dendrograms is obtained by combining thetransitive closures ie maxmin operation the overall approach is described inalgorithm the overall workflow of the proposed approach is summarised in fig in particular for each omic data set xi a fuzzification step is adopted for obtaining thenew data set yi see supplementary material successively adopting a fuzzy similaritymeasure the similarity matrix si is computed and to guarantee the transitive closure ofthe matrix a new matrix ci is computed see algorithm finally all the ci matricesare collected for obtaining the consensus matrix a and the overall final dendrogram seealgorithm in fig we show an example that summarize a realistic agglomeration result we plotin figs 4abc three input hierarchies obtained on datasets that should be combinedin this case four sequences of patients are considered namely s1 s2 s3 and s4 respectively in fig 4d we show the final result by agglomerating dendrograms we observe that 0cciaramella bmc bioinformatics 21suppl page of fig workflow of the fuzzy based hierarchical clusteringthe output hierarchy contains clusters s1 s2 s3 and s1 s2 s3 s4 at different levels andeach of these clusters eg s1 s2 s3 are repeated at least in two out of the three inputdendrograms moreover it is worth stressing that the proposed approach based on theagglomeration of dendrograms can also be applied with commonly used metrics egeuclidean distance in fig we show a comparison between the dendrograms obtainedby using an euclidean metric and a similarity based approach ie Åukasiewicz tnormrespectively in this realistic example we simulate three omic data sets with rows ienumber of patients and columns ie features we split the single datasets in twopartitions or clusters such that the first rows are random samples from a standard normal distribution with variance and the other rows have the same distribution withfig combination algorithm abc input dendrograms d combined hierarchy 0cciaramella bmc bioinformatics 21suppl page of fig crisp hierarchical clustering vs fuzzy based hierarchical clustering a dendrogram of euclidean basedhierchical clustering b dendrogram of similarity based hierachical clusteringalgorithm mintransitive closure input relation si output transitive relation ci sti elaborate compute s if sielse ci sti si ª si ¦ sicid8 si replace si with si si and go to step i and the algorithm terminatesvariance obtaining a sort of an overlap we observe that both methods find two separated clusters but the similarity based approach in fig 5b permits to obtain a perfectseparation of the source partitionsresults and discussionin the following we describe the behaviour of the proposed methodology on multiomicsbenchmark datasetsalgorithm combination of dendrograms input ci ¤ i ¤ l l input similarity matrices dendrograms output similarity matrix dendrogram a aggregate the similarity matrices to a final similarity matrixa aggregate c1 c2 cla let a be the identity matrixb for each ci calculate e a a ª a ¦ cic if a is not changed a a and goto step else goto step 1b create the final dendrogram from aomics datasetswe consider multiomics cancer datasets available from the cancer genome atlastcga tcga is a large multiomic repository of data on thousands of cancerpatients all datasets contain three omics gene expression mirna expression and 0cciaramella bmc bioinformatics 21suppl page of table datasets description three omics are provided for each dataset respectively dna geneexpression mirna and methylationcases dnaoridatasetamlbiccoadgbmkirclihcluscskcmovsarclnrfmirnaorilnmethyorilnmultiomicsorilnrfrfrfthe number of features at each variable selection method is shown ori original variable dimension ln logarithm andnormalisation and rf random forest based on mean decrease gini indexdna methylation1 in table are summarised the main properties of the datasetsnamely acute myeloid leukemia aml breast invasive carcinoma bic colon adenocarcinoma coad glioblastoma multiforme gbm kidney renal clear cell carcinoma kirc liver hepatocellular carcinoma lihc lung squamous cell carcinomalusc skim cutaneous melanoma skcm ovarian serous cystadenocarcinoma ovsarcoma sarc the number of patients ranges from for aml to for bicmultiview clustering algorithmsfor validating the effectiveness of our model we compared it against several categories ofmultiview clustering algorithms2¢ kmeans and spectral clustering techniques ¢ lracluster it is a lowrank approximation based integrative probabilistic model¢ pins perturbation clustering for data integration and disease subtyping pinsis able to address subtype discovery as well as integration of multiple data types thealgorithm is built upon the resilience of patient connectivity and cluster ensembles toensure robustness against noise and biasto fast find the shared principal subspace across multiple data types¢ snf similarity network fusion snf allows for discovery of disease subtypesthrough integration of several types of highthroughput data on a genomic scale snfcreates a fused network of patients using a metric fusion technique and thenpartitions the data using spectral clustering snf appears to be the state of the art inthis area and has proven to be very powerful however the unstable nature ofkernelbased clustering makes the algorithm sensitive to small changes in molecularmeasurements or in its parameter settings¢ mcca multi canonical correlation analysis mcca which extends theapplication of canonical correlation analysis cca to more than two views is oneof the most widely used dimension reduction method for finding linear relationsbetween two or more multidimensional random variables1row data are available at httpacgtcstauacilmulti_omic_benchmarkdownloadhtml2httpsgithubcomshamirlabmultiomicscancerbenchmark 0cciaramella bmc bioinformatics 21suppl page of evaluation metricsin order to assess the performance of each method we adopt three evaluation metricsthat are the logrank test the enrichment of clinical labels in the clusters and the methods runtime the logrank test assumes that if clusters of patients have significantlydifferent survival they are different in a biologically meaningful way for the enrichmentof clinical labels in clusters six clinical labels are considered gender age at diagnosispathologic tumor pathologic metastases pathologic lymph nodes and pathologic stagethe four latter parameters are discrete pathological parameters measuring the progression of the tumor metastases and cancer in lymph nodes and the total progressionpathologic stage enrichment for discrete parameters was calculated using the Ï2 testfor independence and for numeric parameters using kruskalwallis test not all clinicalparameters were available for all cancer types so a total of clinical parameters wereavailable for testing to derive a pvalue for the logrank test the Ï2 test for independence the kruskalwallis test and the statistic for these three tests is assumed to have Ï2distribution preprocessingtcga datasets were preprocessed as follows patients and features with more than missing values were removed and missing values were imputed using knearest neighborimputation the sequence features were logtransformed the features with highestvariance from geneexpression and methylation omics were selected in the mirna omicfeatures with zero variance were filtered all features were then normalized to have zeromean and standard deviation for methylation we selected the features with maximal variance in each dataset and also adopted the standard pipeline proposed in whose procedure filters out the probes from the x and y chromosomes or probes that areknown to have common snps at the cpg sitea further unsupervised variable selection step has been performed by using the meandecrease gini based on random forest the mean decrease in gini is the average of a variable total decrease in node impurity weighted by the proportion of samplesfig mean performance of the algorithms on ten multiomics cancer datasets the xaxis measures thedifferential survival between clusters mean log10 of logranks test pvalue and the yaxis is the meannumber of clinical parameters enriched in the clusters 0cciaramella bmc bioinformatics 21suppl page of fig performance of the algorithms on ten multiomics cancer datasets for each plot the xaxis measuresthe differential survival between clusterslog10 of logranks test pvalue and the yaxis is the number ofclinical parameters enriched in the clusters red vertical lines indicate the threshold for significantly differentsurvival pvalue cid2 reaching that node in each individual decision tree in the forest this is effectively a measure of how important a variable is for estimating the value of the target variable acrossall of the trees that make up the forest a higher mean decrease in gini indicates highervariable importance therefore the most important variables to the model is the highestin the plot with the largest mean decrease in gini values conversely the least importantvariable is the lowest in the plot with the smallest mean decrease in gini values by following this strategy we cutoff all those variables whose importance is zero the numberof variable cutoff at each step is summarised in table experimental resultsin the experiments for all methods the number of searched clusters is selected in therange [ ] to determine the number of clusters for a method we used the elbowmethod to automatically pick out the optimal elbow rather than choose it manually weused as approximation the second derivative of a vector vv [i ] v [i ] 2v[ i] in particular we consider the index i that brings this expression to a maximum or minimum depending on whether v increases or decreases for all methods we adhered tothe guidelines for usage and parameter selection given by the developers in some casestable performance on ten multiomics number of clinical parameters enriched in the clustersfhclustcrisp hclustkmeansspectrallraclusterpinssnfmccaamlbiccoadgbmkirclihcluscskcmovsarcmeans 0cciaramella bmc bioinformatics 21suppl page of table performance on ten multiomics differential survival between clusters log10 of logrankstest pvaluefhclustcrisp hclustkmeansspectrallraclusterpinssnfmccaamlbiccoadgbmkirclihcluscskcmovsarcmeanswhere no information was provided by the authors we devised parameter selection methods we performed the same process pipeline used in for evaluating the performanceof our method all methods were run on a multicore intelr xeonr cpu e52620v3 240ghz with gb ram in the following the obtained experimental results aredescribedfigure shows the average performance for multiomics data and for each singleomicseparately across all cancer types and fig shows the performance on the different cancer datasets all algorithms show quite similar performance in either differential survivalor enriched clinical parameters with respect to survival our fhclust method achievedthe overall best prognostic value sum of log10 pvalues while pins and mcca ranked second and third respectivelyin table the differential survival between clusters mean log10 of logranks testpvalue are reported spectral achieved the highest total number of significant clinical parameters with parameters fhclust along with lracluster and kmeansplaced themselves second with parameters snf achieved the third position with parameterswith respect to survival table fhclust outperformed its competitors achieving parameters mcca pins and snf have achieved good results with and enriched parameters respectivelywe also counted the number of datasets for which a method solution obtains significantly different survival these results are reported in table all methods that weredeveloped for multiomics data had at least four cancer types with significantly differentsurvival in this case fhclust and pins had different cancer subtypes for which itsclustering had significantly different prognosis fhclust spectral clustering and mccahad enrichment in cancer typeson average fhclust pins and mcca had better prognostic value but found lessenriched clinical labels as compared to spectral clustering methodtable for each benchmarked algorithm the number of cancer subtypes for which its clusteringhad significantly different prognosis first row and had at least one enriched clinical label secondrow are shownsignificant different survivalsignificant clinical enrichmentfhclustkmeansspectrallraclusterpinssnfmcca 0cciaramella bmc bioinformatics 21suppl page of table number of clusters chosen by the benchmarked algorithms on ten multiomics cancerdatasetsfhclustcrisp hclustkmeansspectrallraclusterpinssnfmccaamlbiccoadgbmkirclihcluscskcmovsarcmeansthe number of clusters found for each dataset are presented in table ranging from to because of the good methods performance in the previous analysis partitioning thedata into a relatively high number of clusters could indicate that clustering cancer patientsinto more clusters improves prognostic value and clinical significanceconcerning with methods computational burden their run times are reported intable fhclust takes on average seconds per dataset while spectral and snf gotlower timing the worst method takes roughly minutes per dataset see fig finally fig shows the benchmarked methods performance for singleomic datamoreover for each dataset and method the single omic that gave the best results forsurvival and clinical enrichment are also shown these results suggest that fhclust provides better prognostic value and clinical significance on multiomics data compared tothe analysis of singleomic data used separately nevertheless the interested reader mayrefer to the supplementary material for details on additional results concerning singleomics we also stress that the proposed method differently from other methods suchas snf does not need any hyperparameter tuning moreover clustering is embeddedin the data integration and vice versa and the use of fuzzy concepts ie tnormsfrom one hand permits to obtain a generalisation of the clustering approaches whereason the other hand gives the possibility to apply an inference system eg mamdanifor a quantitative and qualitative measure eg high medium low in cancer riskassessmentsin this work we proposed a multiview clustering methodology for identifying patientsubgroups from different omics data in biological and biomedical fields combining theseomics data can significantly increase data mining capabilities one of the main aspects oftable runtime in seconds of the algorithms on ten multiomics cancer datasetsovfhclustcrisp hclustkmeansspectrallraclusterpinssnfmccacoadskcmbicamlgbmkirclihcluscsarcmeans 0cciaramella bmc bioinformatics 21suppl page of fig computational time comparisonsthis methodology is the use of a measure of dissimilarity between sets of observations byusing an appropriate metric and a consensus matrix that is a representative agglomerateinformation of all the dendrograms as emerged from the analysis of the scientific literature to the best of our knowledge our work concerns for the first time a model based onfuzzy logic used for the agglomeration of multiomic data the use of fuzzy logic allowsus to introduce more flexible data mining features also related to approximate reasoningseveral experiments and comparisons have been made on real data eg glioblastomaprostate cancer to assess the proposed methodology the results suggest that fhclustprovides better prognostic value and clinical significance compared to analysis of singleomic data alone fuzzy logic concepts and in particular membership functions permitsfig summarized performance of the algorithms across ten cancer datasets for each plot the xaxismeasures the total differential prognosis between clusters sum across all datasets of log10 of logranks testpvalue and the yaxis is the total number of clinical parameters enriched in the clusters across all cancertypes ac results for singleomic datasets d results when each method uses the single omic that achievesthe highest significance in survival e same with respect to enrichment of clinical labels 0cciaramella bmc bioinformatics 21suppl page of to develop a fuzzy inference model ie mamdani fuzzy cognitive maps for easilyobtaining a model for a quantitative and qualitative risk assessment of the cancer themodel based on approximate reasoning can be particularly useful for embedded devicesin future work it could be possible to improve results for multiomics analysis ina number of ways for instance more accurate feature selection algorithms couldbe adopted for improving the overall performance on one hand the integration oflabelled data could improve the feature selection step on the other hand some specific feature extraction strategies could be adopted indeed approaches based on thesignal analysis of gene expression data eg nonlinear principal component analysis compressive sensing could possibly further improve the performance [ ]in future it is possible to foresee a different weight for each omic data in order toobtain a more robust similarity and parametric similarity measures can be adoptedeg uninorm for generalizing the concept of and and or connections betweenclusterssupplementary informationsupplementary information accompanies this paper at httpsdoi101186s12859020035676additional file supplementary materialabbreviationsfhclust fuzzyhierarchical clustering dna deoxyribonucleic acid rna ribonucleic acid hierarchical clustering hccrisp hc crisp hierarchical clustering fl fuzzy logic tcga the cancer genome atlas aml acute myeloid leukemia bicbreast invasive carcinoma coad colon adenocarcinoma gbm glioblastoma multiforme kirc kidney renal clear cellcarcinoma lihc liver hepatocellular carcinoma lusc lung squamous cell carcinoma skcm skim cutaneousmelanoma ov ovarian serous cystadenocarcinoma sarc sarcoma pins perturbation clustering for data integrationand disease subtyping lracluster low rank approximation based multiomics data clustering snf similarity networkfusion mcca multi canonical correlation analysisabout this supplementthis has been published as part of bmc bioinformatics volume supplement proceedings from the 13thbioinformatics and computational biology international conference bbcc2018 the full contents of the supplement areavailable online at httpsbmcbioinformaticsbiomedcentralcomssupplementsvolume21supplement10authors contributionsac originally designed the methodology ac and dn worked on the developing of the method and the design of theexperiments ac dn and as contributed for interpreting and for analysing the results all authors contributed forwriting the manuscript read and approved the final manuscriptfundingpublication costs are funded by a grant from the dipartimento di scienze e tecnologie università degli studi di napoliparthenope tecniche di machine learning e soft computing per lelaborazione di dati multivariati softmulan piciaramellaavailability of data and materialscode and data of the proposed approach are available on multiomicscancerbenchmark github repositoryethics approval and consent to participateno ethics approval was required for the studyconsent for publicationnot applicablecompeting intereststhe authors declare that they have no competing interestsauthor details1dipartimento di scienze e tecnologie università degli studi di napoli parthenope centro direzionale c4 island naples italy 2hitachi rail sts via argine naples italypublished august 0cciaramella bmc bioinformatics 21suppl page of referencescamastra f di taranto md staiano a statistical and computational methods for genetic diseases an overviewcomput math meth med 20152015 id serra a fratello m fortino v raiconi g tagliaferri r greco d mvda a multiview genomic data integrationmethodology bmc bioinformatics rappoport n shamir r multiomic and multiview clustering algorithms review and cancer benchmark nucleicacids res reddy ck aggarwal cc data clustering boca raton chapman and hallcrc camastra f ciaramella a son lh riccio a staiano a fuzzy similaritybased hierarchical clustering for atmosphericpollutants prediction lncs ciaramella a staiano a on the role of clustering and visualization techniques in gene microarray data algorithmsbora dj gupta ak int j emerg trends technol comput sci napolitano f pinelli m raiconi g tagliaferri r ciaramella a staiano a miele g clustering and visualizationapproaches for human cell cycle gene expression data analysis int j approx reason ciaramella a cocozza sand clustering of genomic data neural netw iorio f miele g napolitano f pinelli m raiconi g tagliafer | Colon_Cancer |
" inhibiting suchtarget protein reduces the virulence of pathogens and reduces thetherapeutic resistance in the case of cancer we first come to the basicunderstanding of the role of grp78 in healthy and diseased cells grp78 in normal versus stressed cellunder normal conditions grp78 is found bound to three essentialenzymes that regulate cell growth differentiation apoptosis and signaling [] these enzymes are activating transcription factor atf6 protein kinase rnalike endoplasmic reticulum kinase perkand inositolrequiring enzyme ire1 which are inactivated throughbinding to grp78 in the er lumen under the pressure of unfoldedproteins in the er grp78 releases atf6 perk and ire1 and theenzymes are activated once activated the enzymes atf6 perk andire1 upregulate transcription of chaperones inhibit the translationand enhance protein folding endoplasmic reticulum assisted degradation erad and other function that have been reviewed byothers [] see also fig if the pressure of the unfolded proteins is not relieved the upr will direct part of the er to autophagyerphagy if it is not enough the whole cell will undergo apoptosis[]on the other hand under the stress of unfolded proteins grp78 canescape the er retention and translocates to the cytoplasm and on thecell membrane and become membraneexposed termed cellsurface corresponding authoremail addresses abdoscicuedueg aelfikyictpit aa elfiky101016jlfs2020118317received may received in revised form july accepted august available online august elsevier inc all rights reserved 0caa elfiky life sciences fig functional aspect of grp78 in normal versus stress condition in normal state left the grp78 is located in the lumen of the endoplasmic reticulum erbound to and inactivating atf6 blue triangle perk red circle and ire1 yellow square enzymes in the stress condition right the enzymes are free to do theirjobs atf6 is translocated to golgi apparatus to be cleaved then again translocated to the nucleus and helps in upregulating chaperones such as grp78 perk inhibitsthe translation and protein synthesis while ire1 enhances the folding and erad under the pressure of the unfolded proteins the grp78 escapes the er retention andtranslocate to the cytoplasm and the cell membrane csgrp78 is subjected to the recognition of pathogenic proteins spike and envelope viral protein and coatproteins of fungi for interpretation of the references to colour in this figure legend the reader is referred to the web version of this csgrp78 fig this csgrp78 characterizes many aggressive types of cancers such as breast ovarian pancreatic and coloncancers [] additionally csgrp78 was reported to facilitate pathogenic entry both viral and fungal infections zika viruszikv dengue virus denv hepatitis c virus hcv human papilloma virus hpv ebola virus ebov middleeast respiratorysyndrome coronavirus mers cov japanese encephalitis virus jevcoxsackievirus a9 and borna disease virus bdv are among viralinfections that reported grp78 association with viral proteins andgrp78 upregulation in infected cells [] additionally recentstudies hypothesized the association of csgrp78 with the spike protein of sarscov2 to help in virus attachment and host cell entry the primary binding viral protein to grp78 is spike proteins in coronaviruses and envelope proteins for the other viruses [] besidesthe spore coat protein homolog coth3 of rhizopus oryzae the causative fungus for mucormycosis is reported to bind to csgrp78 on endothelial cells and the binding is responsible for adherence and invasionof the fungus since the association of the viral or pathogen infection and elevatedlevels of csgrp78 expression researchers are focused on targetinggrp78 to prevent or even weaken the pathogenic infection reducingthe concentration of grp78 over the cell membrane would reduce thenumber of internalized pathogenic ps and hence reduce the infection additionally when we target csgrp78 the pathogen virulence would be diminished at the same time cancerassociated resistance would also be dimensioned which becomes of the highestpriorities in dual diseases such as viral or fungal infections in cancerpatients grp78 associated radio and chemoresistancechemoresistance is the resistance of a tumor to chemotherapy itwas an old observation while the mechanism of grp78inducedchemoresistance in cancer cells was not fully understood twomechanisms may be responsible for the chemoresistance the upr prosurvival branch and the receptormediated activation of the aktpi3kphosphoinositide 3kinase pathway alternatively the proapoptotic action of the upr could be compensated by the activation ofthe aktpi3k pathway resulting in cell survival the extracellular loopof cleft lip and palate transmembrane 1like clptm1l is essentialfor gemcitabine resistance and interaction with grp78 additionally natural products such as isoliquiritigenin a chalconetypeflavonoid were able to reduce the chemoresistance and colonyformingability of oral squamous cell carcinomas it is reported that theprior treatment of acidic stress protects the human dermal microvascular endothelial cells from apoptosis by reduced the cleavage ofcaspase which was supposed to be due to the presence of grp78 onthe membrane of er that suppress caspase activation nonsmallcell lung cancer nsclc and glioblastoma multiformegbm have a low survival rate the overexpressed grp78 on the cellsurface is the primary reason for the radioresistance in nsclc andgbm targeting cellsurface grp78 enhances the apoptosis andreduces cell proliferation colony formation and downregulates thecrucialintracellular phosphatidylinositol3kinaseprotein kinase bmammalian target of rapamycin pi3kaktmtor signaling essentialin the cell cycle growth and survival besides tumor growthis delayed with enhanced efficacy of the radiation treatment upon antigrp78 antibody administration in mice in breast cancer grp78 is overexpressed while the amount of cellsurface grp78 is increased upon the treatment with the antiangiogenicfactor combretastatin a4p additionally cancer cells treated withdoxorubicin showed less resistance when treated with grp78 neutralizing antibodies [] generally elevated levels of grp78 areindicative of cancer aggressivity targeting cancer cellsurface grp78 isa successful strategy to reduce the radioresistance and chemoresistance of tumors 0caa elfiky in this review the focus is not only on some of the previous trials touse antigrp78 to treat cancer but also in the diagnosis see the peptideinhibitors section below grp78 targeting strategiesdifferent strategies are used to reduce the burden of overexpressedcsgrp78 different compounds show binding affinity to csgrp78 once bound to a substrate the csgrp78 will be internalized tothe cell hence the concentration of the membranebound grp78 willbe reduced once the level of grp78 over the cell surface is droppedthe pathogens will not be able to enter the host cell through grp78hence the virulence will be reduced the inhibitory molecules that cantarget csgrp78 include phytochemicals peptides and antibodies andwill be discussed in detail in the next sections the inhibitors competewith the pathogen recognizing proteins such as spike envelope orcoat proteins for the csgrp78 substratebinding domain this domain of the grp78 is reported to be responsible for the binding ofgrp78 to unfolded proteins inside the lumen of the er through itshydrophobic batches phytochemicalsphytochemicals are compounds found in plants and have a varietyof effects on protein function they are derived from fruits vegetables beans grains and some other plants phytochemicalshave a protective role because their antioxidant characteristics whichplay a vital role in the protection of cells against oxidative damage anddecreasing the probability of cancer propagation via the reactiveoxygen species ros which can induce stress in er apoptosis initiated by the er if there is uncontrolled damage in cells wesummarize some phytochemicals crucial in cell stress relief throughinhibiting the master of upr grp78 galangin and 6shogaolgalangin is a flavonol produced from rhizomes of alpinia officinarum which belongs to the ginger family and grows in southeast asiagalangin works as a suppressor for cell proliferation in hepatocellularcarcinoma it raises er stress through the upregulation of the uprtarget genes cebp homologous protein chop grp78 glucoseregulating protein grp94 and cytosolic ca2 er is the primary site for intercellular calcium ions hence rising cytosolic ca2disrupts the function of er chaperones which induce er stress leadingto the activation of upr and subsequent upregulation of grp78 galangin upregulates er stress which inhibits tumor progressionthrough inducing apoptosis 6shogaol is produced by dehydration of 6gingerol and generatedfrom rhizomes of ginger when treating hepatocellular carcinomahcc cellline with 6shogaol cancer cells develop apoptotic phenotypes signs such as nuclear shrinkage and condensation in chromatin activation of chop expression and perk dephosphorylationinitiates reactions of caspase cascade which induce apoptosis in hccsignificant stimulation was observed in er stressrelated proteinswhich induce apoptosis by 6shogaol through rising in the upr expression grp94 grp78 and hsp70 studies proved that exposing cancer cells to 6shogaol and the activator of the perkeif2αpathway salubrinal together for a specific time induce er stress whichleads to cell apoptosis salubrinal alone enhances the phosphorylation of eif2α in the human hepatocarcinoma cell line smmc7721with negligible toxicity this reveals the significant therapeuticeffect of antigrp78 against malignancies fungisulphureuine b is produced from laetiporus sulphureus and tested byglioma cells to detect antiproliferative properties studies revealed thatsulphureuine b provides er stress by raising the level of expression oflife sciences chop caspase12 and grp78 which prevents separation of grp78from perk atf6 and ire1 which initiates upr additionallymushrooms contain pcoumaric acid and caffeic acid that proved itsbinding affinity against grp78 sbd in silico hence suggested to be apossible inhibitor for overexpressed grp78 in cancer cells or cell infected with viruses including sarscov2 grape seeds and skinproanthocyanidins and resveratrol extracted from grapeseedsexposing colorectal cancer cell crc to grape seed extract which has ahigh amount of proanthocyanidins and resveratrol leads to a modification in grp78 and protein disulfide isomerase pdi which have asignificant role in cell apoptosis which leads crc to undergo apoptoticpathway leading to inhibition of the targeted cell to proliferation on the other hand caffeic acid and pcoumaric acid polyphenolsfound in the grape skin have a protective role against photooxidativedamage additionally it has a preexposure protective role for thehuman retinal pigment epithelial cells arpe19 against blue lightassociated apoptosis in a dosedependent manner by promoting grp78expression in contrast grp78 knockdown inhibited this protective role as mentioned before caffeic acid and pcoumaric are suitablebinders to grp78 sbd in silico phytoestrogensestrogen receptorpositive breast cancer cells are responsive tohormonal therapy by blocking the estrogen synthesis leading to estrogenstarvation it was reported that grp78 plays a vital role inresist estrogenstarvation induced apoptosis in breast cancer cellshence it was suggested to dualtarget the grp78 during treating estrogenpositive breast cancer if the expression level of the grp78 ishigh to improve the efficacy and reduce the resistance it wasreported that grp78 interacts with estrogen due to the critical role ofgrp78 in folding the hormonebinding domain of estrogen receptors additionally grp78 targeting was suggested as a therapeuticstrategy to sensitize cancer cells to chemotherapy in endometrial cancerestrogen induced grp78 expression phytoestrogens are found incicer arietinum and include daidzein genistein formononetin andbiochanin a both estrogens estriol and estradiol and the fourphytoestrogens are found to be recognized by grp78 sbd and henceare suggested as possible grp78 inhibitors in silico it was concluded that estrogens and phytoestrogens are the best binders to thegrp78 while the binding affinities range from down to kcalmol this indicates an excellent binding affinity to grp78sbd even better than a selective cyclic peptide pep42 despiteits phytoestrogen activity genistein activates the apoptosis processthrough upr by upregulation of grp78 and cebp homologous proteinchop also termed growth arrest and dna damage gadd153and nuclear translation of gadd153 in hcc cells epigallocatechin3gallateepigallocatechin3gallate egcg is a polyphenol found in greentea and has an antiproliferative effect on breast cancer and melanoma besidesit has an inhibition effect against grp78 functionthrough direct interaction with the atp binding site of grp78 competing against atp binding egcg increases the therapeutic efficacy of temozolomide when exposed to glioblastoma cells in vivo byinhibition of grp78 olive leaf extract and honeybee hive propolisthe olive leaf extracts active ingredient hydroxytyrosol show goodbinding affinity to the grp78 sbd in silico hydroxytyrosolproved its role as a prophylactic agent against myocardial infarctionmediated apoptosis caffeic acid phenethyl ester cape is foundin the hive propolis of the honeybee cape shows in silico binding affinity against grp78 sbd that is comparable to that of the cyclicpep42 a selective grp78 peptide besides cape induces er stress 0caa elfiky in human shsy5y neuroblastoma in an autophagydependent manner peptidesdifferent peptides are used to target the cellsurface grp78 specifically peptides as anticancer drugs have two main types ishort naked peptides to induce apoptosis ii conjugated peptides todeliver an anticancer drug into cells for the first use grp78 serves as areceptor for the peptide and facilitates the internalization of the peptide which can then modulate various pathways peptides to induce apoptosisa gmbp1 peptidemultidrug resistance mdr is drug resistance that happens whencancer cells treated with one anticancer drug develop resistance todifferent drugs that are different from the used drug in structure andfunction an example of binding peptides that use grp78 as areceptor is gmbp1 which is used in reversing gastric cancer mdrgrp78 facilitates gmbp1 internalization into cells through the transferrinrelated pathway b gonadotropinreleasing hormone analogs gnrhagonadotropinreleasing hormone gnrha is a hypothalamus secreted hormone that affects sex hormones testosterone and estrogenmodified gnrha is more efficient than the natural form hence it is usedas a drug depending on the analog gnrha used as a drug againstendometriosis a case in which cells like that lining the inside of theuterus grow outside it in other parts of the body gnrha inhibitsproliferation and induces apoptosis of defected cells by inhibitinggrp78 thus leading to apoptosis conjugated peptidescell targeting is the solution for the nonspecific toxicity of anticancer drugs that affect cancer and healthy cells altogether and thusresulting in severe side effects peptides can target cancer cells anddeliver anticancer drugs into the cell in cancer cells the peptide canbind to the surface a membranebound form of the overexpressedchaperone grp78 to choose the peptide for a particular cancercell in vitro trials are required such as phage display phage display is atechnique for studying molecular interactions such as proteindnaproteinprotein and proteinpeptide utilizing the bacteriophages toencode peptides to genetic information a pool of cyclic peptidestested against the cancer cells and then a peptide is chosen to be usedfor drug delivery a pep42pep42 is a cyclic peptide ctvalpggyvrvc identified by thephage display technique against human melanoma cell line me66524 csgrp78 is the receptor for pep42 and facilitates it's internalization to the cell pep42taxol and pep42doxorubicin conjugates bind to grp78 in highly metastatic human melanoma cellsleading to its death in vitro leading to cancer cell death pep42selectively bind to grp78 and enter the cell and thus make it a powerfultool to deliver anticancer drugs to various cancer cells pep42 was used as a profiler for in silico predicting the csgrp78 andviral proteins of the zika virus human papillomavirus sarscov2and ebola virus b wifpwiql peptidewifpwiql peptide binds to grp78 expressed in breast cancer cellslife sciences surface in the breast and metastatic cells subtilase cytotoxin is a toxinfrom the ab5 toxins family subtilase cytotoxin composed of two subunits suba which is responsible for the toxicity and subb which isresponsible for subtilase cytotoxin internalization to the cell subatoxic effect is that it induces cell apoptosis by cleaving grp78 betweenthe amino acid residues leu416 and leu417 as indicated wifpwiql peptide binds to grp78 over cancer cells csgrp78 wifpwiqlsuba fusion resulting in an efficient anticancer agent wifpwiqlsuba works simultaneously wifpwiql is responsible for grp78recognition and internalization to the cancer cells while suba is responsible for the toxic effect on the cell by cleaving grp78 inside thecell and thus leading to apoptosis wifpwiql liposomes loadedwith doxorubicin are used to target csgrp78 overexpressed overvascular endothelial growth factor vegfactivated human umbilicalvein endothelial cells wifpwiql bound n2hydroxypropylmethacrylamide hpma copolymer aminohexylgeldanamycin conjugates were able to target csgrp78 and hence inhibit human prostatecancer cells additionally the genetic engineered mung beantrypsin inhibitor gbpti that includes the wifpwiql peptide wasable to induce apoptosis in colorectal cancer cells c bone metastasis targeting peptide bmtp78bmtp78 composed of a peptide wifpwiql conjugated withproapoptotic moiety dklaklak2 grp78 facilitates the internalization of bmtp78 into the cytoplasm in vitro trials showed thatbmtp78 induces apoptosis in human and mouse mammary cell lines dklaklak2 after internalization disrupts mitochondrial membranepermeability and thus kills the cell bmtp78 induced dosedependent cytotoxicity in human leukemia and lymphoma cell lines andacute myeloid leukemia patients additionally the grp78receptorbmtp78 system was used to image breast tumors accuratelythe adenoassociated virusm13derived phage aavp can be usedclinically to detect imaging and eradicate targeted therapy of inflammatory breast cancer utilizing csgrp78 as a target d girlrg peptidegirlrg is a peptide identified using phage display and bindsgrp78 girlrg conjugated to paclitaxelencapsulated nanopsspecifically targeted breast cancer and glioblastoma it was predicted in silico that girlrg binds to the atpase domain of grp78girlrg conjugated with poly ethylene glycol peg can efficientlytarget different tumor cell lines including heterotopic cervical ht3esophageal oe33 pancreatic bxpc3 lung a549 and glioma d54 additionally the radiolabeled 111inpeggirlrg show specificity toward cervical esophageal pancreatic lung and brain tumorsusing spect imaging e vap peptidesntrvap vap is a peptide identified using the phage displaytechnique and it binds to grp78 specifically sntrvap couplingwith a sirna for grp78 effectively downregulated its expression vap modified micelles rivap retro inverso isomer of lvapand dvap retro isomer of lvap could effectively achieve gliomatargeted drug delivery through grp78 at the same time it improvedthe therapeutic efficacy of paclitaxel for glioma binding peptides in diagnosticsas we mentioned before peptides could be used for drug delivery itcan be used as a carrier for radiolabels for imaging purposes such as inthe positron emission tomography pet utilizing the same concept oftargeting grp78 over cancer cellsradiolabeled polyethylene glycol peggirlrg is used in targetingmany cancers as heterotopic cervical esophageal pancreatic lung and 0caa elfiky glioma tumors triplenegative breast cancer tnbc resembles of breast cancer cases while the available diagnostic technologyfor its detection is by the invasive needle biopsy for example 68ga aradiolabel for pet imaging can be conjugated with dodecane tetraacetic acid dotavap grp78targeted pet imaging with [68ga]dotavap is a useful and accurate technique for imaging tnbc anddifferentiates it from other cancer types monoclonal antibodiesantibody ab also called immunoglobulin ig is a huge yshapedprotein produced mainly by plasma cells that are used by the immunesystem to neutralize pathogens such as viruses and bacteria the pathogenic molecule that is recognized by the antibody is called an antigen the antibody binds with the antigen with a keylock mechanism once the interaction established the cell bearing the antigentriggers a response such as metabolic inhibition monoclonal antibody mab159mab159 is a highly specific monoclonal antibody against the humangrp78 kd nm when administered mab159 found localized on the membranes of cancer cells but not normal celllinesupon glucose starvation stress mab159 is found more abundant on thecell membrane as the csgrp78 is pi3kakt signaling upstreamregulator through its interaction with crypto and alpha2macroglobulinover the cell membrane it is required for these factors to activate thepi3kakt signaling once bound to csgrp78 mab159 endocytosed and modulate the pi3k pathway leading to inhibition for cellproliferation tumor growth and metastasis at the same time it enhances tumor cell death both in vitro and in vivo the efficacy ofmab159 was examined in various tumor xenograft models includinght29 colon cancer h249 small cell lung carcinoma and a549 lungadenocarcinoma these cells have relatively higher surface grp78 expression compared to healthy cells mab159treatment led to and tumor growth inhibition in thesemodels respectively monoclonal igm antibody sam6the fully human monoclonal igm antibody sam6 was isolatedfrom a gastric cancer patient and it binds to an oglycosylated form ofgrp78 sam6 is internalized via endocytosis and is finally responsiblefor a lethal accumulation of oxidized lipoproteins followed by apoptosisin cancer cells sam6 not only bind to grp78 on the cancer cellmembrane but also it reduces the drug resistance and kills the cancercell human igm antibody patsm6patsm6 specifically binds to primary multiple myelomas cellsstaining the cells by immunohistochemistry reveals binding to grp78of the patsm6 this binding induces apoptosis and complementdependent cytotoxicity α 2macroglobulin α2mα2m is associated with the nterminal region of cellsurface grp78the binding activates akt to suppress apoptotic pathways and promotescell proliferation mouse mab c38 and c107the mouse monoclonal antibody c38 recognizes the cterminaldomain of the murine grp78 exposed on the cell membrane thebinding induces inhibition of the aktpi3k proliferative pathway inmelanoma cells a comparable experiment done on melanomamouse model shows that the antibody c107 also binds to grp78 inboth experiments the binging with the antibody decreases the tumrowth anti grp78cterminal domain ctd antibodies aretested against human prostate cancer cells it significantly reduceslife sciences tumor growth inhibits cell proliferation while promotes apoptosisbesides in the prostate cancer patients the antictd grp78 antibodybinds the cellexpressed grp78 in human prostate cancer cells conclusioncancerliketypes oftriplenegative breastgrp78 a master chaperone protein of the unfolded protein response plays an essential role in cancer chemoresistance and virulenceof the pathogenic infections targeting grp78 was utilized to defeataggressivecanceradditionally inhibiting grp78 overexpressed in viral infections issuggested as a promising strategy to reduce the virulence of manyviruses and fungal infections the present review summarizesthe up to date targeting strategies used to inhibit cellsurface grp78illuminating the potential use of these strategies to defeat both cancerchemoresistance and viral and fungal infectionsdeclaration of competing interestall the authors declare no conflict of interest for this workreferencesmol cell biol l ellgaard a helenius quality control in the endoplasmic reticulum nat rev mj gething j sambrook protein folding in the cell nature h hu m tian c ding s yu the cebp homologous protein chop transcription factor functions in endoplasmic reticulum stressinduced apoptosis andmicrobial infection front immunol im ibrahim dh abdelmalek aa elfiky grp78 a cells response to stress lifesci e little m ramakrishnan b roy g gazit as lee the glucoseregulatedproteins grp78 and grp94 functions gene regulation and applications critrev eukaryot gene expr l brocchieri ec de macario aj macario hsp70 genes in the human genomeconservation and differentiation patterns predict a wide array of overlapping andspecialized functions bmc evol biol am ismail aa elfiky wm elshemey recognition of the gluconeogenic enzyme pck1 via the gid4 e3 ligase an in silico perspective j mol recognit e2821 pc liao sk tan ch lieu hk jung involvement of endoplasmic reticulum inpaclitaxelinduced apoptosis j cell biochem i haas bipa heat shock protein involved in immunoglobulin chain assemblycurr top microbiol immunol j li m ni b lee e barron d hinton a lee the unfolded protein responseregulator grp78bip is required for endoplasmic reticulum integrity and stressinduced autophagy in mammalian cells cell death differ s srivastava g jain s dang s gupta r gabrani phytochemicals targetingendoplasmic reticulum stress to inhibit cancer cell proliferation anticancerplants natural products and biotechnological implements springer pp r ge c kao cell surface grp78 as a death receptor and an anticancer drugtarget cancers u gopal sv pizzo the endoplasmic reticulum chaperone grp78 also functionsas a cell surface signaling receptor cell surface grp78 a new paradigm in signaltransduction biology elsevier pp s luo c mao b lee as lee grp78bip is required for cell proliferation andprotecting the inner cell mass from apoptosis during early mouse embryonic development mol cell biol kt pfaffenbach as lee the critical role of grp78 in physiologic and pathologic stress curr opin cell biol m wang s wey y zhang r ye as lee role of the unfolded protein responseregulator grp78bip in development cancer and neurological disordersantioxid redox signal cancer cells by fak and jnk mol cell biochem tissue factor procoagulant activity cell surface grp78 a new paradigm in signaltransduction biology elsevier as lee grp78 induction in cancer therapeutic and prognostic implicationscancer res m liu b spellberg qt phan y fu y fu as lee the endothelial cellreceptor grp78 is required for mucormycosis pathogenesis in diabetic mice jclin invest h chamolstad je yu z feng sh lee jg kim p yang p62sqstm1sequestosome1 is an nrecognin of the nend rule pathway which modulatesautophagosome biogenesis nat commun x yuan m dong x li j zhou grp78 promotes the invasion of pancreatic aa alhashimi j rak rc austin cell surface grp78 a novel regulator of 0caa elfiky life sciences f visioli y wang gn alam y ning pv rados je nör glucose a choukhi s ung c wychowski j dubuisson involvement of endoplasmic yl tsai as lee cell surface grp78 anchoring and translocation mechanisms ch ji hy kim aj heo sh lee mj lee sb kim the ndegron p mehrbod sr ande j alizadeh s rahimizadeh a shariati h malek pathway mediates erphagy mol cell 1072e9the roles of apoptosis autophagy and unfolded protein response in arbovirusinfluenza virus and hiv infections virulence and therapeutic potential in cancer cell surface grp78 a new paradigm insignal transduction biology elsevier pp yj chang yp huang zl li ch chen grp78 knockdown enhancesapoptosis via the downregulation of oxidative stress and akt pathway afterepirubicin treatment in colon cancer dld1 cells plos one e35123 t chen s xu chronic exposure of cisplatin induces grp78 expression in ovariancancer proceedings of the 4th international conference on biomedical andbioinformatics engineering acm y kim am lillo sc steiniger y liu c ballatore a anichini targetingheat shock proteins on cancer cells selection characterization and cellpenetrating properties of a peptidic grp78 ligand biochemistry z li l zhang y zhao h li h xiao r fu cellsurface grp78 facilitatescolorectal cancer cell migration and invasion int j biochem cell biol z niu m wang l zhou l yao q liao y zhao elevated grp78 expression isassociated with poor prognosis in patients with pancreatic cancer sci rep s tian w chang h du j bai z sun q zhang the interplay betweengrp78 expression and akt activation in human colon cancer cells under celecoxibtreatment anticancer drugs j xie zh tao j zhao t li zh wu jf zhang glucose regulatedprotein grp78 inhibits apoptosis and attentinutes chemosensitivity of gemcitabine in breast cancer cell via aktmitochondrial apoptotic pathway biochembiophys res commun j zhang y jiang z jia q li w gong l wang association of elevatedgrp78 expression with increased lymph node metastasis and poor prognosis inpatients with gastric cancer clin exp metastasis hh chen cc chen ys lin pc chang zy lu cf lin ar12suppresses dengue virus replication by downregulation of pi3kakt and grp78antivir res reticulum chaperones in the folding of hepatitis c virus glycoproteins j virol h chu cm chan x zhang y wang s yuan j zhou middle eastrespiratory syndrome coronavirus and bat coronavirus hku9 both can utilizegrp78 for attachment onto host cells j biol chem s das sv laxminarayana n chandra v ravi a desai heat shock protein on neuro2a cells is a putative receptor for japanese encephalitis virus virology t honda m horie t daito k ikuta k tomonaga molecular chaperone bipinteracts with borna disease virus glycoprotein at the cell surface j virol s jindadamrongwech c thepparit d smith identification of grp bip as aliver cell expressed receptor element for dengue virus serotype arch virol e liberman yl fong mj selby ql choo l cousens m houghton activation of the grp78 andgrp94 promoters by hepatitis c virus e2 envelopeprotein j virol m nain s mukherjee sp karmakar aw paton jc paton m abdin grp78 is an important hostfactor for japanese encephalitis virus entry and replication in mammalian cells j virol s pujhari vm macias rh nissly m nomura sv kuchipudi jl rasgon heatshock protein hsp70 is involved in the zika virus cellular infection processbiorxiv protein and heat shock protein are components of dengue virus receptorcomplex in human cells j virol ac shurtleff ja costantino sr tritsch c retterer kb spurgers s bavarihspa5 is an essential host factor for ebola virus infection antivir res im ibrahim dh abdelmalek me elshahat aa elfiky covid19 spikehostcell receptor grp78 binding site prediction j infect aa elfiky ebola virus glycoprotein gp1host cellsurface hspa5 binding siteprediction cell stress chaperones aa elfiky natural products may interfere with sarscov2 attachment to thehost cell j biomol struct dyn aa elfiky im ibrahim zika virus envelope heat shock protein a5 grp78binding site prediction j biomol struct dyn t gebremariam m liu g luo v bruno qt phan aj waring coth3mediates fungal invasion of host cells during mucormycosis j clin invest wang cy cusack jc jr liu r baldwin as jr control of inducible chemoresistance enhanced antitumor therapy through increased apoptosis by inhibitionof nfkappab nat med c roller d maddalo the molecular chaperone grp78bip in the development ofchemoresistance mechanism and possible treatment front pharmacol wr clarke l amundadottir ma james clptm1lcrr9 ectodomain interaction with grp78 at the cell surface signals for survival and chemoresistance upon j reyesdel valle s chávezsalinas f medina rm del angel heat shock cj dillard jb german phytochemicals nutraceuticals and human health j c hetz the unfolded protein response controlling cell fate decisions under er l su x chen j wu b lin h zhang l lan galangin inhibits pro c bailly mj waring pharmacologic | Colon_Cancer |
" colorectal cancer crc is the third leading cause of cancerassociated mortality the present study aimed to investigate novel biomarkers to predict prognosis and provide a theoretical basis for studies of the pathogenesis and the development of therapies for crc the present study compared mrna expression levels of patients with crc with short and longterm prognosis and of individuals with and without tumors in the cancer genome atlas tcga database differentially expressed genes degs were identified via volcano plot and venn diagram analysis gene ontology go analysis and gene set enrichment analysis gsea were performed to identify the functions of the degs and the degs were further verified using clinical crc samples a total of degs were identified as candidate genes using the tcga database and four degs [defensin 4a defb4a hyaluronan binding protein habp2 oleoylacp hydrolase and tbc1 domain family member 3g] were associated with poor prognosis of patients with crc two degs defb4a and habp2 were upregulated in tumor tissues of patients with crc in the tcga database go and gsea analyses revealed that defb4a was highly associated with immunosuppression participates in myeloid leukocyte differentiation leukocyte proliferation and positive regulation of leukocytemediated immunity and was positively correlated with cd11b cd14 cd45 cd163 and il17a furthermore defb4a expression was significantly upregulated in patients with large tumors advanced cancer stage lymph node metastasis and liver metastasis survival analysis revealed that defb4a upregulation was associated with poor prognosis defb4a correspondence to dr yi zhang biotherapy center the first affiliated hospital of zhengzhou university jianshe east road zhengzhou henan pr chinaemail yizhangzzueducncontributed equallykey words defensin 4a colorectal cancer prognosis immunity biomarkergene knockdown experiments demonstrated that def4ba promotes cell migration these results indicated that defb4a potentially promotes tumor growth by regulating immunosuppressive activity and provided novel insights into the diagnosis and treatment of crcintroductionaccording to the global cancer statistic of colorectal cancer crc is the third most common cancer and the second leading cause of cancerassociated mortality worldwide crc was reported as the fourth most common and fatal cancer in china in patients with crc usually have a low survival rate and poor therapeutic responses and are susceptible to progression and recurrence early diagnosis and effective treatment are critical to improve the survival of patients with crc crc studies have focused on innovative ideas to identify molecular markers used to develop highprecision noninvasive screening tests for crc to increase population compliance and reduce the potentially harmful side effects associated with more invasive techniques diagnostic markers will give an indication of the likely progression of the disease targeting specific molecules in certain patients has facilitated more personalized treatments that help prevent or decelerate cancer progression the present study aimed to determine prognostic factors and novel therapeutic targets to improve the survival of patients with crcprevious studies have focused on the identification of molecules associated with tumor progression through genetic or mrna profiling and screening of patients with colon cancer for example the expression profiles of long noncoding rnas lncrnas were compared at specific tumor stages t0 t1 t2 and t3 in an azoxymethanedextran sodium sulfateinduced primary colon cancer model and upregulation of the lncrna h19 predicted a poor prognosis other studies analyzed microrna mir expression profiles between tumor tissues and matched nontumor tissues obtained from patients with crc for example mir is significantly downregulated in tumor tissues and associated with poor survival of patients with crc and may thus be considered to be a poor prognostic marker of crc furthermore high expression levels of mir 0cwu prognostic role of defb4a in colorectal cancerand mir in serum are associated with poor survival of patients with crc analysis of the cancer genome atlas tcga database revealed that mmp19 is upregulated in patients with crc and is associated with tumor progression however to the best of our knowledge no study has directly screened mrna profiles based on prognosis the present study divided patients with crc into different groups based on prognosis and screened the mrna profiles of the respective groups defensin 4a defb4a also known as bd sap1 defb2 defb4 hbd defb and defb102 belongs to the defensin family comprising cytotoxic peptides secreted by neutrophils which serve important roles in innate immune defense against microbial infections defb4a is upregulated in cutaneous squamous cell carcinoma and basal cell carcinoma it serves an important role in esophageal carcinogenesis both in vivo and in vitro the genomic copy number of defb4a has been analyzed in patients with crohn's disease and controls and an elevated defb4a copy number has been identified as a risk factor for crohn's disease regardless of disease origin however it remains unclear whether defb4a expression is associated with the prognosis of crc furthermore the role of defb4a in the immune system remains unclear the tumor microenvironment serves a significant role in tumor progression various immune elements comprise the tumor microenvironment including bone marrowderived cells such as macrophages cd4 t cells cd8 t cells b cells natural killer cells and dendritic cells myeloid cells can differentiate into macrophages or myeloidderived suppressor cells mdscs which serve a tumorigenic role in the tumor microenvironment mdscs contribute to tumor vascular development by promoting angiogenesis and tumor growth tumorassociated macrophages tams are important regulators of tumorigenesis by inhibiting the antitumor effects of other cells thus promoting tumor growth however it remains unclear whether defb4a has a regulatory effect on the tumor microenvironment or whether it promotes crc progressionto identify candidate target genes that potentially prolong patient survival mrna expression profiles of tissues from crc samples were compared in the tcga database venn analysis was performed to determine candidate genes upregulated in tumor tissues among patients with poor prognosis subsequently immuneassociated pathway enrichment was analyzed using gene ontology go and gene set enrichment analysis gsea and the correlations between candidate target genes and certain immune cells were determined finally clinical samples and crc cell lines were obtained to verify the clinical significance of the identified genes the present results may provide insights into targeted therapy for crcmaterials and methodsacquisition of microarray data microarray data were obtained from tcga httpcancergenomenihgov rnaseq data for samples were included in the dataset project id tcgacoadread including tumor samples from patients with crc and normal tissues from healthy donorsidentification of differentially expressed genes degs tcga data were divided into two groups based on different categories patient prognosis and gene expression in tumor and normal tissues venn analysis of the two groups was performed and genes associated with crc prognosis were identifiedvenn analysis to identify candidate genes associated with patient survival the gene expression profiles in the two groups were analyzed using the venn diagram web tool httpbioinformaticspsbugentbewebtoolsvenngo analysis functional analysis of the degs was performed using go httpwwwgeneontology based on biological processes gsea gsea was conducted using gsea v403 software wwwgseamsigdbgseaindexjsp and the gene used in the present study was downloaded from the molecular signatures database msigdb oftware broadinstitutegseamsigdbindexjsp v40 msigdb curates various gene sets including canonical signaling pathways from biocarta cgapncinihgovcgap_mitelman_retire_noticehtml kyoto encyclopedia of genes and genomes wwwkeggjp pid httppidncinihgov reactome reactome and other pathway databases tcga data were analyzed via gsea and pathways with a false discovery rate fdr were considered significantpatient characteristics tissue samples were obtained from patients with crc at the first affiliated hospital of zhengzhou university zhengzhou china between april and april patients underwent surgical resection or colonoscopy and the samples were verified via pathological analysis the clinical characteristics of the patients are shown in table i a total of men and women were included in the present study the median age was years age range years crc was diagnosed by two pathologists on the basis of pathological assessment the collection of specimens was approved by the institutional ethics committee of the first affiliated hospital of zhengzhou university zhengzhou china approval no sciencelw and informed consent was obtained from each patient with available followup informationreverse transcriptionquantitative pcr total rna was extracted from pairs of tumor and normal tissue samples from patients with crc using trizol reagent invitrogen thermo fisher scientific inc total rna samples µg were incubated at Ëc for min followed by incubation at Ëc for min and Ëc for sec according to the reverse transcription reaction protocol takara biotechnology co ltd the conditions of pcr were as follows Ëc10 min Ëc10 sec Ëc10 sec Ëc10 sec cycles premix ex taq ii roche target gene expression was simultaneously assessed relative to that of gapdh a housekeeping gene and internal control the following primers were used defb4a forward 'ctc ctc ttc tcg ttc ctc ttc a' and reverse 'gca ggt aac agg atc gcc tat' and gapdh forward 'gga gcg aga tcc ctc caa aat' and reverse 'ggc tgtt 0concology letters no of cases table i characteristics of patients with colorectal carcinomacharacteristic percentagesex male female age years ¥ treatment surgery others tumor size mm ¥ pathological type adenocarcinoma others lymph node metastasis yes no tnm stage i ii iii iv liver metastasis negative positive differentiation poor mediumwell others include chemotherapy and radiotherapy gtc ata ctt ctc atg g' the present study compared the expression levels of the target genes in clinical samples using the δδcq method expression levels of defb4a and gapdh were examined for each sample and the relative expression levels of defb4a were determined using the δcq value of defb4a divided by that of gapdh cell transfection sw480 and hct116 cells were seeded into a well plate sw480 and hct116 cells were purchased from chinese academy of sciences cell bank and cultivated with dmemhigh glucose containing fbs hyclone ge healthcare life sciences and penicillinstreptomycin at Ëc in co2 the growth status of the cells was closely observed until they reached a fusion rate of and then cells were transfected with ncsmall interfering rna negative control sinc sense 'uuc ucc gaa cgu guc acg utt' and antisense 'acg uga cac guu cgg aga att' and small interfering rna targeting defb4a sidefb4a sidefb4a sense 'ucc ucu uca uau ucc uga utt' and antisense 'auc agg aau aug aag agg att' purchased from shanghai genepharma co ltd with jetprime polyplus transfection reagent polyplustransfection sa after h the medium was changed to fresh medium and cells were further incubated in co2 for h subsequently cells were collected for the subsequent experimentswound healing assay for the wound healing assay sw480 and hct116 cells were cultured in µl medium with fbs hyclone and the percentage of serum was in line with previous papers subconfluent tumor cells were scraped using a sterile micropipette tip and then serumfree medium was added next cells were imaged at and h using an inverted fluorescence microscope magnification x200 olympus corporation transwell assay in the migration test the transfected cells 1x105 were inoculated into the top chamber microns with µl serumfree medium complete medium µl containing fbs was added to the lower chamber corning inc following incubation at Ëc for h the migratory cells located under the insert were fixed and stained with crystal violet staining solution beyotime institute of biotechnology at room temperature for min and observed using an inverted fluorescence microscope magnification x200 olympus corporationstatistical analysis the Ï2 test was used to compare clinicopathological factors and continuous variables were analyzed via unpaired student's ttest or oneway anova kaplanmeier analysis and the log rank test were performed for survival analysis univariate and multivariate logistic regression models confirmed the associations between defb4a expression and clinical features prism graphpad software inc was used for statistical analysis of all clinical samples anova was followed by tukey's posthoc test and performed using spss for windows spss inc r software version r foundation for statistical computing was used for bioinformatics analysis p005 was considered to indicate a statistically significant difference all experiments were performed in triplicate and data are presented as the mean ± standard deviation resultsgenes associated with poor prognosis to identify genes associated with poor survival in the crc cohort patients were divided into two groups based on os short ¤ days os patients with a survival time of days would be included in short os and long days os fig 1a in total degs fold change were identified using a volcano plot including upregulated and downregulated genes fig 1b and c hierarchical cluster analysis revealed the expression profiles of the degs fig 1d subsequently gene expression profiles in tumor and normal tissues fold change were analyzed and it was observed that genes were upregulated and were downregulated in tumor tissues compared with in normal tissues p005 fdr fold change fig 1eg the venn diagram revealed that 0cwu prognostic role of defb4a in colorectal cancerfigure screening of degs based on tcga a patients with crc were divided into two groups based on whether or not they survived for days in accordance with the rnaseq data from tcga b volcano plot of rnaseq data from tcga the red dots and blue dots represent upregulated and downregulated degs based on a fold change of the volcano plot displays different genes when comparing patients with a prolonged os and those with a short os c a total of upregulated and downregulated genes were identified d hierarchical clustering analysis of the rnaseq data of different genes in short os and long os samples e hierarchical clustering analysis of the rnaseq data of different genes in ca and n samples f using a threshold of p005 false discovery rate and fold change degs were selected using a volcano plot when comparing ca samples with normal colon mucosa samples from tcga g a total of upregulated and downregulated genes were identified h venn diagram representing the distribution of degs in different groups a total of degs were expressed in both patients with ca and patients with a prolonged os crc colorectal carcinoma degs differentially expressed genes os overall survival tcga the cancer genome atlas ca cancer n normal degs were identified in both screening methods fig 1h details are shown in table iivalidated degs are associated with poor prognosis in tcga to determine the prognostic significance of the identified degs their expression levels were determined in cases included in tcga kaplanmeier survival analysis revealed that defb4a hyaluronan binding protein habp2 oleoylacp hydrolase olah and tbc1 domain family member 3g tbc1d3g upregulation was significantly associated with poor survival in patients with crc fig 2a prognostic significance was not observed for kiss1r or5m11 chrnb3 otx2 s100a7a flj43860 and frmd7 in the patients with crc data not shown furthermore defb4a and habp2 were upregulated in tumor tissues fig 2b a previous study have reported low serum expression levels of habp2 in patients with crc therefore defb4a was selected as a candidate marker of poor prognosis in patients with crcgo and gsea to evaluate the biological role of defb4a in crc progression go enrichment and gsea analyses were 0concology letters descriptiontable ii upregulated genes n10 associated with a poor colorectal carcinoma prognosis in the cancer genome atlas databasegene symbol defb4a habp2 olah tbc1d3g kiss1r frmd7 s100a7a otx2 or5m11 chrnb3 defensin beta 4ahyaluronan binding protein oleoylacp hydrolasetbc1 domain family member 3gkiss1 receptorferm domain containing s100 calcium binding protein a7aorthodenticlehomeobox olfactory receptor family subfamily m member cholinergic receptor nicotinic beta subunitgene id performed defb4a was demonstrated to be involved in various biological processes associated functional pathways are shown in fig 3a and b and closely associated with myeloid leukocyte differentiation leukocyte proliferation and leukocyte mediated immunity implying that defb4a potentially regulates the immune system finally the database was searched for expression profiles of defb4a and immunerelated genes and a positive correlation between defb4a expression and the expression of immune markers such as cd11b cd14 cd45 cd163 and il17a was observed fig 3c these results suggest that defb4a is associated with poor prognosis in patients with crc potentially in an immunosuppressive myeloid leukocyte and cytokinedependent mannervalidation in patient samples and clinical relevance of defb4a to further clarify the clinical significance of defb4a expression the present study analyzed tissue samples from patients with crc the associations between their mrna expression levels and clinicopathological variables were observed detailed information of the patients is provided in table iii defb4a expression was significantly upregulated in the crc tumor tissues fig 4a additionally an association between defb4a upregulation and advanced crc stage stage i cases stage ii cases stage iii cases stage iv cases and metastasis m0 cases m1 cases was observed fig 4b and c furthermore defb4a upregulation in the tumor tissues was associated with poor prognosis p00313 fig 4d additionally defb4a upregulation was significantly associated with advanced liver metastasis p0039 stage p0005 high ca72 value p0003 tumor size p0009 and lymph node metastasis p0044 table iii therefore defb4a was considered to be a prognostic marker associated with tumor progression in patients with crc logistic regression analysis was performed to determine whether defb4a can help predict the prognosis of crc univariate analyses revealed that advanced tnm stage [odds ratio or p001] liver metastasis or p003 lymph node metastasis or p004 high ca199 level or1324 p002 a high ca level or p001 and high defb4a level or p002 were associated with the survival of patients with crc furthermore multivariate analyses revealed that advanced tnm stage or p004 histological differentiation or p001 liver metastasis or p001 ca199 level or p001 high ca level or p005 and high defb4a level or p001 were independent prognostic predictors table iv overall these results suggest that defb4a serves an important role in predicting the prognosis of patients with crcdefb4a promotes proliferation and metastasis in crc to explore the biological roles of defb4a in crc defb4a expression was knocked down in hct116 and sw480 cells fig 5a and b a wound healing assay revealed that defb4a knockdown inhibited the migration of hct116 and sw480 cells fig 5c transwell assays demonstrated that the migration of cells was decreased following knockdown of defb4a in sw480 cells compared with that in the nc group fig 5d the number of migratory cells decreased following knockdown of defb4a in sw480 cells fig 5e overall these results suggested that defb4a serves an important role in crc development discussionwith the increasing availability of highthroughput technologies numerous novel biomarkers and therapeutic targets have been identified through transcriptomic analysis of various types of tumor however such studies on biomarkers in crc have not been extensively performed the identification of crc biomarkers may help predict and prolong the survival of patients with crc in the present study mrna profiling of microarray analysis data from the tcga database was performed to identify numerous novel genes associated with poor prognosis in crc a critical role of defb4a in patients with crc was identified the mrna profiles of patients were first compared between the long os and short os groups and between the tumor and normal tissue groups in the tcga database subsequently the present study investigated the association between mrna expression and prognosis defb4a habp2 olah and tbc1d3g were identified as potential predicators of poor prognosis defb4a and habp2 0cwu prognostic role of defb4a in colorectal cancerfigure defb4a is upregulated based on data from tcga and predicts poor prognosis a kaplanmeier curve of four genes defb4a habp2 olah and tbc1d3g derived from data of patients included in the tcga dataset b mrna expression levels of defb4a habp2 olah and tbc1d3g in cancer vs control samples from patients in tcga p005 ns not significant tcga the cancer genome atlas defb4a defensin 4a habp2 hyaluronan binding protein olah oleoylacp hydrolase tbc1d3g tbc1 domain family member 3g ca cancer n normalwere upregulated in crc tissues of patients in the database however habp2 has been reported to be downregulated in the sera of patients with crc p00137 therefore defb4a was considered as a candidate gene for further analysis go and gsea were used to assess the function of defb4a in promoting disease progression and to highlight the role of defb4a in the tumor microenvironment defb4a was involved in myeloid leukocyte differentiation leukocyte proliferation and leukocyte mediated immunity correlation analysis revealed that defb4a expression was positively correlated with immune markers including cd11b cd14 cd45 cd163 and il17a cd11b is expressed on the surface of a number of leukocytes including monocytes granulocytes and macrophages cd14 is expressed on both monocytes 0concology letters figure defb4a is positively correlated with inhibitory immune cells a gene ontology analysis revealed that defb4a is involved in leukocyte proliferation lymphocyte differentiation leukocyte mediated immunity myeloid cell differentiation negative regulation of type i interferon production and interleukin production b gene set enrichment analysis verified the results c correlation between defb4a and cd11b cd14 cd45 cd163 and il17a r and pvalues are indicated defb4a defensin 4a r pearson's correlation coefficientfigure defb4a predicts poor prognosis in colorectal cancer a defb4a mrna expression in groups of cancer tissues and normal tissues are displayed b defb4a mrna levels were compared with respect to tnm stage c defb4a mrna expression levels of patients with different cancer stages d effect of defb4a expression on overall survival in patients with colorectal carcinoma n52 p005 p001 p00001 defb4a defensin 4aand macrophages and cd45 is expressed on leukocytes m2 macrophages may be marked with cd163 and m2 macrophages serve a role in promoting tumor growth the os of patients with nonsmallcell lung cancer and those with esophageal cancer with high m2 macrophage infiltration rates is shorter than those with low m2 macrophage infiltration rates patients with high expression levels of il17a had a poor prognosis in a crc cohort previous studies have suggested that increased il17a promotes crc in various animal models analysis of clinical specimens of patients with crc demonstrated that defb4a expression was associated with 0cwu prognostic role of defb4a in colorectal cancer 0330a total n Ï2 pvaluedefb4a expressiontable iii association between defb4a expression and clinicopathological characteristics of patients with colorectal carcinoma characteristic sex male female age years ¥ site of lesion colon rectum differentiation poor well tumor size cm ¥ pathological type adenocarcinoma others lymph node metastasis no yes liver metastasis no yes stage iii iiiiv cea normal high ca normal high ca normal high afisher's exact test all others were assessed using a Ï2 test defb4a defensin 4ahigh n low n 0009a0003a 0575a0039a poor survival furthermore defb4a expression was upregulated in patients with crc with advanced and metastatic cancer patients with crc with high defb4a expression had poor survival in addition knockdown of defb4a affected the migration ability of crc cells tcga data of patients with crc were used to identify the degs between the long os days and short os days groups in addition mrna expression was compared between tumor tissues and normal tissues in the same database defb4a was highly expressed in tumors and associated with a poor prognosis defb4a upregulation was associated with poor prognosis and defb4a expression was significantly upregulated in patients with large tumors advanced cancer stage lymph node metastasis and liver metastasis another study used the gene expression omnibus database to screen genes that are increased in patients with recurrence hierarchical clustering and pathway analyses revealed that thrombospondin thbs2 and cartilage 0concology letters table iv logistic regression model analysis of liver metastasis predictors in patients with colorectal carcinoma characteristics sex male vs female age vs ¥ years tumor size vs ¥ mm pathological type adenocarcinoma vs others tnm stage iii vs iiiiv differentiation medium vs poor liver metastasis no vs yes lymph node metastasis no vs yes cea vs ¥ ca199 vs ¥ ca724 vs ¥ defb4a high vs low or odds ratio or pvalue univariate ci or pvalue multivariate ci figure defb4a promotes colorectal cancer cell migration a expression levels of defb4a were detected using rtqpcr following transfection of hct116 cells with sinc and sidefb4a b expression levels of defb4a were detected using rtqpcr following transfection of sw480 cells with sinc and sidefb4a c migration ability of cells was examined using a wounding healing assay d representative images were obtained for the transwell assay magnification x200 e proportions of migrated cells after h were quantified defb4a defensin 4a nc negative control rtqpcr reverse transcriptionquantitative pcr si small interfering rna p001 p0001 0cwu prognostic role of defb4a in colorectal canceroligomeric matrix protein comp are associated with the ecmreceptor interaction focal adhesion and tgf signaling pathways the hypergeometric distribution test demonstrated that the association between thbs2 and crc is stronger than that of comp pearson test results indicated that thbs2 might be considered to be a prognostic biomarker for crc to the best of our knowledge this screening method and the hypothesis that defb4a may serve a protumor role through immunosuppression have not been seen in other studies defb4a stimulates keratinocytes to release il and il proinflammatory cytokines serving as deciding factors in the pathogenesis of psoriasis furthermore defb4a induction is required for tolllike receptor tlr activation in monocytes through the convergence of il and vitamin d receptor signaling and exerts direct bactericidal effects against m tuberculosiss the antimicrobial peptides defb4a and camp are inhibited by hsamir leading to suppression of the tlr21induced vitamin d antimicrobial signaling pathway defb4a has been suggested as a biomarker for psoriasis because the clinical efficacy of targeted antibody therapy in psoriasis is associated with the inhibition of defb4a expression defb4a expression can directly be inhibited by anthralin in vitro and in vivo thus benefiting patients with psoriasis however it has remained unclear whether defb4a is involved in the immunoregulation in crc go analysis revealed that defb4a is involved in myeloid leukocyte differentiation leukocyte proliferation and positive regulation of leukocytemediated immunity therefore defb4a may be associated with immunity in crc to the best of our knowledge the present study was the first to report defb4a as a prognostic marker for crc and as an immunoregulatory factor in the tumor microenvironment in patients with crc however a limitation of the present study was that the research cohort was not large enough which may affect the statistical results in addition the specific role of defb4a and immune factors in colon cancer and the underlying molecular mechanism need to be further exploredin conclusion to the best of our knowledge defb4a is upregulated in patients with crc and is closely associated with poor prognosis defb4a regulates immune function and potentially promotes immunosuppression therefore defb4a may be considered as a prognostic marker and immunotherapeutic target for crcacknowledgementsnot applicablefundingthe present study was supported by grants from the national natural science foundation of china grant nos u1804281 and and funding from state's key project of research and development plan grant no 2016yfc1303500availability of data and materialsthe datasets used andor analyzed during the present study are available from the corresponding author on reasonable requestauthors' contributionsyz qw and dw participated in the design and conception of the present study yz qw dw zs jl and wty were involved in data acquisition and analysis of certain clinical data qw dw zz and yw performed the clinical experiments and analysis of the data the manuscript was written by qw and critically reviewed by yz dw zz yw wny and nrm wny ks and nrm were involved in performing and analyzing the cell experiments all authors read and approved the final manuscriptethics approval and consent to participatethe present study was approved by the institutional ethics committee of the first affiliated hospital of zhengzhou university approval no sciencelw and informed consent was obtained from each patient with available followup informationpatient consent for publicationnot applicablecompeting intereststhe authors declare that they have no competing interestsreferences siegel rl miller kd and jemal a cancer statistics ca cancer j clin cidon eu the challenge of metastatic colorectal cancer clin med insights oncol vreeland tj clifton gt herbert gs hale df jackson do berry js and peoples ge gaining ground on a cure through synergy combining checkpoint inhibitors with cancer vaccines expert rev clin immuno siegel rl miller kd fedewa sa ahnen dj meester rgs barzi a and jemal a colorectal cancer statistics ca cancer j clin dickinson bt kisiel j ahlquist da and grady wm molecular markers for colorectal cancer screening gut nikolouzakis tk vassilopoulou l fragkiadaki p mari | Colon_Cancer |
this study hypothesizes that bromelain bl acts as radiosensitizer of tumor cells and that it protects normal cells from radiation effects in vitro and in vivo studies have been carried out to prove that assumption in vitro mtt cell proliferation assay has shown that the irradiated ehrlich ascites carcinoma eac cell line could be sensitized by bl pretreatment in vivo animals were randomly divided into groups group control pbs ip for days group ehrlich solid tumor est bearing mice group est Îradiation fractionated dose gy group est bl mgkg ip daily for days group est bl for days followed by Îirradiation gy the size and weight of tumors in gammairradiated est bearing mice treated with bl decreased significantly with a significant amelioration in the histopathological examination besides bl mitigated the effect of Îirradiation on the liver relative gene expression of poly adp ribose polymerase1 parp1 nuclear factor kappa activated b cells nfκb and peroxisome proliferatoractivated receptor α pparα and it restored liver function via amelioration of paraoxonase1 pon1 activity reactive oxygen species ros content lipid peroxidation lpo and serum aspartate transaminase ast alanine transaminase alt and albumin alb it is concluded that bl can be considered as a radiosensitizer and radioprotector suggesting a possible role in reducing radiation exposure dose during radiotherapykeywordsbromelain tumor Îradiation radiosensitizer radioprotectorsubmitted april revised july accepted july introductionradiotherapy has been used for a long time in treating cancer1 however from the clinical perspective radiotherapy provides inadequate success due to the radioresistance of many tumors as well as the high risk of recurrence and effects on normal cells may occur23 radioresistance occurs as the microenvironment of solid tumors is hypoxic compared with normal tissue4 in addition some tumors have either an intrinsic resistance to ionizing radiation or can attain this property through accumulation of genetic mutations causing an increased survival and proliferation5 thus strategies to improve radiation therapy could include increasing resistance of normal tissues to radiation andor increasing sensitivity of the tumor cells6radiosensitizing agents increase the sensitivity of tumor cells via enhancing the generation of reactive oxygen species ros increasing lipid peroxidation depletion of glutathione which leads to dna damage inhibition of dna repair inhibition of dna synthesis induction of cell cycle arrest induction of apoptosis and inhibition of proliferation7 numerous nutritive cancer chemopreventive compounds having antioxidant properties have been recognized to potentiate radiation therapyinduced cytotoxic 1drug radiation research department national centre for radiation research and technology egyptian atomic energy authority nasr city cairo egypt2biochemistry department alazhar university cairo egyptcorresponding authorhanan a fahmy drug radiation research department national centre for radiation research and technology atomic energy authority p o box nasr city cairo egypt email fahmyhananyahoocomcreative commons non commercial cc bync this is distributed under the terms of the creative commons attributionnoncommercial license creativecommonslicensesbync40 which permits noncommercial use reproduction and distribution of the work without further permission provided the original work is attributed as specified on the sage and open access pages ussagepubcomenusnamopenaccessatsage 0c integrative cancer therapies effects on cancer cells inversely decreasing its toxicity on normal adjacent tissues89 in this regard much research has aimed to develop numerous antioxidant drugs of both natural and synthetic origin tested in both in vitro and in vivo models and also human clinical trials to overcome injuries caused by ir exposure and to induce killing of cancer cells at the same time previous studies have reported that phytochemical soy isoflavones genistein daidzein and glycitein which exhibit anticarcinogenic properties through their antioxidant activities could be used as potent radiosensitizers to enhance the efficacy of radiotherapymediated suppression of the growth and metastatic ability of cancers1011 along parallel lines resveratrol and piperine which possess antitumor activity have been shown to augment ionizing radiation irinduced apoptosis and loss of mitochondrial membrane potential in murine colon carcinoma and melanoma cells via enhancing irinduced ros generation12 moreover pentoxifylline ptx a methylxanthine that possesses antioxidant properties is known for improving tumor tissue oxygenation in murine hypoxic tumors and inhibiting post radiation induced normal tissue injury in mice1314 consequently searching for a natural product possessing anticancer activity that increases radiosensitivity of tumor cells and radioresistance of normal cells may lead to a potential future drug in cancer therapyamong the natural products bromelain bl extract attracts interest due to its anticancer antioxidant as well as antiinflammatory effects1517 bl an extract from pineapple stem ananas comosus belongs to a group of protein digesting enzymes it is a mixture of diï¬erent thiol endopeptidases and other components like phosphatase glucosidase peroxidase cellulase escharase calcium and several protease inhibitors1819 the anticancer activity of bl has been examined in various types of gastrointestinal and breast cancers cell lines in in vivo models bl has shown antimetastatic effect and reduction in local tumor growth2023 it is also used for reducing the severity of such radiation therapy side effects as mucositis skin reactions and dysphagia in patients24 hence this study was aimed to evaluate the radiosensitizing and radioprotective effect of bl using in vivo and in vitro approachesmaterials and methodin vitro studiesmtt cell proliferation assay the growth and viability of ehrlich ascites carcinoma eac cell line were tested in vitro by 345dimethylthiazol2yl25diphenyltetrazolium bromide mtt assay according to freimoser and buch 2526 to verify the antitumor and radiosensitizing effect of bl two plates were designed for this study the first one contained eac cells maintained by serial subculturing at the national cancer institute egypt incubated for hour before irradiation irr gy alone and with different concentrations of bromelain bl in phosphate buffer saline pbs the second one contained eac cells serving as a control and eac with different concentrations of bl each test was seeded in triplicate into a plate at concentration of cellswell containing rpmi media with fbs nahco3 uml penicillin and µgml streptomycin and each plate was incubated for hours at °c in co2 and humidity atmosphere then μl mtt reagent bio basic inc canada was added over the cells in each well and the plate was incubated in the dark for to hours until a purple precipitate was seen and the absorbance was measured at nm the amount of color produced was directly proportional to the number of viable cells viable cell a samples a blanka control a blank the inhibitory concentration ic50 is the dose of a drug which reduces the viability to and was calculated using nonlinear regression analysisfree radical scavenging assay the antioxidant activity of bromelain was evaluated by 1diphenyl2picrylhydrazyl dpph radical assay and its scavenging power was compared with some antioxidants naringin polyphenolic antioxidant garlic oil and glutathione sulfur containing antioxidants about µl of samples mgml dissolved in dist water was added to µl of a solution of dpph g100 ml dissolved in vv methanol after minutes incubation at room temperature in the dark the absorbance was read at nm against a blank µl dist water µl dpphmethanol solution the experiments were done in triplicate according to the method of braca 27 glutathione mgml was used as a standard antioxidant the scavenging percentage of dpph was calculated according to the followscavengin ing equation where b was the absorbance of the blank and a was the absorbance of samples or standard ec50 is defined as concentration of sample that causes dpph loss there values were calculated using nonlinear regression analysisb ab\uf8ee\uf8ef\uf8f0\uf8f9\uf8fa \uf8fbin vivo studiesradiation processing whole body Îirradiation of mice was carried out using gamma cell40 137cesium manufactured by the atomic energy of canada limited ontario canada installed in the national center for radiation research and technology ncrrt cairo egypt the dose rate was gymin during the experimental period daily correction for humidity barometric pressure and temperature were madeanimals adult female swiss albino mice weighing to g obtained from the breeding unit of ncrrt cairo egypt all animal procedures were performed in accordance with the committee of scientific ethics at faculty of 0cmekkawy table sequences of primers for realtime quantitative pcrgeneparp1 nm0074152nfκb nc0000696pparα nc0000816βactin nc0000716forward primerreverse primer²ccatcgacgtcaactacga3²²caatggctacacaggacca3²²actccacctgcagagcaacca3²²gcgtggggacagccgcatctt3²²gtgcgtggtagcatgagtgt3²²cactgtcacctggaaccaga3²²tagatctcctgcagtagcggg3²²atcggcagaaggggcggaga3²pharmacy alazhar university egypt following the guidelines for animal use the animals were housed in colony cages micecage under proper environmental conditions that is hours darklight cycle good ventilation condition and temperature to humidity at the ncrrt animal house fed with standard diet pellets and provided with water ad libitum animals were left week for acclimatization on the lab environment before starting the experimenttumor transplantation the eac cell line was supplied by serial subculturing at the national cancer institute cairo university egypt it was implanted in each donor female swiss albino mice by ip injection of cells22 g b wt and allowed to multiply28 the ehrlich solid tumor est was obtained by the intramuscular inoculation of ml of viable eac in the right lower limb of each mouse29 mice with a palpable solid tumor diameter mm3 that was maintained within to days after inoculation were used in the studyanimal grouping animals were randomly divided into groups mice each group control not bearing tumor received pbs ip for days group ehrlich solid tumor est bearing mice received pbs ip for days group est Îirradiation gy fractionated doses starting days after tumor appearance mm3 and lasting for days group est bearing mice receiving freshly prepared bl dissolved in pbs mgkg ip daily for days according to pilot study starting once est becomes mm3 bl was purchased from merck kgaa co darmstadt germany group est bearing mice received bl as in group hours before Îirradiation as in group mice were anesthetized days after last irradiation dose using urethane mgkg30 blood samples were collected through cardiac puncture and divided into parts edta coated and plain vials at that time they were euthanized by cervical dislocation liver and tumor tissues were dissected out rinsed with icecold saline dried on a filter paper and weighed then homogenized in icecold pbs ph and stored at °c until used for subsequent biochemical analysisestimation of total body tumor and liver weights animals in each group were checked daily for any adverse clinical symptoms and deaths after to days post inoculation with eac body weights were recorded so body weight change could be estimated tumor and liver weights were measured during sample collection and then the tumor inhibitory ratio was calculated by the following formula inhibition ratio aba where a is the tumor weight average of the control and b is that of the treated group also relative liver weight was calculated as liver weighttotal body weight histopathological examination three tumors of each group were collected and fixed in neutral buffered formalin the specimens were dehydrated in ascending grades of ethyl alcohol cleared in xylene and embedded in paraffin wax four micron thick paraffin sections were mounted on clean slides stained with ehrlichs hematoxylineosin he31 and examined using an olympus microscope bx41 hamburg germany histopathological evaluation was done by assessment of necrosis and calculation of tumor area percentage using image analysis software image j 146a nih usa through the following equation of tumor area area of tumortotal area of the field molecular analyses the mrna levels of poly adpribose polymerase parp1 nuclear factor kappa b nfκb and peroxisome proliferatoractivated receptors pparα genes and of the housekeeping gene βactin were measured by real time polymerase chain reaction rtpcr total rna was isolated from liver tissues using qiagen tissue extraction kit qiagen usa in accordance with the manufacturers instructions the extracted rna μg was used for cdna conversion using high capacity cdna reverse transcription kit fermentas usa and μl reaction volume sybr chemistry in applied biosystems thermal cycler usa to amplify pcr under the following conditions °c for denaturation then °c to °c for annealing using primers mentioned in table and °c for elongationresults were expressed using the comparative ct method for relative mrna quantification of target genes normalized to an endogenous reference βactin and a relevant control equal to ct ct is the difference between the mean ctsample and mean ctcontrol where ctsample is the difference between the mean ctsample and the mean ctβactin and ctcontrol is the difference between the mean ctcontrol and the mean ctβactin 0c integrative cancer therapies estimation of lipid peroxidation lpo reactive oxygen species ros and paraoxonase pon1 in liver homogenate liver lipid peroxidation was estimated by measurement of malondialdehyde mda formation using the thiobarbituric acid method of yoshioka 32 a modified technique of vrablic 33 was used to measure the generation of ros by the intracellular conversion of nitro blue tetrazolium nbt to formazan by the action of superoxide anion paraoxonase activity was estimated by using fluorometric assay enzchek® kit invitrogen uk for the anophosphatase activity of paraoxonase based on the hydrolysis of a fluorogenic anophosphate analog34hematological and biochemical analyses whole blood was immediately analyzed for complete blood count with platelet count using the fully automated analyzer abx cobas micros roche germany estimation of serum alanine aminotransferase alt aspartate aminotransferase ast and albumin alb assays follow the recommendations of the international federation of clinical chemistry ifcc but were optimized for performance and stability using the rochehitachi cobas c 311systemstatistical analysis the statistical analysis was performed using oneway analysis of variance anova and the groups were compared by tukeykramer test viability percentage at different concentrations and body weight change analyzed by twoway anova followed by bonferronis posttest graphs were sketched using graph pad prism isi® software usa version software data were presented as mean ± standard error se and p values considered significantresultsin vitro studieseffect of bromelain and gammairradiation blirr on tumor cell growth and viability the radiosensitizing effect of bl on eac cells was determined by performing mtt assay eac cells exposed to gy Îradiation showed high cell viability percentage reflecting a radioresistance of eac cell line while bl treatment showed in vitro cytotoxic activity with ic50 value of mgml however the maximum cytotoxic effect appeared when the eac cells were subjected to bl then Îradiation gy compared to control or irradiated group with ic50 mgml table effect of bromelain and some natural compounds as free radical scavengers the inhibitory percentage of each compound is shown in figure the ec50 value concentration of sample that causes dpph activity loss is a reliable way for estimation of the radical scavenging activity the ec50 value of glutathione referenced antioxidant is mgml while table cytotoxic activity of blirr against eac cell line bromelain concentration mgmleac bl mgmlic50 mgmlviability non irradiated eac irradiated eac48ab59ab60ab69a787a10ab158ab18ab27ab52ab ± ± each value indicates the mean of records statistical analysis carried out by twoway anova followed by bonferroni posttests a significant versus control ehrlich ascites carcinoma eac group where b significant versus irradiated eac group at p ic50 ± se values were calculated by using nonlinear regression analysisbromelain and garlic oil ec50 are almost equal and mgml respectively however the naringin phenolic antioxidant is the least potent one ec50 mgml in this comparisonin vivo studieseffect of bromelain and gammairradiation blirr on tumor weight and volume table shows a significant decrease in tumor weight in groups treated with bl andor Îirradiation as compared to the est nontreated group the more drastic decrease in the tumor weight ratio observed in the combined therapy group bl irr compared with the estirradiated group as well as est group indicates that combination therapy is more significantly effective than single agent therapy the photograph of est xenografts at the time of sacrifice shows the synergistic effect of bl and irr on tumor volume figure effect of bromelain and gammairradiation blirr on tumor histopathological features of est bearing mice histopathological examination of solid tumor sections revealed typical malignant features including sheets of malignant cells infiltrating adjacent muscular tissue the malignant cells show pleomorphism hyperchromatism and mitotic activity while the necrotic cells appear as nonviable homogenous structureless material with degenerated or karyorrhectic nuclei untreated est bearing group shows a deeply stained tumor cells arrow head and areas of necrosis arrow figure 3a also it displays intact cancer cells arrow and giant cells arrow figure 3b and c the estirradiated group shows muscle fibers invaded by deeply stained tumor cells arrow head and large areas of necrosis arrow figure 3d also displays a notable necrosis of cancer cells n figure 3e the bl treated group shows a 0cmekkawy figure dpph 1diphenyl2picrylhydrazyl reduction curve for glutathione bromelain naringin and garlic oil each value represents mean ± se all experiments were replicated timestable tumor weight and inhibition ratio of ehrlich solid tumor estbearing mice treated with gammairradiation irr gy andor bromelain bl mgkggroupsestest irrest blest bl irrtumor weight g ± ± 004a ± 005a ± 005abtumor inhibitory ratio ± 24a ± 14ab ± 204ababbreviations est ehrlich solid tumor est irr ehrlich solid tumor irradiation est bl ehrlich solid tumor bromelain est bl irr ehrlich solid tumor bromelain irradiationthe values shown are mean ± se of data a significant versus est group where b significant versus estirradiated group at p figure a photograph of ehrlich solid tumor est xenografts at the time of scarification showing the effect of bromelain and gammairradation blirr on tumor volume e ehrlich solid tumor e ir ehrlich solid tumor irradiation e br ehrlich solid tumor bromelain e ir br ehrlich solid tumor bromelain irradiation 0c integrative cancer therapies figure photo micrograph of ehrlich solid tumor est xenografts in different animal groups est sections show the degree of tumorogenesis necrosis n regression of tumor by appearance of muscle fibers m and a b c est ehrlich solid tumor d e est irr ehrlich solid tumor irradiation f g est bl ehrlich solid tumor bromelain h i est bl irr ehrlich solid tumor bromelain irradiationwide area of necrosis arrow and n few groups of cancer cells arrow head and muscle fiber m figures 3f and 4g however combined treatment bl irr displays muscle fiber m significant regression of tumor or wide areas of necrotic cancer cells n and few groups of intact cancer cells arrow figure 3h and 3i the tumor area percentage per total tissue area could determine the degree of proliferation as seen in figure there is a great regression of tumor area in the group treated with bl alone or bl and irr compared with untreated est or estirradiated group indicating that combination therapy significantly more effective than single agent therapy 0cmekkawy bl irr shows nonsignificant group change additionally bl irr group significantly upregulated pparα expression compared with est and estirradiated groups indicating that bl might have a hepato as well as radioprotective effect figure effect of bromelain and gammairradiation blirr on the hepatic lipid peroxidation lpo level reactive oxygen species ros content and paraoxonase1 pon1 activity of ehrlich solid tumor est bearing mice lpo in liver tissues significantly increased in all est bearing groups compared to the control group except the combined treated group irr bl succeeded in returning mda lpo measured as mda malondialdehyde level to the normal level however liver ros significantly increased only in untreated and Îirradiated est bearing groups when compared to the control group while a significant decrease in liver ros showed in estirradiated mice treated with bl in comparison with both est untreated and estirradiated groups pon1 activity in liver homogenate was significantly decreased in est untreated and estirradiated groups when compared with the control group bl treated groups revealed significant increases in pon1 when compared with both est untreated and estirradiated groups showing that bl might have a hepato and radioprotective effect figure effect of bromelain and gammairradiation blirr on hematological measurements wbcs and plts were significantly elevated while hgb and hct significantly decreased in the untreated estbearing mice in comparison with control mice however Îirradiation resulted in a significant decrease in wbcs rbcs plt hgb and hct compared with the control mice treatment of the estbearing mice with bl shows a significant amelioration in wbcs plt and hct compared to est untreated mice combined treatment bl irr shows an enhancement in wbcs plt and hct compared to est untreated and gammairradiated est bearing mice table effect of bromelain and gammairradiation blirr on the serum alanine transaminase alt aspartate transaminase ast and albumin alb to investigate the cytoprotective effects of bl against irradiation the levels of serum alt ast and alb were measured figure it was found that alt and ast significantly increased and conversely alb significantly decreased in est untreated and estirradiated groups compared with the control group however estbearing mice treated with bl alone show nearly the same result of alt and alb as control values estbearing mice treated with bl in combination with irradiation initiated a significant decrease in ast and alt as compared with estirradiated group which may reflect a potential hepatic radioprotective effect of blfigure percentage of tumor areatotal tissue area of ehrlich solid tumor est bearing mice treated with gammairradiation irr gy andor bl mgkg the values shown in the plotted area are mean of records from animals ± se significant versus est group where significant versus estirradiated group at p effect of bromelain and gammairradiation blirr on body weight change and relative liver weight regarding the day by day documented recording of body weight bwt illustrated in figure there is almost no change in bwt of bl treated group while it increases significantly in the untreated est group conversely estirradiated groups with or without bl treatment show a significant decrease in bwt when compared with the control group table relative liver weight was compared after normalization to mg body weight untreated estbearing group shows a significant increase in liver weight by hepatomegaly while nonsignificant changes were observed in bl treated groups compared to the normal group table effect of bromelain and gammairradiation blirr on the poly adpribose polymerase parp1 nuclear factor kappa b nfκb and peroxisome proliferatoractivated receptors pparα relative gene expression of ehrlich solid tumor est bearing mice to test the possibility that bl reduces radiation damage to the liver mrna gene expression of parp1 nfκb and pparα was measured in the liver homogenates of est bearing mice and compared to control pbs treated mice the results illustrated in figure show that irr causes significant increases in parp1 and nfκb expression compared to the control group however combined treatment bl irr shows a significant increase in parp1 and nfκb expression compared to control group and a significant attenuation compared to estirradiated groupmoreover all est bearing groups show significant decreases in hepatic pparα relative gene expression compared to the control group except the combined therapy 0c integrative cancer therapies figure effect of bromelain and gammairradiation blirr on body weight during experiment period each value represents the mean of records ± se significant versus control group where significant versus est group and significant versus estirradiated group at p est ehrlich solid tumor est irr ehrlich solid tumor irradiation est bl ehrlich solid tumor bromelain est bl irr ehrlich solid tumor bromelain irradiationtable change in body weight and relative liver weight of control mice and ehrlich solid tumor est bearing mice treated with gammairradiation irr gy andor bromelain bl mgkggroupscontrolestest irrest blest bl irrbody weight change ± ± 101a ± 217ab ± 201b ± 341abrelative liver weight ± ± 026a ± 022a ± 026a ± 026ababbreviations est ehrlich solid tumor est irr ehrlich solid tumor irradiation est bl ehrlich solid tumor bromelain est bl irr ehrlich solid tumor bromelain irradiationeach value represents the mean ± se a significant versus control group where b significant versus est group at p body weight changes percent are related to the initial weight of animalsdiscussionresistance of tumor cells to chemoradiotherapy as well as the damaging effects to nearby normal tissues remains a major obstacle to successful cancer management therefore the current study has been conducted to estimate the effect of bromelain bl as a tumor radiosensetizer and to show to what extent it can protect normal tissue from radiation hazardsradiosensitizers are compounds that when combined with radiation therapy achieve greater cytotoxicity they can be determined in vitro by the mtt assay2635 the present study has found that the radioresistant eac cells could be sensitized when incubated with bl before irradiation it was known previously that in vitro treatment with bl on mouse tumor cell lines resulted in inhibition of cell growth and invasion capacities3637 the anticancer property of bl has been mainly attributed to the protease component through digestion and diffusion in tumor cells38 it may also be due to the bl enhancement of p53 expression as well as another activator of apoptosis eg bax39 in addition it decreases the activity of cell survival regulators such as akt and erk it also deactivates aktdependent proapoptotic regulator foxo3a thus promoting apoptotic cell death in tumors40it is well known that during cancer and radiotherapy excessive energy is used from the host41 ultimately contributing to mechanisms that promote loss of weight as shown in the present study which also showed that bl could return body weight to a normal level by decreasing tumor weight and volume currently the combined therapy bl irr has been shown to be more effective than single agent therapy in reducing tumor volume and weight indicating that bl could possess a radiosensitizing effect in addition the combined therapy has revealed a drastic decrease in tumor area percentage wide areas of necrotic cancer cells and presence of muscle fiber in the histopathological examination compared with the control est and estirradiated groups this seems to be in agreement with other findings of the role of bl in reducing metastasis and local tumor growth2342 in chemically induced mouse skin papillomas topical application of bl reduced tumor formation tumor volume and caused apoptotic cell death39 bl is a hydrolytic enzymatic complex which shows an efficient digestion and diffusion in tumor cells through attacking the glycosidic linkages and hence denatures glycoproteins thus it protects against tumor growth37 another study has demonstrated the use of controlled proteolytic activity on tumor as a successful strategy to increase therapeutic efficacy43 0cmekkawy figure effect of bromelain and gammairradiation blirr on relative gene expression of liver a parp1 b nfκb and c pparα each value represents the mean of records ± se significant versus control group where significant versus est group and significant versus estirradiated group at p est ehrlich solid tumor est irr ehrlich solid tumor irradiation est bl ehrlich solid tumor bromelain est bl irr ehrlich solid tumor bromelain irradiationthe aim of the radiotherapy protocols is to achieve the maximum curative effect on tumor cells with minimal damaging effect on normal cells hence antioxidants and other nutrients which do not interfere with therapeutic modalities for cancer may enhance the killing property decrease side effects and protect normal tissue44for estimation of the antioxidant ability of bl dpph assay was conducted in vitro and the free radical inhibitory action of bl was compared with some antioxidant compounds it was found that bl has a powerful free radical scavenging power bl belongs to thiol proteases in which the catalytic nucleophile is sulfhydryl groups of cysteine residues which in turn accounts for its antioxidant activity45the involvement of ros mda and pon1 are important mechanisms that play a vital role during radiation toxicity the use of antioxidants is an important preventive to decrease the toxic and pathological effects associated with oxidative stress caused by radiation46 the attained results show a hepatic impairment on the third day from exposure to Îradiation elevation of lpo and ros levels and inhibition of pon1 activity compared to normal mice however treatment with bl revealed an amelioration in hepatic damage caused by irradiation these results were in accordance with liu 47 who described the effect of radiation induced ros generation which in turn might attack cell membrane phospholipids and circulating lipids and thus increases production of mda48 lpo acts as a sensitive biomarker for oxidative stress that occurs as part of the pathogenesis of irradiation49 bl has sulfhydryl groups consequently accounting for its antioxidant activity45 thus it could act as ros scavengermeasurement of pon1 postradiotherapy could be an effective clinical biomarker of hepatic and systemic oxidative stress and may be used as an index of the usefulness of radiotherapy50 it has been demonstrated to catalyze hydrolysis of lipid hydroperoxides and lactones51 pon1 protects serum hdl and ldl ps against lipid peroxidation52 in the present study the decreased activity of pon1 upon radiation exposure might be due to its super saturation of lipid hydroperoxides and lactones upon treatment with bl the activity of pon1 was restored near to the normal level hence the pon1 | Colon_Cancer |
"carcinogenesis is a process of somatic evolution previous models of stem and transient amplifying cells in epithelial proliferating units like colonic crypts showed that intermediate numbers of stem cells in a crypt should optimally prevent progression to cancer if a stem cell population is too small it is easy for a mutator mutation to drift to fixation if it is too large it is easy for selection to drive cell fitness enhancing carcinogenic mutations to fixation here we show that a multiscale microsimulation that captures both withincrypt and betweencrypt evolutionary dynamics leads to a different epithelial tissues are metapopulations of crypts we measured time to initiation of a neoplasm implemented as inactivation of both alleles of a tumor suppressor gene in our model time to initiation is dependent on the spread of mutator clones in the crypts the proportion of selectively beneficial and deleterious mutations in somatic cells is unknown and so was explored with a parameter when the majority of nonneutral mutations are deleterious the fitness of mutator clones tends to decline when crypts are maintained by few stem cells intercrypt competition tends to remove crypts with fixed mutators when there are many stem cells within a crypt there is virtually no crypt turnover but mutator clones are suppressed by withincrypt competition if the majority of nonneutral mutations are beneficial to the clone then these results are reversed and intermediatesized crypts provide the most protection against initiation these results highlight the need to understand the dynamics of turnover and the mechanisms that control homeostasis both at the level of stem cells within proliferative units and at the tissue level of competing proliferative units determining the distribution of fitness effects of somatic mutations will also be crucial to understanding the dynamics of tumor initiation and progressionk e y w o r d scancer evolution initiation metapopulation dynamics neoplastic progression simulationmajor findings competition between epithelial units such as colonic crypts tends to suppress initiation of neoplasms by suppressing mutator clones this suppression of initiation is enhanced when crypts have few stem cells and so are likely to go extinct due to stochastic fluctuations in stem cell numbers this is an open access under the terms of the creative commons attribution license which permits use distribution and reproduction in any medium provided the original work is properly cited the authors evolutionary applications published by john wiley sons ltdevolutionary applications wileyonlinelibrarycom eva 2003 2003 0c 2003 2003 2003 2002 2003 2003introductionthe anization of a population into spatially distinct subpopulations can have a dramatic effect on the evolution of that metapopulation hanski gaggiotti this has implications for both the evolution of anisms and for the effect of tissue architecture on somatic evolution and tissue health in multicellular anisms epithelia are typically divided into subpopulations of tissue stem cells along with the transient amplifying cells and differentiated cells that they produce these subpopulations go by different names in different tissues such as crypts in the intestine or more generally epithelial proliferative units cairns first recognized that the division of stem cells into subpopulations such as crypts acts as a tumor suppressor cairns a mutant stem cell with a reproductive or survival advantage may take over a crypt but is generally constrained from expanding beyond that subpopulation unless it breaches the crypt via a process known as crypt fission which tends to duplicate the mutant crypt cell population however by establishing a population size barrier the mutant clone has to overcome the subpopulation structure of the tissue limits the probability that that clone will acquire further carcinogenic mutations yet clones of mutant stem cells can be observed at scales spanning many crypt diameters especially in compromised tissues such as ulcerative colitis and barrett's esophagus maley salk a fundamental question is therefore how the cryptlevel metapopulation dynamics affect the accumulation of somatic mutations during carcinogenesishere we explore the evolutionary dynamics of mutant stem populations that lead to tumor initiation that is the breach of the crypt barrier allowing clonal expansions of crypts across a tissue as well as of mutant stem cells within and out of a crypt while there may be multiple pathways to tumor initiation it has been shown that the inactivation of a single tumor suppressor gene tsg such as the adenomatous polyposis coli apc gene in colon is sufficient to abrogate crypt homeostasis leading to the formation of aberrant crypts and nascent adenomas humphries wright using an agentbased microsimulation model for both stem cell turnover within a crypt and for the cryptpopulation tissuelevel dynamics we study the role of pretumor evolution in tumor initiation this exploration allows for the selection of mutant crypts across the tissue prior to the inactivation of the tumor suppressor genea form of premalignant field cancerizationwhile the stem cell in which the tumor suppressor is inactivated can proliferate beyond the limit of a single crypt due to crypt bifurcationthe evolution of somatic cells is a complex multiscale process depending on the nature of somatic mutations which may either increase or decrease cell fitness stem cell divisions and differentiation or apoptosis as well as subpopulation eg crypt division and extinction rates there is considerable evidence that carcinogenesis involves both an increase in the rate of epigenetic lesions bielas loeb rubin true loeb breivik ji king weisenberger et al and expansions of clones with a relative fitness advantage over their competitor cells cannataro gaffney townsend maley pepper findlay kassen spencer maley vermeulen williams however it continues to be unclear whether the mutator phenotype is a preinitiation phenomenon in carcinogenesis or is more likely to occur during tumor progression in barrett's esophagus another cryptstructured precancer we found evidence that genomic instability precedes genome doubling and transformation martinez the frequency of deleterious versus beneficial mutations in somatic cells is also unknown though the large number of genes in metazoans devoted to differentiation apoptosis and cell cycle control suggests that the frequency of deleterious mutations may be lower in somatic evolution than anismal evolution rajagopalan nowak vogelstein lengauer recent analysis of somatic mutations in cancer found no evidence of purifying selection except in a few essential genes and strong evidence of positive selection with large selective effects williams suggesting that beneficial mutations are more common than deleterious mutations in somatic evolution martincorena although a definitive answer to these questions can only come from further experimental data a theoretical exploration that recognizes the roles of metapopulation dynamics the mutator phenotype and the proportion of deleterious to advantageous mutations in the process of tumor initiation is called for such an exploration will help the identification of factors that drive the tumor initiation processour model integrates previous efforts to characterize the stem cell dynamics within a crypt cannataro mckinley st mary cannataro mckinley st mary frank iwasa nowak komarova komarova cheng loeffler birke winton potten meineke potten loeffler michor frank may iwasa nowak nowak et al pepper sprouffske maley with models of the dynamics of crypt populations cannataro et al chao eck brash maley luebeck kostadinov maley kuhner loeffler bratke paulus li potten totafurno bjerknes cheng mathematical studies of the stem cell population in the crypt niche suggest that epigenetic alterations that increase the rate of genetic lesions mutator mutations and reduce the fitness of stem cells will tend to drift to fixation if the stem cell population is small whereas carcinogenic mutations that increase the proliferation or survival of a stem cell will tend to spread if the stem cell population is large cannataro komarova michor assuming that most nonneutral somatic mutations are deleterious the accumulation of deleterious mutations may lead to senescence of the intestine over time cannataro however competition between crypts of different fitnesses may significantly change the dynamics of the establishment of a mutator clone through a metapopulation dynamic our in silico experiments suggest that there may have been selection at the level of the anism to minimize the number of stem cells within each subpopulation of birtwell 0c 2003 2002 2003 2003its structured epithelium so as to reduce the probability of tumor suppressor gene inactivation and the initiation of carcinogenesisproliferation rate decreased stem cell loss and caused the mutator phenotypethe following equations and assumptions govern the model 2003 2003methodswe implemented a multiscale model of epithelial tissue architecture with stem cells subdivided into crypts under homeostatic control we examined the time required until the two alleles of a tumor suppressor gene tsg were inactivated in at least one stem cell to represent tumor initiation the model was run at least times for every parameter setting crypts were arranged in a flat hexagonal tissue similar to that observed in colon and contained a population of stem cells as well as an implicitly modeled transient amplifying compartment stem cells divided both symmetrically and asymmetrically symmetric division resulted in two daughter stem cells each having the opportunity during the division event synthesis to acquire a mutation asymmetric division did not result in any new stem cells but did provide an opportunity for stem cell mutation stem cell loss due to cell death or differentiation and stem cell gain due to division events were modeled as a stochastic birthdeath process with parameters that were functions of the stem cell fitness and of homeostatic feedback effects in response to deviations of the crypt cell population from its normal target level equations and a flow chart of the model algorithm is shown in figure s1homeostasis operated at two spatial scales within a crypt if the stem cell population dropped below the target level stem cell division rates increased by a parameterized amount equation if the population rose above the target level stem cell loss rates increased equation the level of homeostatic feedback was proportional to the degree of deviation away from the target equilibrium level equations and we also introduced a mechanism for homeostasis on the hexagonal lattice of crypts if all the stem cells in a crypt died the inhibition on stem cell population growth was released from the neighboring crypts when the stem cell population of a neighbor reached twice the equilibrium level we modeled crypt bifurcation by allocating half of its stem cells to a new crypt in the location of the dead neighborwe included both beneficial mutations that increased the division probability or the survival probability of stem cells as well as deleterious mutations that decreased them these can accumulate indefinitely and affect fitness multiplicatively equation we also implemented a genetic instability mutation that increased the clone's mutation rate 100fold bielas herr kennedy knowels schultz preston ji king the frequency of each mutation type those that changed the cell's fitness the proportion of nonneutral mutations that were deleterious as well as the rate of tsg inactivation was set by parameters each mutation affected proliferation survival or mutation rate parameters by a constant factor half of the deleterious mutations decreased stem cell proliferation and half decreased their survival increased cell loss for the beneficial mutations increased the cell's 2003 2003equationsequation time to stem cell losslet t be a random exponential deviate with distribution function fr t and rate parameter r the time to cell loss due to apoptosis or differentiation is the minimum of the time to cell loss due to background cell death or differentiation and the time to cell loss due to crypt feedbacktcell_loss min 01t1 ¼ fbackground_loss t2 ¼ ffeedback_loss 01 equation homeostatic crypt feedback by differentiationwhen the stem cell population within a crypt expands beyond the homeostatic target level kcrypt\\_size the crypt provides homeostatic feedback via a change in the rate of stem cell loss with a rate parameter equal to the base stem cell loss rate multiplied by the crypt feedback multiplier the crypt feedback rate multiplier is used to calculate the time to stem cell loss due to crypt homeostatic feedback the crypt feedback multiplier is equal to raised to the nth power where n is the excess number of stem cells above the crypt size divided by the kcrypt\\_deviation parameter here kcrypt\\_deviation and kcrypt\\_size is a parameter that we varied across experimentsrfeedback\\_cell_loss rbase_cell_loss2max0ncellskcrypt_sizekcrypt_deviationequation homeostatic crypt feedback by proliferationwhen the stem cell population of a crypt drops below kcrypt\\_size the division rate of the remaining stem cells is increased by a factor that depends on the difference between the current number of stem cells ncellsand kcrypt\\_sizerfeedback_division rbase_division2max0kcrypt_sizencellskcrypt_deviationequation fitness mutation effectskï¬tness is a constant factor representing the effect of a single beneficial mutation on fitness as a first approximation we assume that there are many possible mutations that increase and decrease the fitness of a somatic clone by approximately the same amount and so the effect of n beneficial mutations nbeneï¬cial on stem cell fitness is the constant fitness effect raised to the nth power the effect of n deleterious mutations ndeleterious of small effect is just the inverse of kï¬tness raised to the nth power there is a separate mï¬tness calculated for the division probability and the survival probability of a cell because beneficial and deleterious mutations may affect either of those probabilitiesmï¬tness 01kï¬tness 01nbeneï¬cial 03 kï¬tness 03ndeleteriousbirtwell 0c 2003 2003 2003 2002f i g u r e 2003plots of cumulative hazard functions using the kaplanmeier estimator the tissue was x crypts with stem cells per crypt in panels a through d each colored line represents the function for a specific proportion of deleterious mutations a baseline experiment with default parameter values table b mutation rate reduced to 01x of baseline c mutator phenotype reduced to 01x of baseline d each colored line represents a different number of cells per crypt all proportions of deleterious mutations were included 2003 2003assumptionscrypts consist of stem cells and of transient amplifying cellscrypt density is fixed that is the tissue contains a fixed number of crypts arranged on a hexagonal griddrops below the target level the division rate of each stem cell in the crypt is increased when the number of stem cells grows above the target level the cell loss rate of each stem cell in the crypt is increasedcrypts divide to fill vacant slots left by adjacent crypts that have the number of cells in a crypt transient amplifying compartment gone extinct due to loss of the constituent stem cellsis fixedcrypts attempt to maintain a stable population of stem cells through homeostatic feedback when the number of stem cells the extinction of an adjacent crypt suppresses the homeostatic apoptotic signals allowing the stem cell populations in neighboring crypts to expand once that extinct crypt is replaced the normal birtwell 0cta b l e 2003baseline simulation parameterssimulationmaximum simulation duration in daysstem celldivision rate rbase\\_divisionratio of asymmetric divisions to symmetric divisionsstem cell loss rate rbase\\_cell\\_lossmutation rate per stem cell divisionmutation rate maximumtumor suppressor gene mutation ratetumor suppressor gene mutation rate maximumnumber of transient amplifying cells associated with each stem cellcell division minimum time in dayscell loss minimum time in dayscrypttarget equilibrium level of number of stem cells in a crypt kcrypt\\_sizestandard deviation from equilibrium level the crypt uses this value to determine its level of effect on the cell loss and division rates of the stem cells kcrypt\\_deviationbifurcation threshold factor below which a crypt will not bifurcatecrypt cell loss effect multipliercrypt division effect multipliermultiplier to the division effect for each dead neighbor cryptcanceruncontrolled cell proliferation threshold if a crypt has this threshold times the equilibrium number of stem cells it is considered to be experiencing uncontrolled stem cellnumber of tumor suppressor gene hits that mean cancermutation 2003 2002 2003 2003 years x x x also tested x x default varied from percent of nonneutral mutations that are deleteriousthe factor affecting the loss rate of the stem cell from a beneficial mutation kï¬tnessthe factor affecting the division rate of the stem cell from a beneficial mutation kï¬tnessthe factor affecting the cell loss rate of the stem cell from a deleterious mutation kï¬tnessthe factor affecting the division rate of the stem cell from a deleterious mutation kï¬tnessthe factor affecting the mutation rate of the stem cell from a mutator mutation also tested homeostatic controls on stem cell numbers of neighboring crypts are restoredcrypt division is triggered by an expansion of the stem cell population of a crypt to twice its homeostatic level as hypothesized by garcia park novelli and wright as long as there is an empty slot adjacent to the enlarged crypta stochastic birthdeath process governs the scheduling of division and cell loss eventsfitness mutations affect in a multiplicative fashion the rate parameters of the birthdeath processthere is a single mutator phenotype that requires only a single mutator mutation additional mutator mutations have no effect on the mutation ratethe loss of the first allele of the tsg has no effect on stem cell fitness 2003 2003results 2003 2003tsg inactivation depends on the emergence of a mutatorat baseline for comparison our tissue was a 5x5 hexagonal lattice of crypts each crypt having stem cells stem cell loss and symmetric division rates were balanced mutations were acquired stochastically with probabilities defined by proportions starting with deleterious mutations beneficial mutations and mutator mutations and ranging in increments to deleterious beneficial and mutator beneficial versus mutator the incidence of tsg inactivation decreased as the proportion of deleterious mutations increased figure 1a table birtwell 0c 2003 2003 2003 2002reducing the base mutation rate to of baseline we observed a marked decrease in tsg inactivation figure 1b as expected similarly reducing the effect of the mutator mutation to 10x the baseline mutation rate instead of 100x produced a significant decrease in tsg inactivation figure 1c we found that the vast majority of tsg inactivation occurred in stem cells that had previously acquired the mutator phenotype figure s2 this assumes that the mutator phenotype can be caused by a single mutation that is otherwise neutral eg overexpression of dna polymerase beta canitrot or a dominantnegative mutation in p53 de vries though this assumption is easily relaxed 2003 2003proportion of deleterious mutations negatively correlates with tsg inactivationnot surprisingly we found that the proportion of deleterious mutations and the incidence of tsg inactivation were negatively correlated figure cell divisions per time remained roughly constant across all proportions of mutations however as the proportion of deleterious mutations decreased the cost of being a mutator also decreased because it accumulated less mutational burden of deterious mutations this resulted in an increased emergence of crypts with fixed mutator stem cell populations conversely across all experiments we observed progressively less tsg inactivation as the proportion of deleterious mutations approached our maximum of turnover levels we observed a reduction in the average number of stem cells per crypt figure s3 the implemented homeostatic control was unable to maintain the target stem cell population size in the face of high turnover rates essentially there is a lag between depletion of the stem cell pool due to cell death and differentiation and replenishment provided by an increase in stem cell division rates with higher levels of cell loss the simulated crypts spend more time further below the target homeostatic number of stem cells as a result there were fewer total stem cells in the simulation and therefore fewer mutations per time allowing less chance for mutator acquisition and tsg inactivation this may or may not be realistic second as the turnover level increased above our baseline the number of mutator crypts present in the tissue at any given time decreased figure since increased turnover should lead to increased opportunities for mutator mutations to arise the decline in mutator crypts was a surprise however the loss of mutator crypts is due to intercrypt competition as described below increased turnover led to increased stochastic fluctuations in stem cell numbers and thereby increased crypt extinction events the reduction in stem cell numbers and mutator crypts combined to produce a reduction in the overall incidence of tsg inactivation as turnover rates increased above baseline if in reality homeostatic control of stem cell numbers prevents increased crypt extinctions with increased stem cell turnover this result would likely not hold however all things being equal increased cell turnover would be expected to increase stem cell number fluctuations 2003 2003increased stem cell turnover initially increased and then decreased tsg inactivationwe modulated stem cell turnover by varying cell loss and symmetric division rates in unison at lower turnover levels we found that increased cell turnover increased tsg inactivation however at turnover levels 2x and 5x our baseline level the incidence of tsg inactivation declined figure due to two factors first at higher 2003 2003the number of stem cells per crypt had a varying effect on tsg inactivationin general fewer stem cells per crypt reduced the rate of tsg inactivation even though the total number of stem cells in the tissue was held constant figure 1d however there is a tradeoff between tsg inactivation and tissue death at very low stem cells per crypt with only one stem cell per crypt there is no opportunity for homeostatic signals within a crypt to compensate for stem cell loss in f i g u r e 2003this graph represents the proportion of crypts at the end of the run that contained a population of stem cells with the mutator mutation fixed each bar corresponds to a specific proportion of deleterious mutations the proportion of mutator crypts correlates with the risk of tumor initiation as seen in figure birtwell 0cbase cell loss divison rate base cell loss divison rate 2003 2002 2003 2003base cell loss divison rate base cell loss divison rate f i g u r e 2003the effect of changes in stem cell turnover plots of cumulative hazard functions using the kaplanmeier estimator where each colored line represents the function for a specific proportion of deleterious mutations the baseline division and stem cell loss rates were as seen in figure initially turnover correlated positively with the risk of tumor initiation however at higher turnover rates the risk of tumor initiation decreased due to a reduction in the overall number of living stem cells and decreased incidence of mutator cryptsour model tissues with one stem cell per crypt were mostly unviable and died out before tsg inactivation or the predetermined simulation end timeat and cells per crypt we observed a reduction in the incidence of tsg inactivation figure 1d at all but the lowest proportions of deleterious mutations as was predicted by models of the crypt stem cell niche of single crypts komarova michor figure s4 as in other experiments along with a reduction in the incidence of tsg inactivation the frequency of fixed mutator crypts was reduced as well this shows that selection against mutator cells increases as the number of stem cells increases above some threshold in our model as long as the majority of nonneutral mutations are deleterious supporting the s by michor and komarova komarova michor et al when the stem cell populations are large it is very unlikely that a crypt will go extinct and so there is no intercrypt competition in this case the metapopulation dynamics are reduced to the single crypt dynamicsthe increased risk of tsg inactivation associated with increased stem cells per crypt appeared to plateau after approximately stem cells per crypt the total number of cells in the tissue remained constant as did the total stem cell divisions per time figure s5 the average number of mutations per time increased through stem cells per crypt but then reached a temporary plateau figure there was no statistically significant difference between the average number of mutations per time in the and stem cells per crypt cases as the number of stem cells per crypt increased beyond the average mutations per time decreased except when the proportion of deleterious mutations was the incidence of mutator crypts followed a similar trend figure birtwell 0c 2003 2003 2003 2002f i g u r e 2003this graph represents the proportion of crypts at the end of the run that contained a population of stem cells with the mutator mutation fixed as a function of turnover rate each bar corresponds to a specific proportion of deleterious mutations as turnover increased mutator crypts became more rare at higher proportions of deleterious mutationsf i g u r e 2003number of mutations per unit time as a function of cells per crypt and proportion of deleterious mutations where each bar represents a specific proportion of deleterious mutations at most proportions of deleterious mutations mutations per time peaked around stem cells per crypt at deleterious mutations mutations per time reached a minimum at cells per crypt and increased as the cells per crypt grew past we have chosen to explore the case where the total number of stem cells is kept constant assuming that a certain number of selfrenewing tissue stem cells might be required to maintain an epithelial tissue an alternative view is that a fixed number of epithelial units like the crypts might be required to maintain a tissue and that the number of stem cells per crypt could vary by changing the number of differentiated cells produced by each stem cell in this case the risk of tsg inactivation continues to increase with increasing number of stem cells per crypt figure s4 because we held the number of crypts constant but changed the number of cells per crypt in this case the total number of cells in this simulation also changed leading to a large evolving population size of stem cells and thus an increased chance for at least one cell to inactivate both alleles of the tsg 2003 2003partitioning the tissue into crypts imposed a metapopulation dynamicfitness levels measured by the difference between division and loss rates were greater in tissues with smaller crypts even as the total number of stem cells remained constant figure 6a counts of crypt births and crypt life span measurements showed that there was more crypt turnover in smaller crypts figures s6 and s7 this crypt turnover provided an opportunity for fitter phenotypes to spread more easily across the tissue crypt fitness peaked at cells per crypt where there was the most crypt turnover and bottomed out at and cells per crypt after cells per crypt we observed no crypt death and therefore no turnover crypt fitness began to increase again at and cells per crypt through natural selection of stem cells within larger crypts however fitness levels did not increase to the levels seen at cells per cryptthe metapopulation dynamic was observed in the clonal expansion of mutator crypts across the tissue we considered two crypts to have mutator phenotype agreement if they both have a fixed mutator mutation or neither has a fixed mutator we calculated mutator crypt agreement across spatial distance and found that overall agreement decreased as the cells per crypt increased figure 6b further at and cells per crypt closer crypts had increased mutator agreement suggesting clonal expansion of mutator crypts conversely at cells per crypt and above we found that spatial distance correlated less well with mutator agreement indicating that birtwell 0c 2003 2002 2003 2003f i g u r e 2003a crypt fitness measured by the difference between division and cell loss rates across cells per crypt and proportion of deleterious mutations the total number of stem cells remained constant across these experiments the crypt turnover at and cells per crypt allowed fit clones to spread across the tissue resulting in increased overall fitness b mutator agreement by distance class two crypts were in mutator agreement if they both had fixed mutator mutations or neither did overall agreement was higher in the smaller crypts suggesting that mutator clones were able to spread across the tissuewhen there was less crypt turnover mutator crypts arose de novo rather than through cryptlevel clonal expansion 2003 2003discussionin the colon the development of adenomatous polyps frequently involves the inactivation of the apc gatekeeper gene a member of the wntsignaling pathway which represses proliferation and facilitates orderly cell differentiation in the luminal part of the crypts barker goss groden as long as this gatekeeper gene is active mutant stem cell progeny with neoplastic potential is likely eliminated from the crypt and clonal expansion thus averted however when the gatekeeper gene is inactivated the brakes on mutant stem proliferation are removed and mutant cell progeny may undergo focal clonal expansion therefore we asked the question what are the factors that determine the risk of a tsggatekeeper inactivation leading to tumor initiation in a compartmentalized tissue a metapopulationour microsimulations indicate that the base mutation rate figure 1b and the increase in that rate for a mutator clone figure 1c are the main driving forces behind tsg inactivation and thus the initiation of carcinogenesis the proportion of deleterious mutations is important for its effect on selection of the mutator clone we find that if most mutations are assumed to be deleterious then a mutator clone quickly accumulates a large genetic load of deleterious mutations and tends to be driven to extinction in competition with nonmutator cells however if mutations are more likely to be beneficial then a mutator clone can spread more easily which leads to the early inactivation of both alleles of the tumor suppressor gene figure 1a similarly increasing turnover of the stem cells effectively increases the mutation rate and consequently the rate of initiation however when turnover is very high the homeostatic feedback signals cannot maintain the target number of stem cells and so the total stem cell population of the epithelium decreases which in turn decreases the chances for initiation figure since the rate of tsg inactivation is trivially relat | Colon_Cancer |
" in head and neck cancer hnc the relationship between a delay in starting radiotherapy rt andthe outcome is unclear the aim of the present study was to determine the impact of the amount of time beforetreatment intervention tti and the growth kinetics of individual tumors on treatment outcomes and survivalmethods two hundred sixtytwo hnc patients with primary tumors treated with definitive chemo rt wereretrospectively analyzed the tti was defined as the time interval between the date of histopathologic diagnosisand the first day of the rt course volumetric data on tumors were obtained from diagnostic and rt planningcomputer tomography ct scans in order to calculate the tumor growth kinetic parametersresults no significant association between locoregional control or causespecific hazards and tti was found thelog hazard for locoregional recurrence linearly increased during the first days of waiting for rt although this wasnot significant the median tumor volume relative increase rate and tumor volume doubling time was 32dayand days respectively and neither had any impact on locoregional control or causespecific hazards the association between a delay in starting rt and the outcome is complex and does not harm allpatients waiting for rt further research into imagingderived kinetic data on individual tumors is warranted inorder to identify patients at an increased risk of adverse outcomes due to a delay in starting rtkeywords head and neck cancer radiotherapy waiting time treatment delay outcome with new cases and deaths reported in head and neck cancer hnc is the eighth mostcommon and most lethal cancer in men worldwide in addition to surgery and systemic therapy radiotherapy rt is one of the cornerstones for treatment of thiscancer owing to the rising cancer incidence rate in ageing populations and the widening list of indications forirradiation the demands for rt have increased dramatically over the past decades [ ] the increasing complexity of pretreatment diagnostics and rt technology correspondence pstrojanonkoisi1department of radiation oncology institute of oncology ljubljana zaloÅ¡ka si1000 ljubljana slovenia4chair of oncology faculty of medicine university of ljubljana ljubljanasloveniafull list of author information is available at the end of the has led to delays in treatment decisionmaking and thereduction in linear accelerator throughputthat hasresulted in a significant imbalance between the demandfor rt and the availability of rt capacities in manypublically funded health systems this is also the case inslovenia [ ]due to obvious ethical reservations the only way tostudy the impact of delays in starting rt on treatmentoutcomes are retrospective observational analyses of cohorts from different institutions or countries intuitively one would expect that the prolongation of thetime taken before treatment intervention tti is harmful to patients both the likelihood of tumor growth andthe acquisition of a metastatic phenotype increases as afunction of time furthermore advanced tumors aremore difficult to treat than smaller tumors the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cžumer radiation oncology page of indeed a systematic review of pertinent literature fromthe period by chen showed an increasein the risk of local hnc recurrence of for everymonth of delay in definitive rt however certainstudies included in chens metaanalysis and also somemore recent reports negated the association betweenthe delay in definitive rt and the increased risk of treatment failure [] several different biases inherent inretrospective analyses either related to the quality ofdiagnostic procedures and treatment or to the inhomogeneity of the studied population as well as a selectionbias ie patients with fasttumor progression or ahigher burden of symptoms receive priority in treatment and significant variability in the kinetics of individual hnc cases may abolish the effect of tti inoutcomes [] however if patients with advancedor fastgrowing tumors have to wait longer the magnitude of this effect may be overestimated furthermore no compelling relationship between treatmentdelay and prognosis was found in some other cancertypes []in order to determine what would be an acceptabletti in hnc patients treated with definitive rt or concurrent chemoradiation we aimed to analyze the impactof tti and growth kinetics of individual tumors on theoccurrence of localregional failure distant metastasisand survival in the present study of a cohort of slovenepatients with hncmethodsin a retrospective study patients with oral cavity oropharyngeal hypopharyngeal or laryngeal squamous cellcarcinoma scc who were treated with curativeintentdefinitive rt with or without concurrent chemotherapybetween january and december at the institute of oncology ljubljana slovenia were includedpatients with t1n0 or t2n0 glottic cancer were left outof this cohort the period was chosen because of fluctuations in the waiting time for irradiationas a result of intensive renovations and expansion in thedepartment of radiotherapy that took place over thistime span from patients medical and rt charts wecollected information on clinical gender age onset ofsymptoms date and type of disease recurrence anddeath tumor histology site of origin tnm stage andtreatment characteristics rt technique regimen anddose duration of rt type of concurrent chemotherapy[cct] and the number of cycles administered thetnm stage was determined according to the 7th editionof the uicc classification systemfor analysis of the impact of tumor growth kinetics ontreatment outcomes the volumes ml of primary tumors and neck nodal metastases as marked on diagnostic and rt planning computer tomography ct scanswere compared patients with the same basic clinicaldisease and treatment characteristics as indicated abovebut with both sets of ct scans available were selectedfor this part of the study diagnostic ct scans were performed through the acquisition of mm thin ct sections whereas planning ct scans had a slice thicknessof mm both with intravenous iodine contrast enhancements sets with extensive artefacts were excluded forthe purposes of rt planning patients were positionedsupine on the flat tabletop and a fivefixation pointthermoplastic mask was used lymph nodes were considered positive if the smallest diameter was more than cm andor the necrotic center or extracapsular extension was seen if available segmentation was guided bymagnetic resonance imaging mri sets and the resulting contours around the primary tumors and metastaticneck nodes represented a consensus between two radiation oncologists and a radiologist volumes of primarytumors and metastatic neck nodes were separately calculated by a computer software program used for rt planning xio computerized medical systems inc stlouis usa eclipse varian medical systems inc paloalto usa the end points in this part of the study werechanges in the tumornodal volume and tnm stage thecalculation of the primary tumor volume doubling timeand their impact on the treatment outcomestatisticsthe study protocol was approved by the republic ofslovenia national medical ethics committee no for retrospective studies a written consentis deemed unnecessary according to national regulationsbasic descriptive statistics were reported with meansstandard deviations and ranges for numerical variablesand as percentages for categorical variables in patientswith two simultaneous hncs some characteristics werereported in regards to patients while others were reported in regards to tumorsthe survival curves were computed using the kaplanmeier estimator and the aalenjohansen estimator wasused to estimate the cumulative probabilities of competing risks the effect of covariates was analyzed using amultiple cox regression analysis with all the analysesthe data were censored at a fiveyear followupwhen focused on the survival of patients the analyseswere completed with patients as the units and the timewas measured from the first day of therapy until deaththe overall survival os regardless of the cause ofdeath and the absolute risk cumulative probability ofdying due to index cancer were reported in the cox regression only the index cancer deaths causespecifichazard csh were considered to be events of interestin the analyses where locoregional control lrc wasof primary interest the calculations were performed in 0cžumer radiation oncology page of regards to tumors excluding the nonresponders to rtie those with residual local or regional tumors at weeks after rt completion for the latter group weconcluded that it is the radioresistance of tumor cellsthat are responsible for the persistence of the diseaseafter therapy and not that patients had to wait for rtthe followup time was calculated from the last day oftherapy the estimated cumulative probability of localandor regional recurrence distant metastases lrcprobability of being still alive and without local andorregional recurrence and diseasefree survivaldfsprobability of being still alive and without events locoregional and distant failure and deaths were the events ofinterest were reported all the analyses were conductedin regards to the tumors as independent units this assumption was checked in the sensitivity analysis andallowed for gamma frailtythe assumptions of the cox regression were checkeda nonlinear effect a spline with degrees of freedomwas allowed for the numerical variables and the proportional hazards assumption was tested using schoenfeldresidualsthe tti was defined as the time interval between thedate of histopathologic diagnosis and the first day of thert course tumor growth kinetics was expressed as thetumor volume relative increase rate per day and as thetumor volume doubling time in daysthe tumor volume relative increase rate per day was 13 calculated as 12v t 2ðþþv t 1ðt2 t1where vt1 gross tumor volume at time t1 ieon diagnostic ct scans and vt2 gross tumor volume at time t2 ie on planning ct scans it was reported as the percentage increase was reported as per day the tumor volume doubling time was calculated asðv t 2ðv t 1ðln 2ð þ t t þþlnþsince a onetoone relationship existed between thetwo only the tumor volume relative increase rate whichrequires no extrapolation was considered for modellingall analyses were performed using r statistical software version and a pvalue below was considered statistically significantresultsimpact of time to treatment initiationbetween and patients with oral cavity oropharyngeal hypopharyngeal or laryngeal primarysccs were treated with definitive chemo radiotherapywith curative intent there were men and women aged years mean the majority oftumors originated in the oropharynx tumors in patients and were tnm stage iv tumors in patients the tti ranges from to days with mean days the distribution of the tti is only slightlyasymmetric with median and interquartile rangeiqr fig patients were irradiated with dimensionaltechnique or 3dimensionalconformal isocentric technique to the median rt dose gy iqr delivered in gy dailyfractions iqr concurrently to rt patientsreceived chemotherapy consisted of platinbased monochemotherapy patients or mitomycinecbleomycin combination patients the characteristics of patients and their tumors are shown in table treatment outcome and survivalclinical andor radiologic assessment at weeksposttherapy confirmed disease persistence locally andor regionally in cases ie in patients and thesepatients were excluded from further analyses of lrcand dfs thus patients with tumors were analyzed and during followup a local andor regional relapse was recorded in cases with a mediantime from treatment completion of months range months the two and fiveyear probability of localrecurrence after the end of treatment was and respectively whereas the regional relapse was and respectively after threeyears posttherapy we only observed a small increase ie inlocal recurrence probability whereas the probability ofregional relapse was stable after the third year followupthe probability of occurrence of distant metastases at years was and at years it was at the second and fifthyear followup the lrc was confidence interval [ci] and ci respectively and the dfs was ci and ci respectivelyin the fifth year posttreatment out of patients had died the index cancer was the reason in of the cases during the first years posttherapythe probability of cancerrelated death steeply increasedfrom zero to but later the changes were less pronounced at the second and fifth year posttreatment theos independent of the cause of death was ci and ci respectivelyeffect of covariatesin the univariate analysis the following parameters weretested for lrc and csh age gender stage of diseasetype of treatment rt vs rt cct and tti for rtas continuous variable results are presented in table 0cžumer radiation oncology page of fig distribution of ttitable clinical characteristics of patients and their tumorscharacteristicsgenderno femalemaleage yearsatumor locationboral cavityoropharynxhypopharynxlarynxtumor stagebstage istage iistage iiistage ivtreatmentbradiotherapyconcurrent chemoradiotherapyrt duration daysatime to treatment interventiona ± ± ± the same parameters were also included in the multivariate model table the occurrence of locoregional recurrence was significantly related to the diseasestage p whereas the relationship with agewas only of marginal significance p higherage had a lower hazard no significant associationwith the type of treatment could be found p table the log hazard for locoregional recurrenceseemed to linearly increase during the first days oftable effect of covariates on locoregional control and indexcancerspecific hazard n patients with tumors anunivariate analysisparameterlocoregional control cihrpvaluetnm iii vs iiitnm iv vs iiiagettigendertherap cct vs rtcause specific hazardtnm iii vs iiitnm iv vs iiiagettigendertherapy cct vs rt a mean ± sd rangeb eleven patients had two simultaneous primary tumorsci confidence interval tti time to treatment interventioncct concurrent chemoradiotherapy rt radiotherapy 0cžumer radiation oncology page of table impact of tti on locoregional control and indexcancerspecific hazard n patients with tumors amultivariate analysisparameterlocoregional control cihrpvaluetnm iii vs iiitnm iv vs iiiagettigendertherapy cct vs rtcause specific hazardtnm iii vs iiitnm iv vs iiiagettigendertherapy cct vs rt ci confidence interval tti time to treatment interventioncct concurrent chemoradiotherapy rt radiotherapywaiting for rt although the association between thehazard and tti wasregardless ofwhether we made an allowance for nonlinearity ornot fig 2ainsignificantthe hazard of dying due to the index cancer ie cshwas found to be favorably associated with a lower disease stage p and the addition of cct to rtp whereas age gender and tti did not reachthe level of statistical significance table fig 2bassumptions and sensitivity analysisproportional hazards for all included variables and thelinearity assumption for age were analyzed and presented graphically some indication of nonproportionalhazards could be found in gender but allowing for nonproportional hazards did not change the interpretationof the other covariates as a part of the sensitivity analysis the assumption of the dependence of the tumorsbelonging to the same individual was relaxed but nochanges in the interpretation of the results could be observed as an additional confounder the timedependentcovariate duration of rt was considered but its effectdid not prove to be importantimpact of tumor growth kineticsdiagnostic and planning ct scans median interval days range days were available from patientsfive of these patients had two primary tumors the majority of patients were males with a meanage of years range and the patients hadprimary tumors located in the oropharynx table when two ct sets were compared the original tstage of the primary tumor was increased ie upgradedin two cases and the nodal stage was increased in six patients due to the limited number of npositive casesn only volumes of primary tumors werecompared between the two ct sets for the calculationof the tumor volume relative increase rate per dayand tumor doubling time the absolute increase intumor volume ranged from to cm3 per daymedian no increase in volume was observed infive patients ct scans in these five patients were takenfig trend of the hazard for locoregional recurrence a and for index cancerspecific death b 0cžumer radiation oncology page of table characteristics of patients with available diagnostic andplanning ct scanscharacteristicsgenderno femalemaleage yearsatumor locationboropharynxhypopharynxlarynxstagebstage istage iistage iiistage ivtherapybradiotherapyconcurrent chemoradiotherapytumor volume relative increase rate dayatumor volume doubling time daysaa mean ± sd rangeb five patients had two simultaneous primary tumors from days to days apart median days the median tumor volume relative increase rate was perday median range and the mediantumor volume doubling time was days range days no difference was observed when these two parameters were compared between different tumor stagestreatment outcome and survivalamong the tumors the time taken for local andorregional relapse and the occurrence of distant metastaseswere assessed for tumors in which treatment resultedin clinicalradiological eradication of the disease at weeks posttherapy local andor regional relapse wasrecorded in nine cases with a median time fromtreatment completion of months range monthsthe two and fiveyear probability of local recurrenceafter the end of the treatment was and respectively whereas for the regional relapse it was and respectively at years posttherapy only anincrease in the local recurrence probability of wasobserved whereas the probability of regional relapseremained stable the two and fiveyear probability ofdistant metastases was at two and years thelrc was ci and ci respectively and the dfs was ci and ci respectivelyin the yearperiod after the start of treatment outof patients died the index cancer was thereason in cases at two and years posttreatment the os rates were ci and ci respectivelyeventseffect of covariatesdue to the low number of events local andor regionalrecurrence ninecancerspecific deaths events only univariate regression models were fittedthe following covariates were tested in the modelsoverall disease stage initial tumor volume tumor doubling time and relative increase rate day of primarytumor volume measures of tumor kinetics did not showany impact on lrc or csh only an inverse relationshipbetween the initial primary tumor volume and csh wasobserved hr for index cancerspecific death perevery cm3 increase in the volume of primary tumorstable there was no difference in the value of dfsbetween patients with a primary tumor volume relativeincrease rate 1day and 1day p fig discussionwhile it is intuitively anticipated and confirmed by theresults of the metaanalysis that a delay in starting rtwould have a negative impact on the treatment of patients with hnc this association was not confirmed inour study we only found a statistically insignificantupward trend in the risk of locoregional recurrence forthe first days of rt delay in addition the differencesin the growth kinetics between individual tumors whichwere studied in a smaller group of patients were considerable but did not appear to be of significance for theprediction of treatment outcomesobviously the relationship between tti and diseaseprognosis in patients with hnc is more complex than itmight seem at first glance the first negative impact ofwaiting for treatment to begin is the risk that the tumorwill increase in size andor metastasize during this timemaking it harder to treat or resulting in it becoming untreatable [ ] however the time for a tumor to growis only one of the factors that determines prognosis andthe absence of a statistically significant association between tti and the treatment outcome in our study andis thus not surprising []many other reportsmoreover no obvious methodological differences couldbe found between these negative studies and the positive studies that confirmed the association between ttiand treatment outcomes [] in both groups thereare individual studies that have similar sample sizestumor sitestage mix and periods covered speaking infavor of comparable quality of diagnostics therapy andstatistics across the studies 0cžumer radiation oncology page of table effect of stage and tumor kinetics on locoregional control and overall survivalparameterstnm stageiv vs iiiivtuper cm3vtu relative increase rateper dayvtu relative increase rate¤ vs 1dayvtu volume of primary tumorlocoregional controlhr cipvaluecause specific hazardhr cipvalueon the contrary in more recently reported analyses ofthe national cancer registries data an adverse effect ofwaiting for radiotherapy was clearly established []however the results from this type of analysis should betaken with caution not only due to the limitations inherent in the tumor registry data unmeasured confounding selection biasincomplete data and codingerrors but also because the effect of delaying rt oncancerspecific outcomes was not evaluated in additionpatients irradiated in postoperative and definitive settings were not analyzed separately overall the researchmethodology and interpretation used in these studieswere criticized and the magnitude of the effect that theysupposedly demonstrated was questioned in the present study a linear increase in the log hazardfor locoregional recurrence was found during the first days of waiting for rt although it was not statisticallysignificant it is possible that unknown confounders thatwere not accounted in our analysis eg tumor growth kinetics reduced the statistical power of the tti the resultsfrom fortin and naghavi who reported on theincreased risk of locoregional failure with tti daysand days respectively [ ] are the most comparable to our own however optimal ttithresholdsfig impact of primary tumor volume relative increase rate to diseasefree survival in patients with no residual disease at weeks posttherapy 0cžumer radiation oncology page of identified in different studies showed considerable variations pointing to the uncertainty of such calculations andtheir dependence on the characteristics of the analyzedpopulation [ ] the possible role of classical prognostic factors such as location of primary tumor diseasestage and addition of cct is not expected to be relevant inthis respect as in our and other similar studies the statistical significance of tti was verified by multivariateanalysisthe effect of tti on treatment outcomes however isnot only conditioned by the duration of waiting for rtbut also on the rate of tumor growth in hnc a vast heterogeneity in tumor cell kinetics has been observed conditioned by the local milieu from which it arises whichdiffers from patient to patient [ ] historicallydifferent methods were explored to evaluate tumor cellkinetics but did not succeed in providing clinically relevant kinetic parameters [ ] more recently a comparison of two sets of imaging data acquired at twodifferent time points was successfully employed for thispurpose usually volumetric data are extracted from diagnostic and rtplanning ct scans for the calculation ofdifferent parameters reflecting the rate of tumor growtheg tumor volume doubling time or absolutepercentagetumor volume increase per day despite some differencesin the calculation methodology across studies a large variation in the individual values of kinetic parameters wasseen in all of them including ours indicating that all ofthe studied populations represent a unique mix of slowand fast growing tumors [] among our patientsfive had no measurable increase in primary tumor volumeeven when the interval between ct scans was up to days the inverse relationship between kinetic parametersdetermined by the comparison of two ct datasets andtreatment outcomes was implied or even confirmed inseveral smaller studies pointing to potential clinical utilityof imagingderived kinetic data [] in our grouphowever no such association was found a small numberof patients and in contrast to other studies the inclusion of different tumor sites could contribute to the negative result as well as differences in the changes in kineticproperties triggered by rtccr in individual tumors tumor radiosensitivity may also influence the effect oftti on outcome while to some extent it may be evaluated before rt begins eg by molecular profiling identification of hypoxic cells and the determination of hpvstatus in oropharyngeal primary tumors in daily clinicalpractice it is usually not considered when planning treatment [ ] in order to diminish the impact of intrinsic tumor radiosensitivity the patients in our study withresidual disease at weeks postrt were excludedfrom the analysis of lrc we hypothesized that the inability to achieve a complete tumor response to chemoradiation was due to the radiobiological characteristicsof the disease and not the delay in starting rt howeverthe exclusion of these patients from the analysis did notaffect the end resultsour study has weaknesses which are mostly due to itsretrospective nature however for obvious ethical reasons this is an inevitable feature of studying delays inthe initiation of cancer therapy we are aware that thereis always some doubt as to the accuracy of the diagnosticprocedures the staging the quality of planning and theimplementation of rt in the case of retrospective research nonetheless the same restraint exists in the caseof other similar studies and even more so in the case ofanalyses of cancer registry datasets an important feature of our data set is that no patients were lost duringthe followup and in our opinion is free from manyhidden biases that larger studies inevitably bring withsince almost half of our patients had oropharyngeal carcinoma missing information regarding the p16 or human papillomavirus hpv tumor status could be ofimportance howeverin the cohort of oropharyngealcancer patients treated at our institution between and only had a hpvrelated tumor thus we reasonably assume that the impact of p16hpv tumor status on the study results was negligible inaddition in hpvpositive cases perni found nosignificant association between tumor growth velocitycalculated from serial pretreatment ct scanslocaland distant control or os and the same was reportedby chu who measured metabolic growth velocityusing pretreatment petct scans [ ]other drawbacks to consider include technical limitationsin ct scan acquisition the accuracy of presentation of actual tumor volume on ct images and the precision of thedelineation of gross tumor volume to minimize errors inthe estimation of comparative tumor volumes only highquality pairs of ct images were selected for the analysis oftumor kinetics in addition physical findings documentedin clinical records and when available diagnostic mriswere used for this purpose and labelled tumor volumes represented a consensus between two experienced radiationoncologists and a radiologist all dedicated to hnc management like many other studies [ ] the comorbidity burden was not registered in our patientsalthough it should be taken into account when assessingsurvival outcomes finallylags in the prebiopsyperiod were not addressed in our study including patientdelay and delays in referral and diagnostics which may addsignificantly to the total tti these are also costly and potentially fatal but can be successfully reduced by effectivecoordination between providers []sthe relationship between tti and treatment outcomesis multifaceted so the controversy of published results is 0cžumer radiation oncology page of not surprising in this study we found that delays in theonset of rt do not harm all patients as tti is a problem in many public health systems further research iswarranted and should focus on two areas evaluatinglarge population surveys with highquality data andtreatmentrelated outcomes not just os and the prognostic relevance of imagingderived kinetic data of individualtumors which appeared promising in severalsmaller and statistically underpowered studies in orderto obtain a tool to identify patients at increased risk oftreatment failure due to delays in starting rt in a situation without clear knowledge to whom waiting for irradiation is harmful the only possible recommendationcould be that the waiting time for rt should be asshort as reasonably achievable asara abbreviationshnc head and neck cancer rt radiotherapy tti time to treatmentintervention scc squamous cell carcinoma cct concurrent chemotherapyct computer tomography mri magnetic resonance imaging os overallsurvival csh causespecific hazard lrc locoregional control dfms distantmetastasisfree survival dfs diseasefree survival iqr interquartile rangeci confidence interval hpv human papillomavirusacknowledgementsthis study was financially supported by the slovenian research agencyprogram no p30307authors contributionsstudy concepts žumer b strojan p study design žumer b pohar perme mstrojan p data acquisition all authors quality control of data strojan p dataanalysis and interpretation pohar perme m strojan p statistical analysispohar perme m manuscript preparation all authors manuscript editingstrojan p manuscript review žumer b pohar perme m strojan p the authors read and approved the final manuscriptfundingthis study was financially supported by the slovenian research agencyprogram no p3availability of data and materialsthe datasets analyzed during the current study are available from thecorresponding author on reasonable requestethics approval and consent to participatethe study protocol was approved by the republic of slovenia national medicalethics committee no all patients gave consent for using theirdata for study purposes at the start of their treatment for retrospective studies awritten consent is deemed unnecessary according to national regulationsconsent for publicationthe republic of slovenia national medical ethics committee approved thestudy which was conducted in accordance with the ethical standards laiddown in an appropriate version of the declaration of helsinki theneed for consent was waived by the republic of slovenia national medicalethics committeecompeting intereststhe authors declare that they have no competing interestsauthor details1department of radiation oncology institute of oncology ljubljana zaloÅ¡ka si1000 ljubljana slovenia 2institute of biostatistics and medicalinformatics faculty of medicine university of ljubljana ljubljana slovenia3department of radiology institute of oncology ljubljana ljubljanaslovenia 4chair of oncology faculty of medicine university of ljubljanaljubljana sloveniareceived may accepted august referencesbray f ferlay j soerjomataram i siegel rl torre la jemal a global cancerstatistics globocan estimates of incidence and mortality worldwidefor cancers in countries ca cancer j clin world health anization who life expecta | Colon_Cancer |
dysregulation of lncrnas is frequent in glioma and has emerged as an important mechanism involved in tumorigenesis previous analysis of chinese glioma genome atlas cgga database indicated that lbx2as1 expression is one of differentially expression lncrna between lower grade glioma lgg grade ii and iii and glioblastoma multiforme gbm however the function and mechanism of lbx2as1 in glioma has not been evaluated yetmethods here we analyzed the expression of lbx2as1 in gtex data normal brain tcgalgg and tcgagbm rtpcr was performed to detect lbx2as1 in surgery obtained normal brain and glioma cck8 kit and annexin vfitcpi apoptosis detection kit were used to study the function of lbx2as1 on glioma proliferation and apoptosis bioinformatic analysis rna immunoprecipitation rtpcr western blotting and dual luciferase reporter assay were carried out to investigate the target mirna of lbx2as1 the discovered mechanism was validated by the rescue assayresults following study of gtex and tcga data lbx2as1 was significantly elevated in glioma compared with normal brain and in gbm compared with lgg higher expression of lbx2as1 was associated with poor prognosis of patients with glioma expression of lbx2as1 was positively correlated with pathology classification of glioma knockdown of lbx2as1 inhibited cell proliferation and induced cell apoptosis in glioma lbx2as1 have complimentary binding site for tumor suppressor mir4915p and we showed that lbx2as1 sponged mir4915p to upregulate trim28 expression in glioma cells trim28 overexpression attenuated the effect of lbx2as1 knockdown on glioma cellss in lbx2as1 was an increased lncrna in glioma mechanistically lbx2as1 promoted glioma cell proliferation and resistance to cell apoptosis via sponging mir4915pkeywords lbx2as1 mir4915p cell proliferation cell apoptosis glioma trim28 gliomas are the most common primary cancer types derived from the neural ectoderm accounting for approximately half of all brain malignancy gliomas are histologic classified into several groups including grade ii oligodendrogliomas and astrocytomas and correspondence houruizhejldxjlueducn department of neurosurgery chinajapan union hospital of jilin university changchun jilin chinafull list of author information is available at the end of the grade iii anaplastic oligodendrogliomas anaplastic astrocytomas anaplastic oligoastrocytomas anaplastic ependymomas and grade iv glioblastomas gbm according to world health anization who classification [ ] overall gliomas are lethal cancer type and patients with high grade glioma gbm have a median overall survival less than one year the worse prognosis of patients with glioma is partly due to resistance to radiotherapy and chemotherapy induced cell apoptosis and the strong proliferation ability of cancer cells [ ] the authors this is licensed under a creative commons attribution international license which permits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the creative commons licence and indicate if changes were made the images or other third party material in this are included in the s creative commons licence unless indicated otherwise in a credit line to the material if material is not included in the s creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtain permission directly from the copyright holder to view a copy of this licence visit httpcreat iveco mmons licen sesby40 the creative commons public domain dedication waiver httpcreat iveco mmons publi cdoma inzero10 applies to the data made available in this unless otherwise stated in a credit line to the data 0cchen a0et a0al cancer cell int page of long noncoding rnas are rna molecules longer than nucleotides without protein coding potential as part of competing endogenous rna cerna network the function of many lncrnas rely on their interaction with micrornas mirnas to regulate expression of gene with same mirna response elements mres accumulating evidences suggested that lncrnas are implicated in glioma progression via sponging mirnas [] for example lncrna ccat1 sponged mir181b increased mir181b target fgfr3 and pdgfrα and promoted glioma cell proliferation invasion and resistance to cell apoptosis lbx2as1 is a recently identified cancer associated lncrna in several cancer types overexpression of lbx2as1 has been reported in hepatocellular carcinoma gastric cancer nonsmall cell lung cancer and esophageal squamous cell carcinoma and it promoted cancer cell proliferation with different mechanism in different cell [] in nonsmall cell lung cancer lbx2as1 activated notch pathway to facilitate cancer cell proliferation migration and invasion in esophageal squamous cell carcinoma lbx2as1 stabilized zeb1 and zeb2 to promote epithelialmesenchymal transition of cancer cells lbx2as1 is one of aberrantly expressed lncrnas between lgg and gbm from cgga database the biological role of lbx2as1 has not been examined in gliomathe current study revealed that lbx2as1 was a significantly upregulated lncrna in glioma and its expression associated with prognosis of patients with glioma we aimed to explore the biological role and molecular mechanism of lbx2as1 in gliomamaterials and a0methodspatients and a0samplestissue samples including normal brains and glioma tissues were obtained from the therapeutic surgery of patients in the third hospital of jilin university during june to july samples were confirmed histologically by two neuropathologists following the criteria of who classification guidelines written informed consents were provided and the protocol was approved by the third hospital of jilin university institutional review board the collected samples were stored at until rna extractioncell lines and a0culturehuman glioma cell lines u87mg u251mg and a172 were purchased from atcc manassas va human astrocyte cell line nha was bought from the cell bank of the chinese academy of sciences shanghai china cells were maintained in dulbeccos modified eagle medium invitrogen carlsbad ca supplemented with fbs gibco rockville md all cells were cultured in a humid incubator with co2 at rna immunoprecipitation rip assaya magna rip rnabinding protein immunoprecipitation kit millipore bedford ma was used to perform rip in a brief u87mg cell lysate was incubated with magnetic beads preincubated with mouse igg negative control or human antiago2 antibody cat no millipore billerica ma total rnas were then isolated by trizol reagent and detected by rtpcr to measure the enrichment of mir4915p and lbx2as1bioinformatic analysisthe expression of lbx2as1 in gtex tcgalgg and tcgagbm projects were obtained from gepia software httpgepia cance rpkucn which was also used to study the association between lbx2as1 expression and prognosis of patients with glioma the association between lbx2as1 and trim28 was also analyzed by gepia based on tcgalgg project encori software tarb asesysueducn was used to predict target mirnas of lbx2as1 and the association between lbx2as1 and mir4915p expression in tcgalggquantitative realtime polymerase chain reactiontotal rna was extracted from cells and tissues with trizol reagent invitrogen rna concentration was detected by nanodrop thermo fisher scientific wilmington de firststranded cdna was synthesized from rna with primescript rt reagent kit takara dalian china realtime polymerase chain reaction was performed with sybr prime script rtpcr kit takara on an abi fast realtime pcr system applied biosystems foster city ca mrna and lncrna were normalized to actin mirna was normalized to u6 the relative expression of gene was calculated by δδct method primer sequences were listed in table a0cell transfection²uuc ucc gaa cgu guc acg utt3² sicontrol silbx2as11 ²cca uua auu cag caa aca uuc cut t3 and silbx2as12 ²uga uuu uuu aaa gaa aaa ucc aat t3² were designed and synthesized by genepharma suzhou china for transfection sirna was mixed with lipofectamine rnaimax invitrogen in serumfree dmem and added into cultured ²cag cug guu gaa ggg gac caaa3² mir4915p mimic ²agu ggg gaa ccc uuc cau gagg3² and mir4915p inhibitor ²ccu cau gga agg guu ccc cacu3² was synthesized by ribo bio guangzhou china these mirnc mir4915p mimic and inhibitor was mixed with cells mirnc 0cchen a0et a0al cancer cell int page of table sequence of a0rtqpcr primersprimerlbx2as1trim28actinmir4915pu6 snrnasequence²aat tcg cag gaa ggg gag tg3²tgc caa acc tgg gac aaa ct3²tga gac ctg tgt aga ggc g3²²cgt tca cca tcc cga gac tt3²²cat gta cgt tgc tat cca ggc3²²ctc ctt aat gtc acg cac gat3²²ctc aac tgg tgt cgt gga gtc ggc aat tca gtt gag cct cat ²²tcg gca gga gtg ggg aac ccttc3²²ctc aac tgg tgt cgt gga ²²cgc ttc acg aat ttg cgt gtca3²²gct tcg gca gca cat ata cta aaa t3²²cgc ttc acg aat ttg cgt gtcat3²forwardreverseforwardreverseforwardreversestem loopforwardreversestem loopforwardreverselipofectamine invitrogen in serumfree dmem and added into cultured cells the transfection efficiency was detected a0h after transfectionwestern blottingps6k cat and s6k cat antibodies were obtained from cst beverly ca trim28 antibody cat was product of active motif carlsbad ca actin antibody cat kc5a08 was bought from aksomics shanghai china hrpconjugated mouse cat ab6728 and rabbit cat ab6721 antibodies were purchased from abcam cambridge uk ripa lysis buffer thermo fisher scientific was used to extract proteins from cells proteins were separated by an sdspage gel and transferred to pvdf membranes the membrane was blocked in nonfat milk and incubated with primary and secondary antibody ecl substrate thermo fisher scientific was used to develop blotscell proliferation and a0apoptosis assaythe cck8 kit beyotime shanghai china was used to detect cell proliferative ability briefly µl cck8 was mixed with culture medium and sustained for an additional a0h after that the absorbance at a0nm of each well was detected by a microplate reader biorad hercules ca percentage of apoptotic cells was measured with dead cell apoptosis kit with annexin v fitc and pi kit invitrogen by flow cytometry analysis cells were suspended in annexinv binding buffer provided by the kit and stained with annexinv fitc and pi sequentially the cells were then subjected to flow cytometry analysis cells positive for annexin v with or without pi positive were apoptotic cellsdual luciferase reporter assaylbx2as1 trim28 ²utr or their mutant forms were inserted into pmirglo luciferase vector these vectors were then cotransfected with mirnc or mir4915p into cells by lipofectamine after a0 h the relative luciferase activity of each group was measured with the dual luciferase reporter assay system kit promega corp madison wi firefly luciferase was normalized to renilla luciferasebioinformatic analysisthe data were analyzed with graphpad prism software the difference between the groups were examined with students t test or oneway anova analysis followed by tukey test the association between expression of two genes was studied with pearson correlation analysis p value less than was considered as statistically significantresultslbx2as1 was a0overexpressed in a0glioma and a0its expression was a0associated with a0poor prognosis of a0glioma patientswe firstly studied the expression of lbx2as1 in normal brains from gtex project low grade glioma lgg and glioblastoma gbm from tcga project the results suggested that lbx2as1 was 4fold and 10fold highly expressed in glioma tissues from tcgalgg and tcgagbm compared with normal brains from gtex expression of lbx2as1 was nearly 3fold higher in highgrade glioma gbm compared with lowgrade glioma lgg fig a0 1a we collected normal brains and glioma tissues and analyzed lbx2as1 expression by rtpcr it showed that lbx2as1 was approximately 3fold highly expressed in glioma compared with normal brains fig a01b furthermore lbx2as1 was increased in high grade glioma grade iv vs grade iii grade iii vs grade ii fig a01c retrospective analysis of the clinical outcome from tcgalgg and tcgagbm datasets suggested that lbx2as1 high expression group n have a shorter diseasefree survival compared with lbx2as1 low expression group n fig a0 1d meanwhile patients with higher expression of lbx2as1 showed a shorter overall survival time compared with their counterparts fig a01elbx2as1 was a0negatively associated with a0mir5p in a0gliomato study the mechanism of lbx2as1 in glioma we firstly used rtpcr method to detect lbx2as1 expression in a panel of glioma cell lines expression of lbx2as1 was significantly increased in u87mg u251mg and a172 cells in comparison with normal human astrocyte 0cchen a0et a0al cancer cell int page of fig lbx2as1 was an upregulated lncrna in glioma a analysis of lbx2as1 expression in normal brains from gtex project and glioma tissues from tcgalgg and tcgagbm projects b rtpcr detection of lbx2as1 expression in normal brains and gliomas c comparison of lbx2as1 expression in normal brain grade ii glioma grade iii glioma and grade iv glioma d e kaplan meierplotter analysis of association between lbx2as1 expression and diseasefree survival d overall survival e of patients with glioma from tcgalgg and tcgagbm projects p nha fig a0 2a lbx2as1 functioned as a cerna to exert its function on cell behavior we used the following method to screen for target mirnas of lbx2as1 fig a0 2b firstly encori database prediction showed that lbx2as1 have putative binding sites for mirnas pearson correlation analysis suggested that the expression of three of these mirnas mir19115p mir219a23p and mir4915p were negatively correlated with lbx2as1 expression in tcgalgg dataset in these three mirnas mir4915p was an upregulated mirna in tcga data of glioma as reported by qi et a0 al the strong negative correlation between mir4915p and lbx2as1 expression was showed in fig a0 2c pearson r a0 p expression of mir4915p was decreased by more than in collected glioma compared with normal brains fig a02d in contrast to lbx2as1 mir4915p was lowly expressed in high grade glioma compared with those of low grade grade iv vs grade iii fig a02e the negative correlation between lbx2as1 and mir4915p expression was also observed in these glioma tissues fig a0 2f to study whether lbx2as1 interact with mir4915p we firstly transfected mir4915p mimic into u87mg and u251mg cells to elevate mir4915p expression fig a02g full length of lbx2as1 was inserted into luciferase vector pmirglo luciferase reporter assay showed that mir4915p overexpression did repress luciferase activity of pmirglolbx2as1 by around half in u87mg fig a02h and u251mg cells fig a02i more importantly rip assay showed that ago2 antibody enriched both lbx2as1 and mir4915p in u87mg cells fig a0 2j these data implied that lbx2as1 might regulate mir4915p in glioma cellsa mutual regulatory association between a0mir5p and a0lbx2as1 in a0gliomato study the association between mir4915p and lbx2as1 we transfected increasing concentrations a0 nm and a0 nm of mir4915p mimic into u87mg and u251mg cells expression of mir4915p was increased by and 9fold in u87mg cells transfected with a0nm and a0 nm mir4915p mimic respectively fig a0 3a in u251mg cells mir4915p levels were increased by and 45fold after transfection of a0nm and a0nm mir4915p fig a0 3a overexpression of mir4915p decreased lbx2as1 expression by more than half in u87mg and u251mg cells and lower expression of lbx2as1 was observed in cells with high concentration of mir4915p mimic fig a0 3b two independent sirnas targeting lbx2as1 silbx2as11 and 0cchen a0et a0al cancer cell int page of fig lbx2as1 expression was associated with mir4915p levels in glioma a rtpcr detection of lbx2as1 expression in a panel of glioma cell lines u87mg u251mg a172 and normal human astrocyte nha b flow chart of screen for target mirnas of lbx2as1 in glioma c the association between lbx2as1 and mir4915p expression in tcgalgg project was analyzed by pearson correlation analysis d rtpcr detection of mir4915p expression in normal brains and gliomas e comparison of mir4915p expression in normal brain grade ii glioma grade iii glioma and grade iv glioma f the association between lbx2as1 and mir4915p expression in collected glioma samples was analyzed by pearson correlation analysis g the expression of mir4915p was detected in u87mg and u251mg cells transfected with mirnc or mir4915p mimic in u87mg h and u251mg i cells the luciferase activity of pmirglo empty vector as the control group and pmirglolbx2as1 was detected j rip assay was performed with igg and ago2 antibody followed by rtpcr detection of lbx2as1 and mir4915p levels p p silbx2as12 were transfected into glioma cells these sirnas greatly decreased lbx2as1 expression by more than in u87mg and u251mg cells fig a03c in addition knockdown of lbx2as1 increased mir4915p expression in u87mg and u251mg cells fig a0 3d transfection of silbx2as12 which reduced lbx2as1 expression in a relatively larger extent compared with silbx2as11 was more effective in upregulation of mir4915p expression in the cells fig a03d indicating the negative regulation of mir4915p by lbx2as1 we analyzed the complementary site between lbx2as1 and mir4915p and constructed pmirglo vector with mutant lbx2as1 lbx2as1 mut fig a0 3e overexpression of mir4915p repressed luciferase activity of lbx2as1 wt instead of lbx2as1 mut in u87mg fig a03f and u251mg cells fig a03glbx2as1 upregulated trim28 expression in a0gliomaprevious study suggested that mir4915p targeted trim28 to regulate glioma cell proliferation after investigation of trim28 and lbx2as1 expression in tcgalgg and tcgagbm datasets it was found that trim28 was positively correlated with lbx2as1 expression in glioma pearson r p fig a0 4a additionally trim28 was highly expressed in our collected glioma samples compared with normal brains fig a0 4b pearson correlation analysis showed a strong positive correlation between lbx2as1 and trim28 levels in our collected glioma tissues pearson r p fig a04c knockdown of lbx2as1 decreased approximately trim28 mrna expression in u87mg and u251mg cells fig a0 4d trim28 induced dephosphorylation of s6k to control glioma development lbx2as1 downregulation decreased trim28 protein expression and the downstream phosphorylation of s6k ps6k by half in u87mg cells as measured by western blotting fig a04e similarly lbx2as1 knockdown also decreased trim28 and ps6k in u251mg cells to the same extent as in u87mg fig a04f 0cchen a0et a0al cancer cell int page of fig mir4915p mutually regulated lbx2as1 in glioma mir4915p a and lbx2as1 b expression was detected in u87mg and u251mg cells transfected with mirnc or increasing concentration of mir4915p mimic nm nm c lbx2as1 expression was detected in u87mg and u251mg cells transfected with sicontrol or silbx2as11 or silbx2as12 by rtpcr d mir4915p expression was detected in u87mg and u251mg cells transfected with sicontrol or silbx2as11 or silbx2as12 e sequence alignment of lbx2as1 wt lbx2as1 mut and mir4915p luciferase activity was detected in u87mg f and u251mg g cells transfected with lbx2as1 wt or lbx2as1 mut p p lbx2as1 regulated trim28 via a0sponging mir5pwe first transfected mir4915p inhibitor into u87mg and u251mg cells to decrease mir4915p expression in these cells mir4915p inhibitor decreased mir4915p expression in cells fig a05a we found that mir4915p inhibitor could reverse the downregulation of trim28 mrna expression by silbx2as11 in u87mg and u251mg cells fig a05b western blotting further showed that mir4915p inhibitor could reverse the downregulation of trim28 protein expression by silbx2as11 in u87mg and u251mg cells fig a05c luciferase vectors containing wildtype and mutant trim28 ²utr trim28 ²utr wt trim28 ²utr mut were constructed fig a05d in u87mg cells mir4915p inhibitor could reverse the repression of luciferase activity of trim28 ²utr wt by silbx2as11 fig a05e these data collectively demonstrated a lbx2as1mir4915ptrim28 axis in gliomalbx2as1 promoted cell proliferation and resistance to cell apoptosis by repression of mir4915pit is known that aberrant expression of mir4915p and trim28 mediated glioma cell proliferation and survival to investigate the impact of the lbx2as1mir4915ptrim28 axis on cell proliferation we performed cck8 assay in glioma cells transfected with silbx2as1 with or without mir4915p inhibitor knockdown of lbx2as1 inhibited cell proliferation in u87mg cells and the effect of silbx2as1 was reversed upon cotransfection of mir4915p 0cchen a0et a0al cancer cell int page of fig lbx2as1 regulated trim28 expression in glioma a pearson correlation analysis of lbx2as1 and trim28 expression in tcgalgg and tcgagbm projects b rtpcr detection of trim28 mrna expression in normal brains and gliomas c pearson correlation analysis of lbx2as1 and trim28 expression in collected glioma d trim28 mrna expression was detected in u87mg and u251mg cells transfected with sicontrol or silbx2as11 or silbx2as12 by rtpcr trim28 ps6k protein expression was detected in u87mg e and u251mg f cells transfected with sicontrol or silbx2as11 or silbx2as12 by western blotting actin and s6k were internal controls for trim28 and ps6k p p inhibitor fig a0 6a in consistent with u87mg silbx2as11 and silbx2as12 inhibited u251mg cell proliferation which was attenuated by mir4915p inhibitor fig a0 6b with the flow cytometry we also found that lbx2as1 knockdown induced cell apoptosis in u87mg cells and the effect of silbx2as1 was partially rescued by mir4915p inhibitor fig a06c similar results were found in u251mg cells fig a06d these data manifested that lbx2as1 mainly relied on regulation of mir4915p to mediate glioma cell proliferation and survivaldiscussionlbx2as1 is a most recently identified oncogenic lncrna across several cancer types lbx2as1 was reported as a highly expressed lncrna in nonsmall cell lung cancer especially in tumors of advanced stage high expression of lbx2as1 was also found in stomach adenocarcinoma and hepatocellular carcinoma [ 0cchen a0et a0al cancer cell int page of fig lbx2as1 regulated trim28 expression via sponging mir4915p a the expression of mir4915p was detected in u87mg and u251mg cells transfected with mirnc or mir4915p inhibitor b the expression of trim28 mrna was detected in u87mg and u251mg cells transfected with mirnc or mir4915p inhibitor in combination with sicontrol or silbx2as11 by rtpcr c the expression of trim28 protein was detected in u87mg cells transfected with mirnc or mir4915p inhibitor in combination with sicontrol or silbx2as11 by western blotting d sequence alignment of lbx2as1 trim28 ²utr wt trim28 ²utr mut and mir4915p e luciferase activity was detected in u87mg cells transfected with trim28 ²utr wt or trim28 ²utr mut in combination with sicontrol or silbx2as11 and mirnc or mir4915p inhibitor p p see figure on next pagefig lbx2as1 regulated cell proliferation and apoptosis via sponging mir4915p transfection of silbx2as11 or silbx2as12 inhibited cell proliferation and was reversed by transfection of mir4915p inhibitor in u87mg a and u251mg b cells as indicated by cck8 assay transfection of silbx2as11 or silbx2as12 induced cell apoptosis and was partially reversed by transfection of mir4915p inhibitor in u87mg c and u251mg d cells as indicated by flow cytometry analysis p p 0cchen a0et a0al cancer cell int page of 0cchen a0et a0al cancer cell int page of ] here we analyzed lbx2as1 expression in glioma by using data of tcgalgg and tcgagbm projects in combination with normal brains from gtex project similar to observation in other cancer types it was observed that lbx2as1 was increased in glioma especially highgrade glioma gbm we further revealed that patients with high expression of lbx2as1 have a short diseasefree survival and overall survival indicating lbx2as1 could predict poor prognosis of glioma as the published studies showed that lbx2as1 was associated with poor prognosis of patients with hepatocellular carcinoma and nonsmall cell lung cancer [ ] the findings suggested that lbx2as1 might be a predictive biomark for a variety of cancer typeslbx2as1 exerted its procancer functions via acting as a cerna to sponge tumor suppressive mirnas for example lbx2as1 directly interacted with tumor suppressive mir384 to enhance cell proliferation and resistance to cell apoptosis in hepatocellular carcinoma our bioinformatic analysis indicated that lbx2as1 have a putative binding site for mir4915p mir4915p was a tumor suppressor in several cancer types [] aberrant expression of mir4915p was the consequence of upregulated circrna circ_0001361 and lncrna xist in bladder cancer and nasopharyngeal carcinoma respectively [ ] in glioma mir4915p was decreased in cancer tissues and correlated with good prognosis we confirmed mir4915p as a target mirna of lbx2as1 in glioma upregulation of mir4915p induced cancer cell apoptosis to cease cell proliferation [ ] we showed that lbx2as1 mediated glioma cell proliferation and resistance to cell apoptosis downregulation of mir4915p could partially rescue the impact of lbx2as1 knockdown on glioma cell proliferation and apoptosis thus the current data indicated a novel interaction between lbx2as1 and mir4915p and manifested that lbx2as1 promoted glioma proliferation via sponging mir4915p however as the cell apoptosis induced by lbx2as1 knockdown was not fully rescued by mir4915p downregulation we believe there are several other mechanisms underlying the function of lbx2as1 in glioma for example the notch signaling was regulated by lbx2as1 in nonsmall cell lung cancer and the activity of notch signaling determined the cell apoptosis in glioma [ ] it remains unknown whether lxb2as1 uses the same mechanism to control notch signaling in glioma future study will reveal the complexity of signaling network regulated by lbx2as1 in glioma in addition due to the involvement of mir4915p in cancer cell metastasis further studies will be needed to evaluate the effect of lbx2as1 on glioma metastasistrim28 is a cancerassociated e3 ligase in several cancer types upregulation of trim28 was found in glioma and the proproliferative function of trim28 was supported by in vitro and in vivo data [ ] in glioma trim28 mediated degradation of tumor suppressor ampk activated mtorc1 and regulated cell apoptosis trim28 expression was repressed by several noncoding rna in different cell [ ] in the current study in addition to the known mir4915ptrim28 interaction in glioma we further discovered that lncrna lbx2as1 could regulated trim28 via sponging mir4915p in glioma mechanistically trim28 form complex with mage to regulate mtor activity and the downstream phosphorylation of s6k [ ] we found that lbx2as1 not only upregulated trim28 expression but also increased phosphorylation level of s6k in glioma cells therefore the data revealed a lbx2as1mir4915ptrim28 axis in gliomasour results suggested that lncrna lbx2as1 promoted glioma cell proliferation and resistance to cell apoptosis via sponging mir4915p lbx2as1 could be a novel biomarker for patients with gliomaacknowledgementsnoneauthors contributions qc rh and jg performed the experiments and acquired the data qc and yz analyzed data qc and rh designed the study rh supervised the study manuscript was written by rh all authors read and approved the final manuscript funding none availability of data and materialsall data generated or analyzed during this study are included in this published ethics approval and consent to participateinformed consent was obtained from all patients and the study protocol and consent procedures were approved by the third hospital of jilin university review boardconsent for publicationnot applicablecompeting intereststhe authors declare that they have no competing interestsauthor details department of neurosurgery chinajapan union hospital of jilin university changchun jilin china department of gynecology and obstetric the second hospital of jilin university changchun jilin china received may revised july accepted july references ostrom qt bauchet l davis fg deltour i fisher jl langer ce pekmezci m schwartzbaum ja turner mc walsh km et al the epidemiology of glioma in adults a state of the science review neuro oncol 0cchen a0et a0al cancer cell int page of chen r smithcohn m cohen al colman h glioma subclassifications pan sj ren j jiang h liu w hu ly pan yx sun b sun qf bian lg and their clinical significance neurotherapeutics wesseling p capper d who classification of gliomas neuropathol appl neurobiol ostrom qt gittleman h stetson l virk sm barnholtzsloan js epidemiology of gliomas cancer treat res xu h chen b xing j wei z liu c qiu y lin y ren l upregulation of lgmnp1 confers radiotherapy resistance in glioblastoma oncol rep krakstad c chekenya m survival signalling and apoptosis resistance in glioblastomas opportunities for targeted therapeutics mol cancer castrooropeza r melendezzajgla j maldonado v vazquezsantillan k the emerging role of lncrnas in the regulation of cancer stem cells cell oncol dordr zhang z qian w wang s ji d wang q li j peng w gu j hu t ji b et al analysis of lncrnaassociated cerna network reveals potential lncrna biomarkers in human colon adenocarcinoma cell physiol biochem liu x zhu q guo y xiao z hu l xu q lncrna linc00689 promotes the growth metastasis and glycolysis of glioma cells by targeting mir3383ppkm2 axis biomed pharmacother magea6 promotes human glioma cell survival via targeting ampkalpha1 cancer lett wang sn luo s liu c piao z gou w wang y guan w li q zou h yang zz et al mir491 inhibits osteosarcoma lung metastasis and chemoresistance by targeting alphabcrystallin mol ther zhang q li q xu t jiang h xu lg mir4915p suppresses cell growth and invasion by targeting notch3 in nasopharyngeal carcinoma oncol rep xu y hou r lu q zhang y chen l zheng y hu b mir4915p negatively regulates cell proliferation and motility by targeting pdgfra in prostate cancer am j cancer res sun r liu z tong d yang y guo b wang x zhao l huang c mir4915p mediated by foxi1 functions as a tumor suppressor by targeting wnt3abetacatenin signaling in the development of gastric cancer cell death dis 201783e2714 liu f zhang h xie f tao d xiao x huang c wang m gu c zhang x jiang g hsa_circ_0001361 promotes bladder cancer invasion and metastasis through mir4915pmmp9 axis oncogene cheng q xu x jiang h xu l li q knockdown of long noncoding rna xist suppresses nasopharyngeal carcinoma progression by activating mir4915p j cell biochem liao y shen l zhao h liu q fu j guo y peng r cheng l lncrna casc2 denoyelle c lambert b meryetfiguiere m vigneron n brotin e lecerf c interacts with mir181a to modulate glioma growth and resistance to tmz through pten pathway j cell biochem ni w luo l zuo p li rp xu xb wen f hu d lncrna ghet1 downregulation suppresses the cell activities of glioma cancer biomark cui b li b liu q cui y lncrna ccat1 promotes glioma tumorigenesis by sponging mir181b j cell biochem tang lx su sf wan q he p xhang y cheng xm novel long noncoding rna lbx2as1 indicat | Colon_Cancer |
covid19 has had an impact on the provision of colorectal cancer care the aim of the crccovid study is to describe the changes in colorectal cancer services in the uk and usa in response to thepandemic and to understand the longterm impactmethods and analysis this study comprises phases phase is a survey of colorectal units that aims toevaluate adherences and deviations from the best practice guidelines during the covid19 pandemicphase is a monthly prospective data collection of service provision that aims to determine the impactof the service modiï¬cations on the longterm cancer speciï¬c outcomes compared to the national standards phase aims to predict costs attributable to the modiï¬cations of the crc services and additionalresources required to treat patients whose treatment has been affected by the pandemic phase aims tocompare the impact of covid19 on the nhs and usa model of healthcare in terms of service provisionand cost and to propose a standardised model of delivering colorectal cancer services for future outbreaksethics and dissemination this study is a service evaluation and does not require hra approval or ethicalapproval in the uk local service evaluation registration is required for each participating centre in theusa ethical approval was granted by the research and development committee the results of thisstudy will be disseminated to stakeholders submitted for peer review publications conference presentations and circulated via social mediaregistration details nil the authors published by elsevier ltd on behalf of surgical associates ltd this is an open access under the cc byncnd license httpcreativecommonslicensesbyncnd40 introductionthe covid19 pandemic has had a significant impact on theprovision of healthcare worldwide as of the 29th june covid19 has resulted in conï¬rmed cases and corresponding author at department of surgery and cancer imperial collegelondon chelsea and westminster and the royal marsden campus unitedkingdomemail address ckontovounisiosimperialacuk c kontovounisiosdeaths in the uk at a local level hospitals have been forcedto make a number of workforce modiï¬cations and changes toserviceprovision to combat the crisis and maintain standards ofcare for our patients facetoface consultations have beendissolved or minimized in favour of telephone or virtual clinicsprovision of investigations including ct scans and endoscopieshave been significantly reduced and all benign surgical procedurespostponed furthermore the treatment algorithm for conï¬rmed colorectal cancer cases has proved challenging101016jisjp20200700524683574 the authors published by elsevier ltd on behalf of surgical associates ltdthis is an open access under the cc byncnd license httpcreativecommonslicensesbyncnd40 0ca courtney international of surgery protocols in the uk over forty thousand patients are diagnosed with colorectal cancer each year deviation from nice colorectal cancercrc guidelines may lead to significantly poorer outcomes however the current model of cancer services delivery cannot be maintained because of both resource limitation and the potential risksto patients and staff during the pandemic there is a lack of highdependency beds which are being utilized for covid19 patientsthere is the risk of exposing colorectal cancer patients the majority of whom are elderly and have significant comorbidities to thevirus during their treatment within the hospital patients requiringneoadjuvant or adjuvant therapy are at particular risk finallystaff safety must also be considered particularly around aerosolgenerating procedures such as endoscopy and laparoscopic surgery intercollegiate general surgery guidance on covid19 outlinedgeneral principles on the provision of a safe surgical service duringthe pandemic however there has been no speciï¬c guidance todate on how to best modify colorectal cancer crc service provision during the pandemic in the absence of a national consensusthe onus is on individual hospital trusts and multidisciplinaryteams to make very challenging decisions about individual patientcare lack of a uniï¬ed approach may have important consequencesat patient and healthcare institution levelsdelay in cancer diagnosis or treatment due to service modiï¬cation is likely to create an increased demand in resources once thecrisis has passed predicting the economic impact and planningfor this is essentialhow hospitals approach the new constraints on crc care andallocate resources may vary between the uk and usa it is hopedthat gaining insights from both perspectives will improve the problem solving methods and analysis aims and objectivesthe aim of the crc covid study is to describe the changes incolorectal cancer services in the uk and usa in response to thecovid19 pandemic and to understand the longterm impactour primary and secondary objectives relevant to each phase ofthe study are listed in table study designthis is a multicentre service evaluation conducted through aresearch collaborative with the support of the crc covid steeringcommittee all colorectal units continuing to provide cancer services in the uk ireland and the usa have been invited to participate all study and recruitment information is available on thetable primary and secondary study objectiveswebsite crccovid this service evaluation has been endorsedby the royal college of surgeons of england rcsthis service evaluation will be carried out in phases fig phase uses a questionnaire to assess the modiï¬cationsadopted by each colorectal unit in order to continue provision ofthe colorectal cancer services during the covid19 pandemic ithas been developed using an iterative process after research ofall relevant guidelines to construct the standard against which services would be evaluatedthe following guidelines relevant to the management of colorectal cancer have been used as standards for this serviceevaluation nice guidelines colorectal cancer [ng151] nice guideline suspected cancer recognition and referral[ng12] association of coloproctology of great britain irelandacpgbi guidelines for the management of cancer of thecolon rectum and anus [] british society of gastroenterologyassociation of coloproctology of great britain and irelandpublic health england postpolypectomy and postcolorectal cancer resection surveillanceguidelines informal consultations with consultants nurse specialists andpatients have been used to develop the tool and then it has beenmodiï¬ed after clinician review for face validity ï¬ow and relevance the ï¬nal instrument comprises questionsphase investigates the provision of colorectal cancer servicesduring the covid19 pandemic by evaluating the performance ofeach unit against the national bowel cancer audit outcomes all centres participating in phase will be required to registerthis service evaluation as per local protocol prior to commencement of data collection on redcap this will be the responsibilityof the local leadphase of the study will develop a prediction model of the economic burden of the modiï¬cations in cancer service delivery thismodel will be designed jointly by two international businessschools based on previous publications and national statisticsphase will evaluate and compare the impact of the covid19pandemic on the nhs and the usa healthcare using data collectedduring phase and the predictive mode utilized in phase speciï¬cdifferences in modiï¬cations of crc services will be examined recruitmentphase survey has been distributed to all colorectal consultantsin the uk and ireland through personalised emails social mediaand the rcs the recruitment of colorectal cancer units in thephasephase phase phase phase primary objectiveevaluate adherences and deviations from best practiceguidelines on colorectal cancer during covid19pandemicative crc service deliverysecondary objectives 0f describe modiï¬cations to screening process for crc 0f describe modiï¬cations to preoperative intraoperative and postoper 0f demonstrate global effect of covid19 pandemic on crc service provi 0f outline consensus recommendations for sustainable modiï¬cations to 0f predict the impact of modiï¬cations on the incidence and prevalence of 0f plan adjustments to crc service provision after the end of pandemicsion irrespective of the type of healthcare systemdetermine the impact of crc service provision followingmodiï¬cations on longterm cancer speciï¬c outcomescompared to national standards 0f predict the costs attributable to modiï¬cations of crc services during covid19 pandemic 0f predict additional resources required to treat patients whose treatment has been affected by covid19 0f compare the impact of covid19 on the nhs and usa model of healthcare in terms of service provision and cost 0f propose a standardised model of delivering colorectal cancer services for future outbreaksdifferent crc stages in monthscrc services 0ca courtney international of surgery protocols fig phases of crc covidusa will use similar approach all units recruited into phase arerecruited into phase participation in phase is not mandatory inorder to participate in phase data collectionall surveys and data collection follow the gdpr requirementsand comply with caldicott principles individual patient identiï¬able data is not collected in this study study data is collectedand managed using redcap electronic data capture tool hostedat the kennedy institute of rheumatology at the university ofoxford redcap research electronic data capture is asecure webbased software platform designed to support datacapture for research studies providing an intuitive interfacefor validated data capture audit trails for tracking data manipulation and export procedures automated export procedures forseamless data downloads to common statistical packages and procedures for data integration and interoperability with externalsources after the close of the phase data collection period all data setswill be checked for missing data where possible centres will begiven an opportunity to rectify missing data centres where of data is missing will be excluded from data analysis and localleads will be notiï¬ed a nominated data validator will need toensure data accuracy prior to submission if during this processmajor discrepancy is identiï¬ed within the data set the centresdata will be excluded completely from the analysisfor details of the data collected in phase and phase pleaserefer to supplement data analysisphase responses to the survey pertaining to deviations fromdiagnostic and treatment protocols during the covid19 pandemicsee supplement will be converted to a numerical scale where will denote no deviation and will denote complete cessation ofservice provision the scores will be summarized using appropriatesummary statistics and analyzed using unsupervised learningkmeans and hierarchical clustering to identify clusters of homogeneous response to the pandemicphase every month participating centres will report theirdiagnostic and treatment activity see supplement to determine the impact of covid19 on colorectal cancer activity we willuse timeseries methods and data on historical activity and patientoutcomes to estimate a baseline of expected monthly activity that would have taken place in the absence of the pandemicthe baseline and a conï¬dence interval will be estimated atthe national regional and individual nhs trust level the baselineestimate will then be compared to the actual activity as reportedby publicly available data and data collected by this studythe difference between expected and actual activity will providean estimate of the reduction in activityto quantify the impact on patient outcomes associated with theestimated reduction in activity and deviation from standardizedcare protocols we will use estimates of disease progression available in peerreviewed literature [] a similar methodologywill be used to predict the impact of the pandemic on the incidenceand prevalence of different colorectal cancer stages in the following months under different scenarios predictions will be madeat the regional and national level and depending on data granularity at the trust levelphase will estimate the ï¬nancial costs of modiï¬cations to thecrc service provision due to the covid19 pandemic this willallow prediction of the expenditure and the additional resourcesrequired to resume routine services we will base this on the literature regarding the price of treatment at different disease stages and the information about the cost of resource utilizationconsultations diagnostic tests operating theatre time and hospital stay phase will compare the results from phases in theuk with those in the us discussioncancer care and maintaining high standards of diagnosis andtreatment has long been a priority of the nhs and internationalhealth care systems the pandemic has shifted this focus awayfrom the cancer services colorectal cancer patients are particularly 0ca courtney international of surgery protocols vulnerable to the disruption of their care as diagnosis throughendoscopy was stopped due to concerns about virus aerosolysation this study is important because it is the ï¬rst study to ask howindividual units had to modify their services and adapt to the newconstraintsin addition to describing the changes and understandingwhether different units had different approaches we wish to gofurther by understanding the effects of diagnostic and treatmentdelay by prospective data collection of cancer cases referrals diagnosis staging and treatment and comparing them to nationally collected audit data these data will allow us to model the economic impact of thedelay and what resources are required to restore cancer servicesto precovid19 standardsthe strengths of this study are in the multimodal approach tothe issues international collaboration and support from the royalcollege of surgeons our diverse team of management and business academics colorectal surgeons nurse specialists and patientadvisors enable us to have a range of approaches to collect andanalyse the datathe main limitation to the study is nonresponder or samplingbias as we require voluntary participation from colorectal teamswe will ensure that we adjust statistical analysis for any underrepresentation we expect that even with minimal participationuseful models can be generated to understand future resourcerequirements at an individual hospital level the methodologyemployed by other units will demonstrate the utility of the modelin summary this is a novel and important multiphase studythat is vital to understand how to best care for cancer patientsand ensure that the effects of the pandemic are mitigated ethics and disseminationthis study is a service evaluation and does not require hraapproval or ethical approval in the uk departmental approvalhas been granted by the university each participating centre mustseek local permission from their local audit department prior tocommencement of data collection in the usa ethical approvalwas granted by the research and development committeedata for phase will be submitted for publication as soon as theresults become available interim data analysis will be presented tothe royal college of surgeons covid19 research collaborativedata for other phases will be submitted for publication once thedata collection has been completed which is anticipated to be afterthe routine service provision resumes all data will be presented atnational and international conferences circumstances permitting guarantornone research registration numbernoneethical approvalnoneauthor contributionsac designed the study wrote the initial proposal drafted themanuscript based on the study proposal and is part of the auditadvisory groupamh ns nd ow sm sr gm nt mg td bs jee ad ptadvised on the study design and the protocol and is part of thesteering committeeck is a project piall authors read commented on and approved the study designthe protocol and the ï¬nal manuscriptfundingthis research received no speciï¬c grant from any fundingagency in the public commercial or notforproï¬t sectorsdeclaration of competing interestthe authors declare that they have no known competing ï¬nancial interests or personal relationships that could have appearedto inï¬uence the work reported in this paperreferences world health anization the united kingdom who coronovirus diseasecovid19 dashboard covid19whointregioneurocountrygbaccessed june covidsurg collaborative global guidance for surgical care during the covid pandemic br j surg a spinelli g pellino covid19 pandemic perspectives on an unfolding crisisbr j surg british society of gastroenterology endoscopy activity and covid19 bsgand jag guidance apr wwwbsgukcovid19adviceendoscopyactivityandcovid19bsgandjagguidance accessed may nhs england nhs improvement letter to chief executives of all nhs trustsand foundation trusts ccg accountable ofï¬cers gp practices and primary carenetworks and providers of community health services mar wwwenglandnhsukcoronaviruswpcontentuploadssites52202003urgentnextstepsonnhsresponsetocovid19lettersimonstevenspdfaccessed may research uk cancerincidencecancerbowelstatisticswwwcancerresearchukhealthprofessionalcancerstatisticsstatisticsbycancertypebowelcancerincidenceheadingzeroaccessed may royal college of surgeons of england updated intercollegiate general surgeryguidance on covid19 apr wwwrcsengacukcoronavirusjointguidanceforsurgeonsv2 accessed may national institute for health and care excellence nice colorectal cancernice guideline ng151 wwwniceukguidanceng151accessed accessed march national institute for health and care excellence nice suspected cancerrecognition and referral nice guideline ng12 wwwniceukguidanceng12 accessed accessed march c cunningham k leong s clark a plumb s taylor i geh s karandikar bmoran association of coloproctology of great britain ireland acpgbiguidelines for the management of cancer of the colon rectum and anus diagnosis investigations and screening colorectal dis suppl s gollins b moran r adams c cunningham s bach as myint a renehans karandikar v goh d prezzi g langman s ahmedzai i geh association ofcoloproctology of great britain ireland acpgbi guidelines for themanagement ofmultidisciplinary management colorectal dis suppl rectum and anusthe coloncancer of glangman m loughrey n shepherd p quirke association ofcoloproctology of great britain ireland acpgbi guidelines for themanagement of cancer of the colon rectum and anus pathologystandards and datasets colorectal dis suppl k leong j hartley s karandikar association of coloproctology of greatbritain ireland acpgbi guidelines for the management of cancer of thecolon rectum and anus follow uplifestyle and survivorshipcolorectal dis suppl b moran c cunningham t singh p sagar j bradbury i geh s karandikarassociation of coloproctology of great britain ireland acpgbi guidelinesfor the management of cancer of the colon rectum and anus surgicalmanagement colorectal dis suppl md rutter j east cj rees n cripps j docherty s dolwani pv kaye kjmonahan mr novelli a plumb bp saunders s thomasgibson djmtolan s whyte s bonnington a scope r wong b hibbert j marsh bmoores a cross l sharp british society of gastroenterologyassociation ofcoloproctology of great britain and irelandpublic health england postpolypectomy and postcolorectal cancer resection surveillance guidelines gut 0ca courtney international of surgery protocols healthcare quality improvement partnership hqip national bowel canceraudit annual report an audit of the care received by people with bowelcancer in england and wales v20 wwwnbocaukcontentuploads202001nboca2019v20pdf accessed june pa harris r taylor r thielke j payne n gonzalez jg conde researchelectronic data capture redcapa metadatadriven methodology andworkï¬ow process for providing translational research informatics support jbiomed inform pa harris r taylor bl minor v elliott m fernandez l oneal l mcleodg delacqua f delacqua j kirby sn duda re consortium the redcapconsortium building an international community of software platformpartners j biomed inform nhs england and nhs improvement cancer waiting times wwwenglandnhsukstatisticsstatisticalworkareascancerwaitingtimesaccessed june national bowel cancer audit nboca datagov weblink accessed junewwwnbocaukresourcesnbocadatagovweblink cancer research uk incisive health saving lives averting costs an analysis ofthe ï¬nancial implications of achieving earlier diagnosis of colorectal lung andovarian cancer wwwcancerresearchuksitesdefaultï¬lessaving_lives_averting_costspdf accessed may s sun f klebaner t tian a new model of time scheme for progression ofcolorectal cancer bmc syst biol suppl s2 j emery p vedsted new nice guidance on diagnosing cancer in generalpractice br j gen pract hb keshava je rosen mr deluzio aw kim fc detterbeck dj boffawhat if i do nothing the natural history of operable cancer of the alimentarytract eur j surg oncol yh lee pt kung yh wang wy kuo sl kao wc tsai effect of length oftime from diagnosis to treatment on colorectal cancer survival a populationbased study plos one e0210465 d roder cs karapetis i olver d keefe r padbury j moore r joshi dwattchow dl worthley cl miller c holden e buckley k powell dburanyitrevarton k fusco t price time from diagnosis to treatment ofcolorectal cancer in a south australian clinical registry cohort how it variesand relates to survival bmj open e031421 cancer research uk bowel cancersurvivalstatistics wwwcancerresearchukhealthprofessionalcancerstatisticsstatisticsbycancertypebowelcancersurvival accessed june kk turaga s girotra are we harming cancer patients by delaying theircancer surgery during the covid19 pandemic ann surg 0c' | Colon_Cancer |
cancer is a complex and heterogeneous disease with many possible genetic and environmentalcauses the same treatment for patients of the same cancer type often results in different outcomes in terms ofefficacy and side effects of the treatment thus the molecular characterization of individual cancer patients isincreasingly important to find an effective treatment recently a few methods have been developed to constructcancer samplespecific gene networks based on the difference in the mrna expression levels between the cancersample and reference samplesmethods we constructed a patientspecific network with multiomics data based on the difference between areference network and a perturbed reference network by the patient a network specific to a group of patients wasobtained using the average change in correlation coefficients and node degree of patientspecific networks of thegroupresultsnetworks with multiomics data the main differences of our method from previous ones are as follows networksare constructed with multiomics mrna expression copy number variation dna methylation and micrornaexpression data rather than with mrna expression data alone networks are constructed with bothnormal samples and cancer samples of the specified type to extract cancerspecific gene correlations and bothpatient individualspecific networks and patient groupspecific networks can be constructed the results of evaluatingour method with several types of cancer show that it constructs more informative and accurate gene networks thanprevious methodss the results of evaluating our method with extensive data of seven cancer types show that thedifference of gene correlations between the reference samples and a patient sample is a more predictive feature thanmrna expression levels and that gene networks constructed with multiomics data show a better performance thanthose with single omics data in predicting cancer for most cancer types our approach will be useful for finding genesand gene pairs to tailor treatments to individual characteristicskeywordsindividualspecific gene network groupspecific gene network cancer multiomics datacorrespondence khaninhaackr1department of computer engineering inha university incheon southkoreafull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriatecredit to the original authors and the source provide a link to the creative commons licence and indicate if changes weremade the images or other third party material in this are included in the s creative commons licence unlessindicated otherwise in a credit line to the material if material is not included in the s creative commons licence and yourintended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtain permission directlyfrom the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40 the creativecommons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to the data madeavailable in this unless otherwise stated in a credit line to the data 0clee bmc medical genomics 13suppl page of for the past years we have witnessed the rapid development of targeted cancer therapy targeted therapies forcancer work by targeting specific genes proteins or tissues that contribute to cancer growth and survival manytargeted therapies are effective only for patients with specific genetic alterations known as driver mutations thathelp cancer cells form and grow [ ] thus identifying genetic mutations specific to individual patients is ofutmost importance to determine targeted therapies thatcan effectively cure cancer patients while minimizing sideeffects motivated by a massive amount of data generated byhighthroughput technologies several cancer studies usedgene networks to explore gene expression characteristics [] however constructing a patientspecific genenetwork with a single sample obtained from a patient isdifficult because a gene network requires many samples tocompute genegene relationsrecently a few methods have been proposed to construct cancer samplespecific gene networks based onthe difference in the mrna expression levels betweenthe cancer sample and reference samples for exampleliu proposed a method to construct a samplespecific network by computing the difference between areference network from multiple reference samples anda network perturbed by a new sample however a slightchange to the reference samples can result in a significantly different samplespecific network for the samesample due to the small number of reference samples furthermore their samplespecific networks cannot reflectposttranslational modification and epigenetics becausethe networks are built using mrna expression data onlythis paper presents a new method for constructingcancer patientspecific and groupspecific gene networkswith multiomics data using a samplespecific networkand network propagation method network propagation strategies are widely used in recent cancerrelatedresearch li presented a synergy prediction algorithm using network propagation and predicted the drugsynergy in various cancers zhang introduceda propagation algorithm which learns the mutated subnetworks underlying tumor subtypes using a supervisedapproach and classified tumors to known subtypes onbreast and glioblastoma tumors peng identifiedbladder cancerrelated genes by propagating informationfrom seed genes to candidate genes the primary focusof our method is to construct a gene correlation network specific to cancer with multiomics data thus it isdifferent from a typical gene coexpression network thatrepresents coexpression relations between genes frommrna expression data our gene network is not a generegulatory network because our network does not showregulatory relations between genesthe main differences of our method from previous onesare as follows networks are constructed with multiomics mrna expression copy number variation dnamethylation and microrna expression data rather thanwith mrna expression data alone networks are constructed with both normal samples andcancer samples of the specified type to extract cancerspecific gene correlations and both patient individualspecific networks and patient groupspecific networks canbe constructed as shown later in this paper the resultsof evaluating our method with several types of cancershow that it constructs more informative and accuratetargetspecific networks than previous methodsmethodsat the top level our method consists of the followingsteps data processing constructing individualspecific gene networks and constructing a groupspecific gene networks a highlevel description of themethod is given in fig data collection and preprocessingfrom the broad institute tcga gdac firehose we obtained multiomics data of cancer samples of seventypes breast invasive carcinoma brca colon adenocarcinoma coad head and neck squamous cell carcinomahnsc pankidney cohort kipan liver hepatocellular carcinoma lihc lung adenocarcinoma luad andlung squamous cell carcinoma luscthe multiomics data used in this study includemrna expression mrnaseq copy number variationcnv dna methylation and mature mirna expression mirseq data the mrnaseq data were processedusing quartile normalized rsem and then log2transformed the segmented cnv data were convertedto genelevel data using the ensembl api and thecntools package of bioconductor the methylationdata were filtered to select the probe with the mean signal values for each gene the mirseq data were processedby rpm and log2transformed mrnas and mirnas thatwere not expressed in more than of the total sampleswere excluded in further analysis missing expression values of mrnas and mirnas were replaced by the smallestpositive normalized floatingpoint number realmin ofmatlab the number of samples and genes used in thisstudy are available in additional file individualspecific gene networkin each group of tumor samples and normal samples wefirst computed genegene relations by the pearson correlation coefficient pcc selected highly correlated genepairs ie those with pcc and constructed twosample networks one for each group from the tumorsample network we removed edges common to both 0clee bmc medical genomics 13suppl page of fig overview of constructing an individualspecific network and a groupspecific network with multiomics datatumor and normal sample networks and obtained a template reference network for cancer fig 2a the templatereference network consists of highlycorrelated gene pairsthat are specific to cancerwith n reference samples which may be different fromtumor samples used in the template network we computed pcc for every pair of genes in the template reference network and constructed a reference network forthe reference samples for a patient of interest we constructed a network which is a perturbed network byadding a single sample of the patient to the n reference samples a patientspecific network was obtainedby subtracting the reference network from the perturbednetwork 01pcc pccn1 pccnwe computed the difference in the absolute value ofpcc between the perturbed reference network and reference network by eq we also carried out a ztest ofpccn1pccn by eq for a large n we can approximatethe mean μ and standard deviation Ï of pccn1 pccn as and pcc2nn respectively pccchange pccn1 pccnz score pccchange μpccchangeÏ pccchange pccchangepcc2nnthe edges of the patientspecific network were classified into four types correlationgained edges fene pairs whose pccs are increased from the referencenetwork to the patientspecific network correlationlost edges for gene pairs whose pccs are decreased fromthe reference network to the patientspecific network correlationreversed edges for gene pairs whose signs ofpccs are changed from positive to negative or negativeto positive and correlationinvariant edges for genepairs with little change in pccs between the reference andpatientspecific networks ie those with zscore fig 2bthe edges were classified in the following way we firstselected gene pairs with zscore as correlationinvariant type and then selected gene pairs which havedifferent signs of pccs between the reference networkand the patientspecific network as correlationreversedtype the remaining gene pairs were classified into eithercorrelationgained or correlationlost type depending onwhether their pccs are increased correlationgained ordecreased correlationlost from the reference network tothe patientspecific network thus zscore ¥ in bothcorrelationgained and correlationlist gene pairsgroupspecific gene networka groupspecific gene network is useful when analyzinga large number of patientspecific gene networks afterconstructing patientspecific gene networks we obtained 0clee bmc medical genomics 13suppl page of fig process of constructing a patientspecific gene network a template of the cancer reference network obtained by removing edges common toboth networks b patientspecific gene network and four types of edges in the network edges with a zscore represent correlationinvariantgene pairs and edges with zscore ¥ and different signs of pccn and pccn1 represent correlationreversed gene pairs edges with zscore ¥ and 01pcc are correlatedgained gene pairs and those with zscore ¥ and 01pcc are correlationlost gene pairsa gene network specific to a group of patients based onthe average 01pcc and node degree of the patientspecificnetworks fig if the dominant type for a particular edge is correlationgained positive 01pcc in thepatientspecific networks the edge is represented in redin the groupspecific network in contrast if the dominant type for a particular edge is correlationlost negative 01pcc in the patientspecific networks the edgeis represented in blue in the groupspecific network inthe groupspecific network only the dominant type isshown for each edge if nondominant types are shownin addition to the dominant type for each edge the network becomes cluttered and unreadable the node sizeof a groupspecific gene network is proportional to theaverage degree of the nodeintegration of multiomics datato integrate multiomics data we first computed intergene correlations by pcc with four different types ofsingle omics data mrna expression cnv dna methylation and mirna expression separately and selectedsignificant intergene correlations only in mrna expression cnv and dna methylation data we select thetop pcc with pvalue in mirna expression data we selected the top pcc with pvalue due to a smaller number of mirnas inthe data the intergene correlations selected in eachsingle omics data are represented in four correlationmatrices mexpr mcnv mmethyl and mmirna andnormalizedusing the proteinprotein interactions ppis of thestring database we constructed separate weightednetworks from each omics data by eq in eq wexprwcnv and wmethyl denote the weighted networks andppiexpr ppicnv and ppimethyl are subnetworks of a ppinetwork consisting of genes present in each omics datasince the ppi network does not contain information onmirna a weighted network for mirna was not constructedcid3wexpr cid2 ppiexprwcnv mcnv à ppicnv wmethyl cid2 ppimethylwe then integrated the multiomics data by linear xmethylregression using eq in eq yi xcnvand xmirnadenote gene is expression level cnv levelmethylation level and mirna regulator expression levelrespectively βcnvdenote the regression coefficients of gene is expression level on cnv and methylation respectively βmirnais the regression coefficientof gene is expression level on its mirna regulator jsexpression leveland βmethyl mexpr mmethylcid3 à cid2cid3 à cid2cid3iiijiiijyi βcnvii βmethylxcnvixmethyli ncid4j1βmirnaijxmirnaij 01 0clee bmc medical genomics 13suppl page of fig process of constructing a groupspecific gene network from multiple patientspecific gene networks in the groupspecific gene network thenode size and node color are proportional to the average degree and average 01pcc of the node respectively if the dominant type for a particularedge is correlationgained positive 01pcc in the patientspecific networks the edge is represented in red in the groupspecific network if thedominant type for a particular edge is correlationlost negative 01pcc in the patientspecific networks the edge is represented in blue in thegroupspecific networkfrom the regression coefficients and the weighted networks a weight matrix w was derived and normalizedinto w eqs and the weight matrix w is symmetricso wij wji w11 w22 w33 and w44 represent wexprwcnv wmethyl and mmirna respectively the submatrices w21 and w31 contain regression coefficients βcnvand βmethylfor every gene i respectively w41 representsiβmirnaij the submatrices w32 w42 and w43 are emptyi¡¢¢£w w11 w12 w13 w14w21 w22 w23 w24w31 w32 w33 w34w41 w42 w43 w44¤¥¥¦eq and updated iteratively by eq in this iterative process the influence of the seed is propagated tothe neighbors until a mean squared error of st and st¤ à §ªªªª¨ªªªª©if v is a nonseed nv ¥ αif v is a nonseed nv αif v is a seednvnvenvα à nvnvdv st λ à st à w λ à d wheres1 d where nv is the number of neighbors of node v and nv isthe number of seeds in the neighbors the parameter αwhich is a threshold for nv was set to and λ was set to genes with the top st were used in findingcancerrelated genes and in classifying tumor samples andnormal samplesw i j w i jcid11cid12cid12cid13mcid4k1w i k à mcid4k1w k jin network propagation seed genes have greater impactthan nonseed genes on their neighbors thus only thegenes with a high average 01pcc were selected as seedgenes for a groupspecific network and their mirnasregulators extracted from mirtarbase were used asseed mirnas we calculated the cancerrelevance st ofeach gene to reflect the effect of the seed genes and mirnas on neighbors the initial score d was calculated byresultspatientspecific and groupspecific gene networksin this study we constructed patientspecific genenetworks for seven cancer types additional file foreach cancer type we also constructed groupspecific genenetworks as an example fig shows a groupspecificgene network derived from lung squamous cell carcinoma lusc patients 0clee bmc medical genomics 13suppl page of there are three distinct subnetworks in the networkfor the lusc group the subnetwork enclosed in box aof fig contains many hub genes large green nodesthe subnetwork in box b consists of correlationgainededges dark red edges whereas the subnetwork in box ccontains many correlationlost edges dark blue edgescomparison of multiomics data and singleomics datawe performed leaveoneout cross validation loocvto evaluate cancerrelevance score st of a gene and thecontribution of multiomics data to finding cancerrelatedgenes for comparison the cancerrelevance scores werecomputed with multiomics data and single omics dataseparately each seed gene was regarded as a nonseed anda new cancerrelevance score was calculated for the geneseed genes and nonseed genes were considered as positive and negative respectively seed genes included in thetop n genes were considered as true positives and seedgenes not included in the top n genes were consideredas false negatives similarly nonseed genes included infig groupspecific gene network for lung squamous cell carcinoma lusc patients a subnetwork of multiple hub genes large greennodes b subnetwork of correlationgained edges dark red edges c subnetwork with many correlationlost edges dark blue edges 0clee bmc medical genomics 13suppl page of the top n genes and nonseed genes not included in thetop n genes were considered as false positives and truenegatives respectivelywe carried out loocv with different ratios of seedgenes to nonseed genes figure shows the receiver operating characteristic roc curve and the area under thecurve auc of loocv of the cancer relevance of geneson data of breast cancer samples with various seedratios ranging from to enlarged plots of fig are available in additional file for comparative purposes we also computed the cancer relevance of geneswith single omics data as shown in fig multiomicsdata consistently exhibited better performance than singleomics data with any seed ratio between to forlater analysis the seed ratio was set to by default theaverage 01pcc and class label of each gene are available inadditional file indeed the superiority of multiomics data over singleomics data in determining the cancer relevance scoreof genes was observed in all seven types of canceradditional file in seven types of cancer the cancerrelevance score of genes computed with multiomics dataexhibited a good performance auc ¼ thecancer relevance score of genes computed with mrnaexpression data showed the second best performanceauc ¼ in particular the cancer relevancescore computed with mrna expression data showed avery similar performance to that with multiomics inbreast cancer brca the performance of the cancer relevance score computed with cnv auc ¼and dna methylation data auc ¼ alonewas lower than that with mrna expression data auc ¼fig roc curves of the leaveoneout cross validation of the cancer relevance score st of genes with different ratios of seed genes breastcancer samples were used in the leaveoneout cross validation the performance of multiomics data is always better than that of single omics data 0clee bmc medical genomics 13suppl page of evaluation of gene correlations and networksmany networkbased approaches to cancer research havefocused on finding genes that show differential expressions between tumor samples and normal samples genegene correlations ie intergene correlations may bemore helpful than individual genes because intergenecorrelations depend on the expression of neighbor genesin a gene regulatory network to compare the effect ofusing individual genes to that of intergene correlationsie 01pcc we constructed a support vector machinesvm model for classifying cancer samples and normalsamples the svm model was implemented using csvcand rbf kernel and the parameter values of the modelwere determined by the grid search algorithm mrnaexpression levels and 01pccs were used as features ofthe svm models for rigorous validation the test dataused in testing the models were not used in training themadditional file as shown in fig 6a 01pcc showed a better performance than mrna expression levels for six cancertypes except lusc the classification model with 01pccshowed mcc above in six cancer types except hnscwe also examined the effect of different networks on individualspecific networks in the workby liu ppi data with high confidence scoresin the string database were used to construct a network however the ppi data of stringdoes not reflect cancer typespecific characteristicsfigure 6b shows the performance of the classificationmodel with two different networks network from ppi data of string the approachby liu and cancer network ourapproach 01pcc was used as a feature of the classification model except for coad the performance ofthe classification model with the cancer network was better than the model with the string reference network in particular the classification model showed a significant difference for breastcancer brca mcc of vs mcc of detailed results of the classification model are available inadditional file discussionin the analysis of finding cancerrelated genes and genepairs we focused on a subnetwork of genes with a 01pcctable shows the top genes with a high average 01pcc in each groupspecific network of seven cancertypes in breast invasive carcinoma brca fam171a1showed the highest average 01pcc in the groupspecificnetwork fam171a1 is known as a potential biomarkerin triplenegative breast cancer foxc1 is involvedin tumor development and metastasis and associated withpoor prognosis in basallike breast cancer il33 isoverexpressed in various cancers and the serum concentration of il33 is a valuable indicator of poor prognosis inbreast cancer mamdc2 is significantly correlatedwith diseasefree survival of breast cancer patients mterfd1 is closely related to breast cancer recurrencefig results of evaluating features and networks by a validation set a comparison of mrna expressions of genes and 01pcc of genepairs b comparison of the cancer network with the network from ppi data 0clee bmc medical genomics 13suppl page of table top genes with a high average 01pcc in a groupspecific network for seven cancer typesbrcakipanlihccoadhnscfam171a1foxc1il33mamdc2mterfd1hoxa7cttnbp2znf204pjam3frem1gfra2scnn1bddx27rnps1ube2imdficctnnbl1cdk5rap1esf1trmt6cyp2j2barx2znf135ppfia1parlfaddcst6cobloraov1xpo7tfcp2l1kcnq1arl15kctd1oaz2tmem45bhpcal1tmem91sema5begln3slc19a3ecm1cyp2b6fbp1gpaa1f9pahagxt2hsd17b6rnase4luadcldn18adamts8pecam1sftpa1gimap6akr1c1mmeatad2cyp3a5chaf1bluscnsun2cct5fbxo45gpr116slc39a8fxr1inmtveph1crtamwdr53 and hoxa7 plays a critical role in regulating theproliferation of erpositive cancer cells in colon adenocarcinoma coad gfra2 showed thehighest average 01pcc in the groupspecific network itis known to be crucial for enteric neuron survival scnn1b and ddx27 are significantly related to colorectal cancer [ ] no direct relation of rnps1 withcolorectal cancer is known but rnps1 is essential tononsensemediated mrna decay that plays complexfunctions in cancer knockdown of sumo conjugating enzyme ube2i also known ubc9 or e2 inhibitsmaintenance and selfrenewal of colorectal cancer stemcell while overexpression of ube2i increases colorectalcancer cell stemness among the top genes with a high average 01pccin lung adenocarcinoma luad several genes such ascldn18 adamts8 pecam1 and sftpa1 have beenknown to be associated with luad in previous studies[] no direct relation of nsun2 and slc39a8 withlung squamous cell carcinoma lusc has been known sofar however recent studies [ ] reported that nsun2is correlated with survival in other types of squamous cellcarcinomas gao also showed that the epigeneticsilencing of slc39a8 expression by dna methylation isinvolved in the acquisition of resistance against cadmiumin lung cells and the relation between cadmium andlung cancer has received much attention many othergenes in table found in the groupspecific networksfor head and neck squamous cell carcinoma hnscpankidney cohort kipan and liver hepatocellular carcinoma lihc are also directly or indirectly related tocancerin addition to individual genes we identified genepairs of the same type ie either correlationgained orcorrelationlost in most patientspecific networks of thesame type table shows the most frequent gene pairs in breast cancer samples the most frequent gene pairsin other types of cancer are listed in additional file it isinteresting to note that all the gene pairs shown in table include at least one gene in the gene pair mamdc2hoxa7 and that they are correlationgained edges in thegroupspecific network for breast cancer figure showsa subnetwork containing mamdc2 and hoxa7 in thegroupspecific network of breast cancer the subnetworkwas obtained by selecting the edges for which the proportion of the same edge type ie correlationgained or lostis above in the total individualspecific networks ofbreast cancer patients it is interesting to note that all thegene pairs in table are included in the subnetworkto date the actual role of the mamdc2 gene in cancer is not clear but meng reported mamdc2as one of three genes mamdc2 tshz2 and cldn11that are significantly correlated with diseasefree survival of breast cancer patients mamdc2 is known as atarget of mir196a in head and neck squamous cell carcinoma as a member of the family of homeoboxgenes hoxa7 is associated with cell proliferation nerveinvasion distant metastasis and degree of tumor differentiation in several cancers [ ] hoxa7 is regulatedtable the most frequent gene pairs in breast cancersamples all the gene pairs are of a correlationgained type thegenes of table are shown in bold the proportion represents theratio of the gene pairs of the same type ie correlationgained orlost to the total number of patientspecific networksgene pairgene pairsproportion of the gene pairsin total cancer samplesmamdc2hoxa7mamdc2ccl14mamdc2znf204pmamdc2klmamdc2svep1mamdc2coro2bhoxa7meox2hoxa7hoxa9mamdc2sobpmamdc2hoxa9 0clee bmc medical genomics 13suppl page of fig a subnetwork of the groupspecific network of brca which contains mamdc2 and hoxa7 genes in the most frequent gene pairs shown intable are enclosed by a circleby several mirnas including mir196 [] thusboth mamdc2 and hoxa7 are related with mir196but a clear relation among them is to be uncoveredso far most approaches to constructing individualspecific gene networks have been constructed based onthe differential expressions between a small number ofreference samples and a sample of interest howeversuch networks cannot reflect posttranslational modification and epigenetics and are not reliable because a slightchange to the reference samples can result in a significantly different samplespecific network for the samesamplein this paper we presented a new approach to constructing cancer patientspecific and groupspecific networks with multiomics data the main differences ofour method from previous ones are as follows gene networks are constructed with multiomics mrnaexpression copy number variation dna methylationand microrna expression data rather than with mrnaexpression data alone networks can beconstructed with cancer samples of the specified type and both patient individualspecific networks and patientgroupspecific networks can be constructed the resultsof testing our method with several cancer types showedthat it constructs more informative and accurate genenetworks than existing methodsevaluation of our method with extensive data of sevencancer types showed that changes in gene correlations 01pcc between the reference samples and a patient sample is a more predictive feature than mrna expressionlevels and that gene networks constructed with multiomics data are more powerful than those with singleomics data in predicting cancer for most cancer typesmore work is required to validate the genes and genepairs identified in the cancer patientspecific and groupspecific networks however the method for constructingnetworks specific to individual patients or patient groupswith multiomics data should be useful aids in determining effective treatments to individual characteristicssupplementary informationsupplementary information accompanies this paper athttpsdoi101186s12920020007367additional file number of samples and genes in types of canceradditional file roc curve and auc of the cancerrelevance score ofbrca by various seed ratiosadditional file average 01pcc and class label of each gene in typesof canceradditional file roc curve of the cancerrelevance score of each cancertype with the seed ratio of additional file performance of classification of tumor samples andnormal samplesadditional file top gene pairs for each cancer type 0clee bmc medical genomics 13suppl page of abbreviationsauc area under the curve brca breast invasive carcinoma cnv copynumber variation coad colon adenocarcinoma gdac genome dataanalysis center hnsc head and neck squamous cell carcinoma kipan pankidney cohort lihc liver hepatocellular carcinoma loocv leaveoneoutcross validation luad lung adenocarcinoma lusc lung squamous cellcarcinoma mcc matthews correlation coefficient pcc pearson correlationcoefficient ppi proteinprotein interactions roc receiver operatingcharacteristic svm support vector machine tcga the cancer genome atlasacknowledgementsthe authors thank the anonymous reviewers for the constructive commentsthat contributed to improving the final version of the paperabout this supplementthis has been published as part of bmc medical genomics volume supplement selected s from the 15th international symposiumon bioinformatics research and applications isbra19 medical genomicsthe full contents of the supplement are available online at httpsbmcmedgenomicsbiomedcentralcomssupplementsvolume13supplement6authors contributionswl designed and performed all about this study and prepared the initialmanuscript dh helped integrating multiomics data of breast cancer khsupervised the work and wrote the manuscript all authors read and approvedthe final manuscriptauthors informationwook lee is currently working toward his phd degree at the department ofcomputer engineering inha university korea deshuang huang is a directorof the institute of machines learning and systems biology tongji universitychina kyungsook han is a professor at the department of computerengineering inha university koreafundingthis work was supported by the national research foundation of korea nrfgrant funded by the ministry of science and ict nrf2017r1e1a1a03069921and inha university research grant publication of this was funded bythe nrf grant nrf2017r1e1a1a03069921 the funders had no role in thedesign of the study data collection and analysis or writing the manuscriptavailability of data and materialsadditional files are available at httpbclabinhaackrcancernetworkethics approval and consent to participatenot applicableconsent for publicationnot applicablecompeting intereststhe authors declare that they have no competing interestsauthor details1department of computer engineering inha university incheon southkorea 2institute of machine learning and systems biology school ofelectronics and information engineering tongji university shanghaichinareceived may accepted june published august references widakowich c de castro g de azambuja e dinh p awada a reviewside effects of approved molecular targeted therapies in solid cancersoncologist liu s kurzrock r toxicity of targeted therapy implications for responseand impact of genetic polymorphisms cancer treat rev verma m personalized medicine and cancer j personalized medbarabasi al gulbahce n loscalzo j network medicine a networkbasedapproach to hum | Colon_Cancer |
" according to the who most chronic diseases including cancer can be prevented by identifyingtheir risk factors such as unhealthy diet smoking and physical inactivity this research examined the effectiveness ofa theorybased educational intervention on colorectal cancerrelated preventive nutritional behaviors among asample of anizational staffmethods in this interventional study employees of shahid beheshti university of medical sciences wererandomly divided into two groups intervention and control with cluster sampling the data gathering tool was aresearchermade questionnaire containing two parts of 10dimensional information and health belief modelconstructs the educational intervention was conducted for month and in four sessions in the form of classroomlecture pamphlet educational text messages via mobile phones and educational pamphlets through the officeautomation system two groups were evaluated in two stages pretest and posttest data were analyzed usingspss18 software analysis of covariance ancova and independent ttest intergroup comparisonsresults two groups were evaluated for variables such as age sex education level and family history of colorectalcancer and there was no significant difference between the two groups p after the months sinceintervention except for the mean score of perceived barriers which was not significant after intervention the meanscores of knowledge perceived susceptibility perceived severity perceived benefits perceived selfefficacybehavioral intention and preventive behaviors were significantly increased after the intervention in the interventiongroup compared to the control group p implementation of educational intervention based on health belief model was effective for thepersonnel and can enhance the preventative nutritional behaviors related to colorectal cancerkeywords educational intervention health belief model nutritional behavior colorectal cancer correspondence mohtashamghaffarisbmuacir1environmental and occupational hazards control research center schoolof public health and safety shahid beheshti university of medical sciencestehran iranfull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0crakhshanderou bmc medical education page of nearly million new cases of colorectal cancer arediagnosed every year worldwide with nearly half of theaffected patients losing their lives due to the disease approximately of men in and of women in are diagnosed with crc during their life time the incidence of colorectal cancer in iran ranges from to per annually with a death rate of about per hundred thousand and it accounts for approximately of all gastrointestinal cancerrelated deaths according to the latest cancer record in iran colonand rectum cancer ranked third in female cancers andfifth in male cancers the global incidence of crc is predicted to increase by to more than million newcases leading to million cancer deaths by therisk of colon cancer increases with age and is higher inmen than in women various factors are involved inthe development of various types of cancerincludingcolorectal cancer which can be attributed to geneticenvironmental and dietary factors among the riskfactors of colorectal cancer nutritionalfactors areconsidered to be the most important and preventableones so that to of cases can be prevented byproper nutrition [ ] colorectal cancer is also morecommon in iran than in other asian countries [ ]therefore the need to educate people about the nutritionalbehaviors associated with colorectal cancer is becomingmore and more evident theories and models identifyfactors that influence health and behavior which meansthat they can be used to develop programs the most effective training programs are based on the theorydrivenapproaches which are rooted in behaviorchanging modelsalso selecting appropriate model or theory is the first stepin the process of planning a training program [ ] asone of the most widely applied theories of health behaviorthe health belief model hbm posits that six constructspredict health behavior perceived susceptibility perceivedseverity perceived benefits perceived barriers perceivedselfefficacy and cues to action fig the hbmposits that when an individual perceives a serious threatalong with a way to reduce the threat they will be morelikely to take action to reduce the threat the hbmhas been applied to predict a wide variety of healthrelatedbehaviors such as being screened for the early detection ofasymptomatic diseases the model has been applied tounderstand patients responses to symptoms of disease lifestyle behaviors and behaviors related to chronicillnesses which may require longterm behaviormaintenance in addition to initial behavior change the research hypotheses are an intervention based onthe hbm can significantly promote colorectal cancer preventive behaviors the score for each and every constructof the hbm eg perceived awareness and susceptibilityperceived severity perceived benefitsbarriers and perceivedselfefficacy is increased significantly after the interventionin the experimental group as compared to the controlmethodsstudy design and samplingthis interventional study was conducted at shahidbeheshti university of medical sciences tehran iranfrom october to june fig health belief models components and links 0crakhshanderou bmc medical education page of in thisstudy using the sample size formula ¾ z¾2δ2d2 in which δ2 α n ¼ °zd and with an attrition rate of finally women subjects in the experimental and in thecontrol group were considered the random samplingmethod clustering and simple random sampling wasused in this study in order to choose from four facultiesfaculties of shahid beheshti university of medicalsciences four faculties were randomly selected and fromthese four faculties two faculties were assigned as intervention group and were considered as control grouprandom sampling method was used to select samplesfrom each clusterinclusion exclusion criteriabeing under years of age having satisfaction to participate in the study and not having serious diseases including gastrointestinal diseases were the inclusion criteriaalso not willing to continue with the study not completing the questionnaire in full and not attending in morethan two educational sessions were the exclusion criteriameasuresthe researchermade questionnaire was used for datacollection in this study three sources of existed toolsliterature review and expert view were used for itemgeneration this instrument consisted of two main partsas followpart one demographic questions about age gendereducational level and economic statuspart two constructs of the health belief model whichincludes knowledge perceived susceptibility perceivedseverity perceived benefits perceived barriersperceived selfefficacy behavioral intention andbehavior table validity and reliabilityface and content validities were applied for validationphase reliability was confirmed based on methods oftestretest and internal consistency cronbachs alphafor face validity a survey was done on employeesabout the difficulty in understanding the words andphrases the probability of misunderstanding the phrasesand lack of clarity in the meaning of the words somemodifications were made to the tools questions todetermine the content validity of the questionnaire twogastroenterologistsfive health education and healthpromotion specialists and one related expert were askedto complete the questionnaire the initial questionnairehad questions theconstructs of knowledgeperceived susceptibility perceived severity perceivedbenefits perceived barriers perceived selfefficacyintention and behavior had and questions respectively internal consistency was used todetermine the reliability of hbm structures the cronbachs alpha coefficient was for all structures andwas statistically acceptable the retest was used to ensure the reliability of the awareness variable in this way employees completed the questionnaire twice and theicc was obtained also construct validity wasperformed by exploratory analysis method the kmovalue was and bartletts research showed thetable description of study instrumentconstruct knowledge refers to a theoretical or practical understanding of asubject perceived susceptibility refers to subjective assessment of risk ofdeveloping a health problem perceived severity perceived severity refers to the subjectiveassessment of severity of a health problem and its potentialconsequences perceived benefits healthrelated behaviors are also influenced bythe perceived benefits of taking an actionno of items format items truefalsedont know items 5point likert scalestrongly disagree to stronglyagree items5point likert scalestrongly disagree to stronglyagree items5point likert scalestrongly disagree to stronglyagreescoring rangecorrect response dont knowresponse incorrect response strongly disagree disagree noidea agree strongly agree strongly disagree disagree noidea agree strongly agree strongly disagree disagree noidea agree strongly agree perceived barriers healthrelated behaviors are also a function ofperceived barriers to taking action perceived selfefficacy refers to an individuals perception of his orher competence to successfully perform a behavior behavioral intention refers to a persons perceived probability orsubjective probability that he or she will engage in a given behavior items5 point likert scalestrongly disagree strongly agree items5 point likert scalestrongly disagree strongly agree items5point likert scalestrongly disagree to stronglyagreestrongly disagree disagree noidea agree strongly agree strongly disagree disagree noidea agree strongly agree strongly disagree disagree noidea agree strongly agree behavior refers preventative behaviors associated with colorectalcancer items5point likert scalealways to neveralways often sometimes rarely never 0crakhshanderou bmc medical education page of significant correlations among the items Ï2 df p therefore the data were suitable forconducting factor analysisinterventionboth intervention and control groups were pretestedusing the questionnaire the analysis of educational needsdetermined the educational methods educational package and the number of educational sessions was obtainedby the pretestreadabilitycomprehensibility and not complexity of educational contents for participants was obtained by pretesting materialssuch as pamphlets messages etc in a sample of employees who were not included in main researchresults assurance abouteducational intervention based on educational textmassagesover the course of days ten text messages were sentto the employees in the intervention group at am mostof which had been prepared according to the educationalobjectives ofthe constructs of knowledge perceivedsusceptibility perceived benefits perceived barriers andperceived selfefficacycounseling there waseducational pamphletstwo pamphlets were given to the employees during twoseparate sessions along with simultaneous provision ofindividuala possibility ofquestioning and answering any ambiguity regarding thecontent of pamphlets the first pamphlet containedsections on the signs and symptoms of colorectal cancerand the risk factors of this cancer and the secondpamphlet contained sections on methods of preventingthis cancereducational packages in the office automation systemeducational packages were uploaded on the staff automation system for days and the employees were askedto study it during the working hoursthe intervention was conducted month and followup months after the intervention the educationalcontents were taken from the trusted sources of theministry of health complemented by what the staffneeded to know about promoting nutritional behaviorsrelated to the prevention of colorectal cancer the education varied in form across the model constructs forperceived susceptibility the facts and figures of the incident rate of colorectal cancer were presented in theclass and for perceived severityimages of colorectalcancer problems were used also for perceived barrierseducational materials were used to somehow incite theindividuals to analyze the cost of optimal behavioragainst the costs of risks time etc involved in unhealthybehavior the educational content used for perceivedbenefits intended to raise awareness on the usefulness ofhealth promoting behaviors to reduce the risk of illnessor to understand the benefits of healthy behaviors infig the research process is presented in generalethical considerationsat first a permission was obtained from the universityto conduct the study and attend the healthcare centerthe samples were assured about the confidentiality oftheir specifications and information they were also toldthat their information will only be used for the purposeof this study and the data collection the participantswere allowed to enter and leave the study at any timesuitable conditions were provided for a proper understanding of questions and responses for the subjectsafter the end of the intervention period the controlgroup was also trained using the slides that were used totrain the intervention group an informed consent wasobtained from the participants the study on whichthese data analyses are based was approved by theethical board committee of shahid beheshti universityof medical sciencesdata analysisdata were analyzed by spss software kolmogorov smirnovtest was used to check the normality of the data to assessthe effectiveness of intervention on variables of knowledgeperceived susceptibility perceived severity perceived benefits perceived barriers perceived selfefficacy behavioralintention and behavior in the intervention and controlgroups two groups were evaluated in two stages pretestand posttest data were analyzed using spss18 softwareanalysis of covariance ancova and independent ttestintergroup comparisonsthe confidence level of and the significance level of were consideredin this studyresultsthe findings of this study showed no drop out until theend of study the questionnaire was completed in bothgroups in a complete and precise manner homogenizationwas done in the two groups by controlling variables such asage sex level of education and related family history theresults showed no significant relationship within thesevariables p table effectiveness of the educationalintervention in improving knowledge perceived susceptibility perceivedseverity perceived benefits perceived selfefficacybehavioral intention and behavior once age gender andlevel of education factors were adjusted was checkedthrough ancova the results revealed that the intervention was successful in improving constructs of thehealth belief model significantly in participants table the mean score ofintention and behavior in the 0crakhshanderou bmc medical education page of fig schematic diagram of designed interventions for colorectal cancer preventionexperimental and control groups before and after theintervention is presented in fig discussionthe purpose of this study was to investigate the effectsof educationalinterventions on the promotion ofcolorectal cancer prevention nutritional behaviors thekmo and bartletts test p results confirmed the suitability of the model for conducting factoranalysis the kmo is in the range if the value ofthe inedex is near to one the data are suitable for factoranalysis kaiser at least kmo to determinestable demographic and variables in intervention and control groups before the interventionvariablegroupintervention group n n control group n n agegenderlevel of educationfemalemalediplomaassociate degreeundergraduate degreeand higherhistory of specialdiet compliancefamily history of cancerchisquareyesnoyesnop value 0crakhshanderou bmc medical education page of table comparison of intervention and control groups in terms of health belief model constructs before and after the interventionp valueconstructsgroupsbefore interventionmean ± sd ± after interventionmean ± sd ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± meandifference ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± knowledgeinterventioncontrolperceived susceptibilityinterventionperceived severityperceived benefitsperceived barriersperceived self efficacybehavioral intentionbehavioranalysis of covariance ancovacontrolinterventioncontrolinterventioncontrolinterventioncontrolinterventioncontrolinterventioncontrolinterventioncontrol also bartlett test was used to confirm adequacy ofthe samples in the present study the mean score of behavioralconstruct increased after the intervention in the intervention group and there was significant differencebetween the two groups after the intervention in thisregard the results of this study are consistent with thefindings of abood hart roozitalabi alidoosti and davoodi studies behavioral intention is the thought of doing abehavior and is considered as the immediate determinant of that behavior the mean score in this construct aswell increased in the intervention group after the intervention and there was significant difference between thetwo groups after the intervention in the study of braun and gimeno the results were similar tothe results of present study selfefficacy is a keyprerequisite for behavior change there was significantdifference between mean score of perceived selfefficacyconstruct in the two groups after the intervention in thisfig mean scores of intention and behavior in the experimental and control groups before and after the intervention 0crakhshanderou bmc medical education page of regard the results of the study by braun alidoosti and hart are consistent with thisfinding perceived selfefficacy is considered as a strongmotivational source and in fact is an indicator of theability of individuals to anize themselves in pursuit ofcertain goals studies show that individuals with ahigh level of perceived selfefficacy have a greatercommitmentto engage in activities at a time ofchallenges and difficulties and spent more time andeffort on such activities such individuals are morelikely to contribute to maintaining healthy behaviors andretrieve them even after failure and they have strongerintention and motivation this not only improves thetarget adjustment but also ensures achievement andsustainability in pursuit of the goals another important factor is knowledge that can be pointed to itsrole in healthy behaviors this study showed a significant difference in the two group in terms of the meanscore of knowledge after the educationalinterventionthese results are consistent with the findings of roozitalab ho and gimeno studiesalso there was no significant difference in the controlgroup before and afterthe intervention althoughincreasing knowledge is an important step in changingattitudes and behaviors it is not a major contributor tocrc prevention achieving the intention to behave isinfluenced by individual and environmental factors so inaddition to enhancing individual aspects overcomingthe structural and environmental barriers of the healthsystem regarding the use of cancer prevention nutritional behaviors is also vital in the present study themean score of perceived susceptibility and perceived severity constructs showed a significant difference betweenthe intervention and control group after the educationalintervention studies by kolutek wang cengiz and donadiki reportedthe role of beliefs regarding public health threats perceived susceptibility and perceived severity in the healthpromotion behaviors becker believed that onesintention to selfcare is influenced by his or her perception of vulnerability and the severity of disease outcomes therefore the need for interventions to increasethe perception of society about the irreparable complications of diseases caused by unhealthy behaviors malnutrition habits seems necessary in this study there was asignificant difference between the two groups in terms ofthe constructs of perceived benefits after the educationalintervention this result is consistent with the findings ofgrace alidoosti and abood studies also in the present study the mean score of perceived barrier construct decreased after the interventionthis was a good result but it was not statistically significant in the present study the mean score of perceivedbarrier construct decreased after the intervention which isnot consistent with the results of studies by moatari grace and gimeno the study ofrajabi identified some of the most important causes of barriers to nutrition in preventionof cancer such as the difficulty of preventativemeasuresinappropriate economic status and fear ofcancer information therefore strategies that overcome the individual and environmental barriers thataffect nutritional behaviors should be addressed byplanners and policymakerslimitationsthe limitations of this study which could have had a relative effect on its findings include the short duration ofintervention the sample size the inability to follow thelong term effect of the intervention and the selfreportingof the subjects in responding to questions however theuse of this method in such studies is inevitable and maylead to a bias of the researcherdesired report in thisstudy anonymous questionnaire was used to minimizethis biasthe findings of this study confirmed the effectiveness ofhealth belief modelbased education in improvement ofcolorectal cancerrelated preventive behaviors on theother hands interventions based on hbm concepts couldpromote nutritional behaviors related to colorectal cancerprevention consequently offering educational programsincluding public information campaigns workshopsvideos websites exhibitions etc should be used to informpeople about crc symptoms and risk factors alsomodelbased education will have a greater effect on nutritional behaviors improvement by focusing on perceptionsand enhancing beliefs aboutthe applicability oftheprogram and understanding the benefits and barriersabbreviationscrc colorectal cancer hbm health belief modelacknowledgementsthis is a part of an msc dissertation in health education approved by theshahid beheshti university of medical sciences the authors of this paperwould like to express their gratitude and appreciation to all the contributorswho have somehow collaborated on the design guidance andimplementation of this projectauthors contributionsmgh sr as and mm designed the study mm and mgh wrote the firstdraft sr and asm conducted the analyses all authors contributed towriting revising and approved the final manuscriptfundingthis study is sponsored by shahid beheshti university of medical sciences intehran the funding agencies had no role in the design of study datacollection and analysis or presentation of the resultsavailability of data and materialsthe datasets used and analyzed during the current study are available fromthe corresponding author on reasonable request 0crakhshanderou bmc medical education page of ethics approval and consent to participatethe study on which these data analyses are based was approved by theethical board committee of shahid beheshti university of medical sciencesparticipants were provided information about the study and verbalconsented by proceeding to take the survey this implied verbal consent wasapproved by the ethical board committee of shahid beheshti university ofmedical sciencesconsent for publicationnot applicablecompeting intereststhe authors have no conflict of interestsauthor details1environmental and occupational hazards control research center schoolof public health and safety shahid beheshti university of medical sciencestehran iran 2school of public health and safety shahid beheshti universityof medical sciences tehran iranreceived december accepted august screening in general practice in central england j epidemiol communityhealth roozitalab m moatari m gholamzadeh s saberifiroozi m zare n the effectof health belief on participation of the official administrative personnel incolorectal cancer screening programs in shiraz university of medicalsciences govaresh alidosti m sharifirad g hemate z delaram m najimi a tavassoli e theeffect of education based on health belief model of nutritional behaviorsassociated with gastric cancer in housewives of isfahan city daneshvarmed davodi a anoosheh m memarian r the effect of selfcare education onquality of life in patients with esophageal cancer following esophagectomyzums j braun kl fong m kaanoi me kamaka ml gotay cc testing a culturallyappropriate theorybased intervention to improve colorectal cancerscreening among native hawaiians prev med gimenogarca az quintero e nicol¡sp©rez d parrablanco a jim©nezsosa a impact of an educational videobased strategy on the behaviorprocess associated with colorectal cancer screening a randomizedcontrolled study cancer epidemiol bandura a social cognitive theory handbook of social psychologicaltheories london sage bandura a social cognitive theory an agentic perspective annu revpsychol luszczynska a guti©rrezdo±a b schwarzer r general selfefficacy invarious domains of human functioning evidence from five countries int jpsychol ho tv effects of an educational intervention on breast cancer screeningand early detection in vietnamese american women oncol nurs forumkolutek r avci ia sevig u the effects of scheduled observation at homeon health beliefs related to breast and cervical cancer screening andattitudes of married women eur j oncol nurs 201418s25 wang wl hsu sd wang jh huang lc hsu wl survey of breast cancermammography screening behaviors in eastern taiwan based on a healthbelief model kaohsiung j med sci cengiz b bahar z use of the health belief model in screening methodsfor colorectal cancer eur j oncol nurs 201418s27 donadiki e jim©nezgarca r hern¡ndezbarrera v sourtzi p carrascogarrido p de andr©s al jimeneztrujillo i velonakis e health belief modelapplied to noncompliance with hpv vaccine among female universitystudents public health becker mh drachman rh kirscht jp a new approach to explaining sickrole behavior in lowincome populations am j public health ma gx shive s tan y gao w rhee j park m kim j toubbeh jicommunitybased colorectal cancer intervention in underserved koreanamericans cancer epidemiol moatari m roozitalab m saber f zare m gholamzadeh s effect ofeducation on health beliefs on knowledge and participation j res med rajabi r sharifi a shamsi m almasi a dejam s investigating the effectof package theorybased training in the prevention of gastrointestinal cancers publishers notespringer nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliationsreferencesarnold m sierra ms laversanne m soerjomataram i jemal a bray f globalpatterns and trends in colorectal cancer incidence and mortality gut american cancer society colorectal cancer facts and figures available at httpswwwcancercontentdamcancerresearchcancerfactsandstatisticscolorectalcancerfactsandfigures 20172019pdf[accessed ]ansari r amjadi h norozbeigi n zamani f mirnasseri s khaleghnejad amalekzadeh r survival analysis of colorectal cancer in patients underwentsurgical operation in shariati and mehr hospitaltehran in a retrospectivestudy govaresh centers for disease control and prevention cdc colorectal cancer risk byage available at httpwwwcdcgovcancercolorectalstatisticsagehtm[accessed apr ] malekzadeh r bishehsari f mahdavinia m ansari r epidemiology andmolecular genetics of colorectal cancer in iran a review kz aa saadat a jalalian hr esmaeili m epidemiology and survival analysisof colorectal cancer and its related factors trauma monthly winter239ghaffari m mehrabi y rakhshanderou s safarimoradabadi a jafarian szeffectiveness of a health intervention based on who food safety manual iniran bmc public health hosseini sv izadpanah a yarmohammadi h epidemiological changes incolorectal cancer in shiraz iran anz j surg yazdizadeh b jarrahi a mortazavi h mohagheghi ma tahmasebi s nahvijoa time trends in the occurrence of major gi cancers in iran asian pac jcancer prev glanz k rimer bk viswanath k health behavior and health educationtheory research and practice john wiley sons ghaffari m rakhshanderou s safarimoradabadi a torabi s oral and dentalhealth care during pregnancy evaluating a theorydriven intervention oraldis becker mh the health belief model and sick role behavior health educmonogr janz n champion v strecher vj the health belief model k glanz bk rimerjanz nk becker mh the health belief model a decade later health educ qlp o review of translation and cultural adaptation process ofquestionnaires kellar sp kelvin ea munro's statistical methods for health care researchwolters kluwer healthlippincott williams wilkins abood da black dr feral d nutrition education worksite intervention foruniversity staff application of the health belief model j nutr educ behav hart ar barone tl gay sp inglis a griffin l tallon ca mayberry jf theeffect on compliance of a health education leaflet in colorectal cancer 0c" | Colon_Cancer |
" the incidence of thyroid carcinoma is increasing all over the world some studies have suggestedthat the change of adipokines expression can induce thyroid carcinoma however other studies have come to theopposite therefore we studied the relationship between adipokines and thyroid carcinomamethods databasespubmed cochrane library sinomed cnki wanfang and clinical trial registries weresearched a metaanalysis was then performed through a fixed or randomeffects model to calculate i values forheterogeneity analysisresults twentynine s were finally included for analysis the level of serum tumor necrosis factoralpha tnfα [standardized mean difference smd confidence interval ci to i2 p ]and the ratio of tnfα immunoreactivity in tissues [odds ratios or ci to i2 p ]in thyroid carcinoma are significantly higher than those in control the serum interleukin6 il6 in patients withthyroid carcinoma is higher than that in control smd ci to i2 p there is nosignificant difference of the ratio of il6 immunoreactivity in tissues between carcinoma and control or ci to i2 p the ratio of leptin immunoreactivity in tissues is significantly associated with therisk of thyroid carcinoma or ci to i2 p however after analyzing theexpression level of serum adiponectin in three studies no significant difference is found between thyroidcarcinoma and the control p s adipokines tnfα il6 and leptin show a strong relationship between elevated concentrations inserum andor tissue and thyroid carcinoma however the association between adiponectin and thyroid carcinomaneeds further researchkeywords thyroid carcinoma adipokines tnfα il6 leptin metaanalysis correspondence liaolinsdueducn cwc_llsdueducn junyu zhao and jing wen contributed equally to this work1department of endocrinology and metabology the first affiliated hospitalof shandong first medical university shandong provincial qianfoshanhospital jinan china5department of endocrinology and metabology qilu hospital of shandonguniversity cheeloo college of medicine shandong university jinan chinafull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0czhao bmc cancer page of thyroid carcinoma is the most common endocrine malignancy but mostly has good prognosis during the pastdecades a rising incidence of thyroid carcinoma worldwide has aroused the widespread attention of researchers[ ] someone supposed that the growing use of diagnostic imaging and fineneedle aspiration biopsy may bethe main reason but this may be only partial andcan not totally explain the increased incidence of microcarcinoma changes in the incidence of a cancer are notonly associated with increased detection and other unknown risk factors need further explore recently somescientists found that the incidence of thyroid carcinomahas increased along with a marked rise in obesity rateand accumulating evidence of an association betweenobesity and increased thyroid carcinoma risk has beenproposed [] various hypotheses have been supposedto interpret the relaitonship between obesity and thyroidcarcinoma including hyperinsulinemia upregulation ofaromatase activity chronic low grade inflammation altered immune response and dna damage caused byoxidative stress furthermore recent data supportingthe notion that a changed expression of adipokinescaused by obesity can affect the cell proliferation andeven induce a thyroid tumorigenesis [] adipose tissue is a specialized connective tissue composed of fatcells which releases a number of biologically active molecules called adipokines or adipocytokinesincludingleptin adiponectin resistin and many cytokines of theimmune system such as tumor necrosis factoralphatnfα interleukin6 il6 and complement factor dalso known as adipsin adipokines refer to various enzymes hormones cytokines growth factors proteinsand other biological active substances secreted by adipocytes including adiponectin leptin resistin and interleukin the concentration of adipokines such as tnfαil6 and leptin were significantly higher in obese subjects and the elevated levels was linked to obesity andeven positively correlated with body mass index []it is reported that adipokines took part in the biologicalprocesses of insulin sensitivity inflammation and proliferation [ ] which the proliferation have been recognizedthetumorigenesis and development at present many kindsof adipokines have been reported to be associated withthyroid carcinoma rehem ra suggested thatserum leptin levels were higher in welldeffierentiatedthyroid carcinoma patients and a significant drop aftersurgery another envidence showed that adiponectin related with tumor size however the opposite resultswere also found in other studies some researchesreported the expression of adipokines is lower in tumortissue than normal control [] it is clearly that certain confounders such as age sex ethnicity and alsoimportantfactorleadingtoasanheterogeneity in study size methodology and original ofsample should be considered when trying to analyze theassociation between adipokines and thyroid carcinomathese confunding factors above may be the cause of inconsistency results from different researches additionaly the association between adipokines and thyroidcarcinoma are still not well documented therfore theaim of this metaanalysis was to investigate the association between adipokines and thyroid carcinoma andpropose that adipokine as a risk factor for thyroidcarcinomamethodssearching progresswe conducted a search of all studies published until27th july regarding the association between adipokine and thyroid carcinoma eligible casecontrol studieswere found by searching the database of pubmedcochrane library sinomed cnki and wanfang and restricted to published results clinical trial register centers httpwwwclinicaltrialsgov were also searchedthe following search terms adipokine or leptin oradiponectin or resistin or tumor necrosis factoralpha or interleukin6 or complement factor d oradipocytokines or tumor necrosis factorα or tnfα or il6 or adipsin and thyroid cancer or thyroid neoplasm or thyroid tumor or thyroid carcinoma or differentiated thyroid carcinoma or dtc orpapillary thyroid carcinoma or thyroid carcinomapapillary or ptc or thyroid cancer follicular orftc or thyroid carcinoma anaplastic or atc orthyroid cancer medullary or mtc hand searchingwas used to identify appropriate studies including reference lists of eligible s and related previous reviews eligible studies met the following criteria published in english or chinese language studyassessed the association between adipokine and thyroidcarcinoma study designed as the casecontrol study study reported the expression of at least one adipokine either in blood or tissue studies were excluded ifany of the followings were identified insufficient information concerning adipokine or thyroid carcinomaoutcome cannot directly extract or calculate or and95ci the type of study was not a casecontrol designhave not fulltext animal trialsstudy selection and data extractiontwo reviewers screened the studies and extracted dataindependently any disagreement was resolved by discussion or consensus with a third senior reviewer dataincluded the followingfirst author publication yearcountry participant characteristics ie mean age sample size sex ration pathological type of thyroid carcinoma source of controls measured outcomes or the 0czhao bmc cancer page of scores were considered to be of high quality disagreements were resolved by reevaluating and discussing between two reviewersinsearchingthis metaanalysisresultssearch results and characteristics of included studies s regarding the association between adipokine and thyroid carcinoma were searched in therelated database and clinicaltrial websites afterscreening the title and abstracts s were selected for fulltext review finally studies were eligibleprogressincluded and excluded details are all shown in fig eighteen of these studies are published in chinese[ ] and the rest are published in english[] nineteen studies were conducted in chinatwo in india and two in turkey brazil greece iranitaly denmark and serbia each had one study totally there are patients with thyroid carcinomain the case group and controls including healthysubjects patients with benign thyroid diseases or normal thyroid tissue near carcinoma were included inthe control group the sample size ranges from to in the case group while to in the controlgroup all the thyroid carcinoma patients were confirmed by pathologically among these studiesfourteen studies reported papillary thyroid carcinomaptc eight studies reported differentiated thyroidcarcinoma dtcreported differentpathological types in one paper one study reportedmedullary thyroid carcinoma mtc and the restfour studies did not show the pathological detailsthe detailed characteristics ofincluded studies aresummarized in table three studiespercentage of samples show immunoreactivity for adipokines antibody both in the case and control groups thecalculation method is shown below take thyroid cancerfor example the number of samples obtained from thyroid carcinoma that show immunoreactivity for adipokines antibody divided by the total number of thyroidcarcinoma samplesstatistical analysisfor metaanalysis dichotomous outcomes were analyzedby using the odds ratios or computed using the mantelhaenszel method fixed or random models continuousvariables measured on the same scale expressed as a meanvalue and standard deviation were analyzed by usingweighted mean differences wmd otherwise standardized mean difference smd were used for different scaleall results were reported with confidence interval ci i2 was used to assess heterogeneity between studies and i2 values of and representing no lowmoderate and high heterogeneity respectively visual inspection of the funnel plot was done to assess publicationbias the analyses were performed by review manager cochrane collaboration united kingdom httpwwwcochranequality assessment and risk of biasthe methodological quality of casecontrol study wasassessed by the newcastleottawa scale nos supplement table which consists of the three parameterseight questions with nine possible scores selection exposure and comparability a study can be awarded amaximum of one score for each numbered item withinthe selection an exposure categories a maximum oftwo scores can be got for comparability a higher scoremeans better quality in methodology and five or morefig flow chart of the systematic search process 0czhao bmc cancer page of zhao jianqiang chinaptc ftc atcand mtcthyroid adenoma andnormal healthunknownunknowntable characteristic of included studiesfirst authoryearcountrypathologicaltype of thyroidcancersource of controlsl kayser denmark ptc and ftccao guangyao chinaunknownmtrovato italydtc andundifferentiatedcarcinomamultinodular goitersadenomas hashimotosthyroiditis hyperplasticglandsthyroid adenoma andnodular goiternormal thyroid tissues andbenign nodulesmelih akinci wang jingxia zhuangxiaoming yu xiao hou sen snezanazivancevicsimonovic xu xiaocheng xeniprovatopoulou turkeyptchealthy volunteerschinaptc and ftcnormal thyroid tissueschinaptc ftc andmtcthyroid adenoma andnormal healthchinaptcthyroid adenoma andnormal thyroid tissue nearcarcinomachinaptcthyroid adenomaserbiawdtchealthy subjectschinathyroidcarcinomagreeceptcthyroid adenomabenign thyroid disease andhealthy controlssun qinnuan chinaptcnormal thyroid tissue nearcarcinoma and healthycontrolschinaptcthyroid adenomachinaptcthyroid adenomamean age yearfemale outcome indexnumber ofparticipants ncases control casesunknowncontrolcontrolcasesunknownunknownunknownunknownunknowntnfα tissuetnfα tissueil6 tissueil6ãtnfαblood ± ±unknown leptinblood unknown tnfα tissue unknown il6ãtnfαunknownunknownbloodleptintissueunknown unknown leptin ± ± tissuetnfαblood ± ± ± ± ± ± ± il6blood il6blood tnfαbloodandtissue ±unknown unknown leptinunknownunknowntissueadiponectintissueunknown unknown adiponectintissue il6ãtnfαblood ± ± zhang zijie zhong xiuxiu zhang bo hu jinhua snezanazivancevicsimonovic yanlan fan chinadtcchinadtcnormal thyroid tissue nearcarcinomathyroid adenoma andhealthy controls ±serbiaptccontrol subjectsunknownunknownil6bloodchinathyroidcarcinomanodular goitre hashimotosthyroiditis follicular adenomaand adjacent nonneoplasticthyroid tissue samplesunknownunknownleptintissue 0czhao bmc cancer page of table characteristic of included studies continuedfirst authorsource of controlsyearcountrypathologicaltype of thyroidcancerchinathyroidcarcinomabenign thyroid disease andnormal thyroid tissue nearbenign thyroid diseasechinaptcthyroid adenomaturkeyptchealthy volunteersindiaptcindiaptcbenign thyroid diseases andhealthy individualsbenign thyroid diseases andhealthy individualsnumber ofparticipants ncases control cases ±mean age yearfemale outcome indexcontrol ±casescontrol tnfαtissue ± ±tnfα tissue il6bloodunknown unknown tnfαbloodunknown unknown il6bloodwangxinzheng song runbo kemal beksac toral pkobawala toral pkobawala raziyehabooshahab zhang bo zhouxiaodong ma xiaokai marianabonjiornomartins iranmtchealthy subjects ± ± leptinãadiponectinbloodchinadtcnormal thyroid tissue nearcarcinomaunknown unknown leptintissuechinadtchealthy subjects ± ±il6ãtnfαbloodchinaptcthyroid adenomaunknown unknown leptinbrazildtcbenign thyroid nodules andhealthy controls ±tissue il6blood ± ±chinail6 sun zhenhua tissuetnfα tumor necrosis factora dtc differentiated thyroid carcinoma il6 interleukin6 ptc papillary thyroid carcinoma ftc follicular thyroid carcinoma atcanaplastic thyroid carcinoma mtc medullary thyroid carcinoma wdtc welldifferentiated thyroid carcinoma fnac fine needle aspiration cytologynodular goiterptcquality of included studiesthe quality assessment of these studies is assessed bythe nos and the resultis shown in supplementaltable five or more scores are determined as highquality two studies conducted by cao g in and l kayser in only get two scoresshowing a poor quality in methodology the rest studies are assessed as high qualitytnfα and thyroid carcinomatwelve studies reported the expression of tnfα bothin patients with thyroid carcinoma and control subjects[ ] among these sevenstudies [ ] had tested the level ofserum tnfα two studies [ ] had tested the expression of tnfα in tissues and the ratio of tnfα immunoreactivity was tested in four studies [ ] firstly fixedeffect model is used to merge the smdvalues of serum tnfα level however a large heterogeneity is found by the heterogeneity analysis heterogeneity test chi2 p i2 and itmay be due to the different units differenttestingmethods in different researches or other unknown factors then randomeffect model to merge the smd isused and pooled effect size in favor of control group is ci to p fig 2a smdvalues of the expression of tnfα in tissues is mergedby fixedeffected model and the heterogeneity analysisshow a considerable heterogeneity heterogeneity testchi2 p i2 the different unitsand limited numbers of research may be the original ofheterogeneity so the pooled smd with randomeffectmodel of the expression of tnfα in tissues is ci to p fig 2b the pooled orwith fixedeffect model of the ratio of tnfα immunoreactivity in thyroid carcinoma tissues is ci to p however a significant heterogeneity is detected heterogeneity test chi2 p i2 the published by l kayser in with a poor quality in methodology may attributeto this high heterogeneity then randomeffect model ofpooled or is used and pooled effect size in favor of 0czhao bmc cancer page of fig forest plot of the tnfα level and the ratio of tnfα immunoreactivity in tissues in patients with thyroid carcinoma a level of serum tnfα b expression of tnfα in tissue c ratio of tnfα immunoreactivity in tissuecontrol group is ci to p fig 2c in level of serum tnfα and theratio of tnfα immunoreactivity in tissues of thyroidcarcinoma patients are significantly higher than controlsubjects which are without thyroid carcinomail6 and thyroid carcinomaamong the included studies reported the level ofserum il6 in patients with thyroid carcinoma and control subjects [ ] due to thelarge heterogeneity of the merged smd values of serumil6 level by the heterogeneity analysis heterogeneitytest chi2 p i2 randomeffectmodel was used to pooled the smd values and thepooled effect size in favor of control subjects is ci to p fig 3a which meansthat patients with thyroid carcinoma have a significantlyhigher level of serum il6 than control subjects twostudies reported the ratio of il6 immunoreactivity bothin thyroid carcinoma tissue and noncarcinoma tissue[ ] the pooled or of the limited two studies donot show an increased ratio of il6 immunoreactivity inthyroid carcinoma tissues or ci to p and a large heterogeneity always existsheterogeneity test chi2 p i2 fig3b thus the level of serum il6 is higher in patientswith thyroid carcinoma however it needs more clinicaldata to verify the relationship between the expression ofil6 and thyroid carcinoma tissueleptin and thyroid carcinomatwo studies reported the level of serum leptin [ ]and another five studies reported the ratio of leptin immunoreactivity in tissues [ ] because ofthe considerable heterogeneity of the pooled wmd ofserum leptin level heterogeneity test chi2 p i2 and pooled or of the ratio of leptinimmunoreactivity in tissues heterogeneity test chi2 p i2 by the heterogeneity analysis with fixedeffect model randomeffect model is further used to merge the values and analysis howeverthere is no association of higher level of serum leptin 0czhao bmc cancer page of fig forest plot of the il6 level and ratio of il6 immunoreactivity in tissue in patients with thyroid carcinoma a level of serum il6 b ratio ofil6 immunoreactivity in tissuefig forest plot of the leptin level and ratio of leptin immunoreactivity in tissuein patients with thyroid carcinoma a level of serum leptin bratio of leptin immunoreactivity in tissue 0czhao bmc cancer page of with risk of thyroid carcinoma wmd 95ci to fig 4a moreover the pooled or of theratio ofleptin immunoreactivity in tissues from fivestudies is 95ci to fig 4b whichmeans a high ratio of leptin immunoreactivity in tissueis significantly related to thyroid carcinomaadiponectin and thyroid carcinomathree studies reported the expression of adiponectin inthyroid carcinoma including serum and tissue [ ] and the result is summarized in table it could befound that the level of serum adiponectin is not staticallydifferent comparing thyroid carcinoma patients withcontrol subjects p interestinglyit was foundthat the expression of adiponectin in thyroid carcinomatissue is significantly lower than control tissue while theopposite result is found when comparing the ratio ofadiponectin immunoreactivity however there was onlyone study for each result and this may be the reasonwhy the two results are diametrically opposed thus itneeds more clinical studies to confirm in the futurepublication biasthe funnel plot was applied for assessing publicationbias of studies included in the three results includingtnfα fig 5a il6 fig 5b and leptin fig 5c infig 5a and fig 5b almost all studies lies inside the95cis with an even distribution around the verticalindicating no evident publication bias was obtainedthrough the visual distribution of funnel plot howevera potential publication bias was found in fig 5c whencomparing the ratio of leptin immunoreactivity in tissues and that might influence the result of this metaanalysisdiscussioncurrently obesity affects one third of population amongus adults and china has become a big country ofobesity with the incidence ranking first worldwide in theyear of nowadays increasing clinical and experimental studies and documented the closely relationship between malignancies including colon esophaguskidney liver breast endometrium pancreas and prostate as well as nonhodgkins lymphoma and multiplemyeloma and obesityoverweight which affect its occurrence development and prognosis [] becauseof the increasing incidence of thyroid carcinoma duringthe past decades lots of scientists focus on studying therisk factors of thyroid carcinoma it was found that theincidence of thyroid carcinoma has increased along witha marked rising rate of obesity [] furthermore obesity is an independent risk factor for thyroid carcinoma increased insulin resistance elevated serum cholesterol level and upregulated cox2 expression may be thetarget of the correlation between obesity and thyroidcarcinoma it is reported that people with higherbody mass index have a higher concentration of adipokines [] adipokines take part in the followingpathological and physiological processes such as insulinsensitivity inflammation and proliferation [ ] andthese are important in the process of tumorigenesis anddeveloping so adipokines may be one of the targetslinking obesity with thyroid cancer the metaanalysiswas based on previous published studies in previousstudies the analysis of adiponectin and thyroid cancermostly focused on tnf il6 leptin and adiponectinwhile few studies focused on other molecules includingil1 and il8 and we failed to combine statisticstherefore in this metaanalysis only tnf il6 leptinand adiponectin which are the most published adiponectin were analyzedtnfα produced by adipose tissue and inflammatorycells can lead to inflammatory response necrocytosisand assist other cytokines to kill tumor cells and improve the antitumor ability meanwhile tnfα plays animportant role in the process of inflammation insulinresistance diabetes and obesity a moderate amount oftnfα has a protective effect while an excessive amountwill cause damage which may lead to a resistant oftumor cells to tnfassociated apoptosisinduced ligandswhen the microenvironment of apoptosis is maladjustedtnfα has the ability to promote the production ofgranulocytecolony stimulating factor by thyroid fibroblasts which may play an important role in thyroidcancer moreover tnfα can stimulate the vasoactivemediators such as interleukin and prostaglandin and these mediators can promote the proliferation oftumor cells and significantly reduce the immune function tnfα can also induce an increased expression ofvascular endothelial growth factor vegf the laterof that can promote the proliferation of tumor cells andprovide conditions for tumors metastasistable summary of adiponectin expression in thyroid carcinomaserum adiponectin ratio of adiponectin immunoreactivity effect sizewmd or adiponectin in tissue ci confidence interval wmd weighted mean differences or odds ratioswmd 95ci pi2not applicable 0czhao bmc cancer page of fig funnel plots of a tnfα b il6 and c leptin revealed no significant publication bias se smd standard error of standardizedmean differencein surprisingly the results of clinical studies provide evidence for basic research simonovic sz evaluated cytokine profiles determined in supernatants obtained from whole blood cultures in patients with dtc before and days after radioactiveiodine 131itherapy and control subjects andfound that the expression of tnfα in dtc patients ishigher than control subjects and it showed a decreasedlevel after 131i therapy than those before therapy however no statistical difference found for the limited sample size another study conducted by kobawala tp with more patients patients with benign thyroiddisease ptc patients and healthy individuals determined the circulating levels of tnfα and it wasfound that the serum level of tnfα was significantlyhigher in ptc patients than benign thyroid disease patients and the later was also significantly higher thanhealthy individuals furthermore serum tnfα was reported to be a significant prognosticator for overall survival in ptc patients it is a pity thatopposite result wasreported in a casecontrol study that included dtccases and matched cancerfree cohort participantswhich found that tnfa was not associated with thyroidrisk in either gender based on current evidence our metaanalysis suggeststhat tnfα exhibit a strong association with thyroid carcinoma it may because that elevated tnfα may involved in the tumorigenesis and development of thyroidcancer another possible reason is that the tnfα decreased with tumor cells less resulted the activation ofthe immune system by thyroid carcinomathereforemore clincal studies and basic reseaches should be conducted in the futureil6 a multifunctional cytokine plays important rolesin different types of cells including tumor cells it is reported that elevated serum il6 level is closely related tothe tumorigenesis and development of a variety of tumors a strong positive association between theserum il6 and the progression and poor prognosis oftumors in patients with several types of tumor wasalready found [] serum il6 level in thyroid cancer has been evaluated in numerous studies including 0czhao bmc cancer page of in vivo and in vitro studies provatopoulou x found that serum il6 were significantly higher in malignant and benign thyroid diseases compared to healthycontrols however other studies show a different resultthat no significance different of il6 was found betweenthyroid cancer and nonthyroid cancer [ ] a limited sample size different inclusion criteriadifferent population characteristics or different pathological type of thyroid cancer may explain such a difference for in vitro research il6 was also found to beexpressed in thyroid cancer cell lines and a potential roleof il6 in ptc was confirmed indirectly the underlying mechanism may be the followingsbelow tumor cells including esophageal cancerlungcancer colorectal cancer and melanoma were foundhave the function of autocrine il6 which can affect thegrowth and proliferation of tumor cells and participatein the tumor growth and metastasis by acting on themembrane receptors also il6r was found associated with the characterization of thyroid nodules malignancy and tumor aggressivenessin additioniliopoulos d found that src nonsomatic tyrosine kinase family oncogene can induce the normal epithelial cell transformation by activating nfκb and thistransformation contributes to tumorigenesis il6 is considered as an important regulatory factor in this processanother possibility is that the activation of the immunesystem of patients with thyroid cancer leads to an increase in adikopines level in general the data above support that il6 is important for thyroid cancer but the detail mechanism remainto be further studyleptin a circulating hormone secreted by adipocytesexerts its biological effect by combing with its receptorwhich is mainly presented in the hypothalamus meanwhile gene of leptin receptor is also expressed in manyother tissues such as lung liver and kidney it is reported that obesity and overweight can lead to a highlevel of serum leptin which may because that obesity always accompanies with insulin resistance and hyperinsulinemia and insulin further enhance the expression ofleptin moreover leptin acts as a growth factor in a variety of human cellsincluding both normal cells andtumor cells which regulates the process of differentiation proliferation and apoptosis thus stimulate thetumorigenesis and development of tumors through mediatingpathway rhoalimk1cofilinpathway and mapkerk pathway kim wg evaluated the effect of dietinduced obesity on thyroid carcinogenesis in a mouse model that spontaneously develops thyroid cancer thrb pvpv pten mice and found that obesity increases the frequency of anaplasia of thyroid cancer and exacerbatesthyroid cancer progression that were mediated byjakstat3increased activation of the jak2 signaling transducerand activator of stat3 signaling pathway and inductionof stat3 target gene expression leptin is always reported a high expression on solid tumors and it isconfirmed that serum leptin levelis significantly increased in thyroid cancer mainly ptc while otherstudies showed a same results in cancer tissues [ ] yu xiao conducted a clinical studycomparing the level of serum leptin in ptc patientsincluding patients with lymph node metastasis and thyroid adenoma patients in dalian china and foundthat patients with lymph node metastasis have a higherlevel of leptin than those without lymph node metastasisleptin can induce the expression of vascular endothelialgrowth factor and promote neovascularization in tumortissue in addition it can also inhibit the apoptosisthrough bcl2 dependent mechanism meanwhile leptinreceptor exists in all thyroid cancer cells it is overexpressed in ptc and is involved in tumor invasion andlymph node metastasis [ ] thus leptin may be involved in the tumorigenesis and metastasis of thyroidcancer through a complex pathway and a monitoringmay have some significance due to the absence of directevidence elevated leptin levels can also be caused bythyroid carcinoma the cause and effect relationship between leptin and thyroid carcinoma are unclear now andneed further studiescompared to lean women overweightobese womenhad lower serum adiponectin levels and this differencehas statistical significance in addition adiponectinis negatively associated with a variety of benign and malignant tumors especially those associated with obesityand insulin resistance such as leukemia renal carcinoma gastric carcinoma and colon cancer moreover the association of adiponectin with potential tumorlimiting functions has been widely proposed otvos l jr tried in vitro experiments andproved that adiponectin can inhibit the metastasis ofcancer cells mitsiades n measured circulatingadiponectin levels in ptaients with ptc and found thatit is independently and inversely associated with the riskof thyroid cancer as the receptor that binds to adiponectin for biological effects adiponectin receptor hadbeen reported closely correlated with the developmentof ptc adiponectin receptor1 and are higher expression in ptc tissues than that in the surrounding normaltissues and this is thought to be associated with a betterprognosis however other studies have shown different results[ ] and more studies should be done furtherly tosupport the antitumor effect of adiponectin and thepositive correlation between the increased level of adiponectin in circulating blood and the prognosis of thyroid 0czhao bmc cancer page of neoplasms and provide new ideas for the prevention andtreatment of thyroid neoplasmsfrom the above a strong relationship between elevatedconcentrations of adipokines in serum andor tissueand thyroid cancer can be concluded and this may explain why increased incidence of obesity and thyroidcancer are consistent thus targeted drugs for adipokinemay be useful for the treatment of thyroid cancer in thefuturehowever some limitations in our metaanalysis shouldbe taken into account first some data were not normally distributed and were reported in the form of median and quartile and therefore these data werecalculated by formulas second due to | Colon_Cancer |
collagen triple helix repeat containing1 cthrc1 anextracellular matrix ecm protein was identiï¬ed in thescreening of diï¬erentially expressed sequences between balloon injury and normal arteries the evolution of cthrc1can be traced back to at least million years ago and theconserved genes were not found in invertebrates cthrc1has complicated interactions with various intracellular andextracellular matrices in diï¬erent ways of secretion [ ]cthrc1 increases the activity of collagen promoter throughbinding to ligands and could contribute to vascular remodeling by limiting collagen matrix deposition and promoting cellmigration cthrc1 promotes the recruitment of m2macrophages and regulates tgf and notch pathways toaccelerate wound healing in a mouse model of acute woundhealing as a coupling factor cthrc1 can be secretedby osteoclasts and uence bone formation and remodelingby acting on osteoblasts and osteocytes [ ] cthrc1 maypromote il1induced apoptosis of chondrocytes by activating the jnk12 pathway the antiammatoryeï¬ect of cthrc1 expressed on activated synovial cellswas also found in a collagen antibodyinduced arthritismodel besides cthrc1 can regulate physiologicalfunctions such as fat and glycogen synthesis and promoteautonomous activity [ ]therefore as a secreted protein cthrc1 is involved inmultiple pathophysiologies a remarkable eï¬ect is that thehigh expression of cthrc1 promotes tumorigenesis anddevelopment through positive regulation of tumor spreadinvasion migration adhesion and metastasis cthrc1exerts its eï¬ects through several signaling pathways such as 0cmediators of ammationgpa gvp grd gsp gan gip gtp gip grd gfk gek gechydrophobic regiongxy repeatcysteinesnglycosylationnh2signal peptidecollagendomainc c c cc cc ccoohfigure the structure of the cthrc1 protein the construct of cthrc1 contains an nh2terminal peptide for extracellular secretion ashort collagen triple helix repeat of amino acids and a coohterminal globular domain the prolinerich hydrophobic domain liesbetween the 1st and 30th amino acids and serves as a signal peptide for transport to the endoplasmic reticulum cthrc1 comprises acollagen domain between amino acids and and the protein contains cysteine residues corresponding to about cysteine inthe ï¬nal protein what is more its only amino acid posttranslational modiï¬cation is the glycosylation of asparagine at position integrin faktgf wntsrcfak mekerkpi3kakterk hif1α and pkcδerk signaling pathways in this we focus on the advances in the signalingpathways mediated by cthrc1 in tumors the structural characteristics andexpression of cthrc1 the structural features of cthrc1 the cthrc1 geneis located at chromosome 8q223 and it contains ï¬ve exonsin humans and four exons in mice it covers kb onthe direct strand and can be transcribed into kb mrnathe amino acid sequence identity between human and ratcthrc1 proteins was and no homologs were foundin lower species [ ]protein nglycosylationsecreted cthrc1 exists primarily as a dimer kdaand a trimer kda as well as multimers of the trimericcthrc1 kda and kda the construct of cthrc1contains an nh2terminal peptide for extracellular secretiona short collagen triple helix repeat of amino acids and acoohterminal globular domain [ ] similar molecularweight and structural characteristics to adiponectin alsoexplain why cthrc1 can form high molecular weightcomplexes the biological activity of cthrc1 isrestricted to the highly conserved amino acids at thecterminal region and the cterminal region of cthrc1contains a putative nglycosylation site that stabilizescthrc1promotescthrc1 to tether to the cell membrane which promotesactin polymerization and cell polarity a short collagen motif with glyxy repeats presents in c1qtumornecrosis factorαrelated proteins ctrps which appearsto be responsible for the trimerization of protein and renders molecule susceptible to cleavage by collagenase seefigure however dimeric cthrc1 would not be susceptible to cleavage by collagenase [ ] the molecularweight of secreted cthrc1 kda appears to be largerthan that of cellular cthrc1 kda cthrc1 has fourdiï¬erentabout kda to kda the fulllength of cthrc1 accountsfor both secreted and cellular cthrc1 glycosylatedprotein cthrc1 with a signal sequence is related to ecmisoforms with molecular weights ofalsoand contains a variable short collagenlike motif intriguingly cthrc1 plays a role in inhibiting structural proteins unlike other members of the collagen family moreover leclair found that cthrc1 cleaved atthe nterminus by plasmin shows better inhibition of collagen synthesis compared to fulllength cthrc1 in thepac1 cell line these studies suggested that cthrc1might obtain biologicalthrough proteolyticprocessingactivity the expression of cthrc1 cthrc1 is transientlyexpressed by ï¬broblasts in remodeling adventitia and bysmooth muscle cells in the neointima of injured tissuehowever cthrc1 is not detected in normal arteries ininjured arteries and skin the expression of cthrc1 isassociated with myoï¬broblasts and locates in the sites ofcollagen matrix deposition in micethe ï¬rst exon ofcthrc1 was targeted to be replaced with a galactosidase expression construct which demonstrated the expression of cthrc1 in inner ear hair cells there iscthrc1 expression in many mesenchymalderived cellsduring body growth and tissue repair in mouseembryos cthrc1 is expressed in visceral endodermnotochord neuraltube developing kidney and heartabundant expression of cthrc1 is observed in developincluding cartilage primordia growth plateing skeletoncartilagein adultscthrc1 is expressed only in bone matrix and periosteum cthrc1 is also found in the matrix of calcifyingatherosclerotic plaques and mineralized bone of skeletaltissues in humans in other tissues the sites of cthrc1expression overlap considerably with interstitial collagensand transforming growth factor tgf family membersparticularly bone morphogenetic proteins bmps the sitesof cthrc1 expression are characterized by the presence ofactive tgf and abundant collagen synthesis cthrc1mrna expression levels are increased in response to bmp4bmp2 and tgf furthermore tgf signaling could leadto a significant increase in neointimal lesion formation the expression of cthrc1 is also positively correlatedwith tumor lymph node metastasis tumornodemetastasistnm stage and disease prognosis however its potentialand periosteumbone matrix 0cmediators of ammationregulatory mechanisms in the tumor environment have notyet been elucidated the molecules that regulate theexpression of cthrc1cthrc1 is abnormally expressed in several solid tumorsespecially in gastric cancer pancreatic cancer hepatocellularcarcinoma keloid breast cancer colorectal cancer crcepithelial ovarian cancer esophageal squamous cell carcinoma escc cervical cancer nonsmallcell lung carcinoma nsclc melanoma and so on [ ] andmolecules that regulate the expression of cthrc1 includemirnas lncrnas waif1 and dpagt1 mirnas microribonucleic acids mirnas which canregulate gene expression are a class of noncoding singlestranded small rnas of about nucleotides that can inhibitthe mrna translation process by exclusively promoting thedegradation of several mrnas in many tumors mirnas such as mir30c mir9 mir520d5p mir1555pmir98 let7b mir155 mir101 and mir217 can regulate the expression of cthrc1mir30c could regulate cthrc1 at a posttranscriptionallevelin breast cancer it downregulates the cthrc1mediated gsk3catenin signal and inhibits tumor cellproliferation invasion and migration in addition mir30ccan also upregulate baxcaspase9caspase3 a downstreamsignal of cthrc1 inhibiting apoptosis in hepatocellular carcinoma cthrc1 downregulates mir1555p throughthe activation of gsk3involved wntcatenin signalingto promote tumor formation and mir98 dramaticallydownregulates cthrc1 by directly targeting the ²utr ofcthrc1 suppressing hepatocellular carcinoma formation mir9 could inhibit the migration of schwann cell bytargeting cthrc1 following sciatic nerve injury therebyinactivating downstream rac1 gtpase mir520d5pis significantly downexpressed and suppresses cell proliferation migration and invasion by targeting cthrc1 in crc as the second mirna following lin4 in caenorhabditiselegans let7b may directly target cthrc1 and function asa tumor suppressor gene in gastric cancer [ ] in esccmir101 and mir217 could inhibitthe expression ofcthrc1 mir30 could downregulate the expressionof cthrc1 and downstream signal molecules such as mmp and mmp2 to inhibit the invasion and migration ofnsclc cells a recent study found that mir155 downregulation and cthrc1 upregulation were observed incrc moreover overexpression of mir155 can silencedownstream cthrc1 thereby inhibiting cell proliferationand inducing apoptosis of cells to prevent tumor progressionand metastasis in conclusion the negative regulation ofcthrc1 by mirna has the potential to become a noveldirection for cancer treatment in the futurelncrnas metastasisassociated lung adenocarcinomatranscript i malat1 is a large infrequently spliced longnoncoding rna lncrna which could genetically increasecthrc1 activity to regulate lung cancer cell migration the silence of malat1 could also inhibit the expression ofcthrc1 which is a positive regulator of escc furtheranother lncrna named nonmmug014387 could also regulate cthrc1 and activate the wntpcp pathway to promote schwann cell proliferation at the site of injury waif1 wntactivated inhibitory factor waif1 issilenced by promoter hypermethylation in various cancers[ ] lcmsms analysis using liquid chromatographyand mass spectrometry analysis of samples of cthrc1binding membrane proteins indicates that the largest partof cthrc1 binds the waif1 receptor recent researchsuggests that waifi expression is activated by suppressingmethylation of its promoter activated waif1 downregulates the expression of wntcatenin target genes to inhibitthe development of endometrial adenocarcinoma thebinding of cthrc1 to waif1 could promote osteoblast differentiation therefore cthrc1waif1 interactioncan be a potential therapeutic target in the futurepromoter hypomethylation dpagt1 nglycosylation is essential for the migrationpattern of immune cells and its dysregulation is related tovarious diseases including cancer in human escc the overexpression of cthrc1 is associated with hyperglycosylationandincreased nglycosylation is associated with preferential localization ofcthrc1 in wound cells and nglycosylation facilitatesthe promigratory function of cthrc1 dolichylphosphatenacetylglucosaminephosphotransferase dpagt1 thegene that encodes the ï¬rst enzyme and ratelimiting enzymein the assembly of lipidlinked oligosaccharide precursors inthe endoplasmic reticulum is related to the formation ofmature intercellular adhesion complexes as anupstream regulator of nglycosylation status of ecadherindpagt1 could upregulate cthrc1 by increasing proteinturnover indicating that nglycosylation can also stabilizecthrc1 besides tgf and fak could also regulate the expression of cthrc1 in diï¬erent signaling pathways it should behighlighted that cthrc1 not only is the result of tumor progression but also plays a predominant regulatory role in theprogression and metastasis of many solid tumors [ ]in summary many molecules can regulate the expressionand activity of cthrc1 and together with cthrc1 as novelantitumor molecular targets for the treatment of cancer inthe future signaling pathways mediated by cthrc1involved in the progression andmetastasis of tumorthe uence of cthrc1 on various events in tumor progression is based on its regulation of various signaling pathways such as tgf wntintegrin fak srcfakmekerk pi3kakterk hif1α and pkcδerk signaling pathways see figure these properties pathwaysaï¬ected by cthrc1 play an essential role not only in tissueremodeling after injury regulation of ossiï¬cation and other 0cmediators of ammationcthrc1tgfð½tð½r2wnt3alrp56cthrc1wnt5aror12cthrc1cthrc1ð½ð¼ð¼ð½integrincxrc4tð½r1p ppsmad23smad4cthrc1dvlpdvlaxinapcð½cateninck1ð¼gsk3dpagt1stabilizationð½cateninpkcð¿daamrac1rhoajnkrockpsrcfakpaxgrb2rasrafmekerkpfakpi3kaktmtorfra1crebmmpmekerkhifð¼vegfecminvasionpsmad23smad4ð½catenintcflefcjunap1snailcyclin d1g1mg2smetastasisangiogenesisproliferationfigure signaling pathways mediated by cthrc1 involved in the progression and metastasis of tumor tgf signaling pathway isquite complex especially in terms of its eï¬ects which are often contradictory depending on location and time there exists a criticalnegative feedback regulatory loop between tgfsmad23 signaling pathway and cthrc1 wnt signaling includes wntcatenincanonical pathway and cateninindependent noncanonical pathway in the canonical wnt signaling fzd receptor and lrp5lrp6coreceptor are transduced to catenin signaling cascade for the maintenance of stem and progenitor cells in the noncanonical wntsignaling fzd receptor and ror2ptk7ryk coreceptor are transduced to rhoa jnk signaling cascades for the control of tissuepolarity cell adhesion or cell movement the downstream molecules of the wntpcp pathway mainly include the small gtpase familysuch as rac1 rhoa and jnk which play essential roles in cancer cell migration and invasion cthrc1 signal via waif1 canactivate pkcδ which is an essential component of the wntpcp pathway furthermore pkcδ is responsible for the activation of thecthrc1induced erk signaling pathway in cthrc1integrin signaling pathway the upregulation of cthrc1 is related to theprogression and metastasis of several cancers through the activation of several key signaling molecules including src fak paxillin mekerk and rac1 fak promotes cancer cell migration by regulating focal adhesion formation and turnover which involve activation of srcand paxillin fra1 is activated by cthrc1 through the mapkmekerk signaling which leads to the upregulation of cyclin d1 andthat promotes cell proliferation fra1 also induces snail1mediated mmp14 expression to facilitate escc cell invasion migration andmetastasis pi3kakt signaling pathway induces emt change and mmp2mmp9 expression hif1α and vegf are activated bycthrc1 through activating the pi3kaktmtor signaling pathway which promotes tumor angiogenesis cthrc1 also participates intumor cell migration and invasion through hif1αcxcr4 signalsphysiological processes but also in the development of cancerand metastasis negative feedback regulation of cthrc1 and cell typespeciï¬c tgf signaling pathway as the most potentgrowth factor involved in wound healing tgf is releasedby platelets at the site of injury uencing ammatoryresponse angiogenesis reepithelialization ecm and remodeling tgf superfamily members include tgf activin and bmps smad158 mediates bmp signaling whilesmad23 mediates tgf and activin signalingcthrc1 has been reported to have a relationship withthe tgf family since its discovery as their expression sitesoverlap significantly tgf1 and bmp4 can induce thetranscription and expression of cthrc1 in nih3t3 cells cthrc1 can activate tgf signaling via an elevationin smad2smad3 phosphorylation activated smad23 formsa complex with smad4 and accumulates in the nucleus causing an increase in collagen type i deposition during vascularremodeling [] there exists a critical negative feedbackregulatory loop between tgf1 and cthrc1 the conserved region of amino acids in cthrc1 proteincan bind to phosphosmad3 cthrc1 is induced by tgf1 via phosphosmad3 binding to the promoter with subsequenttranscription activation and in turn cthrc1inhibits tgf1 signaling by accelerating proteasomal degradation of phosphosmad3 which inhibits collagen deposition tgf can enhance the migration and invasioncharacteristics of endothelial cells by regulating the secretionand expression of mmp2 and mmp9 therefore inhibiting cthrc1mediated tgf signaling pathways mayeï¬ectively suppress the invasion and angiogenesis of cancercells [ ]however the mechanism of tgf involved in tumorprogression is very complex even in the same tumor typetgf has many diï¬erent roles in tumor progression forexample the activation of nuclear factor of activated tcellsnfats can drive the switch of the tumorsuppressive function of tgf towards tumor progression [ ] tgfincreases the level of cthrc1 in crc cells highly 0cmediators of ammationexpressed cthrc1 promotes epithelialmesenchymal transition emt and tumor metastasis through the smad2smad3 activation of tgf pathway cthrc1 can alsoinhibit the tgfsmad pathway and yap nuclear translocation thereby inhibiting type i collagen synthesis metabolites such as bile acid may induce cthrc1 to activatethe tgfsmad2smad3 pathway to mediate liver ï¬brosisand may progress towards hepatocellular carcinoma in the polyvinyl alcohol sponge model cthrc1 activates tgf and notch pathways to promote the recruitment of m2 macrophages however cthrc1 maydownregulate tgf expression during the late remodelingphase of wound healing tgf regulates the expressionof cthrc1 in a concentrationdependent manner inkeloids and excess cthrc1 reverses collagen synthesis therefore these results of the regulation betweencthrc1 and tgf are not contradictory other than thatcthrc1 has no inhibitory eï¬ect on tgf signaling inendothelial cells these results indicate that the regulation of tgf by cthrc1 may play a role in other interstitial cells of the tumor microenvironment and that thisregulation is cell typespeciï¬c the further exploration ofdetailed molecular mechanism by which cthrc1 activatesthe tgf pathway may resolve these disputes mutual regulation between cthrc1 and wnt pathwaysto promote tumor progression and metastasis wnt familyare secreted glycoproteins include wnt1 wnt1 wnt3awnt4 wnt5a wnt5b wnt6 wnt7a and wnt7b andparticipate in the process of numerous oncogenic and development progress [] wnt5a is a member of the wntprotein family and plays an essential role in the pathologicalprocess of neuropathy and malignant tumors [] wntproteins activate the wntcatenin canonical pathway andcateninindependent noncanonicalamongwhich the planar cell polarity pcp pathway and wntcalcium ca2 pathway are the most widely studied []current reports indicate that cthrc1 is mainly involvedin tumor progression through the canonical wntcateninand noncanonical wntpcp pathwayspathway wntcatenin canonical signaling pathway in thewntcatenin canonical pathway wnt proteins bind tofrizzled fzd receptor and lipoprotein receptorrelatedprotein lrp56 coreceptor in the absence of wntsignaling the cytoplasmic catenin form the destructioncomplex composed with the casein kinase 1α ck1αglycogen synthase kinase 3 gsk3 adenomatous polyposis coli apc and axin which activates the emt topromotethroughcthrc1wntcatenin [ ] the level of cateninis maintained as low by the series of eventsincludingpriming phosphorylation by ck1α at ser45 and subsequently at thr41 ser37 and ser33 by gsk3 [ ]when secreted wnt ligands are accumulated wnt combines with fzd receptor and lrp56 coreceptors lead toactivation of dishevelled dvl protein the activateddvl is phosphorylated and translocated to the fzd recepthe catenintorcausing the dissociation ofand metastases cancerinvasiondestruction complex and the cytosolic accumulation ofcatenin as the cytosolic catenin accumulates rasproteins are accumulated due to the absence of gsk3mediated phosphorylation the stabilized ras proteins atthe plasma membrane activate rafmitogenactivatedprotein kinase mekextracellular signalregulated kinaseerk cascade besides cytosolic catenin subsequently translocates into the nucleus and forms a complexwith the tcell factor tcf or lymphoid enhancer factorlef the complex activates the expression oftargetgenes involving proliferation and transformation such ascmyc cjun ccnd1 gene encoding cyclin d1 epidermal growth factor receptor egfr cd44 cd133 andleucinerich repeatcontaining g proteincoupled receptor lgr5 []the wntcatenin signaling pathway plays an indispensable role in the occurrence and development of manytypes of cancer cthrc1induced nuclear translocation ofcatenin was observed in nclh23 cells and luciferaseassay showed that catenintcf transcriptional activitywas enhanced in contrast the knockdown of cthrc1reduced the catenintcf transcriptional activity whichshows that cthrc1 regulates the invasiveness of nsclccells through the wntcatenin pathway similarlycthrc1 activates snail1 through the wntcatenin signaling pathway to promote emt in epithelial ovarian cancer during the development and metastasis of crccthrc1 may promote the activation of the wnt signalingpathway through anos1 it can also participate in thewntcatenin pathway to regulate the malignant behaviorof hepatocellular carcinoma with gsk3 many cancers usually metastasize to bone in advanced stages cthrc1secreted by osteoclasts promotes basic ï¬broblast growth factor bfgf expression in osteoblasts by stimulating wntcatenin signaling which may induce the development of cancerous bone lesions but not mediate vascular production the constitutive activation of the wntcatenin pathway leads to carcinogenesis in tumors cthrc1 promotes catenin nucleartranslocation and inducestranscription of downstream target genes such as cmycand cyclin d1 in the nucleus reduces cell adhesion and promotes cell proliferation subsequently tumor cell invasion and metastasis occurredinterestingly another reported that catenincould act on the cthrc1 promoter region and promotetranscription nglycosylation stabilizes cthrc1 in oralsquamous cell carcinoma oscc specimens by reducingprotein turnover rate and cthrc1 is positively feedbackregulated by the dpagt1canonical wnt pathway therebyactivating noncanonical wnt pathways to drive tumor cellmigration and invasion in contrast the overexpressionof cthrc1 in hek293t cell and gastrointestinal stromaltumor gist cell significantly inhibited the canonical wntpathway but activated the noncanonical wntpcp pathway[ ] based on the evidence reviewed above it can be indicated that crosstalk between the canonical wntcateninpathway and noncanonical wntpcp pathway and themutual regulation of wntcatenin and cthrc1 acceleratethe process of tumor progression 0cmediators of ammation wntpcp noncanonical signaling pathway earlyreports suggest that cthrc1 activates the pcp pathwayduring inner ear development cthrc1 can interactwith multiple extracellular components of wnt signalingfzd proteins and wntpcp coreceptor ror2 the components form a cthrc1wntfzdror2 complex to activatethe wntpcp pathway selectively and transmit signals fromthe cellsurface complex to the nucleus by dvlrhoarac1jnkatf2cjun cascade promoting cancer cell protrusionsproliferation migration and invasion [ ]cthrc1 is capable of coordinating three small rho gtpasesrac1 rhoa and cdc42 which are the leading performers ofwntpcp to promote cell migration in cervical cancercthrc1 cooperates with e6e7 human papillomavirushpv to activate the noncanonical wntpcp pathway whichaggravates tumor malignancy in pancreatic cancer andhuman urothelial carcinoma wnt5aror2 signaling is associated with emt and promotes tumor cellinvasion andmetastasis [ ] in gist cthrc1 appears to activatethe wntpcp pathway in a dosedependent manner andwnt5apcprho axis determinesinvasionpromoting activity of cthrc1 a recent study demonstrated that cthrc1 could promote erk and jnkphosphorylation by activating pcp signaling pathways inhuman umbilical vein endothelial cells huvecs and promote tumor angiogenesis whatit wasobserved that the paracrine cthrc1 controls the expression of ang2 via noncanonical wnt pathway activationof erkdependent ap1 in huvecs hence overand above that associated with the canonical wntcateninpathway noncanonical wnt signaling pathways interactwith other signaling pathwaysis moretumorthe cthrc1 binds integrin and triggers a series ofsignaling cascades to promote tumor progressionand metastasis integrin faksrc signaling pathway integrins aretransmembrane receptors that promote cellecm adhesion with two subtypes of α and it can participate ina variety of physiological activities such as tumor progression and migration cthrc1 promotes hepatocellular carcinoma cell invasion by activating the rhoarhoassociated kinase rock pathway and facilitates adhesionof hepatocellular carcinoma cells to ecm through induction of integrin 1 expression and activation of focal adhesion kinase fak another study of hepatocellularcarcinoma suggests that cthrc1 inhibits anoikis andincreases tumor cell survival by activating integrin expression cell adhesion to ï¬bronectin is mediatedby integrin 1 previous researches have demonstrated that targeting the integrin 3fak signaling couldenhance the antitumor activity and attenuate cancermetastasiscancernsclc and escc [] guo found that phosphorylated fak was significantly reduced in mice witheoc xenograft tumors and inhibition of fak did notinterfere with integrin 3 expression in vivo howeverthe overexpression of cthrc1 leads to the upregulationincluding melanomaendometrialof integrin 3 in model mice proving that cthrc1 interacts with integrin 3 and promotes fak phosphorylationat tyr397 thereby promoting ovarian cancer cell adhesionmigration and invasion the high level of cthrc1 is connected with the progression and metastasis of pancreatic cancers through the activation of several key signaling molecules including the steroidreceptor coactivator src fak paxillin mek erk andrasrelated c3 botulinum toxin substrate rac1 srcfak signaling cascade takes a regulative role in regulating the formation of protein complexes at focal adhesionsin the migration and metastasis of cancer cells srccan correspond to integrinecm interaction and is recruitedto form the srcfak complex which permits fak to beactive [ ] then fak activates src and paxillin by regulating focal adhesion formation and turnover paxillin a focaladhesion adaptor molecule serves as a scaï¬oldfor the organization and the activation of raf mek anderk [ ] furthermore paxillin can stimulate rac1which is a ras superfamily member of small guanosine triphosphatase gtpase and a critical factor in cytoskeleton reorganization regulation of gene expression and cell proliferationand cellular transformation []erk2mediated paxillin phosphorylation promotes fakadhesion to focal adhesions additionally the inhibitionof fakpaxillin interaction results in decreased phosphorylation of fak and its targets which in turn changes cell adhesion and migration thisinspired thedevelopment of anticancer drugs targeting fak faksrc complex plays essential functions in tgfinduced hepatocyte emt models such as upregulating mmp9 and ï¬bronectin and downregulating ecadherin evidence has mekerk and pi3kakterk signaling pathwaycthrc1 interacts with integrin to trigger a series of signalcascades because src can phosphorylate other fak sites itcan recruit proteins containing src homology sh2domains such as grb2 subsequentlythe downstreamrasmapk pathway and the phosphatidylinositol kinase pi3k akt cascades are activated to participatein cellular response cthrc1 activates fosrelatedantigen1 fra1 through mapkmekerk signalingwhich leads to the upregulation of cyclin d1 and promotescell proliferation fra1 a fos family transcription factoralso induces snail1mediated mmp14 expression to facilitate escc cellinvasion migration and metastasis snail1 transcriptionaltriggeringemt and inducing tumor cell invasion knockingdown cthrc1 will change the phosphorylation level oferk12 and thus regulate the pathological process of endometriosisfrequent upregulation ofcthrc1 observed in human colon cancer cells may bedue to a cpg demethylation event in the exon regionof the gene kim tested the luciferase reporter geneusing the erkresponsive elk1 promoter proposing thatcthrc1 upregulates mmp9 through erk activation further treatment with mek12 inhibitors can reduce tumorcell invasion and erk activation and aggressiveness arereduced by knocking down cthrc1 theis essentialfactorforem 0cmediators of ammationcthrc1 promotes invasiveness and metastasis of hepatocellular carcinoma through the activation of pi3kproteinkinase b pkbakterkcamp responseelementbinding protein creb signaling pathway which inducesemt change and mmp2mmp9 expression cthrc1is highly expressed in hepatitis b virus hbv associatedhepatocellular carcinoma hbv activates nuclear factorkappa b nfκb and creb through the erkcjun nterminal kinase cjnk pathway to stimulate cthrc1expression in addition hypoxiainducible factor 1α hif1α and vascular endothelial growth factor vegf are activated by cthrc1 through activating the pi3kaktmammalian target of rapamycin mtor signaling pathwaywhich promotesis morecthrc1 enhances colony formation migration and invasion of hepatocellular carcinoma cells by downregulatingtumor suppressor p53 and stimulating invasionassociatedfactor mmp9 tumor angiogenesis whatstudies on myocardial infarction mi have also foundthat activation of infarct repair cardiac ï¬broblasts ircfinvolves cthrc1 expression and pi3kakt signaling pathway in ovarian cancer cells gene silencing cthrc1 doesnot alter mmp9 expression or phosphorylate mek theinvasionpromoting eï¬ect of cthrc1 on eoc cells dependson downstream pi3kakt and erk12 signaling dominatedby egfr besides the invasion and metastasis of endometrial cancer are closely related to the upregulation ofcthrc1mediated cx3cr1 in macrophages this processregulates the integrin 3pi3kakt pathway which alsopromotes the recruitment of m2like macrophages cthrc1 activates hifα pathway and contributes totumor angiogenesis as mentioned above cthrc1 in hepatocellular carcinoma can induce hif1α to promote tumorangiogenesis and regulate downstream mmps and tumorsuppressor gene p53 by activating the pi3kakt signalingpathway in human squamous cell carcinoma hif1αoverexpression stimulates vegfc upregulation and induceslymphangiogenesis and tumor cell invasion ding pinpointed that cthrc1 and hif1α were upregulated inthe nucleus of cthrc1 overexpressed gc cells hif1αinhibitors reduced cthrc1induced cxcr4 expressionfurthermore it was found that inhibition of hif1α expression and inhibition of cxcl12cxcr4 signals all alleviatetumor cell migration and invasion therefore cthrc1 canparticipate in tumor cell migration and invasion throughhif1αcxcr4 signals in gc in short cthrc1 canaï¬ect the expression of hif1α which is not only related tolymphangiogenesis but also closely related to tumor progression and invasion a novel signaling pathway the potential role of pkcδerk in tumors protein kinase c δ pkcδ has beenimplicated in various epithelial tumors such as prostatebreast and crc [ ] activated pkcδ causes angiogenesis and tumor growth of prostate tumors by increasingnadph oxidase activity and hif1α expression levels pkcδ can also inhibit the wntcatenin pathwayin colon cancer cells however a recent study illustrated that mek and pkcδ inhibitors could blockcthrc1induced erk phosphorylation and that pkcδphosphorylation was not inhibited by mek inhibition surprisingly inhibition of plc a membraneassociated enzymethat activates pkcδ to promote bone formation in noncanonical wnt signals did not inhibit cthrc1induced alkaline phosphatase alp activity therefore waif1 bound bycthrc1 activates pkcδ and erk to stimulate osteoblastdiï¬erentiation which is a novel signaling pathway unrelatedto the noncanonical wnt pathway therefore pkcδsignal may explain the role of cthrc1 in tumor progressions such as angiogenesis and bone metastasisto put it brieï¬y cthrc1 may be involved in manyother signaling pathways including mirna and lncrnawhich interact with or crosstalk with the tgf wnt andintegrin erk pathways and jointly participate in tumordevelopment and metastasis see table conclusiontumor development and metastasis a complex processinvolving cell adhesion and proteolytic degradation of theecm depends not only on the cancer cells but also on theinteraction between the cancer cells and their microenvironment complementary dna microarray analysis also demonstrated that the cthrc1 gene is expressed in mosthuman solid cancers as we all know cthrc1 is a secretedecm protein which can inhibit collagen deposition and participate in tumor invasion and metastasis even thoughcthrc1 was ï¬rst discovered more than a decad | Colon_Cancer |
dysregulation of lncrnas is frequent in glioma and has emerged as an important mechanism involved in tumorigenesis previous analysis of chinese glioma genome atlas cgga database indicated that lbx2as1 expression is one of differentially expression lncrna between lower grade glioma lgg grade ii and iii and glioblastoma multiforme gbm however the function and mechanism of lbx2as1 in glioma has not been evaluated yetmethods here we analyzed the expression of lbx2as1 in gtex data normal brain tcgalgg and tcgagbm rtpcr was performed to detect lbx2as1 in surgery obtained normal brain and glioma cck8 kit and annexin vfitcpi apoptosis detection kit were used to study the function of lbx2as1 on glioma proliferation and apoptosis bioinformatic analysis rna immunoprecipitation rtpcr western blotting and dual luciferase reporter assay were carried out to investigate the target mirna of lbx2as1 the discovered mechanism was validated by the rescue assayresults following study of gtex and tcga data lbx2as1 was significantly elevated in glioma compared with normal brain and in gbm compared with lgg higher expression of lbx2as1 was associated with poor prognosis of patients with glioma expression of lbx2as1 was positively correlated with pathology classification of glioma knockdown of lbx2as1 inhibited cell proliferation and induced cell apoptosis in glioma lbx2as1 have complimentary binding site for tumor suppressor mir4915p and we showed that lbx2as1 sponged mir4915p to upregulate trim28 expression in glioma cells trim28 overexpression attenuated the effect of lbx2as1 knockdown on glioma cellss in lbx2as1 was an increased lncrna in glioma mechanistically lbx2as1 promoted glioma cell proliferation and resistance to cell apoptosis via sponging mir4915pkeywords lbx2as1 mir4915p cell proliferation cell apoptosis glioma trim28 gliomas are the most common primary cancer types derived from the neural ectoderm accounting for approximately half of all brain malignancy gliomas are histologic classified into several groups including grade ii oligodendrogliomas and astrocytomas and correspondence houruizhejldxjlueducn department of neurosurgery chinajapan union hospital of jilin university changchun jilin chinafull list of author information is available at the end of the grade iii anaplastic oligodendrogliomas anaplastic astrocytomas anaplastic oligoastrocytomas anaplastic ependymomas and grade iv glioblastomas gbm according to world health anization who classification [ ] overall gliomas are lethal cancer type and patients with high grade glioma gbm have a median overall survival less than one year the worse prognosis of patients with glioma is partly due to resistance to radiotherapy and chemotherapy induced cell apoptosis and the strong proliferation ability of cancer cells [ ] the authors this is licensed under a creative commons attribution international license which permits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the creative commons licence and indicate if changes were made the images or other third party material in this are included in the s creative commons licence unless indicated otherwise in a credit line to the material if material is not included in the s creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtain permission directly from the copyright holder to view a copy of this licence visit httpcreat iveco mmons licen sesby40 the creative commons public domain dedication waiver httpcreat iveco mmons publi cdoma inzero10 applies to the data made available in this unless otherwise stated in a credit line to the data 0cchen a0et a0al cancer cell int page of long noncoding rnas are rna molecules longer than nucleotides without protein coding potential as part of competing endogenous rna cerna network the function of many lncrnas rely on their interaction with micrornas mirnas to regulate expression of gene with same mirna response elements mres accumulating evidences suggested that lncrnas are implicated in glioma progression via sponging mirnas [] for example lncrna ccat1 sponged mir181b increased mir181b target fgfr3 and pdgfrα and promoted glioma cell proliferation invasion and resistance to cell apoptosis lbx2as1 is a recently identified cancer associated lncrna in several cancer types overexpression of lbx2as1 has been reported in hepatocellular carcinoma gastric cancer nonsmall cell lung cancer and esophageal squamous cell carcinoma and it promoted cancer cell proliferation with different mechanism in different cell [] in nonsmall cell lung cancer lbx2as1 activated notch pathway to facilitate cancer cell proliferation migration and invasion in esophageal squamous cell carcinoma lbx2as1 stabilized zeb1 and zeb2 to promote epithelialmesenchymal transition of cancer cells lbx2as1 is one of aberrantly expressed lncrnas between lgg and gbm from cgga database the biological role of lbx2as1 has not been examined in gliomathe current study revealed that lbx2as1 was a significantly upregulated lncrna in glioma and its expression associated with prognosis of patients with glioma we aimed to explore the biological role and molecular mechanism of lbx2as1 in gliomamaterials and a0methodspatients and a0samplestissue samples including normal brains and glioma tissues were obtained from the therapeutic surgery of patients in the third hospital of jilin university during june to july samples were confirmed histologically by two neuropathologists following the criteria of who classification guidelines written informed consents were provided and the protocol was approved by the third hospital of jilin university institutional review board the collected samples were stored at until rna extractioncell lines and a0culturehuman glioma cell lines u87mg u251mg and a172 were purchased from atcc manassas va human astrocyte cell line nha was bought from the cell bank of the chinese academy of sciences shanghai china cells were maintained in dulbeccos modified eagle medium invitrogen carlsbad ca supplemented with fbs gibco rockville md all cells were cultured in a humid incubator with co2 at rna immunoprecipitation rip assaya magna rip rnabinding protein immunoprecipitation kit millipore bedford ma was used to perform rip in a brief u87mg cell lysate was incubated with magnetic beads preincubated with mouse igg negative control or human antiago2 antibody cat no millipore billerica ma total rnas were then isolated by trizol reagent and detected by rtpcr to measure the enrichment of mir4915p and lbx2as1bioinformatic analysisthe expression of lbx2as1 in gtex tcgalgg and tcgagbm projects were obtained from gepia software httpgepia cance rpkucn which was also used to study the association between lbx2as1 expression and prognosis of patients with glioma the association between lbx2as1 and trim28 was also analyzed by gepia based on tcgalgg project encori software tarb asesysueducn was used to predict target mirnas of lbx2as1 and the association between lbx2as1 and mir4915p expression in tcgalggquantitative realtime polymerase chain reactiontotal rna was extracted from cells and tissues with trizol reagent invitrogen rna concentration was detected by nanodrop thermo fisher scientific wilmington de firststranded cdna was synthesized from rna with primescript rt reagent kit takara dalian china realtime polymerase chain reaction was performed with sybr prime script rtpcr kit takara on an abi fast realtime pcr system applied biosystems foster city ca mrna and lncrna were normalized to actin mirna was normalized to u6 the relative expression of gene was calculated by δδct method primer sequences were listed in table a0cell transfection²uuc ucc gaa cgu guc acg utt3² sicontrol silbx2as11 ²cca uua auu cag caa aca uuc cut t3 and silbx2as12 ²uga uuu uuu aaa gaa aaa ucc aat t3² were designed and synthesized by genepharma suzhou china for transfection sirna was mixed with lipofectamine rnaimax invitrogen in serumfree dmem and added into cultured ²cag cug guu gaa ggg gac caaa3² mir4915p mimic ²agu ggg gaa ccc uuc cau gagg3² and mir4915p inhibitor ²ccu cau gga agg guu ccc cacu3² was synthesized by ribo bio guangzhou china these mirnc mir4915p mimic and inhibitor was mixed with cells mirnc 0cchen a0et a0al cancer cell int page of table sequence of a0rtqpcr primersprimerlbx2as1trim28actinmir4915pu6 snrnasequence²aat tcg cag gaa ggg gag tg3²tgc caa acc tgg gac aaa ct3²tga gac ctg tgt aga ggc g3²²cgt tca cca tcc cga gac tt3²²cat gta cgt tgc tat cca ggc3²²ctc ctt aat gtc acg cac gat3²²ctc aac tgg tgt cgt gga gtc ggc aat tca gtt gag cct cat ²²tcg gca gga gtg ggg aac ccttc3²²ctc aac tgg tgt cgt gga ²²cgc ttc acg aat ttg cgt gtca3²²gct tcg gca gca cat ata cta aaa t3²²cgc ttc acg aat ttg cgt gtcat3²forwardreverseforwardreverseforwardreversestem loopforwardreversestem loopforwardreverselipofectamine invitrogen in serumfree dmem and added into cultured cells the transfection efficiency was detected a0h after transfectionwestern blottingps6k cat and s6k cat antibodies were obtained from cst beverly ca trim28 antibody cat was product of active motif carlsbad ca actin antibody cat kc5a08 was bought from aksomics shanghai china hrpconjugated mouse cat ab6728 and rabbit cat ab6721 antibodies were purchased from abcam cambridge uk ripa lysis buffer thermo fisher scientific was used to extract proteins from cells proteins were separated by an sdspage gel and transferred to pvdf membranes the membrane was blocked in nonfat milk and incubated with primary and secondary antibody ecl substrate thermo fisher scientific was used to develop blotscell proliferation and a0apoptosis assaythe cck8 kit beyotime shanghai china was used to detect cell proliferative ability briefly µl cck8 was mixed with culture medium and sustained for an additional a0h after that the absorbance at a0nm of each well was detected by a microplate reader biorad hercules ca percentage of apoptotic cells was measured with dead cell apoptosis kit with annexin v fitc and pi kit invitrogen by flow cytometry analysis cells were suspended in annexinv binding buffer provided by the kit and stained with annexinv fitc and pi sequentially the cells were then subjected to flow cytometry analysis cells positive for annexin v with or without pi positive were apoptotic cellsdual luciferase reporter assaylbx2as1 trim28 ²utr or their mutant forms were inserted into pmirglo luciferase vector these vectors were then cotransfected with mirnc or mir4915p into cells by lipofectamine after a0 h the relative luciferase activity of each group was measured with the dual luciferase reporter assay system kit promega corp madison wi firefly luciferase was normalized to renilla luciferasebioinformatic analysisthe data were analyzed with graphpad prism software the difference between the groups were examined with students t test or oneway anova analysis followed by tukey test the association between expression of two genes was studied with pearson correlation analysis p value less than was considered as statistically significantresultslbx2as1 was a0overexpressed in a0glioma and a0its expression was a0associated with a0poor prognosis of a0glioma patientswe firstly studied the expression of lbx2as1 in normal brains from gtex project low grade glioma lgg and glioblastoma gbm from tcga project the results suggested that lbx2as1 was 4fold and 10fold highly expressed in glioma tissues from tcgalgg and tcgagbm compared with normal brains from gtex expression of lbx2as1 was nearly 3fold higher in highgrade glioma gbm compared with lowgrade glioma lgg fig a0 1a we collected normal brains and glioma tissues and analyzed lbx2as1 expression by rtpcr it showed that lbx2as1 was approximately 3fold highly expressed in glioma compared with normal brains fig a01b furthermore lbx2as1 was increased in high grade glioma grade iv vs grade iii grade iii vs grade ii fig a01c retrospective analysis of the clinical outcome from tcgalgg and tcgagbm datasets suggested that lbx2as1 high expression group n have a shorter diseasefree survival compared with lbx2as1 low expression group n fig a0 1d meanwhile patients with higher expression of lbx2as1 showed a shorter overall survival time compared with their counterparts fig a01elbx2as1 was a0negatively associated with a0mir5p in a0gliomato study the mechanism of lbx2as1 in glioma we firstly used rtpcr method to detect lbx2as1 expression in a panel of glioma cell lines expression of lbx2as1 was significantly increased in u87mg u251mg and a172 cells in comparison with normal human astrocyte 0cchen a0et a0al cancer cell int page of fig lbx2as1 was an upregulated lncrna in glioma a analysis of lbx2as1 expression in normal brains from gtex project and glioma tissues from tcgalgg and tcgagbm projects b rtpcr detection of lbx2as1 expression in normal brains and gliomas c comparison of lbx2as1 expression in normal brain grade ii glioma grade iii glioma and grade iv glioma d e kaplan meierplotter analysis of association between lbx2as1 expression and diseasefree survival d overall survival e of patients with glioma from tcgalgg and tcgagbm projects p nha fig a0 2a lbx2as1 functioned as a cerna to exert its function on cell behavior we used the following method to screen for target mirnas of lbx2as1 fig a0 2b firstly encori database prediction showed that lbx2as1 have putative binding sites for mirnas pearson correlation analysis suggested that the expression of three of these mirnas mir19115p mir219a23p and mir4915p were negatively correlated with lbx2as1 expression in tcgalgg dataset in these three mirnas mir4915p was an upregulated mirna in tcga data of glioma as reported by qi et a0 al the strong negative correlation between mir4915p and lbx2as1 expression was showed in fig a0 2c pearson r a0 p expression of mir4915p was decreased by more than in collected glioma compared with normal brains fig a02d in contrast to lbx2as1 mir4915p was lowly expressed in high grade glioma compared with those of low grade grade iv vs grade iii fig a02e the negative correlation between lbx2as1 and mir4915p expression was also observed in these glioma tissues fig a0 2f to study whether lbx2as1 interact with mir4915p we firstly transfected mir4915p mimic into u87mg and u251mg cells to elevate mir4915p expression fig a02g full length of lbx2as1 was inserted into luciferase vector pmirglo luciferase reporter assay showed that mir4915p overexpression did repress luciferase activity of pmirglolbx2as1 by around half in u87mg fig a02h and u251mg cells fig a02i more importantly rip assay showed that ago2 antibody enriched both lbx2as1 and mir4915p in u87mg cells fig a0 2j these data implied that lbx2as1 might regulate mir4915p in glioma cellsa mutual regulatory association between a0mir5p and a0lbx2as1 in a0gliomato study the association between mir4915p and lbx2as1 we transfected increasing concentrations a0 nm and a0 nm of mir4915p mimic into u87mg and u251mg cells expression of mir4915p was increased by and 9fold in u87mg cells transfected with a0nm and a0 nm mir4915p mimic respectively fig a0 3a in u251mg cells mir4915p levels were increased by and 45fold after transfection of a0nm and a0nm mir4915p fig a0 3a overexpression of mir4915p decreased lbx2as1 expression by more than half in u87mg and u251mg cells and lower expression of lbx2as1 was observed in cells with high concentration of mir4915p mimic fig a0 3b two independent sirnas targeting lbx2as1 silbx2as11 and 0cchen a0et a0al cancer cell int page of fig lbx2as1 expression was associated with mir4915p levels in glioma a rtpcr detection of lbx2as1 expression in a panel of glioma cell lines u87mg u251mg a172 and normal human astrocyte nha b flow chart of screen for target mirnas of lbx2as1 in glioma c the association between lbx2as1 and mir4915p expression in tcgalgg project was analyzed by pearson correlation analysis d rtpcr detection of mir4915p expression in normal brains and gliomas e comparison of mir4915p expression in normal brain grade ii glioma grade iii glioma and grade iv glioma f the association between lbx2as1 and mir4915p expression in collected glioma samples was analyzed by pearson correlation analysis g the expression of mir4915p was detected in u87mg and u251mg cells transfected with mirnc or mir4915p mimic in u87mg h and u251mg i cells the luciferase activity of pmirglo empty vector as the control group and pmirglolbx2as1 was detected j rip assay was performed with igg and ago2 antibody followed by rtpcr detection of lbx2as1 and mir4915p levels p p silbx2as12 were transfected into glioma cells these sirnas greatly decreased lbx2as1 expression by more than in u87mg and u251mg cells fig a03c in addition knockdown of lbx2as1 increased mir4915p expression in u87mg and u251mg cells fig a0 3d transfection of silbx2as12 which reduced lbx2as1 expression in a relatively larger extent compared with silbx2as11 was more effective in upregulation of mir4915p expression in the cells fig a03d indicating the negative regulation of mir4915p by lbx2as1 we analyzed the complementary site between lbx2as1 and mir4915p and constructed pmirglo vector with mutant lbx2as1 lbx2as1 mut fig a0 3e overexpression of mir4915p repressed luciferase activity of lbx2as1 wt instead of lbx2as1 mut in u87mg fig a03f and u251mg cells fig a03glbx2as1 upregulated trim28 expression in a0gliomaprevious study suggested that mir4915p targeted trim28 to regulate glioma cell proliferation after investigation of trim28 and lbx2as1 expression in tcgalgg and tcgagbm datasets it was found that trim28 was positively correlated with lbx2as1 expression in glioma pearson r p fig a0 4a additionally trim28 was highly expressed in our collected glioma samples compared with normal brains fig a0 4b pearson correlation analysis showed a strong positive correlation between lbx2as1 and trim28 levels in our collected glioma tissues pearson r p fig a04c knockdown of lbx2as1 decreased approximately trim28 mrna expression in u87mg and u251mg cells fig a0 4d trim28 induced dephosphorylation of s6k to control glioma development lbx2as1 downregulation decreased trim28 protein expression and the downstream phosphorylation of s6k ps6k by half in u87mg cells as measured by western blotting fig a04e similarly lbx2as1 knockdown also decreased trim28 and ps6k in u251mg cells to the same extent as in u87mg fig a04f 0cchen a0et a0al cancer cell int page of fig mir4915p mutually regulated lbx2as1 in glioma mir4915p a and lbx2as1 b expression was detected in u87mg and u251mg cells transfected with mirnc or increasing concentration of mir4915p mimic nm nm c lbx2as1 expression was detected in u87mg and u251mg cells transfected with sicontrol or silbx2as11 or silbx2as12 by rtpcr d mir4915p expression was detected in u87mg and u251mg cells transfected with sicontrol or silbx2as11 or silbx2as12 e sequence alignment of lbx2as1 wt lbx2as1 mut and mir4915p luciferase activity was detected in u87mg f and u251mg g cells transfected with lbx2as1 wt or lbx2as1 mut p p lbx2as1 regulated trim28 via a0sponging mir5pwe first transfected mir4915p inhibitor into u87mg and u251mg cells to decrease mir4915p expression in these cells mir4915p inhibitor decreased mir4915p expression in cells fig a05a we found that mir4915p inhibitor could reverse the downregulation of trim28 mrna expression by silbx2as11 in u87mg and u251mg cells fig a05b western blotting further showed that mir4915p inhibitor could reverse the downregulation of trim28 protein expression by silbx2as11 in u87mg and u251mg cells fig a05c luciferase vectors containing wildtype and mutant trim28 ²utr trim28 ²utr wt trim28 ²utr mut were constructed fig a05d in u87mg cells mir4915p inhibitor could reverse the repression of luciferase activity of trim28 ²utr wt by silbx2as11 fig a05e these data collectively demonstrated a lbx2as1mir4915ptrim28 axis in gliomalbx2as1 promoted cell proliferation and resistance to cell apoptosis by repression of mir4915pit is known that aberrant expression of mir4915p and trim28 mediated glioma cell proliferation and survival to investigate the impact of the lbx2as1mir4915ptrim28 axis on cell proliferation we performed cck8 assay in glioma cells transfected with silbx2as1 with or without mir4915p inhibitor knockdown of lbx2as1 inhibited cell proliferation in u87mg cells and the effect of silbx2as1 was reversed upon cotransfection of mir4915p 0cchen a0et a0al cancer cell int page of fig lbx2as1 regulated trim28 expression in glioma a pearson correlation analysis of lbx2as1 and trim28 expression in tcgalgg and tcgagbm projects b rtpcr detection of trim28 mrna expression in normal brains and gliomas c pearson correlation analysis of lbx2as1 and trim28 expression in collected glioma d trim28 mrna expression was detected in u87mg and u251mg cells transfected with sicontrol or silbx2as11 or silbx2as12 by rtpcr trim28 ps6k protein expression was detected in u87mg e and u251mg f cells transfected with sicontrol or silbx2as11 or silbx2as12 by western blotting actin and s6k were internal controls for trim28 and ps6k p p inhibitor fig a0 6a in consistent with u87mg silbx2as11 and silbx2as12 inhibited u251mg cell proliferation which was attenuated by mir4915p inhibitor fig a0 6b with the flow cytometry we also found that lbx2as1 knockdown induced cell apoptosis in u87mg cells and the effect of silbx2as1 was partially rescued by mir4915p inhibitor fig a06c similar results were found in u251mg cells fig a06d these data manifested that lbx2as1 mainly relied on regulation of mir4915p to mediate glioma cell proliferation and survivaldiscussionlbx2as1 is a most recently identified oncogenic lncrna across several cancer types lbx2as1 was reported as a highly expressed lncrna in nonsmall cell lung cancer especially in tumors of advanced stage high expression of lbx2as1 was also found in stomach adenocarcinoma and hepatocellular carcinoma [ 0cchen a0et a0al cancer cell int page of fig lbx2as1 regulated trim28 expression via sponging mir4915p a the expression of mir4915p was detected in u87mg and u251mg cells transfected with mirnc or mir4915p inhibitor b the expression of trim28 mrna was detected in u87mg and u251mg cells transfected with mirnc or mir4915p inhibitor in combination with sicontrol or silbx2as11 by rtpcr c the expression of trim28 protein was detected in u87mg cells transfected with mirnc or mir4915p inhibitor in combination with sicontrol or silbx2as11 by western blotting d sequence alignment of lbx2as1 trim28 ²utr wt trim28 ²utr mut and mir4915p e luciferase activity was detected in u87mg cells transfected with trim28 ²utr wt or trim28 ²utr mut in combination with sicontrol or silbx2as11 and mirnc or mir4915p inhibitor p p see figure on next pagefig lbx2as1 regulated cell proliferation and apoptosis via sponging mir4915p transfection of silbx2as11 or silbx2as12 inhibited cell proliferation and was reversed by transfection of mir4915p inhibitor in u87mg a and u251mg b cells as indicated by cck8 assay transfection of silbx2as11 or silbx2as12 induced cell apoptosis and was partially reversed by transfection of mir4915p inhibitor in u87mg c and u251mg d cells as indicated by flow cytometry analysis p p 0cchen a0et a0al cancer cell int page of 0cchen a0et a0al cancer cell int page of ] here we analyzed lbx2as1 expression in glioma by using data of tcgalgg and tcgagbm projects in combination with normal brains from gtex project similar to observation in other cancer types it was observed that lbx2as1 was increased in glioma especially highgrade glioma gbm we further revealed that patients with high expression of lbx2as1 have a short diseasefree survival and overall survival indicating lbx2as1 could predict poor prognosis of glioma as the published studies showed that lbx2as1 was associated with poor prognosis of patients with hepatocellular carcinoma and nonsmall cell lung cancer [ ] the findings suggested that lbx2as1 might be a predictive biomark for a variety of cancer typeslbx2as1 exerted its procancer functions via acting as a cerna to sponge tumor suppressive mirnas for example lbx2as1 directly interacted with tumor suppressive mir384 to enhance cell proliferation and resistance to cell apoptosis in hepatocellular carcinoma our bioinformatic analysis indicated that lbx2as1 have a putative binding site for mir4915p mir4915p was a tumor suppressor in several cancer types [] aberrant expression of mir4915p was the consequence of upregulated circrna circ_0001361 and lncrna xist in bladder cancer and nasopharyngeal carcinoma respectively [ ] in glioma mir4915p was decreased in cancer tissues and correlated with good prognosis we confirmed mir4915p as a target mirna of lbx2as1 in glioma upregulation of mir4915p induced cancer cell apoptosis to cease cell proliferation [ ] we showed that lbx2as1 mediated glioma cell proliferation and resistance to cell apoptosis downregulation of mir4915p could partially rescue the impact of lbx2as1 knockdown on glioma cell proliferation and apoptosis thus the current data indicated a novel interaction between lbx2as1 and mir4915p and manifested that lbx2as1 promoted glioma proliferation via sponging mir4915p however as the cell apoptosis induced by lbx2as1 knockdown was not fully rescued by mir4915p downregulation we believe there are several other mechanisms underlying the function of lbx2as1 in glioma for example the notch signaling was regulated by lbx2as1 in nonsmall cell lung cancer and the activity of notch signaling determined the cell apoptosis in glioma [ ] it remains unknown whether lxb2as1 uses the same mechanism to control notch signaling in glioma future study will reveal the complexity of signaling network regulated by lbx2as1 in glioma in addition due to the involvement of mir4915p in cancer cell metastasis further studies will be needed to evaluate the effect of lbx2as1 on glioma metastasistrim28 is a cancerassociated e3 ligase in several cancer types upregulation of trim28 was found in glioma and the proproliferative function of trim28 was supported by in vitro and in vivo data [ ] in glioma trim28 mediated degradation of tumor suppressor ampk activated mtorc1 and regulated cell apoptosis trim28 expression was repressed by several noncoding rna in different cell [ ] in the current study in addition to the known mir4915ptrim28 interaction in glioma we further discovered that lncrna lbx2as1 could regulated trim28 via sponging mir4915p in glioma mechanistically trim28 form complex with mage to regulate mtor activity and the downstream phosphorylation of s6k [ ] we found that lbx2as1 not only upregulated trim28 expression but also increased phosphorylation level of s6k in glioma cells therefore the data revealed a lbx2as1mir4915ptrim28 axis in gliomasour results suggested that lncrna lbx2as1 promoted glioma cell proliferation and resistance to cell apoptosis via sponging mir4915p lbx2as1 could be a novel biomarker for patients with gliomaacknowledgementsnoneauthors contributions qc rh and jg performed the experiments and acquired the data qc and yz analyzed data qc and rh designed the study rh supervised the study manuscript was written by rh all authors read and approved the final manuscript funding none availability of data and materialsall data generated or analyzed during this study are included in this published ethics approval and consent to participateinformed consent was obtained from all patients and the study protocol and consent procedures were approved by the third hospital of jilin university review boardconsent for publicationnot applicablecompeting intereststhe authors declare that they have no competing interestsauthor details department of neurosurgery chinajapan union hospital of jilin university changchun jilin china department of gynecology and obstetric the second hospital of jilin university changchun jilin china received may revised july accepted july references ostrom qt bauchet l davis fg deltour i fisher jl langer ce pekmezci m schwartzbaum ja turner mc walsh km et al the epidemiology of glioma in adults a state of the science review neuro oncol 0cchen a0et a0al cancer cell int page of chen r smithcohn m cohen al colman h glioma subclassifications pan sj ren j jiang h liu w hu ly pan yx sun b sun qf bian lg and their clinical significance neurotherapeutics wesseling p capper d who classification of gliomas neuropathol appl neurobiol ostrom qt gittleman h stetson l virk sm barnholtzsloan js epidemiology of gliomas cancer treat res xu h chen b xing j wei z liu c qiu y lin y ren l upregulation of lgmnp1 confers radiotherapy resistance in glioblastoma oncol rep krakstad c chekenya m survival signalling and apoptosis resistance in glioblastomas opportunities for targeted therapeutics mol cancer castrooropeza r melendezzajgla j maldonado v vazquezsantillan k the emerging role of lncrnas in the regulation of cancer stem cells cell oncol dordr zhang z qian w wang s ji d wang q li j peng w gu j hu t ji b et al analysis of lncrnaassociated cerna network reveals potential lncrna biomarkers in human colon adenocarcinoma cell physiol biochem liu x zhu q guo y xiao z hu l xu q lncrna linc00689 promotes the growth metastasis and glycolysis of glioma cells by targeting mir3383ppkm2 axis biomed pharmacother magea6 promotes human glioma cell survival via targeting ampkalpha1 cancer lett wang sn luo s liu c piao z gou w wang y guan w li q zou h yang zz et al mir491 inhibits osteosarcoma lung metastasis and chemoresistance by targeting alphabcrystallin mol ther zhang q li q xu t jiang h xu lg mir4915p suppresses cell growth and invasion by targeting notch3 in nasopharyngeal carcinoma oncol rep xu y hou r lu q zhang y chen l zheng y hu b mir4915p negatively regulates cell proliferation and motility by targeting pdgfra in prostate cancer am j cancer res sun r liu z tong d yang y guo b wang x zhao l huang c mir4915p mediated by foxi1 functions as a tumor suppressor by targeting wnt3abetacatenin signaling in the development of gastric cancer cell death dis 201783e2714 liu f zhang h xie f tao d xiao x huang c wang m gu c zhang x jiang g hsa_circ_0001361 promotes bladder cancer invasion and metastasis through mir4915pmmp9 axis oncogene cheng q xu x jiang h xu l li q knockdown of long noncoding rna xist suppresses nasopharyngeal carcinoma progression by activating mir4915p j cell biochem liao y shen l zhao h liu q fu j guo y peng r cheng l lncrna casc2 denoyelle c lambert b meryetfiguiere m vigneron n brotin e lecerf c interacts with mir181a to modulate glioma growth and resistance to tmz through pten pathway j cell biochem ni w luo l zuo p li rp xu xb wen f hu d lncrna ghet1 downregulation suppresses the cell activities of glioma cancer biomark cui b li b liu q cui y lncrna ccat1 promotes glioma tumorigenesis by sponging mir181b j cell biochem tang lx su sf wan q he p xhang y cheng xm novel long noncoding rna lbx2as1 indicat | Colon_Cancer |
" the severe acute respiratory syndrome coronavirus sarscov2 has caused the greatest worldwide pandemic since the flu the consequences of the coronavirus disease covid19 are devastating and represent the current major public health issue across the globe at the onset sarscov2 primarily attacks the respiratory system as it represents the main point of entry in the host but it also can affect multiple ans although most of the patients do not present symptoms or are mildly symptomatic some people infected with sarscov2 that experience more severe multian dysfunction the severity of covid19 is typically combined with a set of comorbidities such as hypertension diabetes obesity andor advanced age that seriously exacerbates the consequences of the infection also sarscov2 can cause gastrointestinal symptoms such as vomiting diarrhea or abdominal pain during the early phases of the disease intestinal dysfunction induces changes in intestinal microbes and an increase in inflammatory cytokines thus diagnosing gastrointestinal symptoms that precede respiratory problems during covid19 may be necessary for improved early detection and treatment uncovering the composition of the microbiota and its metabolic products in the context of covid19 can help determine novel biomarkers of the disease and help identify new therapeutic targets elucidating changes to the microbiome as reliable biomarkers in the context of covid19 represent an overlooked piece of the disease puzzle and requires further investigation 0c introduction severe acute respiratory syndrome coronavirus sarscov2 also called 2019ncov arose in the province of wuhan china in december sarscov2 causes severe respiratory disease coronavirus disease covid19 on march the world health anization determined that the outbreak of a novel coronavirus had reached spreading pandemic as of august there are over million confirmed cases worldwide with more than associated global deaths according to johns hopkins university over the last months the global population has been facing problems that impact both world health and global socioeconomics covid19 has been and continues to be the focus of concern in a society threatened by the most destructive pandemic since the flu transmission symptoms vaccines and treatments for covid19 continue to be investigated hiding too many unknowns the immediate strategy to curb the spread of this virus has been to recommend that people carry out a social vaccination which involves restricting social gatherings minimizing public appearances telecommuting implementing social distancing or wear masks as much as possible this is based on the high transmission ability of the sarscov2 via large respiratory droplets and by airborne routes unlike other viruses in the coronavirus family sarscov2 infects people who then have little or no symptoms despite all this asymptomatic covid19 positive individuals can spread the virus this silent transmission capacity is the main reason this virus continues to transmit and infect the global population in an accelerated and uncontrollable way in spite of best efforts to control and curb spread asymptomatic people manage to eliminate the virus without developing typical covid19 symptoms and this suggests to us that the immune system is relevant and may have the key to beat this virus covid19 affects not only the respiratory and cardiovascular systems but also to the central nervous system cns and gastrointestinal system the objective of this review is to examine whether the sarscov2 infection linked to gastrointestinal changes may be linked to 0c specific phases of covid19 associated to inflammation we will explore the studies on microbiome profiling intestinal immune system in the patients with covid19 this review will provide insights for microbiota modification therapy in the early stages of covid19 beyond antiviral and antiinflammatory approaches new therapeutic interventions might be possible through the restoration of the gut microbiome of individuals infected by sarscov2 to mitigate systemic inflammation intestinal damage and limit the effects caused in the cns through the braingut axis early and late stage of the infection of sarscov2 a multitude of epidemiological studies describe different phases in the development of covid19 the early or viral phase shortly after infection marked by a high viral load and a reduced inflammatory activity with hardly any symptoms and which is also associated with gastrointestinal illness figure besides patients with covid19 who had the highest viral load levels at the beginning of the infection and subsequently decreased over time could explain the rapid spread of the disease [ ] during the progressive or late phase of infection covid19 positive individuals develop most severe symptoms like respiratory problems and fever immune cells like neutrophils infiltrating monocytes and macrophages produce inflammatory cytokines and reactive oxygen species also respiratory manifestations such as cough sputum production and shortness of breath remain the most common symptoms following fever or pneumonia [ ] in a study with covid19 patients serum level of cytokines as interleukin il6 and il10 were indicators of disease severity showing that high levels of proinflammatory cytokine storm were associated with more severe disease development some patients with covid19 had severe complications including acute respiratory distress syndrome ards cardiovascular conditions multiple an dysfunction syndromes or septic shock these complications are related to the release of cytokines and hyperinflammation or cytokine storm syndrome figure a person is more infectious in 0c the initial phase when the symptomatology of the disease does not manifest itself with respiratory symptoms and however the presymptomatic transmission may play an important role nevertheless detecting sarscov2 infection in those without fever andor respiratory symptoms is difficult because sarscov2 tests are not usually performed in infected people without these symptoms in summary a subset of the symptoms associated with covid19 during the initial phase are intestinal complications such as vomiting or diarrhea detecting these symptoms might not only lead to slowdown in transmission but also open the door to novel treatments that could reduce the severity of covid19 more studies are necessary to interpret the phases of the disease correctly and especially if the viral load detected is considered infectious and how it is related to respiratory or gastrointestinal symptoms clinical characteristics of covid19 patients with digestive symptoms and intestinal inflammation covid19 is an emerging infection that causes great concern about respiratory manifestations furthermore diarrhea and other gastrointestinal symptoms are frequently observed in patients with covid19 however the significance remains undetermined a viral infection causes an alteration of intestinal permeability resulting in enterocyte dysfunction when we investigate what happened with another coronavirus we found that diarrhea was a frequent symptom in sars patients occurring in intestinal problems were also associated with the severity of the infection patients with diarrhea had an increased need for respiratory assistance and intensive care sarscov was also identified in ileal and terminal colonic biopsies regarding mers several studies showed the presence of diarrhea between to of known cases however a less severe prognosis of the disease was observed in mers patients with diarrhea 0c the first results linking intestinal problems with covid19 were obtained from patients in wuhan china two hundred four patients with covid19 presented at three hospitals indicate that the majority of covid19 patients had typical respiratory symptoms however many patients infected with the coronavirus complained of digestive symptoms such as diarrhea there is no evidence on the efficacy of antidiarrheal drugs but adequate rehydration and potassium monitoring should be performed as in all covid19 patients diarrhea should generate awareness of a possible sarscov2 infection and should be investigated to reach an early diagnosis of covid19 this factor should be considered when suspecting whether the patients are infected instead of waiting for respiratory symptoms to appear which would give us an earlier diagnosis unless the coronavirus tests are also performed on asymptomatic individuals patients with covid19 specifically those with digestive symptoms remained a long time from onset to hospital admission and a worse clinical outcome compared to patients who do not suffer from these symptoms likewise patients with digestive symptoms had an average time of days from the onset of symptoms until admission while patients with respiratory symptoms had an admission time of days this may indicate that those with digestive symptoms waited longer to be diagnosed in the hospital as they did not suspect they were sarscov2 positive in the absence of respiratory symptoms authors also state that patients with digestive symptoms presented other associated clinical manifestations such as anorexia diarrhea vomiting or abdominal pain as the severity of the disease increased gi symptoms became more pronounced but especially the high percentage of patients admitted with symptoms of anorexia one study indicated that covid19 patients experience gastrointestinal gi symptoms such as diarrhea anorexia and nausea however the underlying pathophysiology gastrointestinal is not well understood another study analyzed hospitalized patients and some of them showed mild initial gi symptoms such as diarrhea nausea vomiting and abdominal pain which preceded the characteristic fever and respiratory problems such as dry cough in another study diarrhea 0c was reported in patients and the fecal test remained positive until days after the disease onset in patients the stool test was still positive despite negative respiratory tests another study with patients with low severity of covid19 including presenting with digestive symptoms alone shows that patients with gastrointestinal symptoms had a longer duration between symptom onset and viral clearance and fecal viruspositive than those with respiratory symptoms a systematic metaanalysis of shows results from clinical studies with an incidence rate of diarrhea from as low as and up to of the cases how is this disparity in the data possible more clinical studies are required to elucidate the percentage of covid19 patients that develop intestinal symptoms and if these depend on other factors such as age gender or other comorbidities gastrointestinal symptoms are accompanied by inflammation or intestinal damage there is a loss of intestinal barrier integrity and gut microbes that can activate innate and adaptive immune cells to release proinflammatory cytokines into the circulatory system leading to systemic inflammation some intestinal signaling pathways can regulate inflammation through dendritic cells figure immunomodulation of the innate host immunity through the activation of epithelial receptors could represent a novel therapeutic target to eliminate sarscov2 in the early stage of the infection the viral load of coronavirus appeared in the feces of of the infected patients an interaction between viral shedding of sarscov2 in stool has been reported but its association with infection was yet to be determined identifying fecal coronavirus rna may also lead researchers to ask new questions is there transmission of the coronavirus found in fecal samples what these researchers found is the presence of the coronavirus in the stool but there is controversial studies about if viral load was infectious nor examples of transmission previously a study involving patients with covid19 reported that the median duration of viral shedding was days in survivors range â days cultivable sarscov2 was detected in stool or urine specimens for longer than weeks in three convalescent patients days suggesting that it may remain viable 0c contradictorily high sarscov2 titres detectable in the first week of illness with an early peak observed at symptom onset to day of disease [ ] although sarscov2 rna shedding in respiratory and stool can be prolonged the duration of a viable virus is relatively shortlived the presence of sarscov2 including live virus with infectious capacity in the feces of asymptomatic individuals implies that covid19 could be transmitted through the fecal route sarscov2 shedding in stool samples is detectable over a longer period than in nasopharyngeal swabs donors for fecal microbiota transplant for sarscov2 must be strict and validated to prevent the potential risk of transmission the results show that the elevated levels of fecal calprotectin in patients with covid19 add to the growing evidence that sarscov2 infection causes an inflammatory response in the intestine calprotectin concentrations were significantly higher in covid19 patients who had suffered from diarrhea and with more elevated serum il6 levels in the diagnosis and especially in the followup of covid19related diarrhea the calprotectin measurement could play a potential role in monitoring the disease also diarrhea may be secondary to virusinduced inflammation which in turn is due to the entry of inflammatory cells into the intestinal mucosa including neutrophils and lymphocytes and thus disruption of the gut microbiota viral sarscov2 ps were detected in feces during the second phase of covid19 accompanied by a decrease in the peak of inflammation therefore covid19related inflammatory diarrhea was associated with reduced levels of fecal sarscov2 rna however intestinal damage can manifest after respiratory symptoms as reported in a clinical case there may be a pathogenic role of sarscov2 on the gi in zheijiang china it was detected that half of the covid19 patients tested positive for rtpcr in their feces and intestinal microbial dysbiosis was also identified viral strains alive from feces were isolated indicating potential infectiousness of feces [ ] another analysis conducted from studies in china usa and singapore had also detected 0c sarscov2 rna in fecal specimens of patients at an average of this percentage is according to their clinical forecast intestinal problems or with more severe disease furthermore the fecal test for sarscov2 rna also bears a potential implication for physicians to decide the subsequent isolation strategies for those with positive fecal samples the possibility of the fecaloral route of transmission of sarscov2 should be investigated all candidates for fecal microbiota transplantation and healthy donors should be screened for the virus one could consider studies on the gut microbiome and its therapeutic role in transplanting feces in healthy donors to critically ill covid19 patients still precautions should be taken because infectious sarscov2 ps are known to have been found in covid19 positive feces the use of fecal transplants could be one of the immediate solutions in critically ill patients with a weakened immune system sarscov2 interaction with intestinal ace2 receptors sarscov2 enters cells primarily through binding of protein s to angiotensinconverting enzyme ii ace2 receptors to infected cells the central role of ace2 in the cleavage of angiotensin i to angiotensin ii a peptide involved in vascular homeostasis vasomotor tone and blood pressure regulation ace2 receptors are expressed in various human cells susceptible to viral infection including epithelial cells in the lungs small intestine and colon tubular cells of the kidney neuronal and glial cells in the brain enterocytes vascular endothelial cells smooth muscle cells and cardiomyocytes a very recent study has reported on the expression pattern of ace2 across more than different cell types corresponding to all major human tissues and ans based on rigorous immunohistochemical analysis sarscov2 also uses the receptors for transmembrane protease serine tmrpss2 an enzyme that is expressed in the small intestinal epithelial cells to entry to the infected cells figure the sarscov2 activity could cause ace2 modifications in the gut that increase susceptibility to 0c intestinal inflammation and diarrhea a high coexpression of ace2 and tmprss2 was detected in enterocytes and the esophagus and lungs the coexpression of ace2 and tmprss2 transcripts was highest in the small intestine with expressed in enterocytes and in colon cells as demonstrated by a singlecell rna sequencing study in gi ace2 is not only playing an essential role in intestinal inflammation but also have a significant impact on the composition of the intestinal microbiota ace2 ko mice have been shown to have decreased expression of antimicrobial peptides and exhibited altered intestinal microbial composition which is restored by the administration of tryptophan likewise it is known that there is a connection of transport of ace2 amino acid with microbial ecology in the gut during sarscov2 infection in another recent study using gnotobiotic germfree rats was observed a decrease in ace2 expression in the colon that contributed to the pathology during covid19 there are numerous studies that have shown that by regulating the reninangiotensin system ras which includes ace2 one can modulate systemic inflammation angiotensin ii receptor blockers are widely used compounds that are therapeutically effective in cardiovascular disorders renal disease metabolic syndrome and diabetes under normal healthy conditions homeostasis of rasace2 occurs while a perturbation to this system is observed in cardiovascular disease hypertension and diabetes mellitus a reduction of ace2 is associated with hypertension diabetes and cardiovascular problems which represent the significant comorbidities of covid19 sarscov2 may potentially further upregulate ras in cardiovascular patients and deplete ace2 downregulation of ace2 levels in tissues has been linked to viral replication efficiency and pathogenicity leading to the imbalance of positive and negative regulation of ras rasace2 imbalance in covid19 may greatly exacerbate tissue inflammation and may contribute to more adverse covid19 outcomes in patients with preexisting cardiovascular disease and other comorbidities ace2 receptors may represent a target for covid19 treatments in patients with cardiovascular risk burden susceptible to conditions that worsen asymmetries in rasace2 however suppression of 0c ace2protective roles due to ace2 depletion upon sarscov2 infection is very likely to uphold the poor outcomes observed in covid19 patients especially in those with preexisting conditions ace2 also displays nonrasrelated roles linked with neutral amino acids transport and gut homeostasis ace2 has been implicated in the intestinal epithelium regulation of gut microbiota composition and function in summary ace2 imbalance is likely a key player for the poor outcomes in the covid19 patients with preexisting comorbidities and addresses a possible link for gut microbiota dysbiosis in this interplay the effects of covid19 on the gut microbiome primary inflammatory stimuli trigger the release of microbial products and cytokines which can cause microbial dysbiosis that can induce an inflammatory environment releasing intestinal cytokines into the circulatory system increasing the systemic inflammation of covid19 taken together a combined inflammation can potentially initiate an immune reaction that can create even more harm than the virus itself it is necessary to know the host cytokine pathways and microbiota interactions with cytokine responses in sarscov2 infection to develop novel treatment approaches it is essential to investigate how intestinal bacteria interact in response to sarscov2 infection among covid19 patients an increase of a blood proteomic risk score prs was associated with a risk of becoming a clinically severe infection a recent study has shown more than proteins in bloodrelated to the severity of covid19 these proteins included immune factors that are elevated during systemic inflammation such as creactive protein crp correlations between prs and the intestinal microbiome might be associated with the severity of covid19 this new analysis would allow us to predict if the patient would develop more severe symptoms in the next days or weeks after the infection ruminococcus gnavus was identified in covid19 patients and positively correlated with inflammatory markers while 0c clostridia was negatively correlated another small study of patients hospitalized in hong kong has served to establish a gut microbiome profile in association with covid19 severity and changes in fecal shedding of sarscov2 through the application of indepth shotgun metagenomics analysis the authors investigated longitudinal changes of the gut microbiome in covid19 the abundance of coprobacillus clostridium ramosum and clostridium hathewayi correlated with covid19 severity and it was observed an inverse correlation between the abundance of faecalibacterium prausnitzii an antiinflammatory bacterium during the hospitalization time were detected in covid19 patients bacteroides dorei bacteroides thetaiotaomicron bacteroides massiliensis and bacteroides ovatus which downregulate the expression of ace2 in the murine gut correlating inversely with sarscov2 load in fecal samples this study opens the doors to possible interventions for gut microbiota in hospitalized patients to reduce the severity of covid19 another study of just patients showed that the gut bacteria was associated with fecal sarscov2 load the gut microbiome remained substantially different in hospitalized patients from that of healthy controls a total of bacterial species were identified to be significantly associated with the fecal viral load of sarscov2 across all fecal samples this does not indicate that the gut microbiome is affected for long periods after recovery and future therapeutic interventions would be necessary sarscov2 was detected in feces of hospitalized patients with covid19 and the viral transcriptional activity was analyzed to determine the infectivity range associated with the gut microbiome fecal samples with a signature of high sarscov2 infectivity had higher abundances of bacterial species collinsella aerofaciens collinsella tanakaei streptococcus infantis and manella manii in contrast fecal samples with a signature of lowto no sarscov2 infectivity had higher abundances of scfas producing bacteria parabacteroides merdae bacteroides stercoris alistipes onderdonkii and lachnospiraceae bacterium however live sarscov2 was not isolated from the feces covid19 patients another study 0c of covid19 patients showed a significantly reduced bacterial diversity and a higher relative abundance they compared these results with h1n1 patients who showed lower bacteria diversity compared with covid19 patients specific microbial signatures were identified in covid19 patients which could help identify biomarkers that differentiate them from influenza a h1n1 patients when there is a coinfection despite this the phases of the disease were not established in this study furthermore in another study with covid19 patients was observed a reduced alpha diversity in microbes the metatranscriptional analysis revealed that there were differentially genes associated with immune pathways and cytokine signaling related to the diagnosis and severity of covid19 such as interferongamma signaling despite reporting interesting patterns the studies mentioned above are not without limitations the bioinformatics analyses employed in these studies does not guarantee specieslevel nor strainlevel bacterial identification furthermore no longitudinal analysis has been performed with the same patients to determine if these bacteria change after recovery from covid19 more studies that prospectively include infected but asymptomatic subjects positive for sarscov2 but with different degrees of symptoms would be necessary to establish a correlation with microbial markers or their products monitoring early in the disease during early onset of covid19 and over the longterm will help to delineate the role of changes in the microbiome will be critically important to elucidating this connection further another major limitation is the small number of patients and the absence of information about microbial changes in the context of covid19 that define broad groups of the population stratified by geography ethnicity gender andor age to date there are no metabolomics and metaproteomic analysis studies that are needed to explore the products of these bacteria and their function by identifying microbial metabolites associated with covid19 we can understand what components these bacteria produce during covid19 that help us understand the influence of the communication pathways of the microbiotagutperiphery axis relevant to the association between sarscov2 and 0c hyperinflammation moreover more studies investigating the correlation between sarscov2 and gut inflammation are necessary including histopathology and molecular diagnostic assessments comorbidities as risk factors for covid19 associated with loss of microbial diversity sarscov2 can infect people of all ages but older adults and people with preexisting medical conditions appear to be more vulnerable to becoming seriously ill with the virus there are many hypotheses as to why this occurs still one of the factors could be the loss of microbial diversity associated with aging and with it higher susceptibility to inflammation comorbidities play an essential role in determining the risk of severe complications and death after covid19 infection covid19 comorbidities and risk factors include asthma hypertension smoking male gender or alzheimer's disease or dementia changes in gut microbiota have been previously linked to all of these comorbidities and this deregulation could also be associated with changes in the immune system and the susceptibility to suffer more severe consequences of covid19 and genderrelated differences in vulnerability to complications of covid19 our microbiome changes as we age which favors less diversity and a more significant inflammatory state covid19 appears to be more dangerous in older people men and with comorbidities the gut microbiota evolves during human life the infant microbiota shows reduced diversity and will remain unstable for the first few years of experience until it becomes an adultlike microbiota the gut microbiota within an individual is considered stable throughout adult life in the elderly gut microbiota diversity decreases and dysbiosis increases and is associated with cognitive deficits depression and inflammatory markers a change that is found repeatedly in the microbiota of the elderly is the decrease in the ratio of firmicutes to bacteroidetes [ ] decreased bacterial diversity as well as lower levels in specific bacterial groups have also been observed in very elderly [ ] at the genus and species 0c level the findings vary significantly between studies although bacteroides clostridium and lactobacillus appear recurrently altered in elderly individuals tragically a high rate of covid19 fatalities is associated with groups of people over years old the causes may be the inability to overcome the infection the weakness of the immune system and the reduced microbiome diversity causing the coronavirus to strongly attack this group of the population causing a higher mortality rate another recent study has found significant associations between dietary patterns and measures of gut microbial composition in older men the group of the population with the highest mortality rate from covid19 obesity is also associated with changes in the intestinal flora [ ] and is another risk factor in the severity of covid19 therefore another comorbidity in adults and children in the united states at least of patients who die from covid19 have obesity which is similar to the reported rates of cardiovascular disease in the same highrisk group it is necessary to study the relationship between obesity and the severity of the covid19 disease adipose tissue can serve as a reservoir for the spread of sarscov2 virus clearance and systemic immune activation adipocytes in obese patients express higher levels of ace2 and a reduction or elimination of already inflamed adipose tissue can reduce systemic viral spread viral entry and prolongation in obese individuals there is marked dysregulation of myeloid and lymphoid responses within adipose tissue associated with dysregulation of cytokine profiles obese patients also have heightened levels of proinflammatory adipokines leukotrienes chemerin among others which may exacerbate their risk for cytokine storm syndrome and death the alteration of the immune system causes changes in the intestinal flora and it remains to be seen whether the coronavirus also induces changes in the bacteria that modulate many of these functions of the intestinal brainmicrobiota axis the bacterial intestinal flora plays a critical role in the regulation of neurological functions such as depression or anxiety [ ] surely the results of these studies allow us to know 0c what the role of intestinal flora is in covid19 and its relationship with neurological problems at long term furthermore diabetes is another disease associated with increased severity of symptoms and complications of covid19 and this can be attributed to systemic inflammation and gutmetabolite dysfunction individuals suffering from cardiovascular disease who become infected with sarscov2 are at increased risk of developing a worse prognosis of covid19 and also develop cardiovascular complications including myocardial infarction arrhythmias stroke or heart feature or myocardial suppression cardiovascular disease is accompanied by an imbalance of gut microbiota and a decreased microbiome diversity hypertension is likely to be influenced by diet lifestyle factors and microbiome notably an increase in shortchain fatty acids scfa was previously associated with decreased blood pressure and improved arterial compliance changes in the gut microbiota composition through diet to deal with covid19 the impact of dietary patterns on susceptibility to and severity of infection with the sarscov2 virus has been largely ignored to date the commensal microbiome forms a dynamic environment that can be altered and cause dysbiosis from virus infection but can be positively modulated by diet components and probiotic treatments several studies show than an optimal immune response depends on proper diet and nutrition to control sarscov2 infection [ ] in general malnutrition can compromise the immune response therefore affecting the vulnerability of the response to covid19 consideration of the dietary and nutritional components the factors during viral infection can serve to strengthen the immune system for the prevention of infections and a meaningful and balanced basis for an immune response is an adequate and balanced diet intake of a sufficient amount of protein is crucial for the production of antibodies also a low level of vitamin a or zinc has been associated with an increased risk of infection branchedchain amino acids can maintain the hairy morphology of 0c the intestines and increase intestinal immunoglobulin levels thereby improving the intestinal barrier therefore highquality proteins are an essential component of an antiinflammatory diet nutritional dietary components known to exert antiinflammatory and antioxidant properties include omega3 fatty acids with high antiinflammatory and antioxidant capacity including vitamin c vitamin e and phytochemicals such as carotenoids and polyphenols that are widely present in plantbased foods undoubtedly omega3 fatty acids appear to have the most potent antiinflammatory capability several of these components can interact with cellular signaling components related to antiinflammatory and antioxidant effects an | Colon_Cancer |
" phytolaccaceae species in china are not only ornamental plants but also perennial herbs that areclosely related to human health however both largescale fulllength cdna sequencing and reference genevalidation of phytolaccaceae members are still lacking therefore singlemolecule realtime sequencing technologywas employed to generate fulllength transcriptome in invasive phytolacca americana and noninvasive exotic picosandra based on the transcriptome data rtqpcr was employed to evaluate the gene expression stability in thetwo plant species and another indigenous congener p acinosaresults total of gb and gb clean reads of p americana and p icosandra were generated including and full length nonchimeric flnc reads respectively transcript clustering analysis of flnc readsidentified and consensus isoforms including and highquality ones after removingredundant reads and transcripts were obtained based on structure analysis total and alternative splicing variants and simple sequence repeats ssr as well as and completecoding sequences were detected separately furthermore and lncrna were predicted and and transcripts were annotated respectively subsequently seven reference genes in the two plant species and anative species p acinosa were selected and evaluated by rtqpcr for gene expression analysis when tested indifferent tissues leaves stems roots and flowers 18s rrna showed the highest stability in p americana whetherinfested by spodoptera litura or not ef2 had the most stable expression in p icosandra while ef1α was the mostappropriate one when attacked by s litura ef1α showed the highest stability in pacinosa whereas gapdh wasrecommended when infested by s litura moreover ef1α was the most stable one among the three plant specieswhenever germinating seeds or flowers only were consideredcontinued on next page correspondence yiwangynueducn1yunnan key laboratory of plant reproductive adaption and evolutionaryecology yunnan university kunming china2laboratory of ecology and evolutionary biology state key laboratory forconservation and utilization of bioresources in yunnan yunnan universitykunming chinafull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cliu bmc plant biology page of continued from previous page fulllength transcriptome of p americana and p icosandra were produced individually based on thetranscriptome data the expression stability of seven candidate reference genes under different experimentalconditions was evaluated these results would facilitate further exploration of functional and comparative genomicstudies in phytolaccaceae and provide insights into invasion success of p americanakeywords phytolaccaceae smrt sequencing fulllength transcriptome analysis reference gene evaluation rtqpcr phytolacca americana is a member of the phytolaccaceae family and is native to northeast america becauseof its ornamental and medicinal applications it was introduced into china in unfortunately it hasevolved into an invasive species and spread to mostareas of the country especially in central and southernchina compared to noninvasive exotic congener p icosandra and native congener p acinosa p americana isof interest because it exhibits multiple biological activities such as plant pesticides antimicrobial propertyheavy metal accumulation capacity []in order to investigate the mechanisms of various bioactivities of p americana further transcriptomewidestudy is necessary to facilitate reports have showed thatjasmonic acidinduced and cadmiumtreated transcriptome data of p americana have been obtained by illumina hiseq and illumina hiseq platformrespectively [ ] howeverthese data were bothachieved by second generation sequencing sgs whichcould not produce fulllength transcripts genomic dataof p americana was available at the sra under projectprjna544344 but its raw reads without coding sequences prediction and functional annotation third generation sequencing tgs is known for itskilobasesized long reads and is an outstanding strategyfor better understanding rna processing for exampleit can be used to analyze different transcript isoformsregulated by alternative splicing which greatly increasesthe repertoire of proteins lead to genetic and functionaldiversity and is prevalent in most eukaryotic anisms the long reads could also provide sequence information on genecoding regions for functional analysis atthe transcriptional level and thus can be applied to refine an assembled genome for better annotation however tgs could not quantify gene expression forthe moment and have a relatively high error rate thansgs the combination of tgs and sgs are able to solvethis problem and are highly recommended by most researchers with the transcriptome and genome data availablefunctional genomics research is being performed whichrelied heavily on gene expression analysis reversetranscription quantitative real time pcr rtqpcr hasbeen reported to be a very sensitive and accurate technique to analyze gene expression level but it requiresappropriate reference gene as an internal control tonormalize mrna levels between different samples foran exact comparison of gene expression [ ] anideal reference gene should be expressed at a constantlevel across various experimental conditions howeverstudies have shown that no single reference gene is universal for all experimental conditions [] therefore its necessary to estimate the stability of referencegenes under particular experimental condition beforeusing them for gene expression analysisin the present study to provide highquality and morecomplete assemblies in genome and transcriptome studies of phytolaccaceae a hybrid sequencing approachcombining the sgs and tgs technologies was carriedout first fulllength transcriptome of the invasive plantspecies of pameracana and an noninvasive exotic conicosandra was generated by singlemoleculegener prealtimesmrtsplicingevents simple sequence repeats ssr coding sequencesprotein annotations and long noncoding sequenceswere analyzed respectively at transcription level furtherthe stability of reference genes was evaluated in two phytolaccaceae species mentioned above and one nativecongener p acinosa by rtqpcr in order to facilitatefuture research on functional gene expressionsequencing alternativeresultsto classify the plant species these three phytolaccaceaemembers p americana p icosandra and pacinosa wereidentified by pcr and followed by sequences alignmentbased on sequences of second internal transcribed spacer its2 and the intergenic spacer of photosystem iiprotein d1 gene and trnahis gene of chloroplast genome psbatrnh table s1 the sequences of its2 andpsbatrnh in p americana that we employed had theidentity of with the sequences reported by chen in p icosandra the sequences of its2 had identity and the sequences of psbatrnh had identity with the results of chens in pacinosa 0cliu bmc plant biology page of similarity of its2 and identify of psbatrnh werefound isoforms afterremoving redundantsmrt sequencing data outputusing the pacific biosciences smrt sequencing protocol gb clean reads of invasive species p americana were obtained after preprocessing on the basis offull passes and sequence quality circular consensus sequences ccs with fulllength rate were obtained including fulllengthnonchimeric flnc sequences and highqualityconsensussequences from the high quality consensus isoforms transcripts alternative splicing events ssr complete coding sequences lnc rnasand annotated transcripts in p americana wereachieved similarly gb clean reads in p icosandrawere identified and ccs with fulllength rate flnc sequences as well as highquality consensus isoforms were filtered subsequently transcripts and alternative splicingevents were obtained whats more ssrs complete coding sequences lnc rnas and annotated transcripts were identified in picosandratable transcriptome analysisbased on the structure of achieved transcripts and alternative splicing events were identified in pamericana and p icosandra respectively transcripts of bp in total in p americana wereemployed for ssr analysis based on the sequence lengththat was more than bp including ssrs and ssrcontaining sequences similarly transcripts bp in total in picosandra wereemployed for ssr analysis and ssrs togetherwith ssrcontaining sequences were identifiedtable summary of fulllength transcriptome sequencingclean reads gbccsflncflnc flncccsconsensus isoformhigh quality consensus isoformtranscriptsalternative splicingssrcomplete coding sequenceslncrnaannotated transcriptsp americanap icosandrathe detail information about the number of sequencescontaining more than one ssr the number of ssrspresent in compound formation and the number of different types of ssrs were shown in table in additiontotal of complete coding sequences cds in pamericana and cds in p icosandra were identified by using transdecoder the length distribution ofpredicted proteins was shown in fig s1in pdatabasespecifically nrwith the eight protein databases sequence alignmentswere performed to annotate predicted proteins in total transcripts in p americana and tranicosandra were annotated separatelyscriptstable the number of annotated protein sequencesin p americana was similar with p icosandra under aparticularncbi nonredundant protein analysis revealed that approximately transcripts in p americana and transcripts in picosandra showed the highest sequencesimilarity with beta vulgaris fig go gene ontology assignment also suggested that similar amount ofsequences in the two plant species belonged to the sameterm and many were classified into cell part and cellterm of cellular component catalytic activity and binding of molecular function and metabolic process andcellular process of biological process fig cogclusters of orthologous groups of proteins annotationshowed that a large number of predicted proteins in thetwo plant species were linked to functional class r general function prediction only j translation ribosomalstructure and biogenesis t signal transduction mechanisms g carbohydrate transport and metabolism ando posttranslational modification protein turnoverchaperones fig s2 the result of eggnog evolutionary genealogy of genes nonsupervised orthologousgroup annotation indicated that most of the annotatedproteins in the two plant species were belonging to thefunctional class s function unknown fig s3 kogeukaryotic ortholog groups functional classificationsuggested that r general function prediction only ando posttranslational modification protein turnover andchaperones were the most abundant functional categories in the two plant species fig s4 these results indicated that most of the sequences obtained were trulyfunctional proteins and had a similar functional classification in p americana and its congener picosandraeven though more work is needed to identify sequencesthat regulated or involved in the invasion success of pamericana the annotation of predicted proteins provided necessary information for further studiesbesides the transcripts encoding proteins long noncoding rnas lncrnas were achieved lncrnas arereported to be key regulators in plant biological prolncrna in pameracana andcesses the number ofpicosandra was predicted by cpc coding potential 0cliu bmc plant biology page of table ssrs obtained from transcripts with more than bpsearching itemtotal number of sequences examinedtotal size of examined sequences bptotal number of identified ssrsnumber of ssr containing sequencesnumber of sequences containing more than ssrnumber of ssrs present in compound formationnumber of mono nucleotide ssrnumber of di nucleotide ssrnumber of tri nucleotide ssrnumber of tetra nucleotide ssrnumber of penta nucleotide ssrnumber of hexa nucleotide ssrcalculator cnci codingnoncoding index pfamand cpat coding potential assessment tool respectively in total lncrna in pamericana and lncrna in picosandra were predicted by all these fourmethods fig subsequentlytranscription factorstfs that are key components involved in the transcriptional regulatory system were predicted in p americana tfs of types were filtered and in picosandra tfs of types were predicted thesetwo plant species shared the first types of tfs butthe number of each type tf was not similar especiallyrlkpelle_dlsv c3h snf2 and camk_camklchk1 indicating the particular functions on transcriptregulation fig amplification performance of rtqpcrprimers designed for rtqpcr were evaluated by pcrfirst the primers which produced single ampliconwithout primer dimer were chosen for melting curveanalysis only primers which produced a single fragmentefficiencywereqpcr amplificationchosenforp americanap icosandraassessment the qpcr efficiency of each primer pair wasgenerated from a 10fold serial dilution of pooled cdnaand was shown in table the threshold cycle ct values of each reference genewere employed to evaluate expression level under different experimental conditions fig average ct valuesfor all the seven candidate reference genes ranged from to in which ef1α showed the highest expression level and 28s rrna had the lowest expression levelit was also suggested that ct values of βactin and tubulin fluctuated significantly across all the experimentalsamplesstability of candidate reference genesforto determine the appropriate reference genesnormalization in different experimental conditions theexpression data was analyzed by genorm normfinderand bestkeeper respectively table s2when expression stability of reference genes wereanalyzed in different tissues leaves stems roots andflowers of p americana 18s rrna and ef2 oftable number of proteins annotated via differential protein databasedatabasesp americanaannotated number ¤ length length ¥ p icosandraannotated number ¤ length length ¥ coggokeggkogpfamswissproteggnognrall 0cliu bmc plant biology page of fig homologous species distribution of p americana and p icosandra annotated based on the nr database a p americana b p icosandrapamericana were identified as the most suitable reference genes by genorm and normfinder and 18s rrnawas also suggested by bestkeeper pairwise variation valueof v23 was below the cutoff value of which meansthe combination of two reference genes were most suitable for gene expression normalization fig whentested in picosandra ef1α was recommended for normalizing gene expression analysis not only by genorm butalso by normfinder ef2 was also suggested by genormand bestkeeper in pacinosa ef1α was the best reference gene suggested by genorm and bestkeeper but 18srrna was recommended by normfinder the use of tworeference genes was suitable because pairwise variationvalue of v23 was below when pooled the data of different tissues from pamericana and picosandra togetheref2 was shown to be the most stable gene by all the threemethods when investigated the expression stability ofreference genes in different tissues of pamericana andpacinosa 18s rrna showed the best expression stabilityby genorm and normfinder while ef2 was referred asthe most stable one by bestkeeper however the combination of five reference genes was recommended bygenorm for v56 which was less than when thedata of different tissues from picosandra and pacinosawas put together ef1α was identified as the best oneby genorm and normfinder whereas ef2 was suggested to be the best stability reference gene by bestkeeper when set the data of these three plant speciesas a pool ef1α was suggested to be the most stableone by genorm and normfinder while ef2 was alsorecommended by genorm and bestkeeper accordingto these results it is very important to select the appropriate reference gene when analyze the gene expressionlevel among plant species 0cliu bmc plant biology page of fig classification of the transcripts annotated by the gene ontology gowhen analyzed the data among germinating seeds28s rrna and ef1α were identified as the best reference genes by genorm while 18s rrna was recommended by normfinder and gapdh was suggested bybestkeeper three reference genes were sufficient tonormalize gene expression for v34 was below inflowers only of these three plant species ef1α was confirmed by all the three methods the genorm analysisshowed that the value of v45 was below so fourreference genes in combination were suggested thesefig venn diagram of the number of lncrnas predicted by cpc cnci cpat and pfam a p americana b p icosandra 0cliu bmc plant biology page of fig classification of predicted transcription factorsresults indicated that when focusing on particular tissuesof different plant species the selection of reference genewas also very essentialwhen plants were infested by slitura 18s rrnashowed the most expression stability suggested by genorm and normfinder in different tissues of pamericanawhile ef1α wasby bestkeeper therevealedcombination of two reference genes was suggested bygenorm due to the value of v23 was less than 18srrna was also recommended by genorm in s liturainfested picosandra and ef1α was shown to be themost stable one by normfinder and bestkeeper fourreference genes in combination were recommended bygenorm 18s rrna was also identified as the besttable primers for rtqpcr analysisgene nametubulingene descriptiontubulin ef1αelongation factor 1alphaprimer sequence ²²f gtaaggaagccgagaattgr tcaacaacagtgtcagagaf tgaagaaggtcggatacaatr gtagacatcctggagtgggaphdglyceraldehyde3phosphate dehydrogenasef tggtgctaagaaggttattatcef2elongation factor 18s rrna18s rrnaβactinactin728s rrna28s rrnar2 linear regression coefficientr gagtgaacggtggtcataf gtatcaccatcaagtcaactgr acaatcaaccacaacaaggf acttcctcttctcgtatcattr tgttcagcatagactgtgaf atgctatccttcgtctggr tactcttggctgtctctgf tacgattggttacggacatr ttctcatcaacaacagcatatlength bppcr efficiency r2 0cliu bmc plant biology page of together 18s rrna showed the best stability in genormand normfinder while the expression stability of ef1αwas suggested by bestkeeper in pamericana and picosandra 18s rrna was identified as the best referencegene by all the three algorithms in pamericana andpacinosa 18s rrna and βactin were suggested bygenorm in picosandra and pacinosa while gapdhand ef2 were recommended by normfinder and bestkeeper respectively when take all the data of s liturainfested plant species into account 18s rrna exhibitedthe most stable expression suggested by genorm andnormfinder while ef2 was the gene with the most constant expression identified by bestkeeperdiscussionfulllength transcripts are fundamental resources forstructuralfunctional and comparative genomics research [ ] smrt sequencing has been acknowledged by enabling the generation of multikilobasesequences to improve genome and transcriptome assembly the fulllength cdna sequences generated areable to characterize the posttranscriptional processsuch as alternative splicing lncrna prediction and coding sequences for further gene functional studies basedon the fulllength transcriptome data generated about gb of clean data were obtained for pamericana andpicosandra respectively table accordinglythenumber of ccs flnc consensus isoforms highqualityfig rna transcription levels of seven candidate reference genesin p americana picosandra and p acinosa the expression level ofcandidate reference genes in total samples n was presentedas cycle threshold number ctvalue and explained by box andwhisker plots the asterisks represented the minimum and maximumct value the squares indicated the 25th and 75th percentiles andthe median was represented by a bar across the squarereference gene by genorm in plant species pacinosawhile tubulin was suggested by normfinder and 28srrna was recommended by bestkeeper the combination of three reference genes was appropriate by genorm when analyzed the data oftwo plant speciesfig pairwise variation analyzed by genorm to determine the optimal number of reference genes for accurate normalization a threshold valueof was suggested for valid normalization if the value of vnn pairwise variation is less than then n reference genes in combinationare recommended for gene normalization if the value of vnn is more than then vn 1n should be taken into account pam pamericana pic p icosandra pac p acinosa lsrf different tissues of leaves stems roots and flowers gs germinating seeds of these three plantspecies f flowers of these three plant species lsr different tissues of leaves stems and roots i infested by s litura of third instar 0cliu bmc plant biology page of isoforms transcripts alternative splicing events ssrscomplete coding sequeces lncrnas and annotated transcripts were analyzed providing basic transcriptomic information for further studiesreports have showed that fulllength transcriptome ofzea mays have greatly helped in refining gene annotation and revealed the complexity of gene expression inmaize similar analysis has also been conducted inshum bicolor whats more the world expansioncapability of cydia pomonella has been informed according to its genome information molecularmechanism of rapid growth and invasive adaptation ofan invasive species mikania micrantha has also been investigated according to itsreference genome therefore the fulllength transcriptome data of pamericana and picosandra will contribute to the genomic research and provide insights into invasive mechanism ofpamericana through comparative genomics study inphytolaccaceae speciesgenereliesonanalysisexpressionaccurate relative quantification of rtqpcr for furtherrobustnormalization by stably expressed reference genes tominimize error in the experimental process therefore suitable reference genes for the normalization ofrelative gene expression data in three phytolaccaceaespecies pamericana picosandra and pacinosa weresought under a diverse set of conditions these resultsdemonstrated the importance of validating referencegenes under the relevant experimental conditions forexamplein different tissues leaves stems roots andflowers of pamericana 18s rrna and ef2 were recommended to be the bestsuited reference genes and similar results were found in s liturainfested pamericanahowever even though the appropriate reference genesin picosandra were ranked according to the analyzed results of the three methods all the pairwise variationvalues were above the cutoff value of while thecombination of 18s rrna βactin ef1α and ef2 weremost suitable in s liturainfested picosandra ef2 andef1α have been considered as the ideal reference genesin pacinosa whereas the combination of 18s rrna βactin and gapdh were recommended after s litura infestation researches have also showed that no singlereference gene is stably expressed among different tissues of an anism such as the reference gene selectionin amygdalus persica solanum lycopersicum and glycine max [ ] whats more our results alsosuggested that reference genes identified based on transcriptome data should be confirmed by experimentalevidence in jainduced transcriptome of p americana28s rrna showed stable expression between exogenousjatreated and control plants ja signal pathway ofplants can be induced by lepidopteran herbivores infestation however 18s rrna and ef2 were identifiedas the most stable expression reference genes in pamericana after s litura infestationin order to conductthe gene expression analysisamong different plant species of phytolaccaceae the dataof the three plant species were also compared togetherwhen compared the data in germinating seeds of threeplant species various genes were recommended by thethree methods the combination of plant species underother experimental conditions showed that the pairwisevalues of almost all the combination were higher thanthe cutoff value of exceptthe combination ofpamericana and pacinosa where five reference geneswere recommended for data normalization as well as thecombination of sliturainfested pamericana and sliturainfested picosandra where three reference geneswere suggested these results indicated that no particular gene was expressed constantly across different plantspecies even though these plants are congeners therefore reference genes should be employed appropriatelyunder the relevant experimental conditionsthe research has provided transcriptomewide fulllength isoforms of pamericana and picosandraproviding insights into invasive success of pamericanaguidelines for selecting appropriate reference genesunder different tissues in one plant species or amongvaried plant species were recommended further no particular gene was expressed constantly under differentexperimental conditions indicating the necessity of reference gene identification these results would facilitatethe exploration of functional and comparative genomicsstudies in phytolaccaceae to better understand plantbiologymethodsplant and insect materialsplants of p americana °²n °²e p icosandra°²n °²e and p acinosa °²n °²eused in this study which was named m k and q firstwere collected in yunnan china sampling was permitted when conducted complying with locallegislationthe formal identification of the samples were conductedby chao chen botany major of laboratory of ecologyand evolutionary biology state key laboratory for conservation and utilization of bioresources in yunnanyunnan university according to flora of china vol5 flora of north america vol43 chinese virtual herbarium httpwwwcvhaccn and global plants on jstor httpplantsjstor dna identification was also employed according tothe its2 region of nuclear ribosomal dna one of themost widely used dna fragments in plant molecularsystematics at the generic and species levels and the 0cliu bmc plant biology page of chloroplast psbatrnh intergenic region all voucher specimens were maintained at an experimental fieldof laboratory of ecology and evolutionary biology statekey laboratory for conservation and utilization of bioresources in yunnan yunnan universitytissues of leaves stems roots and flowers from oneindividual plant of p americana or p icosandra werecollected individually from the wild in yunnan provinceand no permission is needed for collecting theses samples each sample was flash frozen in liquid nitrogen andstored at °c for further experimentsshop101732681taobaocomthird instar larvae of spodoptera litura were purchased from henan jiyuan baiyun industry co ltdchinaand then werereared on artificial diet in a climate chamber h at °c with light and h at °c without light for further usefor reference gene evaluation seeds of p americanap icosandra and p acinosa were collected first from thewild in yunnan province and no permission is neededthe seeds were sown separately in agar plates andcultivated in the climate chamber after d five germinating seeds of one plant species were collected togetheras one sample for subsequent experiments each plantspecies have three replications two weeks later othergerminating seeds of each species were transplanted intoplastic pots cm diameter and cm height withsoil jiangsu peilei matrix technology development coltd china and cultivated with adequate water in artificial chambers with same conditions as described abovefour months later leaves stems roots and flowers ofeach plant species were collected individually simultaneously six larvae s litura of third instar were employedto infest on p americana p icosandra or p acinosawith one insect per leaf control treatments were herbivore free after h infestation leaves stems and rootsof these three plant species were harvested individuallyall samples collected were flash frozen in liquid nitrogenand stored in °c for subsequent assays and threereplicates were conducted for each treatmentnucleic acid extraction and assaysgenomic dna was isolated from the leaves of differentplant species following protocols provided by dnaquickplant system tiangen biotech co ltd beijing chinathen it was employed as the pcr template for plantspecies identificationpurekitplanttotal rnas from different tissues was prepared usingrnapreppolysaccharides polyphenolicsrich tiangen biotech co ltd beijingchina according to the manufacturers instructionsthe rna quality and purity were measured by using ananophotometer n60 implen germany and the agilent bioanalyzer system agilent technologies causa samples only with a ratio of to a ratio between and and a rin value morethan were chosen for the sequencing library construction an equal amount of total rnas from four different tissues of the same plant species were mixed asone sample for fulllength transcriptome sequencingtotal rnas from the samples collected for referencegene evaluation was also extracted individually as described above for each sample cdna was prepared byusing μg of total rna following the recommendedinstructions of fastquant rt kit with gdnase tiangenbiotech co ltd beijing chinapacbio cdna library preparation and smrt sequencingfulllength cdna was synthesized by using the smarter¢ pcr cdna synthesis kit clontech ca usathe generated cdna was then reamplified using pcrafter end repairing smrt adaptor with a hairpin loopstructure was ligated to the cdna via exonucleasedigesting the cdna library was constructed after quality measurement of the cdna library smrt sequencingwas performed using the pacific bioscience sequel platform following the provided protocolillumina cdna library construction and secondgenarationsequencingthe extracted mrna was purified using oligo dtattached magnetic beads fragmentation was conducted inthe nebnext first strand synthesis reaction bufferfirststrand cdna was acquired based on the randomhexamers and then the secondstrand cdna was synthesized with dntps rnase h and primestar gxldna polymerase the synthesized cdna was purifiedwith ampure xp beads after end repairing adding polya and adaptor ligation ampure xp beads were used forsize selection the generated cdna was then amplifiedfor building cdna libraries the qualified libraries werepair end sequenced on illumina nova platformquality filtering and error correction of long readsraw smrt sequencing reads were filtered by removingpolymerase reads less than bp and sequence accuracyless than after removing adaptor subreads were obtained clean data was produced with subreads morethan bp ccss were produced from clean data withparameters of full passes and accuracy over after examining the coexistence of ² and ² adaptorsand poly a tail fulllength transcripts were selectedduring the processes of library preparation the chimericsequences formed by the direct linkage of two cdnatemplate strands due to the low concentrations ofadaptor or smrtbell are called artificial chimeric sequences the nonchimeric sequences in the fulllength 0cliu bmc plant biology page of transcripts are the fulllength nonchimeric flncsequencesas smrt sequencing generates a high error rate it isnecessary to perform error correction iterative clustering was used first to obtain consensus isoforms and thefulllength consensus sequences from iterative clusteringfor error correction were refined using quiver [ ]moreover the raw illumina sgs reads were filtered toremove adaptor sequences and low quality reads anderror correction of lowquality isoforms was conductedusing the sgs reads with the software proovread inbriefly the short reads of illumina rnaseq data weremapped to the low quality isoforms and then the basein the low quality isoform was replaced by the particularbase that had the maximum number | Colon_Cancer |
" the activation of the nfκb pathway plays a crucial role in the progression of breast cancer bca andalso involved in endocrine therapy resistance on the contrary to the canonical nfκb pathway the effect of thenoncanonical nfκb pathway in bca progression remains elusivemethods bca tumor tissues and the corresponding cell lines were examined to determine the correlation betweenrelb and the aggressiveness of bca relb was manipulated in bca cells to examine whether relb promotes cellproliferation and motility by quantitation of apoptosis cell cycle migration and invasion rnaseq was performedto identify the critical relbregulated genes involved in bca metastasis particularly relbregulated mmp1transcription was verified using luciferase reporter and chip assay subsequently the effect of relb on bcaprogression was further validated using bca mice xenograft modelsresults relb uniquely expresses at a high level in aggressive bca tissues particularly in triplenegative breastcancer tnbc relb promotes bca cell proliferation through increasing g1s transition andor decreasing apoptosisby upregulation of cyclin d1 and bcl2 additionally relb enhances cell mobility by activating emt importantlyrelb upregulates bone metastatic protein mmp1 expression through binding to an nfκb enhancer elementlocated at the ²flanking region accordingly in vivo functional validation confirmed that relb deficiency impairstumor growth in nude mice and inhibits lung metastasis in scid micekeywords relb emt mmp1 cancer progression and breast cancer according to the latest statistic data the numbers ofnew cases and deaths of breast cancer bca accountedfor and of total cancers worldwide sincethe past decade bca incidence has been consistently increasing in developing countries such as south america correspondence yxu4696njmueducn tangjhnjmueducn2jiangsu cancer hospital jiangsu institute of cancer research theaffiliated cancer hospital of nanjing medical university baiziting nanjing p r china1department of general surgery the first affiliated hospital with nanjingmedical university guangzhou road nanjing p r chinafull list of author information is available at the end of the asia and africa owing to improved early diagnosisand advanced therapy strategies the current death ratesof bca have appreciatively decreased in the western developed countries including the united states howeverdistantan metastasis associated with endocrine therapy resistance still remains as a large obstacle to successful control of advanced bca [ ] to data bca patientswith distant metastasis at the time of diagnosis appearedto be worse prognosis with a 5year survival rate of since metastasis is the main cause of death ofbca patients the key for improving the bca survival rateis to accurately evaluate the metastatic potential for the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cwang cell communication and signaling page of guidance of clinical implementation of the individualizedtreatment plan current therapeutic strategies for bcamainly consisted of surgical reduction and radiotherapybased locally therapy with anticancer drugs providedsystemic therapy including chemotherapy endocrinetherapy and targeted therapy however the comprehensive treatment strategy is still limited and a large numberof patients eventually developed to more aggressive malignant forms that are resistant to the most of treatments[ ] thus the discovery of new biomarkers for surveillance of the disease progression and novel molecular targets for improving therapeutic efficiency are urgentlyneededthe reoccurrence ofnuclear factorκb nfκb a family of transcriptionfactors regulates immune responses and inflammation it has been welldocumented that nfκb plays a crucial role in bca endocrine therapy resistance in particular nfκb activation is thought to promote bcaprogression toward erindependent phenotypes especially fortriplenegative bcatnbc after hormone treatment [ ] mammals express five nfκb membersincluding nfκb1 p50p105 nfκb2 p52p100 rela p65 relb and crel[ ] based on the components of the signaling cascade the nfκb signaling can be activated via either canonical pathway p50rela or noncanonical pathwayp52relb [ ] the canonical nfκb signaling pathway is regarded as the central regulator of the inflammatory response but it has been extensively studied in thecontext of hormone disorders autoimmunity obesityand cancer [ ] nevertheless the molecular basisfor the activation of the noncanonical nfκb pathway awell as its role in cancer progression have been recentlyreceiving increased attention tissuematerials and methodsbca patient tissue samples and immunohistochemistryihcthe first affiliated hospital with nanjing medical university nanjing china has collected fresh tumor tissuesfrom bca patients prior to treatment and noncancerousbreast tissues the ethics committee of nanjing medicaluniversity has approved the study protocol with writteninformed consents obtained from all the patients participated in this study a total of bca cases including cases with lymphatic metastasis were selected to examine the correlation between the level of relb and bcapathological grade or stage the tissues were fixedparaffinembedded slides theslides weredewaxed by xylene and then ethanol washing off excessliquid the slides were further rehydrated by rinsingwith dh2o for ihc the slides were soaked in bsabuffer for h and then incubated with 400x diluted primary antibodies within bsa buffer at °c overnightthe slides were washed and then incubated with biotinylated secondary antibody at room temperature for min after washing the slides with 1x bps a dabsubstrate kit cell signaling tech usa was used toobserve immunostaining images under a microscopethe intensity of ihc staining was scored as negativescore weak medium and strong totalcell positivity was scored as the percentage of positivecells including no positive cell and respectively the hscore was calculated using pi i for all slides inwhich pi represented the percentage of positive and irepresented the staining intensityrelb has been originally identified in bcells and therelbbased noncanonical nfκb signaling has beenrecognized to impair antigen presentation by dcs whichis associated with skin inflammation and excessive immune cell infiltration to various ans in the past decade [] recently relb has been implicated incancer progression particularly in sex hormonerelatedcancers including bca prostate cancer pca and cervical cancer [] we have reported that relb ishighly expressed in advanced pca and enhances radioresistance speculating that the activation of noncanonicalnfκb pathway contributes to pca malignancy from androgen receptorto arindependentphenotypes [ ] the present study further demonstrated that relb highly expresses in advanced bcawhich is necessary for sustaining bca malignant progression mechanistically relb promoted bca cell proliferation emt and metastasis through transcriptionalupregulation of several oncogenic proteinsincludingbcl2 cycline d1 and mmp1ardependentlinesthe selected human breast cellcell culture and gene manipulationalllines were purchased from the american type culture collectionatcc usa and grown in the recommended mediaincluding normal breast epithelial cellline mcflines mcf7 and t47d10a erpositive bca celland tnbc cellmdamb231 and bt549relb was ectopically expressed in erpositive cellswith low constitutive relb conversely relb wasknocked out from the tnbc cells using crisprcas9approach briefly several grnas were designed usingthe optimized crispr design tool doublestrand oligo dna for each grna was cloned into theecorisite in pgl3u6 spsgrna vector the constructwas cotransfected with 331sp cas9 plasmid intomdamb231 and bt549 cells singlecell colonieswere selected using puromycin and neomycin andfurther validated by western blots dna was extractedfrom the candidate clones and confirmed by dna sequencing prior to t7en1 digestion 0cwang cell communication and signaling page of cell proliferationcell proliferation was determined using cell countingand colony formation assay respectively bca cells wereseeded into 96well plates at cellswell and treatedwith cck8 reagent dojindo mol tech japan andcell viability was measured as the optical density at nm for colony formation assay bca cells were seededinto 6well plates at cellswell and cultured for weeks the cells were fixed in paraformaldehyde for min stained with crystal violet beyotime biotech china for h after rinsing three times with1xpbscolonies were photographed andcountedcelltheflow cytometryflow cytometry was used to analyze apoptosis and cellcycle for apoptotic cell qualification à cells wereseeded in 6well plates and cultured for h the cellswere collected and washed three times with cold pbsand then stained with μm annexin vfitc and μmpi dojindo mol tech in a supplied binding buffer for min at room temperature and dark condition apoptotic cells were counted using a bd facscalibur flowcytometer bd biosciences usa and the percentage ofapoptotic cells was calculated by the number of preapoptotic and apoptotic cells divided by the amount oftotal cells for cell cycle analysis the cultured cells weredetached washed and fixed with ethanol overnightat °c the cells were then washed times with coldpbs and treated with μl propidium iodide bd biosciences usa for min the cell cycling phases weredetermined by flow cytometrycell motilitycell movability was analyzed using wound healing assayand transwell assay respectively bca cells were platedinto 6well plates at à cellswell with serumfreedmem for h after rinsing with à pbs three timesthe cultured cells were separated using pipette tips tocreate three parallel wounds the wells were thenwashed several times with pbs to remove floating cellsthe cells were continuatively cultured in the completedmedia the wound closure was monitored at and h using a microscope and the wound surface area wasquantified in addition transwell assay corning usawas used to quantify cell migration and invasion abilitiesfor migration assay the cells were plated into the upperchambers without coated membrane at à cellswellfor invasion assay the chamber inserts were coated with μl serumfree medium mixed with matrigels bdbiosci usa and solidified at °c for h the cellswere placed into the upper chamber at cellswell inboth assays the cells were plated in μl serumfreemedium in the upper chambers and μl mediumsupplemented with fbs was placed into the lowerchambers after culturing the cells for h the invadedcells on the lower surface were fixed in paraformaldehyde stained with crystal violet and counted using amicroscoperna sequencingtotal rna was isolated from bca cells using a totalrna isolation kit invitrogen mrna was converted tocdna libraries and added adapters for sequencing theprocedure of ngs sequencing on illumina hiseq platform was conducted by vazyme biotech co ltd nanjing chinachromatin immunoprecipitation chipa chipit system active motif usa was used toquantify relb binding to the nfκb enhancer elementlocated at the ²flanking region of the human mmp1gene briefly chromatin isolated from bca cells werepulled down using a relb antibody cell signalingusa unprecipitated chromatin was used as input control and chromatin pulled down by an igg antibodyserved as a negative antibody control the pulled downthe enhancer fragment was quantified using a quantitative pcr with the genespecific primerswestern blottingcytosolic and nuclear proteins were extracted from cellsand tumor tissues using a ripa lysis buffer containingpmsf and then quantified using a bca assay kit keygen biotech china the extracted proteins μg were separated on sdspage gels and thentransferred to pvdf membranes the membranes weresubsequently incubated overnight at °c with theprimary antibodies against relb bcl2 cyclin d1 and βactin santa cruz biotech usa against er ecadherin vimentin snail slug twist cell signalingtech usa thereafter the membranes were washedthree times with tbst buffer and incubated at roomtemperature for h with hrpconjugated secondaryantibody santa cruze biotech the immunoblots werevisualized using an enhanced chemiluminescence detection system biorad usa the intensities of blots werequantified using quantity one software and protein expression was normalized by loading controls such as βactin and gapdhanimal experimentthe effects of relb on tumorigenesis and metastasiswere validated using bca cells bearing mouse xenografttumor experimental models all animal studies wereconducted according to the institutional animal careand use approved by the research committee ofnanjing medical university no iacuc1711030 five 0cwang cell communication and signaling page of weekold female balbc athymic nude mice beijingvital river lab animal tech co ltd china wereused for studying tumor growth and fiveweekold female scid mice nanjing medical university chinawere used for studying tumor metastasis respectivelyfor the tumor growth experiment à bca cellswere subcutaneously implanted into the right axilla ofmice tumor volume was measured using digital calipersevery other day and calculated using a standard formulav à ab2 a and b represent the diagonal tumorlengths the mice were executed when tumor volumereached to mm3 and tumor tissues were removedfor tumor metastasis study bca cells were injectedinto mice through tail vein and assessed for lung metastasis the mice were sacrificed at weeks and the number of metastatic lung nodules was countedusage of tcga databasethe tcga bca dataset was analyzed to assess the association of rela or relb expression with bca occurrenceand the correlation between the mrna level of rela orrelb and ernegative bca patient survival ratestatistical analysisdata were presented as the mean ± standard deviationsd from at least three replicates significant differencesbetween the experimental groups were analyzed by unpaired students ttest oneway analysis of varianceanova followed by dunnetts or bonferronis multiple comparison test was performed using prismgraphpad san diego usa statistical significance wasaccepted at p additional materials and methods are in additional fileresultsrelb is correlated with bca aggressivenesswe analyzed the bca cohort in tcga database toexamine whether the expression level of relb is correlated to bca progression and patient survival the statistical analytic data indicated that the mrna levels ofrela and relb in tumor tissues were higher than theirlevels in normal breast tissues particularly the differencein relb levels appeared to be highly associated with bcaprogression fig 1a consistently the expression ofrela and relb was also associated with the overall survival of ernegative bca patients notable compared torela the high level of relb led to lower survival ratesfig 1b to assess the correlation between nfκb andbca normal breast tissues and tumor tissues from theselected bca patients with different stages were analyzedby ihc additional file table s1 compared to thenormal breast tissue control the levels of four membersof the nfκb family increase in bca tumor tissuesespecially in the ernegative phenotype notably the expression of relb is apparently correlated to the aggressiveness of bca fig 1c subsequently we furtherexamined the levels of relb in tnbc tissues vs erpositive bca tissues by western blots as expected thehigh levels of relb were detected in all the tnbc tissues suggesting the relb is inversely related to er inbca fig 1dfurthermore several bca cell lines correspondent tothe different stages of bca progression vs a normalbreast epithelial cell line mcf10a were examined consistently the high constitutive levels of the nfκb members were detected in bca cell lines particularly relbuniquely expressed at high levels in tnbc celllinesfig 2a accordingly the nfκb activity was consistently elevated in bca cells especially in tnbc cellsfig 2b furthermore erpositive mcf7 cells vstriplenegative mdamb231 cells were used to measure cytoplasmic and nuclear levels of the nfκb members as expected all the members highly increased inmdamb231 cells compared to mcf7 cells importantly the nfκb upstream ikkα and its phosphorylatedlevels also heighten in tnbc cells fig 2c the distribution of the nfκb members was further confirmed byconfocal microscope fig 2d taken together these results indicated that the high level of relb is associatedwith aggressiveness of bca suggesting that relb maycontribute to the advanced bcarelb promotes bca cell proliferationto investigate the potential mechanism by which relbregulates downstream gene expression involved in bcaprogression relb was ectopically expressed into mcf7and t47d cells with low levels of constitutive relbfig 3a in parallel relb was knocked out from tnbccells mdamb231 and bt549 using a crisprcas9gene edition system additional file figure s1 fig 3bthe nfκb activity was slightly elevated in the relboverexpressed ernegative cells but significantly reduced in relbknocked out tnbc cells fig 3c and dconsistently the elevated relb in erpositive cells led toincreased cell colony number and the cell proliferationrate fig 3e g and i conversely cell colony formationand the cell proliferation rate significantly decreased inrelbknocked out tnbc cells fig 3f h and jrelb promotes g1s transition and inhibits apoptosisto elucidate the role of relb in bca cell proliferationthe cell cycling process and the cell apoptotic rate wereanalyzed by flow cytometry the enforced expression ofrelb in mcf7 cells resulted in decreasing g1 phase butincreasing s and g2m phases which enhances the acceleration of cell division fig 4a in contrast knockout of relb in mdamb231 cells led to an increasing 0cwang cell communication and signaling page of fig see legend on next page 0cwang cell communication and signaling page of see figure on previous pagefig the correlation between relb and aggressiveness of bca a b tcga database geo accession gse11121 was analyzed to examine thecorrelation between mrna expression profiles of rela and relb and bca occurrence and over survival of patients c bca tumor tissues withdifferent pathological grades and normal breast tissues were screened by ihc using nfκb antibodies the luminal group was characterized aserpositive and her2negative lowgrade genotype her2 group was assessed as a middlegrade genotype with high tumor growth tnbcgroup was determined as a high grade d the levels of relb in tnbc tumors vs erpositive bca tumors were quantified βactinnormalizedimages were plottedfig the nfκb activation in bca cell lines a several corresponding cell lines were selected to confirm the relationship between nfκb proteinsand the aggressiveness of bca cells b the relative nfκb activity was quantified in the bca cell lines c cytoplasmic and nuclear levels of nfκbproteins in erpositive mcf7 and tnbc mdamb231 cells were quantified accordingly the nfκb upstream phosphorylated ikkα levels in thetwo cell lines were measured d the cellular distribution of the nfκb proteins in the two cell lines were further examined using aconfocal microscope 0cwang cell communication and signaling page of fig see legend on next page 0cwang cell communication and signaling page of see figure on previous pagefig determination of relbenhanced bca cell survival and growth a relb was ectopically expressed in erpositive bca cells by stablytransfected a human relb cdna construct b relb was knocked out in tnbc cells using a crisprcas9 gene edition system c d the relative nfκb activity was quantified in relbmanipulated bca cells e f the survival rates in the relbmanipulated bca cells were examined by colonyformation assay gj the proliferation rates in the relbmanipulated bca cells were quantified by cck8 assay mean ± sd was representative ofthree independent experiments carried out in duplication p p show the significances between two groups as indicatedg1 phase but decreasing s phase fig 4b furthermoreapoptosis was examined in the relbmanipulated bcacells although the elevated relb in mcf7 cells unlikelychanged the cell apoptotic rate the depletion of relb inmdamb231 cells obviously increased apoptotic cellrates fig 4c and d consistently the overexpression ofrelb in mcf7 cells resulted in upregulation of cyclind1 but no effect on bcl2 expression fig 4e additionally knock out of relb in mdamb231 cells led to suppression of cyclin d1 and bcl2 suggesting that relbsustains tnbc cell growth via activation of prosurvivaland antiapoptotic pathways fig 4f moreover relb depletion led to increases in p21 and p27 but decreases incmyc cyclin e1 and bclxl additional file table s2additional file figure s2relb enhances bca cell mobility by enhancing emtit has been widely recognized that bca progression isgenerally through the erdependent phenotype to erindependent malignancyimportantly emerging evidence demonstrated the inverse relationship between erand nfκb in bca cells consistently the expression of relb appeared to be inversely correlated to erlevels in the tested cell lines additional file figures3a furthermore er elementsdriven luciferase reporter gene expression construct was constructed toexamine whether relb is able to regulate the er response expectedly the erreporter response was remarkably reduced due to the increased relb in mcf7cells suggesting that relb negatively regulates er signaling additional file figure s3bfurthermore to testify whether relb contributes toestrogendeprived bca progression the effect of relb onthe metastatic ability of bca cells was examined sincethe emt process has been well documented in bca metastasis several emt markers were measured in ourpanel of bca cell lines the elevated relb in erpositivecells led to decreasing ecadherin but increasing snail islug i twist i and vimentin fig 5a in parallel knockout of relb in tnbc cells resulted in decreasing thoseemt markers while ecadherin was not detectable intnbc cells fig 5b accordingly the effect of relb onthe cell motility was analyzed by quantifying cell capacities of wound healing migration and invasion as expected the enforced expression of relb in mcf7 cellssignificantly enhanced the cell capacity for wound recovery compared to a vehicle control fig 5c consistentlythe elevated relb in mcf7 cells also resulted in enhancing cell migration and invasion fig 5e subsequentlyknock out of relb in mdamb231 cells led to decreases in those capacities fig 5d and f these resultssuggest that relb promotes erindependent bca progression mainly through activating emt processrelb upregulates bone metastasis associated proteinmmp1to identify the important relbregulated genes involvedin bca metastasis we applied rnaseq analysis usingtotal rna isolated from relbknocked out mdamb cells vs the parent cells the results indicated thatthe mrna expression profiles of numerous genes werealtered in the relbknocked out cells fig 6a and b importantly multiple genes relating to metastatic signalingpathways were downregulated fig 6c and table among of them mmp1 an activator in bca bone metastasis was highly related to the level of relb toassess the effect of relb on mmp1 expression we verified the expression level of mmp1 increased in relboverexpressed mcf7 cells but decreased in relbknocked out mdamb231 cells suggesting that relbmay directly regulate the mmp1 gene fig 6dto elucidate how relb regulates mmp1 expressionwe identified a putative nfκb element located in the²finking region of the human mmp1 gene for verifying this nfκb element functional response to transcriptional activation by relb a ²flanking region containingthe nfκb enhancer element and the core promoter inthe human mmp1 gene was cloned into a pgl4 vectorto drive the luciferase reporter gene expression compared to the basic vector control the reporter gene response driven by the mmp1 enhancer highly increasedin relboverexpressed mcf7 cells but significantly decreased in relbknocked out mdamb231 cellsfig 6e moreover to confirm that relbmediated transcriptional activation through binding to the nfκb enhancer element located at the ²flanking region of themmp1 gene a 240bp fragment containing the nfκbenhancer element was pulled down using a specific relbantibody and amplified using pcr the amount ofpulleddown dna fragment from mcf7 cell chromatinwas less than the level from mdamb231 cell chromatin fig 6ffurthermore to validate whether the increased mmp1can further promote emt mmp1 was manipulated by 0cwang cell communication and signaling page of fig the effect of relb on cell cycle and apoptosis a b the cell cycling process in relbmanipulated bca cells was analyzed using flowcytometry c d preapoptotic and apoptotic cells in the relbmanipulated bca cells were quantified by flow cytometry e f the levels of bcl2 andcyclin d1 proteins were measured by western blots statistical significance between two groups as described in fig 0cwang cell communication and signaling page of fig the effect of relb on emt a b the emtassociated markers in relbmanipulated bca cells were measured by western blots c d the cellwound healing abilities were analyzed e f the cell a migration and invasion capacities were examined using a transwell assay statisticalsignificance between two groups as described in fig 0cwang cell communication and signaling page of fig see legend on next page 0cwang cell communication and signaling page of see figure on previous pagefig relbmediated transcriptional regulation mrna expression profiles in the relbknocked out mdamb231 cell vs the parent cell wereexamined using a rnaseq platform a the distribution of mrna profiles in the relbdeprived cell was illustrated b cluster analysis of rnaseqdata indicates updownregulated mrna profiles heatmap c kegg pathway enrichment analysis was performed to assess emtmetastasisrelating mrna profiles d the protein level of mmp1 was quantified in the relbmanipulated bca cells e a ²flanking region of the human mmp1gene mmp1 containing the nfκb element underline and the core promoter region was cloned in pgl4 vector to drive the luciferasereporter gene expression as illustrated the enhancer activity modulated by relb in bca cells was estimated by normalized reporter responses f asmall dna fragment containing the nfκb element was pulled down from chromatins using a relb antibody and then quantified by pcr iggserves as a negative antibody control statistical significance between two groups as described in fig overexpressing in mcf7 cells but silencing in mdamb231 cells the elevated mmp1 in mcf7 cells led toa slight decrease in ecadherin but increases in snail twist1 and vimentin in contrast the silence of mmp1in mdamb231 cells resulted in decreasing snail twist slug and vimentin regardless of the undetectable of ecadherin in the cells additional file figures4a in addition to determine the functional contribution of mmp1 to emt under the relb regulationmmp1 was either ectopically expressed in relbknockedout mbamb231 cells or wassilenced in relboverexpressed mcf7 cells intriguingly the elevatedmmp1 in relbknocked out mbamb231cells resultedin partially restored the emt proteins while the silenceof mmp1 in relboverexpressed mcf7 cells was ableto abrogate the elevated emt proteins except for no effect on twist expression additionalfile figures4brelbmediated bca progression was validated in micetwo mouse xenograft tumor experimental models wereused to validate the effects of relb on tumor growth andmetastasis in vivo relbknocked out mdamb231 cellsand the parent cells were subcutaneously injected intothe front leg area of nude mice one week after injection tumors were formed and continuously grew toreach the maximum volume mm3 compared toinjection of the control cells tumor formation by therelbknocked out cells was approximately week late month after injectionthe tumor volumes in thecontrol group reached the maximal volume howeverthe tumor growing speed and tumor weight wereremarkably reduced in the group injected with the relbknocked out cellsfig 7ac according to relbknocked out bcl2 and cycline d1 in the tumor tissueswere significantly reduced fig 7d which just reflectedto in vitro results as shown in fig furthermore the reduction in the metastatic capacityof the relbknocked out mdamb231 cells was validated by injecting the cells through tail vents of scidmice six weeks after the injection the tumor occurredon the lung of mice injected with the control cells however the incidence of metastatic lung tumors was dramatically reduced in the relbknocked out groupfig 7e the excised tumor tissues were analyzed byihc analysis using the relative antibodies compared tothe control group tumor aggressiveness stain ki67 wassignificantly reduced in the relbknocked out groupconsistently the levels of mmp1 and emt markerswere also reduced in relbknocked out group fig 7fadditionally tumor images by hematoxylin and eosinhe staining confirmed that the metastasized tumorstable mrna expression profiles in mdamb231 cellsgene namevimrelbkoctrlcdh11zeb1snai1mmp1mmp9mmp28junhsp70dmbt1smad2log2relbkoctrlupordowndowndowndowndowndowndowndownupupupunchangedp value500e05500e05500e05500e05vim vimentin cdh11 cadherin zeb1 zinc finger eboxbinding homeobox snai1 snail1 mmp1 matrix metallopeptidase mmp9 matrix metallopeptidase mmp28 matrix metallopeptidase jun jun protooncogene ap1 transcription factor hsp70 heat shock protein dmbt1 deleted in malignant brain tumor tumor suppressor smad2 a member in tgfbsmad signaling family 0cwang cell communication and signaling page of fig see legend on next page 0cwang cell communication and signaling page of see figure on previous pagefig validation of relbenhanced tumor growth and metastasis in mice a relbknocked out mdamb231 and the parent cells weresubcutaneously injected into the right axilla of female nude mice and allowed to form tumors thirty days after bca cell implantation tumortissues were removed out prior to the execution the excised tumor tissues were photographed two out of five mice injected with relbknockedout cells didnt form the tumor b c tumor growth rates and the excised tumor weights were measured and plotted d total proteins wereextracted from the tumor tissues and immunoblotted with the relating antibodies βactin served as an internal control e female scid mice wereinjected with relbknocked out mdamb231 and the parent cells through the tail vein after weeks the mice were sacrificed and lungmetastasis was examined the excised lungmetastasized tumors indicated by arrows were photographed f the metastatic lung tissues werescreened by ihc with relative antibodies incorporated with ki67 staining the relative images were plotted g he staining was applied toconform the lung metastatic tissues and the numbers of micrometastasis in the two groups were plotted statistical significance between twogroups as described in fig were obviously reduced in the relbknocked out groupcompared to the control group fig 7g it should benoted that the detection of low levels of relb in therelbknocked out group reflected the constitutive mouserelb additionallythe enforced expression relb inmcf7 cells was still insufficient to tumor metastasizeto the lung altogether these results suggest the noncanonical nfκb pathway may serve as a major contributor to the progression of malignant bcadiscussionit has been widely recognized that bca is | Colon_Cancer |
" in head and neck cancer hnc the relationship between a delay in starting radiotherapy rt andthe outcome is unclear the aim of the present study was to determine the impact of the amount of time beforetreatment intervention tti and the growth kinetics of individual tumors on treatment outcomes and survivalmethods two hundred sixtytwo hnc patients with primary tumors treated with definitive chemo rt wereretrospectively analyzed the tti was defined as the time interval between the date of histopathologic diagnosisand the first day of the rt course volumetric data on tumors were obtained from diagnostic and rt planningcomputer tomography ct scans in order to calculate the tumor growth kinetic parametersresults no significant association between locoregional control or causespecific hazards and tti was found thelog hazard for locoregional recurrence linearly increased during the first days of waiting for rt although this wasnot significant the median tumor volume relative increase rate and tumor volume doubling time was 32dayand days respectively and neither had any impact on locoregional control or causespecific hazards the association between a delay in starting rt and the outcome is complex and does not harm allpatients waiting for rt further research into imagingderived kinetic data on individual tumors is warranted inorder to identify patients at an increased risk of adverse outcomes due to a delay in starting rtkeywords head and neck cancer radiotherapy waiting time treatment delay outcome with new cases and deaths reported in head and neck cancer hnc is the eighth mostcommon and most lethal cancer in men worldwide in addition to surgery and systemic therapy radiotherapy rt is one of the cornerstones for treatment of thiscancer owing to the rising cancer incidence rate in ageing populations and the widening list of indications forirradiation the demands for rt have increased dramatically over the past decades [ ] the increasing complexity of pretreatment diagnostics and rt technology correspondence pstrojanonkoisi1department of radiation oncology institute of oncology ljubljana zaloÅ¡ka si1000 ljubljana slovenia4chair of oncology faculty of medicine university of ljubljana ljubljanasloveniafull list of author information is available at the end of the has led to delays in treatment decisionmaking and thereduction in linear accelerator throughputthat hasresulted in a significant imbalance between the demandfor rt and the availability of rt capacities in manypublically funded health systems this is also the case inslovenia [ ]due to obvious ethical reservations the only way tostudy the impact of delays in starting rt on treatmentoutcomes are retrospective observational analyses of cohorts from different institutions or countries intuitively one would expect that the prolongation of thetime taken before treatment intervention tti is harmful to patients both the likelihood of tumor growth andthe acquisition of a metastatic phenotype increases as afunction of time furthermore advanced tumors aremore difficult to treat than smaller tumors the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cžumer radiation oncology page of indeed a systematic review of pertinent literature fromthe period by chen showed an increasein the risk of local hnc recurrence of for everymonth of delay in definitive rt however certainstudies included in chens metaanalysis and also somemore recent reports negated the association betweenthe delay in definitive rt and the increased risk of treatment failure [] several different biases inherent inretrospective analyses either related to the quality ofdiagnostic procedures and treatment or to the inhomogeneity of the studied population as well as a selectionbias ie patients with fasttumor progression or ahigher burden of symptoms receive priority in treatment and significant variability in the kinetics of individual hnc cases may abolish the effect of tti inoutcomes [] however if patients with advancedor fastgrowing tumors have to wait longer the magnitude of this effect may be overestimated furthermore no compelling relationship between treatmentdelay and prognosis was found in some other cancertypes []in order to determine what would be an acceptabletti in hnc patients treated with definitive rt or concurrent chemoradiation we aimed to analyze the impactof tti and growth kinetics of individual tumors on theoccurrence of localregional failure distant metastasisand survival in the present study of a cohort of slovenepatients with hncmethodsin a retrospective study patients with oral cavity oropharyngeal hypopharyngeal or laryngeal squamous cellcarcinoma scc who were treated with curativeintentdefinitive rt with or without concurrent chemotherapybetween january and december at the institute of oncology ljubljana slovenia were includedpatients with t1n0 or t2n0 glottic cancer were left outof this cohort the period was chosen because of fluctuations in the waiting time for irradiationas a result of intensive renovations and expansion in thedepartment of radiotherapy that took place over thistime span from patients medical and rt charts wecollected information on clinical gender age onset ofsymptoms date and type of disease recurrence anddeath tumor histology site of origin tnm stage andtreatment characteristics rt technique regimen anddose duration of rt type of concurrent chemotherapy[cct] and the number of cycles administered thetnm stage was determined according to the 7th editionof the uicc classification systemfor analysis of the impact of tumor growth kinetics ontreatment outcomes the volumes ml of primary tumors and neck nodal metastases as marked on diagnostic and rt planning computer tomography ct scanswere compared patients with the same basic clinicaldisease and treatment characteristics as indicated abovebut with both sets of ct scans available were selectedfor this part of the study diagnostic ct scans were performed through the acquisition of mm thin ct sections whereas planning ct scans had a slice thicknessof mm both with intravenous iodine contrast enhancements sets with extensive artefacts were excluded forthe purposes of rt planning patients were positionedsupine on the flat tabletop and a fivefixation pointthermoplastic mask was used lymph nodes were considered positive if the smallest diameter was more than cm andor the necrotic center or extracapsular extension was seen if available segmentation was guided bymagnetic resonance imaging mri sets and the resulting contours around the primary tumors and metastaticneck nodes represented a consensus between two radiation oncologists and a radiologist volumes of primarytumors and metastatic neck nodes were separately calculated by a computer software program used for rt planning xio computerized medical systems inc stlouis usa eclipse varian medical systems inc paloalto usa the end points in this part of the study werechanges in the tumornodal volume and tnm stage thecalculation of the primary tumor volume doubling timeand their impact on the treatment outcomestatisticsthe study protocol was approved by the republic ofslovenia national medical ethics committee no for retrospective studies a written consentis deemed unnecessary according to national regulationsbasic descriptive statistics were reported with meansstandard deviations and ranges for numerical variablesand as percentages for categorical variables in patientswith two simultaneous hncs some characteristics werereported in regards to patients while others were reported in regards to tumorsthe survival curves were computed using the kaplanmeier estimator and the aalenjohansen estimator wasused to estimate the cumulative probabilities of competing risks the effect of covariates was analyzed using amultiple cox regression analysis with all the analysesthe data were censored at a fiveyear followupwhen focused on the survival of patients the analyseswere completed with patients as the units and the timewas measured from the first day of therapy until deaththe overall survival os regardless of the cause ofdeath and the absolute risk cumulative probability ofdying due to index cancer were reported in the cox regression only the index cancer deaths causespecifichazard csh were considered to be events of interestin the analyses where locoregional control lrc wasof primary interest the calculations were performed in 0cžumer radiation oncology page of regards to tumors excluding the nonresponders to rtie those with residual local or regional tumors at weeks after rt completion for the latter group weconcluded that it is the radioresistance of tumor cellsthat are responsible for the persistence of the diseaseafter therapy and not that patients had to wait for rtthe followup time was calculated from the last day oftherapy the estimated cumulative probability of localandor regional recurrence distant metastases lrcprobability of being still alive and without local andorregional recurrence and diseasefree survivaldfsprobability of being still alive and without events locoregional and distant failure and deaths were the events ofinterest were reported all the analyses were conductedin regards to the tumors as independent units this assumption was checked in the sensitivity analysis andallowed for gamma frailtythe assumptions of the cox regression were checkeda nonlinear effect a spline with degrees of freedomwas allowed for the numerical variables and the proportional hazards assumption was tested using schoenfeldresidualsthe tti was defined as the time interval between thedate of histopathologic diagnosis and the first day of thert course tumor growth kinetics was expressed as thetumor volume relative increase rate per day and as thetumor volume doubling time in daysthe tumor volume relative increase rate per day was 13 calculated as 12v t 2ðþþv t 1ðt2 t1where vt1 gross tumor volume at time t1 ieon diagnostic ct scans and vt2 gross tumor volume at time t2 ie on planning ct scans it was reported as the percentage increase was reported as per day the tumor volume doubling time was calculated asðv t 2ðv t 1ðln 2ð þ t t þþlnþsince a onetoone relationship existed between thetwo only the tumor volume relative increase rate whichrequires no extrapolation was considered for modellingall analyses were performed using r statistical software version and a pvalue below was considered statistically significantresultsimpact of time to treatment initiationbetween and patients with oral cavity oropharyngeal hypopharyngeal or laryngeal primarysccs were treated with definitive chemo radiotherapywith curative intent there were men and women aged years mean the majority oftumors originated in the oropharynx tumors in patients and were tnm stage iv tumors in patients the tti ranges from to days with mean days the distribution of the tti is only slightlyasymmetric with median and interquartile rangeiqr fig patients were irradiated with dimensionaltechnique or 3dimensionalconformal isocentric technique to the median rt dose gy iqr delivered in gy dailyfractions iqr concurrently to rt patientsreceived chemotherapy consisted of platinbased monochemotherapy patients or mitomycinecbleomycin combination patients the characteristics of patients and their tumors are shown in table treatment outcome and survivalclinical andor radiologic assessment at weeksposttherapy confirmed disease persistence locally andor regionally in cases ie in patients and thesepatients were excluded from further analyses of lrcand dfs thus patients with tumors were analyzed and during followup a local andor regional relapse was recorded in cases with a mediantime from treatment completion of months range months the two and fiveyear probability of localrecurrence after the end of treatment was and respectively whereas the regional relapse was and respectively after threeyears posttherapy we only observed a small increase ie inlocal recurrence probability whereas the probability ofregional relapse was stable after the third year followupthe probability of occurrence of distant metastases at years was and at years it was at the second and fifthyear followup the lrc was confidence interval [ci] and ci respectively and the dfs was ci and ci respectivelyin the fifth year posttreatment out of patients had died the index cancer was the reason in of the cases during the first years posttherapythe probability of cancerrelated death steeply increasedfrom zero to but later the changes were less pronounced at the second and fifth year posttreatment theos independent of the cause of death was ci and ci respectivelyeffect of covariatesin the univariate analysis the following parameters weretested for lrc and csh age gender stage of diseasetype of treatment rt vs rt cct and tti for rtas continuous variable results are presented in table 0cžumer radiation oncology page of fig distribution of ttitable clinical characteristics of patients and their tumorscharacteristicsgenderno femalemaleage yearsatumor locationboral cavityoropharynxhypopharynxlarynxtumor stagebstage istage iistage iiistage ivtreatmentbradiotherapyconcurrent chemoradiotherapyrt duration daysatime to treatment interventiona ± ± ± the same parameters were also included in the multivariate model table the occurrence of locoregional recurrence was significantly related to the diseasestage p whereas the relationship with agewas only of marginal significance p higherage had a lower hazard no significant associationwith the type of treatment could be found p table the log hazard for locoregional recurrenceseemed to linearly increase during the first days oftable effect of covariates on locoregional control and indexcancerspecific hazard n patients with tumors anunivariate analysisparameterlocoregional control cihrpvaluetnm iii vs iiitnm iv vs iiiagettigendertherap cct vs rtcause specific hazardtnm iii vs iiitnm iv vs iiiagettigendertherapy cct vs rt a mean ± sd rangeb eleven patients had two simultaneous primary tumorsci confidence interval tti time to treatment interventioncct concurrent chemoradiotherapy rt radiotherapy 0cžumer radiation oncology page of table impact of tti on locoregional control and indexcancerspecific hazard n patients with tumors amultivariate analysisparameterlocoregional control cihrpvaluetnm iii vs iiitnm iv vs iiiagettigendertherapy cct vs rtcause specific hazardtnm iii vs iiitnm iv vs iiiagettigendertherapy cct vs rt ci confidence interval tti time to treatment interventioncct concurrent chemoradiotherapy rt radiotherapywaiting for rt although the association between thehazard and tti wasregardless ofwhether we made an allowance for nonlinearity ornot fig 2ainsignificantthe hazard of dying due to the index cancer ie cshwas found to be favorably associated with a lower disease stage p and the addition of cct to rtp whereas age gender and tti did not reachthe level of statistical significance table fig 2bassumptions and sensitivity analysisproportional hazards for all included variables and thelinearity assumption for age were analyzed and presented graphically some indication of nonproportionalhazards could be found in gender but allowing for nonproportional hazards did not change the interpretationof the other covariates as a part of the sensitivity analysis the assumption of the dependence of the tumorsbelonging to the same individual was relaxed but nochanges in the interpretation of the results could be observed as an additional confounder the timedependentcovariate duration of rt was considered but its effectdid not prove to be importantimpact of tumor growth kineticsdiagnostic and planning ct scans median interval days range days were available from patientsfive of these patients had two primary tumors the majority of patients were males with a meanage of years range and the patients hadprimary tumors located in the oropharynx table when two ct sets were compared the original tstage of the primary tumor was increased ie upgradedin two cases and the nodal stage was increased in six patients due to the limited number of npositive casesn only volumes of primary tumors werecompared between the two ct sets for the calculationof the tumor volume relative increase rate per dayand tumor doubling time the absolute increase intumor volume ranged from to cm3 per daymedian no increase in volume was observed infive patients ct scans in these five patients were takenfig trend of the hazard for locoregional recurrence a and for index cancerspecific death b 0cžumer radiation oncology page of table characteristics of patients with available diagnostic andplanning ct scanscharacteristicsgenderno femalemaleage yearsatumor locationboropharynxhypopharynxlarynxstagebstage istage iistage iiistage ivtherapybradiotherapyconcurrent chemoradiotherapytumor volume relative increase rate dayatumor volume doubling time daysaa mean ± sd rangeb five patients had two simultaneous primary tumors from days to days apart median days the median tumor volume relative increase rate was perday median range and the mediantumor volume doubling time was days range days no difference was observed when these two parameters were compared between different tumor stagestreatment outcome and survivalamong the tumors the time taken for local andorregional relapse and the occurrence of distant metastaseswere assessed for tumors in which treatment resultedin clinicalradiological eradication of the disease at weeks posttherapy local andor regional relapse wasrecorded in nine cases with a median time fromtreatment completion of months range monthsthe two and fiveyear probability of local recurrenceafter the end of the treatment was and respectively whereas for the regional relapse it was and respectively at years posttherapy only anincrease in the local recurrence probability of wasobserved whereas the probability of regional relapseremained stable the two and fiveyear probability ofdistant metastases was at two and years thelrc was ci and ci respectively and the dfs was ci and ci respectivelyin the yearperiod after the start of treatment outof patients died the index cancer was thereason in cases at two and years posttreatment the os rates were ci and ci respectivelyeventseffect of covariatesdue to the low number of events local andor regionalrecurrence ninecancerspecific deaths events only univariate regression models were fittedthe following covariates were tested in the modelsoverall disease stage initial tumor volume tumor doubling time and relative increase rate day of primarytumor volume measures of tumor kinetics did not showany impact on lrc or csh only an inverse relationshipbetween the initial primary tumor volume and csh wasobserved hr for index cancerspecific death perevery cm3 increase in the volume of primary tumorstable there was no difference in the value of dfsbetween patients with a primary tumor volume relativeincrease rate 1day and 1day p fig discussionwhile it is intuitively anticipated and confirmed by theresults of the metaanalysis that a delay in starting rtwould have a negative impact on the treatment of patients with hnc this association was not confirmed inour study we only found a statistically insignificantupward trend in the risk of locoregional recurrence forthe first days of rt delay in addition the differencesin the growth kinetics between individual tumors whichwere studied in a smaller group of patients were considerable but did not appear to be of significance for theprediction of treatment outcomesobviously the relationship between tti and diseaseprognosis in patients with hnc is more complex than itmight seem at first glance the first negative impact ofwaiting for treatment to begin is the risk that the tumorwill increase in size andor metastasize during this timemaking it harder to treat or resulting in it becoming untreatable [ ] however the time for a tumor to growis only one of the factors that determines prognosis andthe absence of a statistically significant association between tti and the treatment outcome in our study andis thus not surprising []many other reportsmoreover no obvious methodological differences couldbe found between these negative studies and the positive studies that confirmed the association between ttiand treatment outcomes [] in both groups thereare individual studies that have similar sample sizestumor sitestage mix and periods covered speaking infavor of comparable quality of diagnostics therapy andstatistics across the studies 0cžumer radiation oncology page of table effect of stage and tumor kinetics on locoregional control and overall survivalparameterstnm stageiv vs iiiivtuper cm3vtu relative increase rateper dayvtu relative increase rate¤ vs 1dayvtu volume of primary tumorlocoregional controlhr cipvaluecause specific hazardhr cipvalueon the contrary in more recently reported analyses ofthe national cancer registries data an adverse effect ofwaiting for radiotherapy was clearly established []however the results from this type of analysis should betaken with caution not only due to the limitations inherent in the tumor registry data unmeasured confounding selection biasincomplete data and codingerrors but also because the effect of delaying rt oncancerspecific outcomes was not evaluated in additionpatients irradiated in postoperative and definitive settings were not analyzed separately overall the researchmethodology and interpretation used in these studieswere criticized and the magnitude of the effect that theysupposedly demonstrated was questioned in the present study a linear increase in the log hazardfor locoregional recurrence was found during the first days of waiting for rt although it was not statisticallysignificant it is possible that unknown confounders thatwere not accounted in our analysis eg tumor growth kinetics reduced the statistical power of the tti the resultsfrom fortin and naghavi who reported on theincreased risk of locoregional failure with tti daysand days respectively [ ] are the most comparable to our own however optimal ttithresholdsfig impact of primary tumor volume relative increase rate to diseasefree survival in patients with no residual disease at weeks posttherapy 0cžumer radiation oncology page of identified in different studies showed considerable variations pointing to the uncertainty of such calculations andtheir dependence on the characteristics of the analyzedpopulation [ ] the possible role of classical prognostic factors such as location of primary tumor diseasestage and addition of cct is not expected to be relevant inthis respect as in our and other similar studies the statistical significance of tti was verified by multivariateanalysisthe effect of tti on treatment outcomes however isnot only conditioned by the duration of waiting for rtbut also on the rate of tumor growth in hnc a vast heterogeneity in tumor cell kinetics has been observed conditioned by the local milieu from which it arises whichdiffers from patient to patient [ ] historicallydifferent methods were explored to evaluate tumor cellkinetics but did not succeed in providing clinically relevant kinetic parameters [ ] more recently a comparison of two sets of imaging data acquired at twodifferent time points was successfully employed for thispurpose usually volumetric data are extracted from diagnostic and rtplanning ct scans for the calculation ofdifferent parameters reflecting the rate of tumor growtheg tumor volume doubling time or absolutepercentagetumor volume increase per day despite some differencesin the calculation methodology across studies a large variation in the individual values of kinetic parameters wasseen in all of them including ours indicating that all ofthe studied populations represent a unique mix of slowand fast growing tumors [] among our patientsfive had no measurable increase in primary tumor volumeeven when the interval between ct scans was up to days the inverse relationship between kinetic parametersdetermined by the comparison of two ct datasets andtreatment outcomes was implied or even confirmed inseveral smaller studies pointing to potential clinical utilityof imagingderived kinetic data [] in our grouphowever no such association was found a small numberof patients and in contrast to other studies the inclusion of different tumor sites could contribute to the negative result as well as differences in the changes in kineticproperties triggered by rtccr in individual tumors tumor radiosensitivity may also influence the effect oftti on outcome while to some extent it may be evaluated before rt begins eg by molecular profiling identification of hypoxic cells and the determination of hpvstatus in oropharyngeal primary tumors in daily clinicalpractice it is usually not considered when planning treatment [ ] in order to diminish the impact of intrinsic tumor radiosensitivity the patients in our study withresidual disease at weeks postrt were excludedfrom the analysis of lrc we hypothesized that the inability to achieve a complete tumor response to chemoradiation was due to the radiobiological characteristicsof the disease and not the delay in starting rt howeverthe exclusion of these patients from the analysis did notaffect the end resultsour study has weaknesses which are mostly due to itsretrospective nature however for obvious ethical reasons this is an inevitable feature of studying delays inthe initiation of cancer therapy we are aware that thereis always some doubt as to the accuracy of the diagnosticprocedures the staging the quality of planning and theimplementation of rt in the case of retrospective research nonetheless the same restraint exists in the caseof other similar studies and even more so in the case ofanalyses of cancer registry datasets an important feature of our data set is that no patients were lost duringthe followup and in our opinion is free from manyhidden biases that larger studies inevitably bring withsince almost half of our patients had oropharyngeal carcinoma missing information regarding the p16 or human papillomavirus hpv tumor status could be ofimportance howeverin the cohort of oropharyngealcancer patients treated at our institution between and only had a hpvrelated tumor thus we reasonably assume that the impact of p16hpv tumor status on the study results was negligible inaddition in hpvpositive cases perni found nosignificant association between tumor growth velocitycalculated from serial pretreatment ct scanslocaland distant control or os and the same was reportedby chu who measured metabolic growth velocityusing pretreatment petct scans [ ]other drawbacks to consider include technical limitationsin ct scan acquisition the accuracy of presentation of actual tumor volume on ct images and the precision of thedelineation of gross tumor volume to minimize errors inthe estimation of comparative tumor volumes only highquality pairs of ct images were selected for the analysis oftumor kinetics in addition physical findings documentedin clinical records and when available diagnostic mriswere used for this purpose and labelled tumor volumes represented a consensus between two experienced radiationoncologists and a radiologist all dedicated to hnc management like many other studies [ ] the comorbidity burden was not registered in our patientsalthough it should be taken into account when assessingsurvival outcomes finallylags in the prebiopsyperiod were not addressed in our study including patientdelay and delays in referral and diagnostics which may addsignificantly to the total tti these are also costly and potentially fatal but can be successfully reduced by effectivecoordination between providers []sthe relationship between tti and treatment outcomesis multifaceted so the controversy of published results is 0cžumer radiation oncology page of not surprising in this study we found that delays in theonset of rt do not harm all patients as tti is a problem in many public health systems further research iswarranted and should focus on two areas evaluatinglarge population surveys with highquality data andtreatmentrelated outcomes not just os and the prognostic relevance of imagingderived kinetic data of individualtumors which appeared promising in severalsmaller and statistically underpowered studies in orderto obtain a tool to identify patients at increased risk oftreatment failure due to delays in starting rt in a situation without clear knowledge to whom waiting for irradiation is harmful the only possible recommendationcould be that the waiting time for rt should be asshort as reasonably achievable asara abbreviationshnc head and neck cancer rt radiotherapy tti time to treatmentintervention scc squamous cell carcinoma cct concurrent chemotherapyct computer tomography mri magnetic resonance imaging os overallsurvival csh causespecific hazard lrc locoregional control dfms distantmetastasisfree survival dfs diseasefree survival iqr interquartile rangeci confidence interval hpv human papillomavirusacknowledgementsthis study was financially supported by the slovenian research agencyprogram no p30307authors contributionsstudy concepts žumer b strojan p study design žumer b pohar perme mstrojan p data acquisition all authors quality control of data strojan p dataanalysis and interpretation pohar perme m strojan p statistical analysispohar perme m manuscript preparation all authors manuscript editingstrojan p manuscript review žumer b pohar perme m strojan p the authors read and approved the final manuscriptfundingthis study was financially supported by the slovenian research agencyprogram no p3availability of data and materialsthe datasets analyzed during the current study are available from thecorresponding author on reasonable requestethics approval and consent to participatethe study protocol was approved by the republic of slovenia national medicalethics committee no all patients gave consent for using theirdata for study purposes at the start of their treatment for retrospective studies awritten consent is deemed unnecessary according to national regulationsconsent for publicationthe republic of slovenia national medical ethics committee approved thestudy which was conducted in accordance with the ethical standards laiddown in an appropriate version of the declaration of helsinki theneed for consent was waived by the republic of slovenia national medicalethics committeecompeting intereststhe authors declare that they have no competing interestsauthor details1department of radiation oncology institute of oncology ljubljana zaloÅ¡ka si1000 ljubljana slovenia 2institute of biostatistics and medicalinformatics faculty of medicine university of ljubljana ljubljana slovenia3department of radiology institute of oncology ljubljana ljubljanaslovenia 4chair of oncology faculty of medicine university of ljubljanaljubljana sloveniareceived may accepted august referencesbray f ferlay j soerjomataram i siegel rl torre la jemal a global cancerstatistics globocan estimates of incidence and mortality worldwidefor cancers in countries ca cancer j clin world health anization who life expecta | Colon_Cancer |
" mirnas regulate a multitude of cellular processes and their aberrant regulation is linked to humancancer however the role of mir4255p in lung cancer lca is still largely unclear here we explored the role ofmir4255p during lca tumorigenesismethods cell proliferation was evaluated by cell counting kit8 and colony formation assay western blot and realtime pcr were accordingly used to detect the relevant proteins mirna and gene expression luciferase reporterassays were used to illustrate the interaction between mir4255p and ptenresults we demonstrate that mir4255p is overexpressed in lca tissue and enhances the proliferative and colonyformation capacity of the lca cell lines a549 and ncih1299 through predictive binding assays pten wasidentified as a direct gene target and its exogenous expression inhibited the procancer effects of mir4255pthrough its ability to downregulate pten mir4255p activated the pi3kakt axis we conclude that mir4255p promotes lca tumorigenesis through ptenpi3kakt signalingkeywords mir4255p lung cancer pten pi3kakt signaling pathways lung cancer lca is a leading cause of cancer relatedmortality across the globe lca is prevalent in males and asymptomatic during early disease stages as manyas in every cases are at an advanced stage iii or ivwhen diagnosed and the 5year survival rates remainlow particularly for those with metastatic lca improved lca therapeutics is thus urgently requiredmicrornas mirnas regulate many cell processesincluding differentiation metabolism and tumorigenesis[] emerging evidence suggests that mirnas are keyplayers during lca tumorigenesis [] the aberrantexpression of mir4255p is linked to hepatocellular carcinoma hcc gastric cancer gca and colorectal correspondence kjhhev163comcardiothoracic surgery the fourth affiliated hospital zhejiang universityschool of medicine shangchen road no1 of yiwu zhejiang chinacancer crc [] here we report the upregulationof mir4255p in lca and highlight its contribution tolca development we further identify pten as a novelmir4255p target that is inhibited in lca to promoteptenpi3kakt signalingmethodspatient specimenslca samples n and adjacent healthy tissue at least cm from the resection margin were collected from thefourth affiliated hospital zhejiang university school ofmedicine the study was fully supported by the institutional review board of the fourth affiliated hospitalzhejiang university school of medicine no2015009all participants provided consent for sample analysisand anything about their identities will not be includedin the data the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0czhou bmc pulmonary medicine page of lca cell lines cell culture and cell transfectionhuman lung cancer cell lines a549 ncih1299 ncih460 hcc827 and normal human lung epithelial cellline beas2b were obtained from the cell bank of thechinese academy of sciences shanghai china a549 hcc827 cells were cultured in dmem plus fbsand penstrep ncih1299 ncih460 cells weregrown in rpmi1640 plus fbs at °c in co2beas2bs were cultured in clonetics¢ mediathe mir4255p mimics and negative control mirnanc were chemically synthesized by shanghai genepharma co ltd songjiang shanghai china lipofectamine invitrogen eugene or usa was usedfor transfection according to the manufacturers protocol pi3k activity was assayed as previously described pi3k inhibitor ly294002 was obtained from abcamto analyze the effects of mir4255p on pi3kakt indicated lca cells were cultured in the presence or absence ofthe workingconcentrations of ly294002 were μm for experiments with ly294002 treatmentsindicated lca cellswere pretreated with ly294002 for h prior to exposure to proteasome inhibitorsthe drugs for h at °ccell growth assayslca viability was assessed using cell counting kit8from shanghai haling biotechnology co ltd shanghai china as per the manufacturers protocols brieflyafter incubating the transfected cells for one full daythey were collected after trypsinization and seeded cellswell into 96well plates ten microliters ofcck8 solution were added per well and kept for h at °c the absorbance of the mixture was estimated in amicroplate readerinchercules usa at nmfrom biorad laboratoriescolony formation assaythe colony formation assays were performed as previous each group of treated cells per well wasseeded into cm culture dish and cultured for weeksfinally colonies were stained using crystal violet andthe number of cell colonies was countedqrtpcr analysistotal rna was isolated by trizol® reagent from invitrogen thermo fisher scientific inc and a nanodropnanodrop technologies thermo fisher scientific incwas used to estimate its quality and concentration theexpression of mir4255p was done by reverse transcription using the mirx¢ mirna firststrand synthesis kitfrom takara biotechnology co ltd dalian chinaand quantitative evaluation of the synthesized cdnawas done by quantitative pcr rtqpcr using themirx¢ mirna qrtpcr tb green® kit from takaraforwardbiotechnology as an endogenous control the small nuclear rna u6 normalized the expression of mir4255pthe δδcq system was used to evaluate all genes expressions and the primer sequences were shown as fol² ggggagttaglows mir4255pgattaggtc3² reverse ² tgcgtgtcgtggagtc3² u6 forward ²ctcgct tcggcagcaca² reverse ²aac gct tca cga att tgc gt3²pten forward ² tggattcgacttagacttgacc ² reverse ² aggatattgtgcaactctgcaa² gapdh forward ²catcaccatcttccaggagcg3² reverse tgaccttgcccacagcctt3²to getdual luciferase assaysthe design and synthesis of pten fragments containingbinding sites for wt wildtype and mut mutant onmir4255p was done by shanghai genepharma thesewere cloned into the target expression vector pmirglo dualluciferase from promega corporation wiusathe reporter plasmids wtpten andmutpten one night prior to transfection seeding ofcells confluence was done in plates with wells transfection of these cells was done with reporterplasmids harboring wt or mut in the presence ofmirnc or mir4255p mimic post h of transfectionluciferase activity of the cells was estimated as per instructions using the dualluciferase reporter assay system from promega corporation promega fitchburgwi usa the data normalization was done by the activity of renilla luciferasewbcell lysates ripa lysates were resolved on sds pageand transferred to pvdf membranes membraneswere blocked for h in milk plus tbstincubated with primary antibodies against pten dilution cat no ab170941 abcam pi3k dilution cat no ab32089 abcam pakt dilution cat no ab81283 abcam akt dilution cat no ab32505 abcam pakt dilution cat no ab81283 abcam actin dilution cat no ab8226 abcam at °c overnights andlabeled with hrpconjugated secondarys for h atroom temperature bands werevisualized usingchemiluminescent hrp substrate and analyzed usingimage lab tm softwarestatistical analysesdata analysis was performed using spss treatmentgroups were compared using a oneway anova pvalues were taken as significant experiments wereperformed on at least three occasions data representthe mean ± sd 0czhou bmc pulmonary medicine page of resultsmir4255p is upregulated in lcawe compared mir4255p levels in paired lcaand normal lung tissue samples by qrtpcr analysismir4255p was upregulatedspecimensfig 1a and expressed to high levels in a549 ncih1299 ncih460 and hcc827 cells compared tonormal human lung epithelial cellline beas2bfig 1b consistent with previous findings in othercancer types previous results indicated that mir4255p is upregulated in lcain lcamir4255p enhances proliferation and inhibits apoptosisin lca cellsto dissect the role of mir4255p in lca its expressionwas manipulated using mir4255p mimics figure 2acshows that mir4255p upregulation enhanced cell survival meanwhile enhanced cell colony formation abilityfig 2d and e taken together above results indicatedmir4255p is thus an oncogene in lca cellsmir4255p targets ptenfrom targetscan pten was identified as a predictedmir4255p target fig 3a in pten ²utr reporterassays mir4255p suppressed wt pten expressionfig 3bc but had little effect on mutated pten ²utr fragments fig 3bc the levels of pten werelower in cells transfected with mir4255p mimics fig3de mir4255p also negatively related pten mrnalevels in lca tissue p r2 fig 3f thesedata implicate pten as a cellular target of mir4255pmir4255p promotes lca via ptento define whether mir4255p regulate pten in lcawe firstly overexpression pten in lca fig 4a wbanalysis demonstrated that mir4255p reduces ptenlevels which could be recovered by exogenous expression fig 4b cell viability assays showed that mir4255p enhances lca proliferation which could be reversedby pten transfection fig 4cd suggesting mir4255pmediates its activities via pten this indicates that mir4255p targets pten to mediate its protumor effectsmir4255p activates ptenpi3kakt signalingit has been reported ptenpi3kakt signaling wasclosely related to cell proliferation and apoptosis [] next we explored the effect of mirna4255p onptenpi3kakt signaling as shown in fig 5aincomparison to nc groups pten was down regulated inresponse to mir4255p mimics whilst pi3k and paktlevels increased in addition nsclc cells were treatedwith the pi3kakt inhibitor ly294002 or ly294002 mir4255p mimics mimic fig 5b kinase activity assays further showed that pi3k activity in nsclc transfected with ly294002 was significantly lower than thattransfected with mimic control p and pi3k activity in nsclc cells transfected with both ly294002and mir4255p mimic was significantly higher than thattransfected with ly294002 p take together thisimplicates ptenpi3kakt signaling in the protumorigenic effects of mir4255pdiscussionthe poor prognosis of lca highlights the need for urgent therapeutic strategies mirnas are novel targets forfig mir4255p in highly expressed in lca a qrtpcr of mir4255p b qrtpcr of a549 ncih1299 ncih460 hcc827 and beas2b cellsdata mean ± sd p p p 0czhou bmc pulmonary medicine page of fig mir4255p promotes lca a mir4255p expression in a549 and ncih1299 cells b c cck8 assays in mir4255p mimic transfectedcells d e colonyforming assay in mir4255p mimic transfected cells the raw data of all colony formation experiments was listed insupplemental table data mean ± sd p p p fig mir4255p targets pten in lca a predicted binding of mir4255p and pten b c relative luciferase activity of ptenwt ptenmut inlca cells expressing mir4255p mimic d pten mrna levels are significantly lower after transfection with mir4255p mimic e pten expressionassessed by wb fulllength blotsgels are presented in supplementary figure f mir4255p and pten negatively correlate in lca tissue datamean ± sd p p p 0czhou bmc pulmonary medicine page of fig mir4255p promotes lca growth by targeting pten a qrtpcr of pten in indicated lca cell lines b wb analysis of pten expression fulllength blotsgels are presented in supplementary figure c d cck8 analysis of cell viability in the indicated cell lines with empty vector mir4255p mimic andor pten in a549ncih1299 cells data mean ± sd p p p fig effects of mir4255p on ptenpi3kakt a wb analysis of pten pi3k pakt and akt in lca cells fulllength blotsgels are presented insupplementary figure b the pi3k kinase activity was determined in nsclc cells transfected with ly294002 was significantly lower than thattransfected with mimic control and the pi3k kinase activity in nsclc transfected with both ly294002 and mir4255p mimic was significantlyhigher than that transfected with ly294002 data mean ± sd p p p 0czhou bmc pulmonary medicine page of cancer therapies and their dysregulation occurs in lcatissue [] duan showed that mir203 bindsto zeb2 to suppress emt yuan showed thatmir30a inhibits eya2 migration and invasion andli showed that mir1304 inhibits lca cell divisionthrough heme oxygenase1 the cellular roles ofmir4255p in lca are poorly understoodin thepresent work we further explore the underlying mechanisms of mir4255pinduced lca cell progressionin the present study we confirmed that mir4255p isoverexpressed in lca cell lines and tissues implicating arole in lca tumorigenesis upregulating mir4255plevels enhanced the cell survival and colony formationability of lca cells in vitro implicating it as a novel lcaoncogene in the mechanism using the algorithms targetscan website tools we identified pten as the potential target of mir4255p furthermore we performedluciferase reporter assays and the results showed thatmir4255p may directly target pten3utr the resultof qrtpcr and western blot also confirmed that overexpression of mir4255p could suppress the expressionlevel of pten all the above suggested that pten was apotentialtarget of mir4255p moreovermir4255p also negatively related pten mrna levelsin lca tissue rescue experiment indicated that exogenous pten expression inhibited the procancer effects ofmir4255p pten was downregulated in lca tissuepten is a tumor suppressor with wellcharacterizedphosphatase activity pi3kakt promotes cell cycleprogression inhibits apoptosis and is known to be overactive in a multitude of human cancers [ ] ptencan suppress pi3kakt signaling and thus displays anticancer effects upon assessment of the molecularmechanisms of mir4255p in lca cells its procancereffects were found to be mediated through manipulationof the ptenpi3kakt signaling axisfunctionalin we highlight mir4255p as an oncogenein lca that promotes an oncogene phenotype by inhibiting pten these findings enhance our knowledge of therole of mir4255p and reveal new therapeutic strategiesfor the diagnosis and treatment of lca angiogenesisand metastasis biological experiments will clarify thefunctions and roles of mir4255p in lcasupplementary informationsupplementary information accompanies this paper at httpsdoi101186s12890020012610additional file supplement figure s1 uncropped images of blotsand gels related to fig supplement figure s2 uncropped imagesof blots and gels related to fig supplement figure s3 uncroppedimages of blots and gels related to fig table s1 the raw data of allcolony formation experimentsabbreviationslca lung cancer pten gene of phosphate and tension homology deletedon chromsome ten pi3k phosphatidylinositol 3kinase nsclc nonsmallcell lung cancer gca gastric cancer crc colorectal cancerhcc hepatocellular carcinoma emt epithelialmesenchymal transitionacknowledgementsnot applicableauthors contributionsjsz zsy syc and qf did patient recruitment and assessment andperformed the experiments jhy and cjh were responsible for statisticalanalysis jsz zsy and syc interpreted the data and wrote the manuscriptall authors contributed to the subsequent drafts and approved the finalversionfundingthis research was funded by zhejiang provincial natural science foundationof chinaexploration program lq19h020010 core talent program ofdepartment of health of zhejiang province 2013rca018 zhejiangprovincial natural science foundation of chinageneral programly15ho2004 the funders had no role in the design of the study andcollection analysis and interpretation of data and in writing the manuscriptavailability of data and materialsthe datasets used andor analyzed during the current study are availablefrom the corresponding author on reasonable requestethics approval and consent to participateethics approval for this study was obtained from the fourth affiliatedhospital zhejiang university school of medicine no2015009 all patientsgave written informed consent for participation in the studyconsent for publicationnot applicablecompeting interestsall authors declare no conflicts of interestreceived march accepted august referenceschen w zheng r zuo t zeng h zhang s he j national cancer incidenceand mortality in china chin j cancer res aberle dr berg cd black wc the national lung screening trialoverview and study design radiology ni js zheng h huang zp microrna1973p acts as a prognosticmarker and inhibits cell invasion in hepatocellular carcinoma oncol lettzhang tj wang yx yang dq downregulation of mir186 correlateswith poor survival in de novo acute myeloid leukemia clin lab wang b teng y liu q microrna153 regulates nrf2 expression and isassociated with breast carcinogenesis clin lab lin h huang zp liu j mir4943p promotes pi3kakt pathwayhyperactivation and human hepatocellular carcinoma progression bytargeting pten sci rep yang j li j le y zhou c zhang s gong z pfklmir128 axis regulatesglycolysis by inhibiting akt phosphorylation and predicts poor survival inlung cancer am j cancer res wang r chen x xu t mir326 regulates cell proliferation andmigration in lung cancer by targeting phox2a and is regulated by hotairam j cancer res qin q wei f zhang j wang x li b mir134 inhibits nonsmall cell lungcancer growth by targeting the epidermal growth factor receptor j cellmol med hu h xu z li c mir145 and mir203 represses tgfbetainducedepithelialmesenchymal transition and invasion by inhibiting smad3 in nonsmall cell lung cancer cells lung cancer 0czhou bmc pulmonary medicine page of fang f song t zhang t cui y zhang g xiong q mir4255p promotesinvasion and metastasis of hepatocellular carcinoma cells through scaimediated dysregulation of multiple signaling pathways oncotarget cristobal i madozgurpide j rojo f garciafoncillas j potential therapeuticvalue of mir4255p in metastatic colorectal cancer j cell mol med zhang z wen m guo j clinical value of mir4255p detection and itsassociation with cell proliferation and apoptosis of gastric cancer pathol respract huang z xing s liu m deng w wang y huang z huang y huang x wuc guo x pan x jiang j feng f li t mir26a5p enhances cellsproliferation invasion and apoptosis resistance of fibroblastlikesynoviocytes in rheumatoid arthritis by regulating ptenpi3kakt pathwaybiosci rep meng j liu gj song jy chen l wang ah gao xx wang zj preliminaryresults indicate resveratrol affects proliferation and apoptosis of leukemiacells by regulating ptenpi3kakt pathway eur rev med pharmacol sciliu hy zhang yy zhu bl feng fz yan h zhang hy zhou b mir21regulates the proliferation and apoptosis of ovarian cancer cells throughptenpi3kakt eur rev med pharmacol sci yu g wang c song x liu s zhang y fan l yang y huang y song jformaldehyde induces the apoptosis of bmcs of balbc mice via the ptenpi3kakt signal transduction pathway mol med rep sun xh wang x zhang y hui j exosomes of bonemarrow stromal cellsinhibit cardiomyocyte apoptosis under ischemic and hypoxic conditions viamir4865p targeting the ptenpi3kakt signaling pathway thromb res mou x liu s mir485 inhibits metastasis and emt of lung adenocarcinomaby targeting flot2 biochem biophys res commun su tj ku wh chen hy oncogenic mir137 contributes to cisplatinresistance via repressing casp3 in lung adenocarcinoma am j cancer resliu y wang f xu p mir590 accelerates lung adenocarcinoma migrationand invasion through directly suppressing functional target olfm4 biomedpharmacother yang l luo p song q fei x dnmt1mir200agolm1 signaling pathwayregulates lung adenocarcinoma cells proliferation biomed pharmacother duan x fu z gao l direct interaction between mir203 and zeb2suppresses epithelialmesenchymal transition signaling and reduces lungadenocarcinoma chemoresistance acta biochim biophys sin shanghai yuan y zheng s li q overexpression of mir30a in lungadenocarcinoma a549 cell line inhibits migration and invasion via targetingeya2 acta biochim biophys sin shanghai li cg pu mf li cz microrna1304 suppresses human nonsmall celllung cancer cell growth in vitro by targeting heme oxygenase1 actapharmacol sin li dm sun h ptenmmac1tep1 suppresses the tumorigenicity andinduces g1 cell cycle arrest in human glioblastoma cells proc natl acad sciu s a bellacosa a chan to ahmed nn akt activation by growth factors is amultiplestep process the role of the ph domain oncogene li jw wang xy zhang x gao l wang lf yin xh epicatechin protectsagainst myocardial ischemiainduced cardiac injury via activation of theptenpi3kakt pathway mol med rep qin y huo z song x chen x tian x wang x mir106a regulates cellproliferation and apoptosis of colon cancer cells through targeting theptenpi3kakt signaling pathway oncol lett publishers notespringer nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations 0c" | Colon_Cancer |
acute myeloid leukemia aml is the most common type ofacute leukemia in adults caused by the clonal expansion ofundiï¬erentiated myeloid progenitor cells although mostpatients with aml can achieve complete remission by inductionchemotherapy the recurrence rate remains high and thus is themain factor aï¬ecting the outcomes of aml patients relapseoften develops from minimal residual disease in the protectivebone marrow bm microenvironment a comprehensiveunderstanding of the bm microenvironment is conducive to thediagnosis and personalized treatment of aml leukemic cellscytogenetics and molecular aberrations are the main factorsfor risk stratiï¬cation in patients with aml in additionthe bm microenvironment also plays a very important role thein the homing and survival ofbm microenvironment contains various componentssuchas immune cells stromal cells endothelial progenitor cellsextracellular matrix growth factors and cytokines theinteraction between leukemic cells and bm microenvironmentaï¬ects resistance to chemotherapy in aml the modulationof bm microenvironment in aml is currently undergoingpreclinical research and early clinical trials molecular inhibitorssuch as cxcr4 inhibitors vla4 inhibitors and eselectininhibitors are currently undergoing clinical trials immunecells and stromal cells are important components of the bmmicroenvironment and are also the main uencing factorsof leukemia development the estimate program isa common method to explore the microenvironment of manytumors recently it has also been used to explore theprognostic genes in the microenvironment of aml patients most studies have focused on diï¬erentially expressedgenes degs however the interaction and relationship betweengenes are open to investigate moreover the coding genes wereextensively explored but regions that encoded lncrnas andmirnas were less wellstudiedweighted gene coexpression network analysis wgcnais an algorithm commonly used in systems biology toexplore the correlation between gene sets and the clinic functionalization is achieved by constructing a freescalecoexpression gene network wgcna can identify highlyrelated genes and aggregate them into the same genetic modulecurrently wgcna is used in multiple ï¬elds such as cancer andnervous system or to identify potential biomarker candidates ornew therapeutic targets from genetic data the competition endogenous rna cerna hypothesis was anew regulatory mechanism between noncoding rna ncrnaand messenger rna mrna proposed by salmena in in this theory crosstalk between cernas is achieved byabbreviations aml acute myeloid leukemia auc area under curve bmbone marrow cam cell adhesion molecules cerna competing endogenousrnas deg diï¬erentially expressed gene david the database for annotationvisualization and integrated discovery go gene ontology icam1 intercellularadhesion molecule il10ra interleukin receptor subunit alpha keggkyoto encyclopedia of genes and genomes ppi proteinprotein interactiontcga the cancer genome atlas tlr6 toll like receptor tlr8 toll likereceptor tom topological overlap matrix wgcna weighted correlationnetwork analysisimmunerelated cerna network of amlcompetitively combining shared mirnas in recent yearsthe cernas hypothesis has attracted widespread attention in thestudy of molecular and biological mechanisms of tumorigenesisand development for example previous studies have foundthat lncrnarelated cernas were involved in the biologicalprocesses of glioblastoma and breast cancer theresearch on cernas of leukemia was generally based on thediï¬erential genes screened by leukemia and normal controls but no known module based on cernas network related tomicroenvironment in leukemia has been set upinformation ofthe aml cohortin this study mrnas mirnas and lncrnas data andclinicalfrom the cancergenome atlas tcga database were used to calculate theimmune and stromal scores of these aml cases using theestimate algorithm diï¬erentially expressed mrnas andlncrnas were applied to wgcna to identify the modulesmost relevant to the aml immune microenvironment thenthe immunerelated lncrnamirnamrna cerna networkwas established to screen genes with clinical signiï¬cance theseï¬ndings will help to better understand the role oftumormicroenvironment in aml and shed light on the developmentand progression of amlmaterials and methodsdata acquisitionall data sets of aml patients were downloaded from tcgadatabase1 the data used in this study met the following criteria excluding samples combined with other malignancies samples with lncrnas and mirnas and mrnas detection datafinally all lncrnas mrnas and mirnas expression proï¬lesof aml specimens and the corresponding clinical followupdata were downloadedidentiï¬cation of differentially expressedgenesthe estimate algorithm2 was used to calculate the immunescores and stromal scores of aml samples diï¬erentiallyexpressed genes degs such as lncrnas mirnas and mrnaswere identiï¬ed between high and low score groups stratiï¬ed bythe median value of immune scores and stromal scores usinglimma package all q values use fdr to correct the statisticalsigniï¬cance of the multiple test lncrnas and mrnas withlog fc and fdr were regarded as diï¬erentiallyexpressed while mirnas with log fc and fdr were regarded as diï¬erentially expressed then all the degs wereentered into r version auckland nz united states forcluster analysis based on the expression value of each sample inits respective data set the results were expressed in a clustergrameach column represents a sample and each row represents theexpression level of a given gene1portalgdccancergov2sourcefenetprojectsestimateprojectfrontiers in oncology wwwfrontiersinaugust volume 0cwang immunerelated cerna network of amldavid3 wasgo and pathway enrichment analysesthe database for annotation visualization and integrateddiscoveryenrichedbiological themes of degs functions particularly go geneontology terms and kegg kyoto encyclopedia of genes andgenomes pathway enrichment p was set as thecutoï¬ criterionapplied to analyzeweighted gene coexpression networkanalysiswgcna is an algorithm for identiï¬cation of gene coexpressionnetworksthrough highthroughput expression proï¬les ofmrnas or lncrnas with diï¬erent characteristics pairwisepearson correlation analysis was used to evaluate the weightedcoexpression relationship between all datasettopics in theadjacency matrix in this study wgcna was used to analyzemrnas and lncrnas to obtain the mrnas or lncrnas mostrelevant to aml immune microenvironmentcerna network construction andanalysisaccording to the results of wgcna we selected all mrnasand lncrnas in the most relevant module turquoise anddiï¬erentially expressed mirnas to construct a cerna networkbrieï¬y the associated cerna network in aml was constructedfollowing three stages prediction of lncrnamirna inorder to make lncrnas and mirnas map into the interactionssuccessfully we used the miranda4 and pita5 to get targetedlncrnas that mirnas may regulate prediction of mirnamrna three online databases miranda4 targetscan6 andmirwalk7 were used simultaneously for target mrna prediction construction of lncrnamirnamrna cerna networkthe cerna network was constructed based on the negativelyregulating target relationships of mirnamrna and mirnalncrna correlation pairsppi network constructionthe retrieval of interacting genes string database8 wasutilized to construct a proteinprotein interaction ppi networkof the degs identiï¬ed in the cerna network the interactingpairs with a conï¬dence score greater than were considered assignificant and were retained the degree represents the numberof interaction partnerssurvival analysiskaplanmeier plots weretherelationship between the overall survival of aml patientsand the expression level of mrnas lncrnas and mirnasthe statistical signiï¬cance of the correlation was tested by theconstructed to illuminatelogrank test and p was considered significant theanalysis was conducted using the r package of survivalstatistical analysisgraphpad prismtm san diego ca united states or rversion auckland nz united states software was usedfor all data analyses diï¬erences across groups were comparedusing kruskalwallis test for continuous variables diï¬erenceswere considered significant when p resultsimmune and stromal scores areassociated with aml clinicalparameterswe obtained gene expression proï¬les and clinical informationof aml patients from tcga database supplementarymaterial among them were male and were female with a median age range years oldaccording to the fab classiï¬cation there were cases of m0 m1 cases m2 cases m3 cases m4 cases m5 cases m6 cases and m7 case the estimate algorithm was used tocalculate the immune scores and stromal scores of aml patientsthe median immune score was range from to and the median stromal score was range from to we analyzed the relationship between immune scores andstromal scores and clinical parameters of aml patients caseswith m4 subtype aml had the highest immune scores while caseswith m3 subtype had the lowest immune scores p figure 1a similarly m4 cases had the highest stromal scoreswhereas m0 subtypes had the lowest p figure 1baccording to cytogenetics aml patients were divided intothree groups favorable intermediatenormal and poor therewas an obvious correlation between the cytogenetic risk andthe immune scores p figure 1cfavorable vsintermediate p favorable vs poor p intermediate vs poor p but no significant correlationbetween the cytogenetic risk and the stromal scores was observedp figure 1dscoreand high immunestromalusing the median immune or stromal score as a thresholdaml patients were divided into two groups with lowimmunestromalscoresurvival analysis showed that the survival rate of aml patientswith low immune scores was significantly higher than that ofpatients with high immune scores p figure 1ehowever there was no significant diï¬erence in survival betweenpatients with low stromal scores and those with high stromalscores p figure 1f3httpdavidncifcrfgov4httpwwwmicrorna5genieweizmannacilpubsmir07mir07_exehtml6httpwwwtargetscan7http12920671508tringdbidentiï¬cation of differentially expressedgenes based on immune scoresand stromal scoressetting the cutoï¬ criteria as log fc and fdr we identiï¬ed mrnas figure 2a and lncrnasfrontiers in oncology wwwfrontiersinaugust volume 0cwang immunerelated cerna network of amlfigure immune and stromal scores are associated with aml clinical parameters ab distribution of immune scores a and stromal scores b for aml fabsubtypes cd the correlation between immune scores c and stromal scores d and aml cytogenetic risk ef kaplanmeier survival curve based on immunese and stromal scores f aml acute myeloid leukemiafigure heatmap of differentially expressed genes in the high and low immunestromal score groups a mrnas b lncrnas and e mirnas based onimmune scores c mrnas d lncrnas and f mirnas based on stromal scoresfigure 2b based on immune scores and mrnasfigure 2c and lncrnasfigure 2d based onstromal scores setting the cutoï¬ criteria as log fc and fdr we identiï¬ed and mirnas based onimmune scores figure 2e and stromal scores figure 2frespectively the degs oflow vs high immunethefrontiers in oncology wwwfrontiersinaugust volume 0cwang immunerelated cerna network of amlfigure top go terms in each of biological process were performed for functional enrichment clustering analysis a top25 significant go terms based onupregulated genes in immune scores b top25 significant go terms based on upregulated genes in stromal scores c top25 significant go terms based ondownregulated genes in immune scores d top25 significant go terms based on downregulated genes in stromal scores go gene ontologyscore or stromal score groups were illustrated in the heatmap figure aml and thus require further research to determine theirbiological contributionfunctional enrichment analysis of degsbased on the david the databasefor annotationvisualization and integrated discovery gene annotation tool weperformed go analyses of both upregulated and downregulateddegs the top go biological process indicated that theupregulated degs based on immune or stromal scores wereprimarily enriched in neutrophil degranulation regulationof immune response signal transduction and ammatoryresponse figures 3ab while the downregulated degs basedon immunestromal scores were primarily enriched in rrnaprocessing regulation of translation regulation of transcriptionand cell diï¬erentiation figures 3cd subsequently weperformed kegg kyoto encyclopedia of genes and genomespathway enrichment and interrelation analysis kegg analysisrevealed that the upregulated degs were mainly enriched ininfection osteoclast diï¬erentiation nodlike receptor signalingpathway hematopoietic cell lineage and cell adhesion moleculescams pathways figures 4ab while the downregulateddegs were mainly enriched in ribosome metabolism pi3kaktsignaling pathway transcriptional dysregulation in cancer andmirnas in cancer figures 4cd above analyses revealedthatthese degs play a vital role in the development ofconstruction of weighted correlationnetwork analysis and identiï¬cation ofkey modulesbased on the results of survival analysis degs based on immunescores were selected for subsequent analysis the best β valuein the lncrnamrna coexpression network was which wascalculated using the diï¬erential lncrnas diï¬erentialmrnas and their expression data in leukemia samples nextthe method of dynamic tree cutting was used to producecoexpression modules finally modules of lncrnamrnacoexpression networks were generated and the heat map plot oftopological overlap matrix tom was shown figure 5a eachmodule was calculated and plotted with its corresponding clinicalcharacteristics correlation analysis showed that turquoisemodule displayed the highest relationship with aml immunescores r which included mrnas and lncrnasfigure 5b these mrnas were further used to performthe gene enrichment analysis the genes were most relatedto neutrophil degranulation immune response ammatoryresponse signal transduction and tolllike receptor signalingpathway figure 5c in addition genes were highly enriched ininfection osteoclast diï¬erentiation nodlike receptor signalingfrontiers in oncology wwwfrontiersinaugust volume 0cwang immunerelated cerna network of amlfigure top kegg pathway analysis were performed for functional enrichment clustering analysis a top25 significant kegg pathways based onupregulated genes in immune scores b top25 significant kegg pathways based on upregulated genes in stromal scores c top25 significant kegg pathwaysbased on downregulated genes in immune scores d top25 significant kegg pathways based on downregulated genes in stromal scores kegg kyotoencyclopedia of genes and genomespathway metabolic pathways and hematopoietic cell lineage bykegg analysis figure 5dcerna network constructionsince the turquoise module showed the highest relationshipwith aml immune scores we selected lncrnas and mrnas inthe turquoise module and diï¬erentially expressed mirnasbased on immune scores to construct a cerna network firstlybased on the pita and mircode online database that matchespotential mirnas with lncrnas a total of lncrnamirnapairs contained lncrnas and mirnas then we searchedfor the mrnas targeted by the diï¬erentially expressed mirnasusing three target gene prediction websites miranda mirwalkand targetscan using these websites we detected and target mrnas respectively based on the venn intersectionanalysis target mrnas were selected subsequently wematched the predicted target genes with the mrnas in theturquoise module then we constructed the cerna networkby integrating the mirnalncrnamrna interactions at lasta ï¬nal lncrnamirnamrna cerna network was constructedwith lncrnas mirnas and mrnas figure 6aproteinprotein interaction ppinetwork analysisto further explore the interplay among the mrnas in cernawe constructed a ppi network based on the string theretrieval of interacting genes online database figure 6b inthe network tlr8 toll like receptor icam1 intercellularadhesion molecule tlr6 toll like receptor and il10rainterleukin receptor subunit alpha had higher degrees and respectively supplementary table the genes encoding these proteins have been conï¬rmed tobe associated with immune microenvironment and leukemiaprogression association between mrnas mirnasand lncrnas in cerna and overall amlsurvivalwe further analyzed the prognostic values of mrnas mirnasand lncrnas in the cerna network subjects were dividedinto highexpression and lowexpression cohorts accordingto the median value ofthese genes for overall survivalfrontiers in oncology wwwfrontiersinaugust volume 0cwang immunerelated cerna network of amlfigure wgcna was used to analyze genetic modules a cluster diagram of coexpression network modules based on topological overlap in mrna andlncrna b study the relationship between each module with their corresponding clinical characteristics c go analysis showed the gene symbols and geneinteractions in the turquoise module d kegg analysis was used to study the pathway enrichment in the turquoise module wgcna weighted correlation networkanalysis go gene ontology kegg kyoto encyclopedia of genes and genomesthrough the package survivalthe highexpression and lowexpression cohorts were splitfor the logrank testin rsoftware out of lncrnas ac0099485 cmahp cta331p31 fcgr2c grk6p1 linc00539 linc01272 mipepp3psmb8as1 rp11266l95 rp11320g101 rp11421f163rp11439e1910 rp11792a83 and stag3l2 out of mirnas hsamir125b5p and hsamir3383p and out of mrnas agpat3 ankrd27 cbx2 ccnd2cd300lb cyth1 erg gdf11 igf1r kiaa0513 kiaa0930larp1 lfng lpcat1 nrep nudt16 pou2f2 ppm1hptafr qsox2 rab3d ralgps2 siglec7 slc43a2 srsf6tnfaip2 tns3 trib1 zbtb5 znf70 and znrf1 wereassociated with overall survival according to the logrank testp representative genes are shown in figure age ¥ years vs years and risk group favorablevsintermediatenormal vs poor were also associated withoverall survival according to the logrank test p and respectively the above mentioned lncrnas mrnas and mirnas were brought into further multivariatecox proportional hazard regression analysis with age and riskgroup finally mrnas ccnd2 erg lpcat1 nudt16ralgps2 tnfaip2 and znf70 lncrnas cmahp fcgr2cpsmb8as1 rp11266l95 rp11320g101 rp11792a83 andstag3l2 and mirnas hsamir125b5p and hsamir3383p were independently associated with overall survival table discussionin recent years studies about the roles of gene mutationsand chromosomalin the occurrence anddevelopment of aml and their prognostic values have madesignificant progress however the bm microenvironmentwhich also plays an important role in the pathophysiologytranslocationsfrontiers in oncology wwwfrontiersinaugust volume 0cwang immunerelated cerna network of amlfigure cerna network construction and proteinprotein interaction network a a lncrnamirnamrna cerna network was constructed by lncrnas mirnas and mrnas b a ppi network was constructed based on the string database the rectangle represents micrornas the circle represents mrnasand the triangle represents lncrnas the red represents upregulation in ishigh and the green represents downregulation in ishigh the size of the dot representsthe regulatory capacity of the mrna and larger points indicate stronger regulatory capability cerna competing endogenous rnas ppi proteinprotein interactionprocess in aml are poorly understood therefore mosttreatments previously targeted only tumor cells butfewtargeted the tumor microenvironmentindepth researchon the microenvironment of leukemia will help to furtherunderstand the mechanism of leukemia development and mayï¬nd new targets for microenvironment treatment this studyscreened microenvironmentrelated genes based on the tcgadatabase and further established microenvironmentrelatedlncrnamirnamrna cerna networks through wgcnafirst we calculated the immune scores and stromal scores ofaml patients based on the estimate algorithm and found thatthese scores were related to the fab typing of aml in additionimmune scores were significantly correlated with cytogenetic riskand overall survival estimate is a new algorithm to inferthe level of stromal and immune cells in tumor tissues andtumor purity using gene expression data high immunescores in the bm samples from patients with poor prognosisindicated that more immune cells were recruited in their bmfrontiers in oncology wwwfrontiersinaugust volume 0cwang immunerelated cerna network of amlfigure correlation between mrnas mirnas and lncrnas in cerna and overall aml survival in tcga kaplanmeier survival curves with the logrank testwere performed for the representative mrnas mirnas and lncrnas cerna competing endogenous rnas aml acute myeloid leukemia tcga the cancergenome atlasmicroenvironment this may be due to that aml cells activelyshape the bm environment and immune cells to promote diseaseprogression through cellular structural and functional changes however there was no significant correlation betweenstromal scores and cytogenetic risk or survival of aml patientssuggesting that the proportion of stromal cells were comparablein diï¬erent group possible explanation may be that stromal cellsplay an important role in solid tumors while its role inleukemia is not as strong as in solid tumors then we identiï¬ed diï¬erentially expressed mrnas mirnasstromaland lncrnas based on the immune scores orscores functional enrichment analysis indicated thatthesedegs were mainly involved in immune and ammatoryresponses consistent with these results previous studies haveshown that the biology of the immune system is essentialfor the formation of a complex bm microenvironment in recent yearsthe immunologicalcharacteristics of aml has increased and the developmentof eï¬ective aml immunotherapy strategies has attractedwidespread attention the understanding ofin recent years important advances in cerna coexpressionnetwork research have developed rapidly the disruption offrontiers in oncology wwwfrontiersinaugust volume 0cwang immunerelated cerna network of amltable multivariate cox proportional hazard regression analysis of lncrnas mrnas and mirnasgenesccnd2erglpcat1nudt16ralgps2tnfaip2znf70cmahpfcgr2cpsmb8as1rp11266l95rp11320g101rp11792a83stag3l2hsamir125b5phsamir3383phr 95ci pthe cerna network balance is a major cause for tumorigenesis therefore understanding the complex interactions betweendiï¬erent cerna networks will lead to an indepth understandingof gene regulatory networks and has implications for cancertreatment in addition the lncrnamirnamrna cernanetwork can predict the prognosis of the disease for examplea lncrnamirnamrna cerna network was established basedon rnaseq data of breast cancer from tcga which consistsof mirnas lncrnas and mrnas multiplexcox regression analyses showed that four of these lncrnasadamts9as1 linc00536 al3914211 and linc00491 havesignificant prognostic value wang identiï¬ed lncrnas mirnas and mrnas from a database toconstruct a lncrnamirnamrna cerna network inthe network a univariate and multivariate cox proportionalhazard regression analysis was used to establish a survivalmodel with target mrnas hoxa9 insr krit1 mybspry2 ube2v1 wee1 and znf711 where auc area undercurve is indicating the sensitivity and speciï¬city ofprognostic prediction however the screening of diï¬erentialgenes in this study was based on leukemia patients andnormal people and did not focus on tumor microenvironmentso far there is no cerna research based on the leukemiamicroenvironment in our research the screening of diï¬erentialgenes was based on the immune score then wgcna wasused to identify the modules most relevantto the amlimmune microenvironment then using wgcna and mirnaprediction websites a lncrnamirnamrna cerna networkconsisting of lncrnas mirnas and mrnaswas constructedsubsequently we built a ppi network predicting theinteraction among the proteins encoded by the degs inthe cerna network tlr8 icam1 tlr6 and il10ra hadhigher degrees tlr8 and tlr6 are members of the tolllike receptor family which is upstream to the transcriptionittransportationthe innate immune system andfactor nfκb and part ofplays an importantin progression of aml roleicam1 is one of the cams a large class of transmembraneproteinsinvolved in the binding of cells to another cellor extracellular matrix and involved in cell proliferationdiï¬erentiation movementandtissue structure the protein encoded by il10ra is areceptor for interleukin which has been shown to mediatethe immunosuppressive signal ofinterleukin and thusinhibits the synthesis of proammatory cytokines and isreported to promote survival of progenitor myeloid cellsthrough the insulin receptor substrate2pi3kakt pathway these results indicated that this novel cerna networkwere closely associated with immune microenvironment andprogression of amlapoptosisfurthermore lncrnas mirnas and mrnas withprognostic signiï¬cance were screened out which could be usedas biomarkers for prognosis among the genes with prognosticsigniï¬cance in our module of immunerelated cerna networkthere were aml related reports about cbx2 ccnd2 ergigf1r larp1 lfng nudt16 pou2f2 ptafr rab3dsiglec7 srsf6 tnfaip2 trib1 zbtb5 and znrf1 themost reported of which were erg ccnd2 and ifg1r ergtranslocation was involved in the occurrence and developmentof aml and high expression of erg was a poor prognosticfactor for patients with normal karyotype aml ccnd2mutations were more common in cbfaml and it was also afrequent mutation event in t aml ccnd2 leadedto increased phosphorylation of retinoblastoma proteins leadingto significant cell cycle changes and increased proliferation ofaml cell lines nicolas chapuis found that igf1spontaneous lesions played a key role in pi3kakt activation ofaml cells providing strong evidence for targeting igf1r as apotential new therapy for aml the functions of agpat3ankrd27 cd300lb cyth1 gdf11 kiaa0513 kiaa0930lpcat1 nrep ppm1h qsox2 ralgps2 slc43a2 tns3and znf70 in aml have not been reported we identiï¬ed lncrnas with clinical signiï¬cance among them only cmahpwas reported to be related to mllpositive aml andother lncrnas have not been reported in leukemia twomirnas including hsamir125b5p and hsamir3383p havebeen reported to be associated with a variety of cancers but no studies have been reported related to aml all theseunreported mrnas mirnas and lncrnas may be potentialnovel biomarkers or therapeutic targets for amlis importantto note that our current research haslimitations we selected the target data from the tcga publicdatabase only through the biological algorithm method weshould further verify the results of this in clinical patientsin further studyconclusionin summary a comprehensive bioinformatics analysis wasperformed on the aml dataset in tcga with an emphasison the immune microenvironment using wgcna andfrontiers in oncology wwwfrontiersinaugust volume 0cwang immunerelated cerna network of amlmirna prediction programs an immunerelated lncrnamirnamrna cerna network was established and degs withprognostic value were further identiï¬ed further studies of thesegenes are needed in the clinic and may provide new insights intothe pathogenesis of aml this study increases our understandingof the complex interactions between aml tumor cells and the bmmicroenvironment and may provide novel prognostic factors andtherapeutic targetsdata availability statementall data sets of aml patients were downloaded from the cancergenome atlas tcga database portalgdccancergovauthor contributionsyfl cw and zj conceptualization and design sw dataacquisition and writing original draft sw ly yx and dzmethodology sw and cw data analysis and interpretationyjl and yfl writing review and editing yfl cw and zjproject administration all authors contributed to the andapproved the submitted versionfundingthis work was supported by the national natural sciencefoundation of china grant numbers and u1804191and the henan medical science and technology research projectgrant number acknowledgmentswe thank the tcga database for the availability of the datasupplementary materialthe supplementary materialonline202001579fullsupplementarymaterialfor this can be foundwwwfrontiersins103389foncatreferences ferrara f schiï¬er ca acute myeloid leukaemia in adults lancet doi 101016s0140673612617279 zeng z shi yx samudio ij wang ry ling x frolova o targetingthe leukemia microenvironment by cxcr4 inhibition overcomes resistanceto kinase inhibitors and chemotherapy in aml blood doi 101182blood200805158311 shafat ms gnaneswaran b bowles km rushworth sa the bone marrowmicroenvironmenthome of the leukemic blasts blood rev doi 101016jblre201703004 bullinger l döhner k döhner h genomics of acute myeloid leukemiadiagnosis and pathways j clin oncol doi 101200jco ayala f dewar r kieran m kalluri r contribution of bonemicroenvironment to leukemogenesis and leukemia progression leukemia doi 101038leu2009175 uy gl rettig mp motabi ih mcfarland k trinkaus km hladnik lm a phase study of chemosensitization with the cxcr4 antagonist plerixaforin relapsed or refractory acute myeloid leukemia blood doi 101182blood201110383406 rashidi a uy gl targeting the microenvironmentin acute myeloidleukemia curr hematol malig rep doi 101007s11899 austin r smyth mj lane sw harnessing the immune system in acutemyeloid leukaemia crit rev oncol hematol doi 101016jcritrevonc201604020 yehudairesheï¬ s attiasturgeman s sabbah r gabay t musallam rfridmandror a abnormal morphological and functional nature ofbone marrow stromal cells provides preferential support for survival of acutemyeloid leukemia cells int j cancer doi 101002ijc yoshihara k shahmoradgoli m martÃnez e vegesna r kim h torresgarciaw inferring tumour purity and stromal and immune cell admixture fromexpression data nat commun doi 101038ncomms3612 yan h qu j cao w liu y zheng g zhang e identiï¬cation of prognosticgenes in the acute myeloid leukemia immune microenvironment based ontcga data analysis cancer immunol immunother doi101007s00262019024087 huang s zhang b fan w zhao q yang l xin w identiï¬cation ofprognostic genes in the acute myeloid leukemia microenvironment aging doi 1018632aging102477 ni j wu y qi f li x yu s liu s screening the cancer genome atlasdatabase for genes of prognostic value in acute myeloid leukemia front oncol doi 103389fonc201901509 langfelder p horvath s wgcna an r package for weighted correlationnetwork analysis bmc bioinformatics doi yao y zhang t qi l zhou c wei j feng f integrated analysis of coexpression and cerna network identiï¬es ï¬ve lncrnas as prognostic markersfor breast cancer j cell mol med doi 101111jcmm14721 spiers h hannon e schalkwyk lc smith r wong ccy odonovan mc methylomic trajectories across human fetal brain development genomeres doi 101101gr180273114 liu q jiang c xu j zhao mt van bortle k cheng x genomewide temporal proï¬ling of transcriptome and open chromatin of earlycardiomyocyte diï¬erentiation derived from hipscs and hescs circulat res doi 101161circresaha116310456 salmena l poliseno l tay y kats l pandolï¬ pp a cerna hypothesisthe rosetta stone of a hidden rna language cell doi101016jcell201107014 karreth fa pandolï¬ pp cerna crosstalk in cancer when cebling rivalriesgo awry cancer discov doi 10115821598290cd13 zhang k li q kang x wang y wang s identiï¬cation and functionalcharacterization of lncrnas acting as cerna involved in the malignantprogression of glioblastoma multiforme oncol rep doi103892or20165070 wang jd zhou hs tu xx he y liu qf liu q prediction ofcompeting endogenous rna coexpression network as prognostic markers inaml aging doi 1018632aging101985 ritchie me phipson b wu d hu y law cw shi w limma powersdiï¬erential expression analyses for rnasequencing and microarray studiesnucleic acids res 43e47 doi 101093nargkv007 kanehisa m goto s kegg kyoto encyclopedia of genes and genomes nucleicacids res brenner ak bruserud ø functional tolllike receptors tlrs are expressed | Colon_Cancer |
"what clinicians know about the diagnosis treatment and prevention of disease originates from studies mostly done on male cells male mice and men1 historically for multiple reasons including the purported safety of women and their offspring women of childbearing age were excluded from clinical trials as a result medical research and care have been centred on male physiology the assumption was that male and female cells and animals were biologically identical and evidencebased medicine was defined by clinical trials done predominantly in men1 in the us national institutes of health nih mandated the inclusion of women in nihfunded clinical trials but many investigators did not follow this mandate and many of those who did include women did not analyse the results by sex23 minimising the effectiveness of this policy preclinical research and drug development studies have also predominantly used male animal models and cells4 it is not surprising that a us government accountability office report found that eight of the ten prescription drugs withdrawn from the market between and posed greater health risks for women than for men most funding agencies from europe and north america have implemented policies to support and mandate researchers to consider sex and gender at all levels of medical research8 still the field of sexbased biology and medicine is often viewed as a specialised area of interest rather than a central consideration in medical research essential for the success of clinical care and translational science is awareness by clinicians and researchers that the diseases they are treating and studying are characterised by differences between women and men in epidemiology pathophysiology clinical manifestations psychological effects disease progression and response to treatmentthis review explores the role of sex biological constructs and gender social constructs as modifiers of the most common causes of death and morbidity and articulates the genetic biological and environmental determinants that underlie these differences we aim to guide clinicians and researchers to better understand and harness the importance of sex and gender as genetic wwwthelancetcom vol august biological and environmental modifiers of chronic disease ultimately it is a necessary and fundamental step towards precision medicine that will benefit women and mensex as a genetic modifier of biology and diseasesex differences in disease prevalence manifestation and response to treatment are rooted in the genetic differences between men and women genetic sex differences start at conception when the ovum fuses with a sperm cell carrying an x or a y chromosome resulting in an embryo carrying either xx or xy chromosomes this fundamental difference in chromosome complement eg genes outside the testisdetermining sry gene generates ubiquitous sex differences in the molecular makeup of all male and female cells9 first the y chromosome carries genes that exhibit subtle functional differences from their xlinked homologues eg zfy vs zfx and uty vs utx and also carries genes with no homologue at all eg sry in addition in men the x chromosome carries only maternal imprints ie epigenetic modifications made by the parent in generating the sex cellswhich alter the expression of genes in the offspring as women have x chromosomes from both parents they carry maternal and paternal imprints which target a different set of genes random inactivation of one of the x chromosomes in female cells which prevents sex differences in x chromosome gene dosage causes another degree of sex difference in gene expression as some of these xlinked genes escape inactivation in women those genes are often expressed at higher levels in women than in men9 sexspecific gene expression due to genomic search strategy and selection criteriawe searched pubmed for papers published in english between jan and june using sex or gender and the name of the disease of interest as search terms although we tried to cite seminal studies when necessary because of space limitation representative reviews were often selectedlancet diabetes discovery sexbased medicine laboratory section of endocrinology john w deming department of medicine tulane university school of medicine and southeast louisiana veterans health care system medical center new orleans la usa prof f mauvaisjarvis md barbra streisand womens heart center cedarssinai smidt heart institute los angeles ca usa prof n bairey merz md national heart lung institute imperial college london london uk prof p j barnes md department of pharmacology and department of neurology college of medicine center for innovation in brain science university of arizona tucson az usa prof r d brinton phd department of medical epidemiology and biostatistics and center for gender medicine karolinska institutet stockholm sweden prof jj carrero phd channing division of network medicine and the division of pulmonary and critical care medicine department of medicine brigham and womens hospital harvard medical school boston ma usa d l demeo md neuroscience institute and department of biology geia state university atlanta ga usa prof g j devries phd department of psychiatry university of colorado school of medicine anschutz medical campus aurora co usa prof c n epperson md division of oncology department of medicine washington university school of medicine st louis mo usa prof r govindan md w harry feinstone department of molecular microbiology and immunology the johns hopkins bloomberg school of public health baltimore md usa prof s l klein phd department of biomedical metabolic and neural sciences university of modena andreview 0creggio emilia azienda ospedalierouniversitaria di modena ospedale civile di baggiovara modena italy prof a lonardo md department of psychiatry department of psychology and department of obstetrics gynecology university of illinois at chicago chicago il usa prof p m maki phd department of neurology mcgovern medical school university of texas health science center houston tx usa prof l d mccullough md berlin institute of gender medicine charituniversittsmedizin berlin berlin germany prof v regitzzagrosek md department of cardiology university hospital z¼rich university of z¼rich switzerland prof v regitzzagrosek center for womens health research divisions of general internal medicine and cardiology university of colorado school of medicine aurora co usaimprinting extends to autosomes as well and these imprinted genes exhibit sexspecific and tissuespecific expression in humans10 thus fundamental sex differences deriving directly from genetic heterogeneity between the x and y chromosome complements and parentoforigin inher itance exist at the molecular level in all human cells these sex differences persist throughout life and are independent of sex hormones figure arguably the greatest source of differences between men and women comes from the y chromosomal sry gene which directs the development of a testis in men the ensuing developmental surge of testicular testosterone permanently masculinises the reproductive tract and the anisation of brain circuits affecting male behaviour at puberty1112 in humans the first surge occurs at the end of the first trimester of pregnancy because it alters cellular gene expression and tissue structure in multiple ans of men via epigenetic mechanisms this testosterone surge is also paramount in programming sex differences in physiology and susceptibility to diseases that will manifest in adulthood after this initial testicular testosterone surge gonadal hormone concentrations remain low until puberty which triggers lasting sex differences in circulating oestrogens and testosterone concentrations after puberty cells with androgen or afemale sexbrandom x chromosomeinactivation and escapexxxxxxxxxxxxxxxxxxxy chromosome complementmale sexsryctesticular testosterone surgefetal testistestosteroneohogenetic diï¬erences of male and female cellsepigenetic programming of male cellsfigure genetic causes of sex differencesa genetic sex differences start with cells carrying either xx or xy chromosome complement eg genes outside the testisdetermining sry gene which generates ubiquitous sex differences in the molecular makeup of all male and female cells b random inactivation of one x chromosome in female cells causes another level of sex differences in gene expression some xlinked genes escape inactivation in female individuals and have a higher expression in female than male individuals c the y chromosomal sry gene directs the development of a testis in male individuals which produces a surge of testicular testosterone at the end of pregnancy the testosterone surge programmes cellular gene expression and tissue structure in multiple ans of male individuals via epigenetic remodelling the combination of these genetic and developmental events programmes sex differences in physiology and susceptibility to diseases that will manifest in adulthoodoestrogen receptors will be affected differ ently in men and women the combination of all genetic and hormonal causes of sex differences aforementioned culminates in two different biological systems in men and women that translate into differences in disease predisposition manifestation and response to treatment therefore sex is an important modifier of physiology and disease via genetic epigenetic and hormonal regulations figure gender as a determinant of patients and doctors behaviour and as a modifier of health disease and medicinegender according to the global health definition refers to the socially constructed norms that impose and determine roles relationships and positional power for all people across their lifetime13 gender interacts with sex the biological and physical characteristics that define women men and those with intersex identities13 gender is not a binary term it includes the understanding that in many people traits of masculinity or femininity coexist and are expressed to different degrees gender attributes are fluid more than two thirds of women and men report genderrelated characteristics traditionally attributed to the opposite sex1415 in transgender people gender identity differs with the sex they were assigned at birth so far transgender people have generally been underrepresented in clinical studies to date although this underrepresentation is changing gender is an equally important variable as biological sex in human health and influences the behaviour of communities clinicians and patients1415 gender roles represent the behavioural norms applied to men and women in society which influence individuals everyday actions expectations and experiences including diet perceived stress smoking and physical activity and affect health and disease susceptibility gender identity describes the fluidity of how a person perceives oneself as a woman or a man which affects feelings and behaviours gender relations refer to how we interact with or are treated by people on the basis of our ascribed gender institutionalised gender reflects the distribution of power between men and women in the political educational and social institutions in society and shapes social norms that define perpetuate and often justify different expectations and opportunities for women and men1617 as such the distribution of genderrelated charac teristics within populations of men and women can influence health differently than biological sex together these gender constructs determine access to health care helpseeking behaviours and individual use of the healthcare system being perceived as a man or a woman triggers different responses from clinicians who might diagnose and suggest interventions differently according to gender as such gender largely determines the use of preventive measures and referral for or acceptance of invasive therapeutic strategies genderrelated behaviours contribute to risk exposure and preventive behaviour in several wwwthelancetcom vol august review 0cdiseases this postulation is well exemplified in the cardiovascular field in which women often underestimate their risk compared with men and seek consultation later than men in the clinic for treatment of myocardial infarction1819 in the genesispraxy prospective study mortality year after an acute coronary event was more strongly associated with gender than with biological sex1415 similarly control of cardio vascular risk factors hypertension diabetes depres sive symptoms was better predicted by gender than by biological sex1415 therefore including a gender dimension in clinical studies and practice will contribute to the understanding of different clinical manifestations and outcomes of diseases in women and men1617 although beyond the scope of this review it is also important to consider that regarding health and disease gender intersect with race or ethnicity and age20 sex and gender are fundamentally and frequently reciprocally interrelated in biology and disease24 sex influences behaviours eg towards more aggressive or caring phenotypes on the other hand genderrelated behaviours eg smoking lifestyle perceived stress and pain and nutritional habits might produce epigenetic modifications that modulate gene expression and biological phenotypes figure summarises how sex and gender are interrelated in biology and diseasesex and gender differences in major chronic diseaseshaving established the importance of sex and gender in disease we will summarise their influences on the most common causes of death and debilitating diseases in the usa as an example figure note that these sex and gender disparities are relevant to other highincome countries as well as lowincome and middleincome countries where the burden of these diseases becomes increasingly like those in highincome countries26 in most diseases efforts to separate the effects of sex and gender are still incomplete so that we just refer to the differences among women and men because current knowledge about pathophysiology diagnosis and treatment of disease is primarily based on men as representative of the human species this review focuses on how women differ from men we discuss some key aspects regarding the dimensions of men in a dedicated sectionheart diseaseepidemiology pathogenesis manifestations and diagnosisheart disease is the leading cause of death in the usa in heart disease accounted for · of all deaths for men and for · of all deaths for women figure ischaemic heart disease and heart failure are major contributors to heart disease mortality and have important sex and gender differences for example heart failure disproportionately contributes to coronary heart disease mortality in women27 potentially due to undiagnosed ischaemic heart disease in women the strength of the association with cardiovascular risk factors differ by sex biological sexsex chromosomesepigeneticeï¬ectssex hormonesohohohobehaviour of patients and doctorssocietygender constructslifestylenutritional habitsexerciseperceived stresssmokingdiseasepathophysiologymanifestationresponse to treatmentdisease perceptionhelpseeking behaviouruse of health caredecision makingtherapeutic responsesex and gender diï¬erences in health disease and medicinefigure interrelation between sex and gender in health diseases and medicinebiological sex causes sex differences through genetic and hormonal influences in disease pathophysiology clinical manifestations and response to treatment sex also influences behaviours towards more aggressive or caring phenotypes on the other hand genderrelated behaviours eg smoking lifestyle perceived stress and nutritional habits produce epigenetic modifications that modulate the expression of biological sex gender constructs determine patients perception of disease helpseeking behaviour and individual use of health care gender constructs also influence decision making and trigger different therapeutic responses from providers biased by gendersystolic blood pressure and hypertension smoking and diabetes are associated with higher hazard ratios for myocardial infarction in women than in men28ischaemic heart disease is the most recognised example for integrating the concept of gender and sex which shape divergent or distinct disease outcomes compared with men women suffering from ischaemic heart disease are older this difference is historically believed to be due to the protection of endogenous oestrogens29 although contemporary study refutes this simplistic explanation30 and associations cannot be inferred to be causation still women suffering from ischaemic heart disease are underdiagnosed3132 and less likely to have a prehospital diagnosis of myocardial infarction33 the reasons for this disparity reflect the intersection between sex and gender first biological sex differences exist in the pathogenesis of ischaemic heart disease whereas men are more likely to be affected by obstructive coronary artery disease of large vessels than women coronary microvascular dysfunction36 leading to chronic myocardial ischaemia without obstructive coronary artery disease has a higher prevalence in women than men37 a metaanalysis reported that following acute myocardial infarction both sexes most often presented with chest pain but compared with men women were more likely to present with pain between the shoulder blades nausea or vomiting and shortness of breathprof j g regensteiner phd department of medicine department of paediatrics and department of neuroscience washington university school of medicine st louis mo usa prof j b rubin md center for the study of sex differences in health aging and disease geetown university washington dc usa prof k sandberg phd division of gastroenterology duke university medical center durham nc usa a suzuki md and durham va medical center durham nc usa a suzukicorrespondence to prof franck mauvaisjarvis diabetes discovery sexbased medicine laboratory section of endocrinology john w deming department of medicine tulane university school of medicine new orleans la usa fmauvaistulaneeduwwwthelancetcom vol august review 0cmale individualsother·heart disease·protection might disappear after menopause45 by contrast testosterone induces adverse cardiac remod elling in the male heart44chronic liver disease ·inï¬uenza and pneumonia ·suicide ·alzheimers disease ·type diabetes ·stroke ·cpd ·injuries ·female individualsother·septicaemia ·chronic kidney disease ·inï¬uenza and pneumonia ·type diabetes ·injuries ·alzheimers disease ·stroke ·cpd ·cancer·heart disease·cancer·figure percent distribution of the ten leading causes of death by sex usa adapted from heron25 cpdchronic pulmonary diseasesecond a gender bias appears to be responsible for the absence of recognition of ischaemic heart disease presentation in women38 men and women with ischaemic heart disease who score high on feminine roles and personality traits on questionnaires designed to ascertain aspects of gender are at an increased risk of recurrent ischaemic heart disease independent of female sex39heart failure affects of adults aged years and older and more women than men in absolute numbers4041 heart failure occurs at an older age and with less ischaemic causes in women than in men however hypertension and diabetes predispose older women to heart failure to a greater extent than men heart failure with preserved ejection fraction a form of heart failure with normal systolic function is twice as prevalent in women as in men by contrast heart failure with reduced ejection fraction affects more men than women women who have heart failure with preserved ejection fraction have smaller and stiffer hearts than men inflammation and the resulting fibrosis play a sexspecific role in the pathogenesis of heart failure with preserved ejection fraction under stress premenopausal womens hearts develop less inflammation resulting in less fibrosis than mens hearts4243 this difference is partially driven by sex hormones as oestrogens produce antiinflammatory actions on endothelial and immune cells and promote cardioprotective effects in premenopausal women44 this response to treatmentcompared with men women suffering from ischaemic heart disease are less likely to receive evidencebased treatment3132 and when suffering from acute myocardial infarction they are less likely to receive reperfusion33 an stelevation myocardial infarction registry revealed that compared with men women exhibit delayed reperfusion leading to higher mortality46 women suffering from acute myocardial infarction treated by male emergency physicians have a higher mortality rate than those treated by female physicians38 additionally male physicians are more effective at treating female patients with acute myocardial infarction when they work with female colleagues and when they have experience in treating female patients38 this treatment disparity between women and men can be corrected by improving emergency recognition of stelevation myocardial infarction in women and acceleration of percutaneous coronary intervention which equilibrates gender mortality47guidelines for the treatment of heart failure are similar for women and men24 however evidence suggests that optimal survival in women occurs with lower doses of β blockers angiotensin receptor blockers and angiotensin converting enzyme inhibitors than in men48 finally fewer women undergo heart transplantation than men although women are more frequently donors suggesting a referral bias could exist41cancersepidemiology pathogenesis manifestations and diagnosiscancers are the second leading cause of death dominated by lung cancer accounting for · of deaths in men and · of deaths in women figure more men develop cancer than women49 with few exceptions eg meningioma thyroid cancer lung cancer in nonsmokers nonreproductive cancers exhibit a male predominance though for some cancers oropharynx larynx oesophagus and bladder the male versus female incidence ratios can be higher than a male predominance in cancers that affect both sexes is evident around the world in all races and at all ages50 survival is also shorter for men than women across multiple cancer types the higher cancer risk in men is partially explained by gender constructs like dietary habits or risk behaviours such as smoking and alcohol consump tion51 it is unlikely to be the only cause after appropriate adjustment for these risk factors adult men still have a higher cancer risk than women52 moreover a similar male bias in incidence and survival is seen in paediatric cancers before puberty and the adoption of highrisk cancerpromoting behaviours eg smoking53the universal male predominance in cancer incidence and differential outcomes argues for a fundamental role wwwthelancetcom vol august review 0cin inutero tumour suppressors of sex in addition to gender in cancer biology sexspecific biology includes genetic differences xx vs xy chromosomes the incomplete xinactivation in female individuals which results in biallelic expression of xencoded female cells54 y chromosomeencoded oncogenes such as the rnabinding motif on y chromosome in male cells55 and the chromatin remodelling effects of testicular testosterone in male cells56 these mechanisms have an influence on several of the hallmarks of cancer57 including metabolism growth regulation58 angiogenesis and immunity which all contribute to cancer predisposition59 a crucial example is the male predisposition to glioblastoma which is the most common form of brain cancer in glioblastoma there is a cellintrinsic predisposition of male astrocytes a subtype of glial cell to malignant transformation60 after puberty sex hormones produce additional epigenetic and acute effects on cells that further influence sex disparities in cancer for example the increased frequency and aggressive phenotype of hepatocellular carcinoma in male individuals has been linked to the stimulatory effects of androgens in male individuals and the protective effects of oestrogens in female individuals61 importantly the biology of cancer is not the same across histological and genetic diagnostic groups or even within single histol ogical subtypes thus the interaction between sex gender and cancer mechanisms cannot be expected to be constant take colon cancer the second leading cause of cancerrelated death for example although women have a lower overall incidence of colon cancer than men they have a higher incidence of rightsided colon cancers which have the worst outcomes62 tumours from women with rightsided colon cancers exhibit a distinct molecular signature of energy metabolism compared with those of women with leftsided colon cancers63 this molecular signature is not observed when comparing tumours from men with rightsided colon cancers to men with leftsided colon cancers thus overall the male predisposition to cancer is probably the consequence of genetic program ming of male cells and the effect of sex hormones after puberty interacting with genderspecific behaviours to establish cancer risksresponse to treatmentin the future cancer prevention and treatment will be improved by sexspecific and genderspecific approaches for example immune checkpoint inhibitors can improve survival for men with advanced melanomas and nonsmallcell lung cancers more than for women64 the molecular subtyping of glioblastomas based on sexspecific transcriptomes has the potential to enhance chemotherapy in a sexspecific manner65 in colon cancer sex differences in xenobiotic metabolism regulatory networks might underlie greater treatment response in women than men and require modification of approaches for men with colon cancer66 other elements of cancer metabolism are also ripe for novel sexspecific targeting in treatment cellular nutrient partitioning is sexually dimorphic so approaches such as ketogenic diets or glutaminase inhibition might be associated with substantial sexspecific responses67 furthermore data from models of development ageing and cancer all indicate that molecular pathways such as the enzyme phosphatidylinositol 3kinase and tumour suppressors such as p53 and retinoblastoma protein are sexually dimorphic and require sexspecific targeting545968chronic pulmonary diseaseepidemiology pathogenesis manifestations and diagnosischronic pulmonary disease is the third leading cause of death for women · of deaths and the fourth for men · figure it is mostly accounted for by chronic obstructive pulmonary disease and to a lesser extent by asthma chronic obstructive pulmonary disease is characterised by irreversible airflow limitation and is associated with previous exposure to smoking or air pollutants women are overrepresented among individuals with chronic obstructive pulmonary disease especially among those with earlyonset disease or those who have never smoked69 women are also twice as likely to have chronic obstructive pulmonary disease with chronic bronchitis and women with severe chronic obstructive pulmonary disease have as much emphysema as men countering the misconception of emphysema as a male form of chronic obstructive pulmonary disease70 the female lung is more susceptible to chronic obstructive pulmonary disease than the male lung and women develop symptoms of the disease at a younger age with less tobacco exposure than men71 the genetic epidemiology of chronic obstructive pulmonary disease copdgene study72 suggests that earlyonset chronic obstructive pulmonary disease might originate in utero in susceptible women from alterations in lung development potentiated by maternal asthma and smoking genetic factors or hormonal influences future studies should focus on the contribution of maternally inherited factors such as mitochondrial and x chromosome genes to understand disease pathogenesis it is important to consider gender constructs as well smoking advertising campaigns targeting women rose in the 1960s and the resulting higher smoking rates influenced womens risk for developing chronic obstructive pulmonary disease73 from a disease severity vantage point chronic obstructive pulmonary disease exacerbation rates are also higher in women than men especially at a younger age74 additionally despite the burden of symptomatology and increased rates of hospitalisations and deaths women with chronic obstructive pulmonary disease are often misdiagnosed and disproportionally suffer from comorbid conditions including anxiety and depression therefore physicians should consider chronic obstructive pulmonary disease in the differential diagnosis of women with pulmonary symptoms regardless of tobacco or pollutant exposure historieswwwthelancetcom vol august review 0casthma characterised by variable airflow obstruction and chronic airway inflammation also affects men and women differently asthma is more prevalent in prepubertal boys than girls regarding asthma both male biological sex lung development and atopy and male gender constructs related to outdoor play and indoor pet exposure are factors contributing to the devel opment of asthma and sex versus gender contributions could be difficult to separate75 from puberty onwards more female than male individuals have asthma with an increased severity in middleaged women and a higher mortality rate76 this phe nomenon could be secondary to gender differences eg symptoms perception or healthseeking behaviours however biological sex plays a crucial role in asthma and sex hormones have a major impact on female asthma symptoms and severity after puberty75 worsening of asthma occurs in women before menstruation and is known as premenstrual asthma premenstrual asthma is more common in women with severe rather than mild asthma obesity rather than normal weight and a long rather than a short duration of asthma77 premenstrual asthma is hypoth esised to be due to a fall in progesterone and patients with a severe disease respond to progestogens78 during pregnancy approximately a third of asthmatic women exhibit a worsening of asthma a third show an improvement and the remainder are unaffected79response to treatmentthe menopausal transition represents a pivotal time of accelerated decline in lung function in women with chronic obstructive pulmonary disease and thus represents a sexspecific window for treatment intensification these observations also suggest that oestrogens protect from chronic obstructive pulmonary disease women exhibit greater expression of m2 over m3 muscarinic receptors and accordingly show greater improvements in lung function than men in response to the muscarinic anticholinergic bronchodilator ipatroprium80 pooled analyses of drug studies also suggest that women experience a greater improvement in quality of life than men after treatment of chronic obstructive pulmonary disease with a β2adrenoreceptor agonist combined with an anti cholinergic drug eg indacaterol and glycopyrronium81unlike chronic obstructiv | Colon_Cancer |
" excessive perioperative fluid administration may result in iatrogenic endothelial dysfunction andtissue edema transducing inflammatory markers into the bloodstream colloids remain longer in the circulationrequiring less volume to reach similar hemodynamic endpoints compared to crystalloids thus we tested thehypothesis that a goaldirected colloid regimen attenuates the inflammatory response compared to a goaldirectedcrystalloid regimemethods patients undergoing moderate to highrisk open abdominal surgery were randomly assigned to goaldirected lactated ringers solution n or a hydroxyethyl starch n fluid regimen our primaryoutcome was perioperative levels of pro and antiinflammatory cytokines secondary outcome was perioperativelevels of white blood cell count wbc creactive protein crp procalcitonin pct and lipopolysaccharidebindingprotein lbp measurements were performed preoperatively immediate postoperatively on postoperative day onetwo and fourresults the areas under the curve of interleukin il p il p il p and tumor necrosisfactor α p levels did not differ significantly between the groups wbc crp and pct values were alsocomparable lbp although significantly higher in the crystalloid group remained in the normal range patientsassigned to crystalloids received a median iqr amount of ml of crystalloid patients assigned tocolloids received ml of crystalloid and ml of colloid cytokine and inflammatory marker levels did not differ between goaldirected crystalloid and colloidadministration after moderate to highrisk abdominal surgerytrial registration clinicaltrialsgov nct00517127 registered 16th august correspondence barbarakabonmeduniwienacat1department of anaesthesia general intensive care medicine and painmedicine medical university of vienna spitalgasse vienna austriafull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cobradovic bmc anesthesiology page of is crucialintroductionvolume replacementin the perioperativeperiod and has great impact on postoperative outcome fluid restriction may cause hypotension and hypoperfusion leading to an dysfunction on the otherhand excessive fluid administration leads to destructionof the endothelial surface layer and consequently to tissue edema with harmful side effects [ ]fluidgdtbasedtherapygoaldirectedonoptimization of flowrelated hemodynamic parametersimproves clinical outcome in low to highrisk surgicalpatients compared to fixed fluid protocols [ ] specifically gdt enhances cardiac performance and gutmicrocirculation while avoiding iatrogenic hyperhydration [ ] in addition to hypervolemia the inflammatoryaggravatesdegradation of the endothelial barrier the socalled glycocalyx inflammation leads to cytokine release andmay thus worsen outcome for example high postoperative interleukin il levels are independently associatedwith postoperative complications response dueto surgicaltraumaso far in most previously performed gdt studieshemodynamic algorithms were based on colloid bolusadministration to improve hemodynamic variables colloids better maintain the intravascular oncotic pressure and provide a higher volume effect when used incase of hypovolemia goaldirected colloid administration reduces intraoperative fluid requirement and improves cardiac performance compared to crystalloids[ ] whether this translates into better outcomespecifically in a decreased postoperative inflammatoryresponse is still a matter of research the comparisonbetween colloid versus crystalloid based fluid regimenswas still lacking therefore we tested the primary hypothesis that perioperative levels of pro and antiinflammatory cytokines il il il and tumor necrosis factor alpha tnf α are reduced by goaldirectedcolloid versus crystalloid administration during the firstfour postoperative days in patients undergoing moderateto highrisk open abdominal surgery in addition wemeasured white blood cell wbc count creactive protein crp procalcitonin pct and lipopolysaccharidebinding protein lbp levelsmaterials and methodsthis prospective randomized controlled trial was conducted at the department of anesthesia intensive caremedicine and pain medicine medical university ofvienna vienna austria the institutional review boardof the medical university of vienna approved it as partof a large multicenter outcome trial evaluating the effectof goaldirected crystalloid and colloid on postoperativecombined morbidity and complications the ethicalcommittee of medical university of vienna viennaaustria provided ethical approval for this trial the trialwas conducted in accordance with the declaration ofhelsinki and good clinical practice and registered atclinicaltrialsgov nct00517127 and eudract a written informed consent was obtainedfrom all patients the authors have followed the applicable consort guidelinesfor this single center substudy consecutive eligiblepatients were included patients aged to yearsundergoing elective moderate to highrisk open abdominal surgery with american society of anesthesiologistsasa physical status iiii were included we excludedpatients with severe obesity body mass index bmi kgm cardiac insufficiency ejection fraction ef coronary artery disease with angina severe chronicobstructive pulmonary disease autoimmune diseases coagulopathies renal insufficiency creatinine clearance mlmin or renal replacement therapy symptoms of infection or sepsis and preoperative crp higher than mgdl allpatientsreceivedprotocolpreoperativelyantimicrobialprophylaxis using a single dose of a 2nd generationcephalosporine according to our clinicalstandardsanesthetic management wasstandardized standardmonitoring included electrocardiography ecg invasiveblood pressure surveillance pulse oximetry and esophageal core temperature monitoring a central venouscatheter was inserted when deemed clinically necessarywe used balanced anesthesia with sevoflurane none ofour patients received locoregional anesthesia accordingto patients requirements additional fentanyl and nondepolarizing neuromuscular blocking were administeredventilatory rate was adjusted to maintain endtidal carbon dioxide partial pressure etco2 of mmhgnormothermia was maintained with forced air warmingpatients were randomized to crystalloid lactatedringers solution or colloid hydroxyethyl starch voluven fresenius kabi germany grouprandomization was based on computergenerated codesto conceal allocation sealed opaque envelopes wereopened only shortly before induction of anesthesiaall patients were given mlkg oflactatedringers solution during induction of anesthesia followedby mlkg per hour for maintenance normalized toideal body weight ibw throughout surgery we calculated ibw according to the robinson formula thereafter the randomized fluid crystalloid or colloidwas esophageal dopplerguided cardiac q deltex medical group plc chichester uk according to a standardalgorithm this method is based on corrected aorticflow time ftc as well as stroke volume sv and allowsdistinguishing whether a patient is a fluid responder or 0cobradovic bmc anesthesiology page of not if mean arterial pressure map was below mmhg and no signs of hypovolemia were detected vasopressors were administratedpatients were transferred to postanesthetic care unitpacu or intensive care unit icu at the discretion ofthe attending anesthesiologist fluid management wasstandardized for the first postoperative hours in whichpatients received mlkg ibw crystalloid per hourmeasurementsdemographic and morphometric data were recorded aswell as asa score medical history type of surgery andpreoperative laboratory values duration of anesthesiaand surgery were recorded we also recorded intraoperative fluid requirements estimated blood loss transfusion requirements and urinary output for evaluation ofanesthetic management the total amount of fentanylendtidal sevoflurane concentration core temperatureandnotedhemodynamic parameters such as map heart ratehr ftc sv and cardiac output co were recorded at10min intervals the application of phenylephrine usewas notedicu admission werepostoperativethe primary outcomes were the areas under the curveaucs of postoperative levels of pro and antiinflammatory cytokines il il il and tnf α andtheir differences between the crystalloid and the colloidgroup secondary outcomes were aucs of wbc crppct and lpb and their differences between the groupsall blood samples for parameteranalyses were obtainedbefore surgery as baseline values t0 immediately postoperatively t1 as well as on postoperative days onetwo and four t2 t3 and t4 respectively for analysisof il il il and tnf α blood samples were centrifuged within h at g for min and plasma wasimmediately stored at °c for later enzymelinkedimmunosorbent assay elisa analyses the serum concentrations of il il il and tnf α were determined according to the manufacturersinstructionshuman sil6 instant elisa human il8nap1 instant elisa and human sil10 instant elisaebioscience vienna austria wwwebiosciencecom human tnf α duoset rd systems minneapolis minnesota wwwrndsystemscom for that purpose opticaldensity was measured with a victor microplate readerat a wavelength of nm multiple testing of sampleson different plates revealed an intraassay variability of for il for il for il for tnf α andan interassay variability of for il for il for il and for tnf αfor investigation of wbc crp pct and lpb separateblood samples were obtained their analysis took placeimmediately after blood sampling as routine laboratoryanalysessample size calculation and statistical analysissample size calculations for our trial were based on thestudy of steppan and colleagues they observed amean standard deviation sd of pgml in il h after surgery in abdominal surgery patients assuming a similar coefficient of variation sdmean for each of the four cytokines primarily plannedfor evaluation in our study we calculated a total of patients in order to obtain a power to detect a reduction in any of the cytokines at an overall significance level with powergroups were primarily compared for balance in patients demographic data intraoperative characteristicsand postoperative variables absolute standardized differences asd were calculated for patients baseline covariates subsequent measurements ofintraoperativeparameters were first averaged within each patient andthen averaged among the patients in each treatmentgroup for descriptive analysis normal distribution wasassessed with qq plots and kolmogorowsmirnow testsnormally distributed variables were with unpaired twotailed ttests otherwise the in case of normally distributed values wilcoxon ranksum test was used for notnormally distributed continuous data paired comparisons between baseline data and postoperative data wereperformed with paired sample ttest or wilcoxonsignedrank test as applicable nominal data were analyzed with chisquare or fishers exact test for low expected cell counts data were presented as means ± sdmedians iqr or as numbers percentage as applicableadjustment for multiple testing was performed with thebonferroni method a p value was considered statistically significantanalysis was conducted with spss software version armonk ny ibm corp r for macintosh version321 r core team r a language and environmentfor statistical computing r foundation forstatistical computing vienna austria was used to calculate asdresultsa total of patients were included between november and october in the colloid group and in the crystalloid group fig at t0 all values weremeasured overall in the crystalloid group and inthe colloid group of the preplanned blood sampleswere collected and analyzedpatients baseline characteristics did not differ exceptfor height bmi with a slightly higher bmi in the colloidgroup type of surgery and crp also higher in the colloid group table duration of anesthesia and surgerywere comparable between both groups patients assignedto crystalloid administration received a median of ml crystalloids whereas patients assigned 0cobradovic bmc anesthesiology page of fig consort patient flow chartto the colloid group received ml ofcrystalloid solution and ml of colloidsblood loss transfusion requirements and urinary outputdid not differ between the groups anesthetic management map and hr did not differ between the groupsftc sv and co were significantly higher in the colloidgroup compared to the crystalloid group ftc ms versus ms p sv ml versus ml to p and co ± lmin versus ± lmin p thenumber of patients requiring vasopressor support wascomparable between groups the incidence of postoperative icu admissions did not differ in the crystalloid versus in the colloid group p table baseline values of il il il and tnf α in thecrystalloid group were comparable to values in the colloid group il pgml versus pgml p il pgml versus pgml p il pgml versus pgml p tnf α pgml versus pgml p immediatepostoperative values of il and were significantlyhigher compared to baseline values in both groupsp for all measurements while tnf α did notshow any significant increase in the crystalloid p and the colloid group p fig aucs of il il il and tnf α did not differ significantly between the groups table wbc values at baseline were gl in thecrystalloid versus gl in the colloidgroup p crp pct and lbp baseline valueswere also comparable in both groupscrp mgdl versus mgdl p pct ngml versus ngml p lbp mcgl versus mcgl p immediate postoperative values of wbc and pctwere significantly higher compared to the baseline valuesin both groups p for all measurements fig 0cobradovic bmc anesthesiology page of table baseline characteristicsage yrsweight kgheight cmbmi kgm gender no menwomenasa score no iiiiiimedical history no pulmonary diseasecardiovascular diseasediabetes type idiabetes type iitype of surgery no colorectalliverpancreaticcrystalloidsn ± ± ± ± colloidsn ± ± ± ± asd preoperative laboratory valuescrp mgdl ± ± patient characteristics data are presented as means ± sd or as counts for thecategorical outcomesabbreviations asd absolute standardized differences absolute difference inmeans or proportions divided by the pooled sd asd values of and represent small median and large differencesbmi body mass index m male f female asa american society ofanesthesiologists crp creactive protein sd standard deviationthe aucs for wbc crp pct and lbp for the timeperiods from t1 to t4 did not differ significantly between the groups fig table however lbpshowed significantly higher levels in the crystalloidgroup in the immediate postoperative period comparedto the colloid group mcgl versus mcgl p at all other postoperativetime points there were no significant differences betweenthe groups fig discussionthis trial is a substudy of a large multicenter randomized trial evaluating the effect of goaldirected crystalloidversus goaldirected colloid fluid administration on acomposite of serious complications after moderate tohighrisk open abdominal surgery the overall trial concluded that colloids did not decrease the composite ofmajorincomplicationsare ourresultsconcordance as they did not show any differences inperioperative pro and antiinflammatory markers between a crystalloid and a colloid fluid regimendespite multimodal care and enhanced recovery programs it still remains challenging to blunt the inflammatory response to surgery systemic inflammationafter abdominal surgery impairs outcome and thereforemany attempts have been made to alter the inflammatory response several factors influence the perioperative inflammatoryresponse such as the underlying disease type and invasiveness of surgery as well as type of anesthesia [] themost important factor is the magnitude of surgical traumaand tissue damage which induce proliferation and activation of immune competent cells in turn triggering cytokine and inflammatory marker release so far veryfew trials have specifically investigated the influence offluid therapy and differences in terms of the type of fluidon the extent of inflammatory marker releaseto investigate the potential influence of goaldirected hydroxyethyl starch versus a lactated ringers solution fluid regimen on inflammatory response pro il il and tnf α and antiinflammatory cytokine il serum levels were measured during the perioperativeperiod additionally we measured wbc crp pct andlbpgenerally the most commonly measured biomarkersare crp and wbc if levels of crp are above mgdl after postoperative day four a postoperative infection can be suspected crp levels in our studygroups increased on the first postoperative day droppingon the fourth postoperative day to nearly mgdl inboth study groupswbcs are an imprecise marker to detect postoperativecomplications after major abdominal surgery amore sensitive parameter in predicting postoperativecomplications after major abdominal surgery is il surgical trauma and hypoperfusion of the colon aremain sources of il release in colorectal surgery noblett demonstrated that gdt during elective colorectal surgery significantly reduced il levels in comparison to a control group yates showed no differencesof il and il levels between goaldirected colloidand crystalloid fluid therapy during the first h in asubgroup of patients undergoing colorectal surgery although patients in the crystalloid group received significant more volume amount as compared to the colloid groupthere was no significant difference inhemodynamic variables our patients showed similar courses of il and il levels in the immediatepostoperative period in contrast to the trial of yateswho measured cytokine levels up to the first h aftersurgery we extended our measurement period to fourpostoperative days we showed comparable circulating 0cobradovic bmc anesthesiology page of table intraoperative dataduration of anesthesia minduration of surgery minfluid managementtotal fluid intake mlacrystalloid mlcolloid mlestimated blood loss mltransfusion yesno urinary output mlanesthesia managementfentanyl mcgtwa et sevoflurane core temperature °cicu admission yesno hemodynamictwa map mmhgtwa hr beatsmintwa ftc mstwa sv mltwa co lmin phenylephrine yesno crystalloidsn ± ± [ ] ± ± ± ± colloidsn ± ± [ ] ± ± ± ± p value ° ° °° °°intraoperative data are presented as means ± sd medians iqr or as counts for the categorical outcomes means were compared with an unpaired twosided ttests ormannwhitneyu tests as appropriate medians with wilcoxon ranksum tests and counts with chisquare or fishers exact tests ° represents statisticalsignificance p abbreviations et end tidal icu intensive care unit twa time weighted average map mean arterial pressure hr heart rate ftc corrected flow time sv strokevolume co cardiac output sd standard deviationa total fluid intake includes baseline fluid boluses antibiotics analgesics and additional fluid administered at the discretion of attending anesthesiologistfig ad pro and antiinflammatory cytokines il il il and tnf α over time a il b il c il and d tnf α data arepresented as medians iqr abbreviations il interleukin tnf α tumor necrosis factor alpha pod postoperative day 0cobradovic bmc anesthesiology page of table areas under the curve of inflammatory markerspcrystalloidsn colloidsn auc il pgml 1dauc il pgml 1dauc il pgml 1dauc tnf α pgml 1d auc wbc gl1dauc crp mgdl 1dauc pct ngml 1dauc lbp mcgl 1dtable areas under the curve of il il il and tnf α as well as wbccrp pct and lbp are presented as medians iqr medians were comparedwith wilcoxon ranksum testsabbreviations il interleukin tnf α tumor necrosis factor alpha wbc whiteblood cells pct procalcitonin lbp lipipopolysaccharidebinding protein il and il levels between a gdt crystalloid and colloid administration these surrogates of inflammatoryresponse imply that gut perfusion during surgery waswell preserved with both types of fluid and suggest thatthe type of fluid might be of minor importance as longas the fluid is administered in a goaldirected fashionthe fact that tnf α levels in both groups remainedstable over the entire measured period further supportsour theory the course of tnf α levels during the perioperative period was in accordance with the study ofin which fluid therapy was guided withszakmanypicco versusin majorvenous pressurecentralabdominal surgery in patients at risk for postoperativecomplications as tnf α per se triggers glycocalyxdegradation we anticipate that tnf α did not influence glycocalyx shedding and thus possible fluid shifts inour study populationpct is an early predictive marker for systemic inflammation after abdominal surgery values above ngml are associated with postoperative complicationssuch as pneumonia or anastomotic leakage in ourstudy median pct levels did not exceed ngml atany measured time point these results are in concordance with our main study where infectious complications rate were held low and did not differ between thegroups moreover as pct production can also beinduced by tissue hypoperfusion we might assume thatgoaldirected fluid administration contributed to lowpct values by optimizing cardiac performance furthermore we measured lbp a prognostic markerfor bacterial infections patients in the crystalloidgroup showed significantly higher levels immediatelyafter surgery however the measured values remainedwithin the normal range therefore this difference ismost likely not to of clinical importancethe vascular endothelium is one of the earliest sitesinvolved in the inflammatory response syndrome an adequate perioperative fluid management has a major impact on the integrity of the glycocalyx with goaldirected fluid management individualized and time appropriate fluid resuscitation can be achieved enablingfig ad inflammatory markers wbc crp pct and lbp over time a wbc b crp c pct and d lbp data are presented as medians iqrabbreviations wbc white blood cells crp creactive protein pct procalcitonin lbp lipopolysaccharidebinding protein pod postoperative day
represents significant difference in lbp in the immediate postoperative period between the crystalloid and the colloidgroup p 0cobradovic bmc anesthesiology page of preservation of endothelial surface layer and sufficientan perfusion thus improving postoperative outcomesafter major surgery patients in the colloid group received significantly lessfluid ml confirming the previously publishedfluid sparing effect of colloids however clinical significance of this difference may be questionable during aperioperative period of nearly five hours further ourhemodynamic data showed significantly higher values ofsv and co with colloid administration though the absolute difference of ml in sv most likely has onlylimited clinical relevance it might very well be that thesignificant differences in sv and co are the result ofour number of patients included in this substudyassurrogatesfirst limitation of our study is that we measured inflammatory markers that reflect the inflammatory responseand not direct markers ofglycocalyx degradation like syndecan1 therefore wecannot draw any s about the preservation ofthe endothelial surface layer in our patients secondlywe did not control postoperative fluid management during the postoperative followup period a further limitation isthe time between patient enrolment andsubmission of our current results due to the fact thatthe main trial has been published recently a delay of oursubmission occurred nevertheless our results canstill be extrapolated to current clinical practicein summary goaldirected hydroxyethyl starch administration did not attenuate the inflammatory responseexpressed by cytokine levels of il il il and tnfα in patients undergoing moderate to highrisk open abdominal surgery wbc crp and ptc values did not differ between the different fluid regimes as wellabbreviationsasa american society of anesthesiologists asd absolute standardizeddifference auc area under the curve bmi body mass index co cardiacoutput crp creactive protein ecg electrocardiography ef ejectionfraction elisa enzymelinked immunosorbent assay etco2 endtidalcarbon dioxide ftc corrected flow time gdt goaldirected therapyhr heart rate ibw ideal body weight icu intensive care unitil interleukin iqr interquartile range lbp lipopolysaccharidebindingprotein map mean arterial pressure pacu postanesthetic care unitpct procalcitonin picco pulse contour cardiac output spss statisticalpackage for the social sciences sv stroke volume tnf α tumor necrosisfactor alpha twa time weighted average wbc white blood cell countacknowledgementsassistance with this we thank bianca tudor md department ofanesthesia intensive care medicine and pain medicine medical university ofvienna spitalgasse vienna austria for her support in laboratoryskills performing elisasauthors contributionsall authors have read and approved the manuscript mo patientrecruitment data acquistion and prepratation of the manuscript ak studyprotocol writing and preparation of the manuscript bk study protocolwriting and preparation of the manuscript statistical analyisis gr dataanalysis revision of the manuscript ok data analysis preparation of themanuscript oz patient recruitment data acquisition data managementab patient recruitment data acquisition revsion the manuscript cr dataacquistion revision of the manuscript as patient recruitment revision ofthe manuscript ef study protocol writing and preparation of the manuscriptfundingmedical university of viennapartially funded by fresenius kabi deltex medical provided oesophagealdoppler monitors and disposablesthe sponsors were not involved in protocol development data acquisitionor data analysisavailability of data and materialsthe datasets used andor analysed during the current study are availablefrom the corresponding author on reasonable requestbarbarakabonmeduniwienacatethics approval and consent to participatethe main trial was approved by the local ethics committee of the medicaluniversity of vienna in ek and was registered atclinicaltrialsgov nct00517127 and eudract a writteninformed consent was obtained from all patients prior to participationconsent for publicationnot applicablecompeting intereststhe authors declare no competing interestsauthor details1department of anaesthesia general intensive care medicine and painmedicine medical university of vienna spitalgasse vienna austria2department of outcomes research and general anesthesiologyanesthesiology institute euclid avenue cleveland clinic cleveland ohusa 3department of anesthesiology and general intensive care franziskushospital nikolsdorfergasse vienna austria 4department ofgynecology klinik ottakring montleartstrasse vienna austria5department of surgery medical university of vienna spitalgasse vienna austriareceived july accepted august referencesbrandstrup b tonnesen h beierholgersen r effects of intravenousfluid restriction on postoperative complications comparison of twoperioperative fluid regimens a randomized assessorblinded multicentertrial ann surg myles ps bellomo r corcoran t restrictive versus liberal fluidtherapy for major abdominal surgery new engl j med chappell d jacob m hofmannkiefer k conzen p rehm m a rationalapproach to perioperative fluid management anesthesiology lowell ja schifferdecker c driscoll df benotti pn bistrian brpostoperative fluid overload not a benign problem crit care med sun y chai f pan c romeiser jl gan tj effect of perioperative goaldirected hemodynamic therapy on postoperative recovery following majorabdominal surgerya systematic review and metaanalysis of randomizedcontrolled trials critical care calvovecino jm ripollesmelchor j mythen mg effect of goaldirected haemodynamic therapy on postoperative complications in lowmoderate risk surgical patients a multicentre randomised controlled trialfedora trial br j anaesth noblett se snowden cp shenton bk han af randomized clinical trialassessing the effect of doppleroptimized fluid management on outcomeafter elective colorectal resection br j surg 0cobradovic bmc anesthesiology page of alphonsus cs rodseth rn the endothelial glycocalyx a review of thevascular barrier anaesthesia pearse rm harrison da macdonald n effect of a perioperativecardiac outputguided hemodynamic therapy algorithm on outcomesfollowing major gastrointestinal surgery a randomized clinical trial andsystematic review jama publishers notespringer nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliationskimberger o arnberger m brandt s goaldirected colloidadministration improves the microcirculation of healthy andperianastomotic colon anesthesiology kolarova h ambruzova b svihalkova sindlerova l klinke a kubala lmodulation of endothelial glycocalyx structure under inflammatoryconditions mediat inflamm rettig tc verwijmeren l dijkstra im boerma d van de garde emnoordzij pg postoperative interleukin6 level and early detection ofcomplications after elective major abdominal surgery ann surg gan tj soppitt a maroof m goaldirected intraoperative fluidadministration reduces length of hospital stay after major surgeryanesthesiology jacob m chappell d rehm m clinical update perioperative fluidmanagement lancet feldheiser a pavlova v bonomo t balanced crystalloid comparedwith balanced colloid solution using a goaldirected haemodynamicalgorithm br j anaesth orbegozo cortes d gamarano barros t njimi h vincent jl crystalloidsversus colloids exploring differences in fluid requirements by systematicreview and metaregression anesth analg kabon b sessler di kurz a effect of intraoperative goaldirectedbalanced crystalloid versus colloid administration on major postoperativemorbidity a randomized trial anesthesiology robinson jd lupkiewicz sm palenik l lopez lm ariet m determination ofideal body weight for drug dosage calculations am j hosp pharm steppan j hofer s funke b sepsis and major abdominal surgery leadto flaking of the endothelial glycocalix j surg res wilmore dw from cuthbertson to fasttrack surgery years of progress inreducing stress in surgical patients ann surg desborough jp the stress response to trauma and surgery br j anaesth veenhof aa sietses c von blomberg bm the surgical stress responseand postoperative immune function after laparoscopic or conventional totalmesorectal excision in rectal cancer a randomized trial int j color dis gilliland he armstrong ma carabine u mcmurray tj the choice ofanesthetic maintenance technique influences the antiinflammatory cytokineresponse to abdominal surgery anesth analg wichmann mw huttl tp winter h immunological effects oflaparoscopic vs open colorectal surgery a prospective clinical study archsurg watt dg han pg mcmillan dc routine clinical markers of themagnitude of the systemic inflammatory respon | Colon_Cancer |
section all disclosure information for gie editors can befound online at httpwwwgie contentconï¬ictoï¬nterest cme editors and their disclosures are as followsprasad g iyer md associate editor for cmeconsultingadvisoryspeaking olympus research supporttakeda pharmaamit rastogi md associate editor for cmeconsultingadvisoryspeaking olympuskarthik ravi md cme editordisclosed no relevant ï¬nancial relationshipswilliam ross md cme editorconsultingadvisoryspeaking boston scientiï¬c olympusara sahakian md cme editordisclosed no relevant ï¬nancial relationshipsbrian weston md cme editordisclosed no relevant ï¬nancial relationshipsall cme activities including their associated sare copyrighted by the asge gastrointestinal endoscopy volume no wwwgie 0ccme activitycontinuing medical education questions september question objectivedemonstrate triaging advanced gi endoscopy procedures during the covid19 pandemictriaging advanced gi endoscopy procedures during the covid19 pandemicquestion due to the resurgence of covid19 in your area healthofï¬cials issue a mandate to cease elective proceduresbased on the ï¬ndings of the current study a consensuswas reached that all of the following advanced gi procedures can be safely deferred for weeks exceptpossible answers aea ercp for asymptomatic choledocholithiasisb radiofrequency ablation for barretts esophagus withhighgrade dysplasiac endoscopic mucosal resection for barretts esophaguswith nodular highgrade dysplasiad endoscopic mucosal resection for a cm adenomatous colon polyp with highgrade dysplasiae eus for incident pancreatic and common bile duct dilation on ct or mri normal liver function testslookup sawhney ms bilal m pohl h triaging advanced gi endoscopy procedures during the covid19 pandemic consensus recommendationsusing the delphi method gastrointest endosc question objectivedescribe the learning curve for ablative therapy for dysplastic barretts esophagus and the effect of center volumes onoutcomeslearning curves and the uence of procedural volume for the treatment of dysplastic barrettsesophagusquestion a 53yearold man returns for surveillance endoscopy ofc3m5 nondysplastic barretts esophagus initially diagnosed years prior the patient has been compliant with twicedailyproton pump inhibitor therapy and denies any heartburnacid regurgitation or dysphagia he has no other significantmedical history you perform an egd which again reveals ac3m5 barretts segment without any nodularity biopsyspecimens are obtained and subsequently reveal multifocallowgrade dysplasia which is subsequently conï¬rmed by anexpert gi pathologist after a subsequent discussionregarding ablative therapy with radiofrequency ablationrfa versus continued surveillance the patient wishes topursue ablation you discuss referral to a new colleague whojoined your practice several months earlier the colleague isthe ï¬rst endoscopist to perform rfa at your institution andhas treated a total of patients the patient inquireswhether he would be more likely to have a successfuloutcome if he is referred to a moreexperienced endoscopistat a highvolume center which of the following is truepossible answers ada the patient is more likely to achieve complete remission of intestinal metaplasia crim if he undergoesablation at a center that has treated more than patientsb the patient is more likely to achieve complete remission of dysplasia crd if he undergoes ablation at acenter that has treated more than patientsc the patient is more likely to achieve crim if he undergoes ablation by an endoscopist who has treatedmore than patientsd the patient is more likely to achieve crd if he undergoes ablation by an endoscopist who has treatedmore than patientslookup lipman g markar s gupta a learning curves and the uence of procedural volume for the treatment of dysplastic barretts esophagusgastrointest endosc wwwgie volume no gastrointestinal endoscopy 754e1 0ccme examquestion objectiveexplain the utility of a new gi bleeding prediction score for patients with upper gi bleedingthe horibe gi bleeding prediction score a simple score for triage decisionmaking in patients withsuspected upper gi bleedingquestion you are called to the emergency department to see apatient who has just arrived with the complaint of blacktarry stools the emergency department physician has notyet seen the patient but the nurse relays vital signs labresults and history sufï¬cient to calculate a harbingerscore according to the study by horibe in thismonths issue this score is used to assess risk of which ofthe followingpossible answers ada deathb need for blood transfusionc need for endoscopic interventiond presence of highrisk stigmatalookup horibe m iwasaki e bazerbachi f horibe gi bleeding prediction score a simple score for triage decisionmaking in patients with suspectedupper gi bleeding gastrointest endosc question objectivereport the longterm efï¬cacy and safety of eusguided hepaticogastrostomy for treatment of malignant biliary obstructionlongterm outcomes of a long partially covered metal stent for eusguided hepaticogastrostomy inpatients with malignant biliary obstructionpossible answers ada success rate of eushgs is less than b adverse events occur in less than of casesc recurrent biliary obstruction occurs in about onethirdof patientsd prior biliary drainage has no impact on the likelihood ofrecurrent biliary obstructionquestion a 58yearold man is transferred to your hospitalformanagement of a pancreatic head mass causing obstructive jaundice and gastric outlet obstruction an endoscopic ultrasound with ï¬neneedle aspiration of the massconï¬rmed pancreatic adenocarcinoma and a contrastenhanced computed tomography scan demonstratednumerous liver lesions compatible with metastatic disease a duodenal stent was placed which precluded anercp procedure for biliary drainage biliary drainage isnecessary before chemotherapy can be initiated variousoptions are discussed with the patientincluding eusguided hepaticogastrostomy eushgs according tothe current study by nakai which of the following isaccuratelookup nakai y sato t hakuta r longterm outcomes of a long partially covered metal stent for eusguided hepaticogastrostomy in patients withmalignant biliary obstruction with video gastrointest endosc 754e2 gastrointestinal endoscopy volume no wwwgie 0ccme activitycontinuing medical education answers september question correct response erationale for correct responsethe covid19 pandemic has necessitated many elective procedures to be rescheduled in order to mitigate the spreadof infection and conserve vital resources choosing which advanced endoscopic procedures should be performed or safelydeferred during the covid19 pandemic may be a complex decision14 the aim of the current study was to provideguidance for triaging advanced endoscopic procedures using a modiï¬ed delphi method the delphi method is a validatedand structured technique to obtain expert consensus which is useful in the present situation in which outcome data arelimited156 the following important patient outcomes were used avoidance of deathprolongation of life avoidance of cancercancer progression avoidance of major surgery andor hospitalization and improvement orpalliation of symptoms the following procedural timing categories were used timesensitive emergent schedulewithin week timesensitive urgent schedule within to weeks or nontime sensitive defer for weeks andthen reassess the timinga prespeciï¬ed consensus threshold of was achieved in of advanced endoscopy indications reviewed by expert gastroenterologists one hundred percent consensus was achieved in of indications in of indications to consensus was achieved the only indication for which consensus could not be achieved was incidentallyfound pancreatic duct dilation mm and common bile duct dilation mm on ct scan or mri with normal liverfunction teststhis study provides a decisionmaking framework for endoscopists to determine the timing for endoscopic proceduresduring and after the pandemic the decision to perform endoscopy during the covid19 pandemic needs to balance therisks associated with delaying the procedure in the individual patient with the risk of viral exposure to patients and healthcare providerstakehome message triaging advanced gi procedures during the covid19 pandemic may be facilitated by thecurrent consensus recommendations using the delphi method individual practice and patient factors must also beconsideredreferences sawhney ms bilal m pohl h triaging advanced gi endoscopy procedures during the covid19 pandemic consensus recommendations usingthe delphi method gastrointest endosc sultan s lim jk altayer o aga institute rapid recommendations for gastrointestinal procedures during the covid19 pandemic gastroenterology epub mar joint gi society message on covid19 available at httpsgi20200315jointgisocietymessageoncovid19 accessed march gastroenterology professional society guidance on endoscopic procedures during the covid19 pandemic available at httpswebï¬lesgilinksmediajoint_gi_society_guidance_on_endoscopic_procedure_during_covid19_final_impending_3312020pdf accessed april hsu cc sandford ba the delphi technique making sense of consensus pract assess res eval donohoe h stellefson m tennant b advantages and limitations of the edelphi technique implications for health education researchers am j healtheduc question correct response crationale for correct responseendoscopic eradication therapy for dysplastic barretts typically uses endoscopic mucosal resection of visible lesionswith subsequent ablation of the ï¬at barretts segment with rfa this approach has demonstrated high clinical successwith estimates that crd is achieved in over and crim in over of treated patients1 however studies assessingthe learning curve associated with attaining proï¬ciency with endoscopic therapy of dysplastic barretts and the effect ofcase volumes at centers on outcomes have yielded unclear results23 consequently current guidelines suggesting minimum numbers of cases to achieve competence for individual endoscopists or reï¬ecting quality for centers are based onlimited evidence4wwwgie volume no gastrointestinal endoscopy 754e3 0ccme answersin this months issue of gie lipman and colleagues5 report results from a retrospective study examining patientstreated in the uk rfa registry to assess crd crim and dysplasia recurrence based on endoscopist experience andcenter case volumes5 a total of consecutive patients were identiï¬ed from centers which were further divided into patients treated at lowvolume centers patients treated patients treated at mediumvolume centers patients treated and patients treated at highvolume centers patients treated rates of crd and crim at months did not differ based on center volumes however dysplasia recurrence washigher in patients treated at lowvolume centers further analysis using a riskadjustment cumulative risk sum curve wasdone to assess the effect of the learning curve on individual endoscopists a significant reduction of crd was seen at cases whereas a similar reduction of crim was seen at cases takehome message in conclusion this study suggests that the learning curve for rfa may be relatively short withfewer than cases required to achieve competency further center volume may have a very limited effect on outcomesfuture prospective studies are still required to assess the optimal number of training cases required to achieve endoscopicablative competencyreferences haidry rj dunn jm butt ma radiofrequency ablation and endoscopic mucosal resection for dysplastic barretts esophagus and early esophagealadenocarcinoma outcomes of the uk national rfa registry gastroenterology pasricha s cotton c hathorn ke effects of the learning curve on efï¬cacy of radiofrequency ablation for barretts esophagus gastroenterology fudman di lightdale cj poneros jm positive correlation between endoscopist radiofrequency ablation volume and response rates in barrettsesophagus gastrointest endosc fitzgerald rc di pietro m ragunath k british society of gastroenterology guidelines on the diagnosis and management of barretts oesophagusgut lipman g markar s gupta a learning curves and the uence of procedural volume for the treatement of dysplastic barretts esophagus gastrointest endosc question correct response drationale for correct responsefor decades gastroenterologists have been searching for a straightforward method to determine whether a patientpresenting with an upper gastrointestinal bleed is at high risk of needing hospital admission therapeutic interventions ordeath multiple scoring systems have been proposed but systematic reviews have found them all ï¬awed to various degrees12 however one of the betterknown scores proposed by blatchford has been recommended in recentguidelines to assess risk of requiring an intervention34 yet the quest for a more predictive model continues with somegroups turning to machine learning methods for an answer5in this months issue of gie horibe 6 report on a simple 3factor assessment to predict the likelihood of ï¬ndinghighrisk stigmata hrs on endoscopy which they call harbinger because their median time to endoscopy was only hours the prevalence of hrs was quite high up to even in patients deemed to be low risk for stigmata predictablythe intervention rate was more than double that in a large international trial vs comparing harbinger toother scoring systems including blatchfords the authors found their model to be superior this outperformance couldhave been predicted because the other models were designed to detect risk of other endpoints such as mortality or needfor intervention not hrsin addition the performance of the model was not consistently strong across the study institutions because auc valuesfor harbinger ranged from to between institutions with no overlap in conï¬dence intervals between the topand low scores6 concerns about how harbinger would perform in other settings are reasonable as factors like access toppis varies across marketsin a resourceconstrained world the ability to predict need for interventions would seem more helpful than predictingendoscopic appearance although hrs are linked to the need for interventions the rapid time to endoscopy skews thelinkage by ballooning the prevalence of hrs many of these patients are likely to have far less ominous endoscopic appearances if the endoscopy was done hours after presentationreferences ramaekers r mukarram m smith cam the predictive value of preendoscopic risk scores to predict outcomes in emergency department patientswith upper gastrointestinal bleeding a systematic review acad emerg med de groot nl bosman jh siersema pd prediction scores in gastrointestinal bleeding a systematic review and quantitative analysis endoscopy blatchford o murray wr blatchford m a risk score to predict need for treatment for uppergastrointestinal hemorrhage lancet barkun an almadi m kuipers ej management of nonvariceal upper gastrointestinal bleeding guideline recommendations from the internationalconsensus group ann int med 754e4 gastrointestinal endoscopy volume no wwwgie 0ccme answers shung dl au b taylor ra validation of a machine learning model that outperforms clinical risk scoring systems for upper gastrointestinalbleeding gastroenterology horibe m iwasaki e bazerbachi f horibe gi bleeding prediction score a simple score for triage decisionmaking in patients with suspected uppergi bleeding gastrointest endosc stanley aj laine l dalton hr comparison or risk scoring systems for patients presenting with upper gastrointestinal bleeding international multicenter prospective study bmj 2017356i6432question correct response crationale for correct responsebiliary drainage options may be limited by gastric outlet obstruction or surgically altered anatomy eusguided hepaticogastrostomy eushgs provides an effective alternative to ercp and percutaneous drainage in these complex cases1additionally eushgs has been compared to ercp for primary biliary drainage and outcomes were found to be comparable2 however potential adverse events of eushgs such as stent migration and peritonitis can be severe and longterm data thus far have been lacking3in the current issue of gie nakai and colleagues4 present a retrospective study of patients who underwent eushgs for drainage of malignant biliary obstruction partially covered metallic stents ranging in length from to cm wereused to create the hepaticogastrostomy technical and functional success rates were and respectively adverseevents occurred in of patients with the most common being fever and abdominal pain peritonitis occurred in andcholangitis in of patients no cases of stent dislodgement were reported recurrent biliary obstruction rbo occurredin of patients in a median time of months short length of the intragastric portion of the stent and prior biliarydrainage were both associated with recurrent biliary obstruction of the patients who had rbo underwent successfulreintervention with eushgstakehome message eusguided hepaticogastrostomy is an effective alternative for longterm biliary drainage recurrences can be successfully managed with repeat hepaticogastrostomyreferences nakai y isayama h yamamoto n safety and effectiveness of a long partially covered metal stent for endoscopic ultrasoundguided hepaticogastrostomy in patients with malignant biliary obstruction endoscopy paik wh lee th park dh eusguided biliary drainage versus ercp for the primary palliation of malignant biliary obstruction a multicenter randomized clinical trial am j gastroenterol giovannini m eusguided hepaticogastrostomy endosc ultrasound 20198s35s9 nakai y sato t hakuta r longterm outcomes of a long partially covered metal stent for eusguided hepaticogastrostomy in patients with malignant biliary obstruction with video gastrointest endosc wwwgie volume no gastrointestinal endoscopy 754e5 0c' | Colon_Cancer |
" african americans aa are at high risk for colorectal cancer crc studies report a increasein crc risk with physical inactivity obesity and metabolic syndrome activation of the wntcatenin ctnnb1signaling pathway plays a critical role in colorectal carcinogenesis accumulating evidence also indicates a role ofwntctnnb1 signaling in obesity and metabolic diseasesaim to examine the association between obesity catenin expression and colonic lesions in african americansmethods we reviewed the pathology records of colorectal specimens from to crcs advanced adenomas and normal colon tissues tissue microarrays tma were constructed from these samplesimmunohistochemistry ihc for ctnnb1 catenin clone catenin1 was performed on the constructed tmasthe ihc results were evaluated by pathologists and the nuclear intensity staining was scored from to bmisex age location of the lesion and other demographic data were obtainedresults positive nuclear staining in normal advanced adenoma and crc was and respectively p crc was asso ciated with positive status for nuclear ctnnb1 intensity adjusted or 95ci p for positive nuclear staining compared to noncrc samples normal or advanced adenoma nuclearstaining percentage has a fair diagnostic ability for crc with an auc of 95ci overweightobese patients bmi did not show a significant difference in p nuclear ctnnb1 staining positive in normal weight vs positive in overweightobese the association between nuclear intensityand crc was not different between normal and overweight patients p for interaction the positive nuclearctnnb1status in crc stage iii and iv of all crc was not different from stage i and ii vs respectively p continued on next page correspondence b_shokranihowardedu hashktorabhowardedu1department of medicine department of pathology and cancer centerhoward university college of medicine geia avenue nwwashington dc usafull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cshokrani bmc gastroenterology page of continued from previous page in our study advanced adenoma and crc were associated with activation of catenin in physically fitoverweight and obese patients thus obesity and wntcatenin pathway seem to be independent in africanamerican patients wntcatenin signaling pathway has a potential to be used as an effector in coloncarcinogenic transformation whether or not bmi is a modifier of this pathway needs to be investigated furtherkeywords catenin colorectal cancer advanced adenoma african americans colorectal cancer crc is one of the most commoncancers in the industrialized world lifestyle and epidemiological factors associated with an increased risk ofcrc include physical inactivity obesity and metabolicsyndrome in the united states approximately twothirds of the adult population are overweight or obesewhich represents a putative risk factor for multiple target an malignancies including crc there is evidence to suggest that excess adiposity is associated with up to greater risk of crc comparedwith normal weight individuals and that physical activity may decrease colorectal cancer risk althoughexcessive accumulation of white adipose tissue wat isthe key feature of adiposity obesity is clinically definedby a bmi over kgm2 which does not take fat contentinto account it is also known that most crcs arise froma genetic and morphological adenoma to carcinomatransition also it is widely accepted that both crcsand colorectal adenomas cras share similar etiologicalcauses which explains why cras which are amongstthe mostfindings in all crcscreening participants are present in more than ofgeneral asymptomatic populations consequentlyrisk algorithms have been applied to use bmi as a predictor variable to stratify individuals according to theirrisk of colorectal neoplasia however the underlyingmechanisms that might explain the association and themagnitude ofthe connection between excess bodyweight and crc remain unclearfrequent pathologicalin the obesitycancer relationship multiple biologicalprocesses including insulininsulinlike growth factorigf1 insulin resistance sexual hormones estrogensand proinflammatory cytokines tnfα il6 and crpactively participate all these elements create a favorable environment for carcinogenesis and a decrease inapoptosisas a separate molecular pathway activation of thewnt signaling pathway plays a critical role in colorectalcarcinogenesis wnt ligands are a family of proteinsfor normal cell development that are importantcatenin ctnnb1 is a major mediator of the wntpathway that is traditionally classified into canonical catenindependent and noncanonical cateninindependent wnt canonical pathway utilizes a group ofcell surface receptors called frizzled frz to activateseveral pathways the most important one involving catenin and apc in the absence of wnt signalingapc causes degradation of catenin preventing its accumulation in the cytoplasm by forming a complex withcatenin which leads to the phosphorylation and eventually destruction of catenin by the proteasome signaling by wnt blocks this process allowing cateninto migrate from the cytoplasm to the nucleus once inthe nucleus catenin upregulates cmyc cyclin d1and other genes which increase cellular proliferation therefore continuous wnt signaling can be seenin cells with loss of apc metabolic syndromeassociated conditionssuch asobesity and type ii diabetes are influenced by geneticand functional variations in the wnt signaling pathway wnt signaling when activated represses the terminal differentiation during adipogenesis whereby preadipocytes take on the characteristics of mature adipocytes a cascade of transcriptional events like the induction of catenin ensues which in turn inducesenhancer binding proteinα cebpa and peroxisomeproliferatoractivated receptorÎ pparg the excessive accumulation of wat features adiposity butobesity does not take fat content into account recently genetic factors linked to fat mass and adipositywere reported to be associated with increased obesityrisk in young obese individuals wholeexome sequencing revealed rare gainoffunction mutations inctnnb1catenin the cateninregulated transcription of an adipocytederived chemokine calledserum amyloid a3 saa3 leads to the formation of a catenintcf complex in mature adipocytes that promotes the proliferation of preadipocytes in wat andthereby increases obesity and the risk for metabolic syndrome other data also suggest that obesity and lack ofphysical activity are associated with a higher risk forcolorectal cancer [ ] these findings have importantimplications especially in the obese and physically inactive african american population that may haveunderlying predisposing mutations to colorectal cancer the aim of this study is to assess the catenin expression profile in colorectal premalignant and malignant lesions in correlation with obesity as determined by 0cshokrani bmc gastroenterology page of body mass index bmi or waist circumference wc inafrican american populationmethodspatients and clinical datacolorectaltissue samples submitted to the surgicalpathology laboratory at howard university hospitalfrom january to december were retrieved from the pathology archive system powerpath¢a total of samples were included in the study consisting of tissue samples from crc n advancedadenoma n and normal colon n patientsdata included age sex height weight and waist circumference body mass index bmi was calculated in thestudy table the protocol of this study was approvedby the howard university institutional review boardirbtissue microarray tma constructionhematoxylineosin stained slides he slides weremade from paraffinembedded blocks the he slideswere reviewed by two pathologists to confirm the pathological diagnosis and to mark areas of interest multipleareas from more than one block were marked to ensurea good representability of the sample on the tma fivetma paraffin blocks each containing cores of mmin diameter each and mm distance from each otherwere constructed tissuespecific and an system controls were included in each tmaimmunohistochemical ihc analysis of ctnnb1 cateninthe constructed tmas were stained for catenin theimmunostaining was carried out as follows dako monoclonal mouse antihuman betacatenin cateninc1intended for laboratory application to identify qualitatively by light microscopy catenin positive cells in normal and neoplastic tissues was used at a dilution of using the envision dab code k4006 detectionsystem the deparaffinized tmas were treated prior tothe ihc staining procedure target retrievalinvolvedimmersion of tissue sections in a preheated buffer solution and maintaining heat in a water bath °cfor greater adherence of tissue sections to glass slidessilanized slides dako code s3003 were used targetretrieval solution code s1700 or 10x concentratecode s1699 using a 20min heating protocol was usedthe cellular staining pattern of anticatenin is mainlymembranous especially at the cellcell boundaries positive nuclear staining and diffuse cytoplasmic staining arealso reported in cancer cells fig evaluation and assessment of the catenin expressiontwo pathologists interpreted the ihc slides cateninexpression status was assessed based on the pattern ofstaining nuclear cytoplasmic and membranous intensity to and percentage of staining to the staining would be considered negative if there wasweak or no nuclear expression or positive if there wasmoderate or strong nuclear expressionstatistical analysisdistribution of continuous and categorical variables weretested by kruskalwallis and chisquare test betweendifferent groups respectively we used logistic regression analysis to test association between the staining andrisk of crc after adjusting for age and gender areaunder the curve auc was calculated for variables withsignificant association with crc using receiver operative characteristics curve all statistical analyses wereperformed by stata statacorp college station txresultsepidemiological characteristics and bmi in normaladvanced adenoma and crcthe bmi was calculated for individual patients and normal subjects as represented in table crc patientswere older p while our healthy normal population was mostly overweight higher bmi was moreclosely associated with advanced adenoma and crchowever the differences were not significant table advanced adenomas and crcs were associated withpositive nuclear ctnnb1we assessed whether alterations in wntctnnb1 catenin signaling plays any role colon carcinogenesispositive catenin nuclear stains were seen in normaladvanced adenomas and crcs were and respectively p table based on the designationtable epidemiological characteristics and bmi in normal advanced adenoma and crc patientsage median iqrmale n bmi kgm2 median iqroverweight n normaln advanced adenoman crcn p value 0cshokrani bmc gastroenterology page of fig immunostain for catenin in three individuals normal a à advanced adenoma b à and cancer c d à and à respectively showing membranous staining in the normal cytoplasmic and membranous staining in adenoma with no evidence of nuclearexpression arrow showing lack of nuclear staining and nuclear and cytoplasmic staining in cancer arrow showing nuclear stainingof n intensity which is associated with higher risk ofcancer crcs were associated with positive status fornuclear ctnnb1 intensity age gender adjusted or 95ci p for positive nuclearstaining compared to noncrc samples normal or advanced adenoma fig nuclear staining percentagehas also a fair diagnostic ability for crc with an auc of 95ci table fig overweight and obese patients show a trend withpositive nuclear ctnnb1 expressionpositive nuclear ctnnb1 staining was in normalweight and in overweightobese bmi patientsthis difference pointed to trend that was not statisticallysignificantassociation between nuclear intensity and crc in normaland overweight patientsthe association between nuclear intensity and crcwas not statistically significant different between normal weight and overweight patientsinteraction tables and the positive nuclearctnnb1 status in crc stage iii and iv of allcrc was not different from stage i and ii vs respectively p p fortable catenin nuclear and cytoplasmic expression tabulated as intensity and percentage in normal advanced adenoma andcrcnormaln cnc intensity n intensity advanced adenoma n crcn c cytoplasmic n nuclearc intensity and n intensity mean intensity and aboveoverall p valuep value for advanced adenoma vs normalp value for crc vs other 0cshokrani bmc gastroenterology page of fig catenin nuclear and cytoplasmic expression in normal advanced adenoma and crcdiscussionone of the important risk factors in colorectal cancer isobesity [ ] catenin is an ecadherin binding proteinthat mediates cellcell adhesion and plays a role inthe canonical wnt signaling pathway that controls thecoordinated expansion and differentiation of the intestinal crypt stem cells degradation of catenin byphosphorylation followed by alteration of destructioncomplex apc gsk3 and axin results in inactivation if wnt pathway in our study we found thatwas associated with an increased adjusted or of 95ci p for positive nuclear staining compared to noncrc samples normal or advanced adenomathe gatekeeper gene apc is a negative regulator of catenin and is mutated in approximately of sporadic and hereditary colon cancers there are severalmutations that can cause an accumulation of cateninin tumor cells such as mutations of the apc gene pointmutations in gsk3 or mutations in catenin gene itself []our positive nuclear staining in crc and its association with the positive status for nuclear ctnnb1intensity compared to noncrc samples are in contrastto a study by brabletz which showed that catenin is localized in the cytoplasm and membrane ofthe tumor cells whereas in our study it was mainly concentrated in the cytoplasm and the nucleus they alsomentioned that there was positive nuclear staining at theinvasive front as catenin is involved in tumor progression such is not the case in our studyindeed evenwhen considering nuclear staining in our specimensthere was no statistically significant differences betweenstage iiiiv cases staining versus stages iii crc caseslevels of staining the fact that catenin is expressedearly in the african american specimens analyzed heremight partially explain the aggressive nature of crc inthis population in addition we showed that there is uniform membranous staining in normal and increasingcytoplasmic and nuclear staining in advanced adenomasand crcs this confirms that the decrease in membranous staining begins with dysplastic changes leading to atable association of bmi with catenin nuclear intensity in advanced adenoma casesadvanced adenoma with catenin expression n in intensity and no nuclear intensity n bmi median interquartileoverweight n nuclear intensity negativen nuclear intensity n p value 0cshokrani bmc gastroenterology page of table association of bmi with catenin nuclear intensity in crc casescrc with catenin expression with high nuclear intensity and without negativebmi median interquartileoverweight n nuclear intensity negativen nuclear intensity n p valueprogressive disappearance atcrcsthe membrane levelinas we mentioned above a major risk factor for crc isobesity which continues to expand as a pandemicworldwide the american cancer society cancerprevention study ii states that there is an increased incidence of crc esophageal adenocarcinoma and othercancers with obesity in our study we showed that of advanced adenoma patients and of crc patients were overweight with bmi in comparison toadvanced adenoma the percentage was lower in cancerperhaps due to the late stage of cancer and weight lossin the interim table there are several mechanismsby which obesity is believed to promote crc this includes increase in leptin levels that cause an increase ingrowth and proliferation of colon cancer cells altered adipokine levels altered gut microbiome apartfrom increased steroid hormones and growth factors insulin is however the established biochemical linkand the main pathway involved is pik3aktmtorpathway elevated igf1 and insulin act through the insulin receptors and phosphotidylinositide3 kinase in addition to the above findings we also found thatoverweight and obese patients bmi did not showa significantly increased expression p of nuclearctnnb1 positive in normal weight vs positive in overweightobese morikawa found that inobesepositivity wasnuclear ctnnb1patientsassociated with significantly better cancerspecific survival suggesting that wnt signaling acts as a switch andwhen it is on adipogenesis is repressed kennell demonstrated that activated frz1 frizzled promotes catenin stabilityinhibits apoptosis and adipogenesisross also showed that wnt signaling acts as a molecular switch that controls adipogenesis upregulationof wnt signaling maintains preadipocytes in an undifferentiated state and when wnt signaling is prevented theydifferentiate into adipocytes [ ]although in our study there was no association between nuclear intensity and crc between normal andoverweight patients p for interaction there is accumulating evidence to show that the state of chronicinflammation incited by obesity might play a role in promoting colorectal carcinogenesis [ ] of the manymarkers tnfα is important [ ] as it activateswnt signaling through the induction of gsk3 phosphorylation resulting in increased nuclear localization ofcatenin in addition to tnfα other humoralagents associated with obesity might also be contributingto the activation of wnt signaling like il1 and adiponectin which is decreased in the obese state and is notan inflammatory cytokine that can modulate gsk3catenin signaling pathway although multiplemechanisms may be operating in parallel and contributing to the protumorigenic milieu wnt is a pivotaltumorigenic pathway aberrations of which isfig the putative relationship between obesity and colorectal cancer evolution pathways by cellular ctnnb1 status based on the data by thecurrent study our study suggests that there is no association between obesity and ctnnb1 expression 0cshokrani bmc gastroenterology page of important in the evolution of most sporadic crc insummary there is positive nuclear staining in crcs which was associated with the positive status fornuclear ctnnb1 intensity adjusted or 95ci p for positive nuclear staining compared to noncrc samples normal or advanced adenoma this shows that advanced adenomas and crcswere associated with activation of catenin in physically fit overweight and obese patients fig advanced adenoma and crc were associated with activation of catenin in physically fit overweight andobese patients thus participation of obesity and wntpathway seem to be independent crc factors in africanamerican patientsinflammationdriven activation ofwnt signaling as a potential pathway linking obesity tothe development of crc needs to be investigated furtherin the african american population this might provideinsights into the identification of new therapeutic targetsto reduce the burden of obesityassociated crcabbreviationscrc colorectal cancer wat white adipose tissue aa african americansacknowledgementsnot applicableauthors contributionsconceived and designed experiments bs ha performed experiments bsel ha hb analyzed data ha hb mn th aa and zs contributed reagentsmaterialsanalysis tools zs ha hb bs and el wrote and edited manuscriptbs and ha provided statistical analysis mn all authors have read andapproved the manuscriptfundingthis project was supported in part by grant from the national institute onminority health and health disparities of the national institutes of healthunder award numbersg12md007597 the funder had no role in designing or execution of thisstudyavailability of data and materialsall data generated or analyzed during this study are included in thispublished ethics approval and consent to participatethis retrospective and chart review study was conducted according to theworld medical association declaration of helsinki and was approved by theinternal review board of howard university since the chart review was donethrough unidentifiable approach no consent form needed for this studyconsent for publicationnot applicablecompeting intereststhe authors declare that they have no competing interests related to thismanuscriptauthor details1department of medicine department of pathology and cancer centerhoward university college of medicine geia avenue nwwashington dc usa 2division of pulmonary allergy and criticalcare medicine university of pittsburg pittsburg pa usareceived april accepted august referencesresearch iafco estimated cancer incidence mortality and prevalenceworlwide in lyon globocan haggar fa boushey rp colorectal cancer epidemiology incidencemortality survival and risk factors clin colon rectal surg oxentenko as body size and incident colorectal cancer a prospectivestudy of older women cancer prev res phila ma y obesity and risk of colorectal cancer a systematic review ofprospective studies plos one 201381e53916slattery ml physical activity and colorectal cancer sports med wong mc lam ty tsoi kk hirai hw chan vc ching jy chan fk sung jja validated tool to predict colorectal neoplasia and inform screening choicefor asymptomatic subjects gut sung jjy wong mcs lam tyt tsoi kkf chan vcw cheung w ching jyla modified colorectal screening score for prediction of advanced neoplasiaa prospective study of subjects jgastroenterolhepatol liu z dietinduced obesity elevates colonic tnfalpha in mice and isaccompanied by an activation of wnt signaling a mechanism for obesityassociated colorectal cancer j nutr biochem najdi r holcombe rf waterman ml wnt signaling and coloncarcinogenesis beyond apc j carcinog macdonald bt tamai k he x wntbetacatenin signaling componentsmechanisms and diseases dev cell denysenko t wntbetacatenin signaling pathway and downstreammodulators in low and highgrade glioma cancer genomics proteomics willert k nusse r betacatenin a key mediator of wnt signaling curr opingenet dev vella a camilleri m pharmacogenetics potential role in the treatment ofdiabetes and obesity expert opin pharmacother christodoulides c adipogenesis and wnt signalling trendsendocrinol metab world health anization [httpwwwwhointmediacentrefactsheetsfs311en] chen m lu p ma q cao y chen n li w zhao s chen b shi j sun yshen h sun l shen j liao q zhang y hong j gu w liu r ning g wangw wang j ctnnb1betacatenin dysfunction contributes to adiposity byregulating the crosstalk of mature adipocytes and preadipocytes sci adv20206eaax9605 morikawa t kuchiba a yamauchi m meyerhardt ja shima k nosho kchan at giovannucci e fuchs cs ogino s association of ctnnb1 betacatenin alterations body mass index and physical activity with survival inpatients with colorectal cancer jama morikawa t prospective analysis of body mass index physical activityand colorectal cancer risk associated with betacatenin ctnnb1 statuscancer res ashktorab h kupfer ss brim h carethers jm racial disparity ingastrointestinal cancer risk gastroenterology kaler p augenlicht l klampfer l activating mutations in betacatenin incolon cancer cells alter their interaction with macrophages the role of snailplos one 201279e45462enzo mv the wntbetacatenin pathway in human fibroticlikediseases and its eligibility as a therapeutic target mol cell ther stamos jl weis wi the betacatenin destruction complex cold springharb perspect biol 201351a007898 chung gg tissue microarray analysis of betacatenin in colorectalcancer shows nuclear phosphobetacatenin is associated with a betterprognosis clin cancer res miyoshi y activation of the betacatenin gene in primaryhepatocellular carcinomas by somatic alterations involving exon cancerres fukuchi t betacatenin mutation in carcinoma of the uterineendometrium cancer res brabletz t variable betacatenin expression in colorectal cancersindicates tumor progression driven by the tumor environment proc natlacad sci u s a 0cshokrani bmc gastroenterology page of berger na obesityassociated gastrointestinal tract cancer from beginningto end cancer calle ee the american cancer society cancer prevention study iinutrition cohort rationale study design and baseline characteristicscancer frezza ee wachtel ms chirivainternati m influence of obesity on the riskof developing colon cancer gut berger na obesity and cancer pathogenesis ann n y acad sci guo s insulin signaling resistance and the metabolic syndrome insightsfrom mouse models into disease mechanisms j endocrinol 20142202t1t23 ross se inhibition of adipogenesis by wnt signaling science kennell ja macdougald oa wnt signaling inhibits adipogenesis throughbetacatenindependent and independent mechanisms j biol chem liu z yingka y inflammation driven activation of wnt pathway a potentialmechanism responsible for obesity associated colorectal cancer obes resopen j tilg h moschen ar adipocytokines mediators linking adipose tissueinflammation and immunity nat rev immunol fischerposovszky p wabitsch m hochberg z endocrinology of adiposetissue an update horm metab res oguma k activated macrophages promote wnt signalling throughtumour necrosis factoralpha in gastric tumour cells embo j wang y adiponectin modulates the glycogen synthase kinase3betabetacatenin signaling pathway and attenuates mammary tumorigenesis ofmdamb231 cells in nude mice cancer res renehan ag roberts dl dive c obesity and cancer pathophysiologicaland biological mechanisms arch physiol biochem publishers notespringer nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations 0c" | Colon_Cancer |
clinical manifestations of sarscov2 infection include more frequently fever and cough failure can occur in persons with additional comorbidities liver dysfunction is one of the most striking affections among patients suggesting that sarscov2 may represent a new king of liver aggressor however the molecular process underlying this phenomenon is 0cstill unclear in this work we overview the most recent findings between the molecular biology of the virus pathogenic mechanisms and its relationship to liver disease observed in patients abbreviations aado2 ace2 aih alt ast covid19 ggt gi gtex alveolararterial oxygen gradient angiotensinconverting enzyme autoimmune hepatitis alanine transaminase aspartate aminotransferase coronavirus infectious disease gamma glutamyl transpeptidase gastrointestinal genotypetissue expression metabolicassociated fatty liver disease nonstructural proteins open reading frame preproofcomplex disease in many severely ill patients in other infected subjects an infection is keywords sarscov2 liver liver impairment covid19 ace2 mafld nsp orf introduction sarscov2 is the etiological agent of the disease known as covid19 which causes a disease characterized by pneumonia cough fever occasional diarrhea headache cardiac injury and in some patients liver alterations covid19 has been found to be an extremely reported to be so severe that it can lead to a disproportionate and mortal reaction of the immune system known as a cytokine storm all of these factors make covid19 highly unpredictable it is what specialists call a multisystem disease 0caround the world cases of liver dysfunction denoted by elevated hepatic enzymes in serum such as ast aspartate aminotransferase and alt alanine transaminase have been documented among patients infected with sarscov2 there is still no certainty whether the covid19related liver damagedysfunction is due mainly to the viral replication per se drugs toxicity or other coexisting comorbidities whether sexrelated difference could help to explain why infected men are more healthy or harmful relationship between the liver and its viral aggressor in this paper we describe a brief overview of the implications for researchers in the field of it is important to understand how liver function can be altered by direct infection with this predisposed to develop covid19associated liver dysfunction than infected women to analyze if there is any genetic predisposition related to impaired liver function during the respiratory virus which mechanisms of viral pathogenesis are involved to evaluate disease and of course the crosstalk between viral and cellular proteins that mediate this preproofliver disease of the most recent findings between the molecular biology of the virus this emerging viral illness is typically characterized by fever dry cough myalgia headache sore throat diarrhea and may be aggravated with shortness of breath and respiratory failure the angiotensinconverting enzyme ace2 the functional receptor of the spike glycoprotein of sarscov2 is widely distributed in the anism historically hamming and colleagues reported ace2 expression in the surface of lung alveolar epithelial cells enterocytes of the small intestine arterial and venous endothelial pathogenic mechanisms and its relationship to liver disease observed in patients clinical characteristics and liver injury in patients with covid19 0ccells and arterial smooth muscle cells posterior transcriptomic and proteomic analyses confirmed their findings and added high ace2 expression in adipose tissue bone marrow duodenum endometrium heart kidney small intestine smooth muscle testis and thyroid ace2 is also expressed in liver but in lesser extent one of the most worrisome severe cases of covid19 regarding the gastrointestinal gi tract and liver over covid19associated liver injury is defined as any liver damage that occurs during disease progression andor covid19 treatment in patients with or without a history of previous complications is the unusual formation of blood clots in many patients with covid19 even those who were receiving anticoagulants researchers at mount sinai in new york published studies suggesting that clots in the lungs play an important role in the most of covid19 patients develop gi symptoms such as anorexia diarrhea nausea and vomiting and a significant proportion present with altered liver function tests preproofdecreased albumin levels are associated with severe infection and poor prognosis still ast elevation is the most common abnormality in patients presenting with covid19 observed more frequently in men and is mainly documented in more severe cases liver disease in general the incidence of increased liver biochemical markers in hospitalized patients with covid19 mainly ast and alt and slightly elevated bilirubin varies between to of cases the increase in liver enzymes is there are no reports of acute or subacute liver failure in patients with covid19 the largest cohort study that included cases of covid19 from china showed that had preexisting chronic liver disease lei and colleagues reported that impaired liver function was related to mortality in covid19 patients elevated ast was more frequent and significant than the increase of alt in severe 0chospitalized patients moreover elevated ast was shown to be associated with highest mortality risk in the study reported by yijin wang they found that of covid patients had elevated ast activity the median levels of alt were ul vs ul respectively ast were ul vs ul respectively in abnormal and normal aminotransferase groups liver enzymes abnormality were associated with disease severity protein levels in addition they found by ultrastructural examination of coronavirus ps in hepatocytes in covid19 cases sarscov2 infected hepatocytes displayed as well as a series of laboratory tests including higher alveolararterial oxygen gradient aado2 higher gamma glutamyl transpeptidase ggt lower albumin and lower total conspicuous mitochondrial swelling endoplasmic reticulum dilatation and glycogen granule decreased histological findings showed apoptosis and binuclear hepatocytes preproofshowed a disease course similar to that reported in non immunosuppressed population coronavirus the interaction of its proteins with cellular proteins and consequently the immunosuppressive therapy for autoimmune hepatitis aih developing covid19 taken together both ultrastructural and histological evidence indicated a typical lesion of viral infection all these findings by different reports demonstrates that sarscov2 infection in liver is a crucial cause of hepatic impairment in covid19 patients however alteration of cellular metabolism that give rise to systematic alterations and metabolic report from alessio gerussi demonstrated that patients under today the cellular and molecular mechanisms that are altered by infection with this changes are still unknown 0cmolecular biology of sarscov2 coronaviruses are enveloped viruses that contain a positively polarized unsegmented rna genome belonging to the coronaviridae family and the order of nidovirales they are distributed in humans and other mammals the size of the sarscov2 virions is approximately to nm in diameter [] sarscov2 has a genome that consists polymerase rdrp which is nsp12 and is responsible of the replication and transcription of the virus which are encoded by the various genetic loci on the genome at the center of the virion lies a nucleocapsid composed of the genomic rna and the nucleocapsid protein the virus glycoprotein s consists of two subunits s1 which is at the amino terminus and that encodes for structural proteins and a larger region that encodes two open reading frames orf 1a and 1b which together encode for the nonstructural virus proteins from nucleocapsid protein which is within the phospholipid bilayer and nonstructural proteins nsp1 to nsp16 the virions have a structural sspike protein outer spiky glycoprotein mmembrane protein a type iii transmembrane glycoprotein nof nucleotides encoding amino acids and it is composed of a region preproofvirus as well as protein m which is a type iii transmembrane glycoprotein and participates in the cellular membrane rearrangements for the replication and transcription complexes among the encoded proteins is an rnadependent rna provides the receptor binding site and s2 which is at the carboxyl terminus responsible for membrane fusion the envelope protein e has a role in the assembly and release of the nonstructural proteins have several functions during de viral cycle for example nsp 0cthe virus enters the cell by endocytosis through the interaction between envelope glycoprotein s with the cell receptor ace2 and with the participation of the type ii transmembrane serine protease tmprss2 once it enters the cell the n protein with viral genome are released within the cytoplasm then cellular proteases degrade the capsid and the virus genome is left free next the polyprotein containing the viral proteins that are how does the virus select which cells to infect viruses can infect only certain species of hosts and only permissive cells within that host permissive cells make all the necessary proteins and viral factors to allow virus to replicate once a virus gets inside a cell it hijacks the cellular processes to produce virally encoded proteins that will replicate the viruss genetic material viral replication may cause exocytosis will translate into viral proteins this entire process will occur in the cell cytoplasm the processed to form the replication complex is translated and then the complementary strand of negative sense pregenomic rna is synthesized to be used as a template to replicate the structural proteins that will be synthesized in the endoplasmic reticulum membrane to assembly the viral p and finish the cycle through the release of the viruses by positive sense viral genome furthermore the replication and transcription complex will lead to a series of smaller positive sense subgenomic rnas these are the ones that preproofboth sarscov and the new sarscov2 are very similar in structure and pathogenicity but the major structural protein s protein is slightly different between them compared to other beta coronaviruses the presence of a furinlike cleavage site in sarscov2 enables the s protein priming and facilitates an increase on the efficiency of the spread of sarscov2 as is reported wide world 0cbiochemical changes producing cell damage called cytopathic effects like other coronaviruses sarscov2 requires cellular receptors to initiate its internalization to the cells that carry these factors li sarscov2 uses the angiotensinconverting enzyme ace2 as a host cell receptor sarscov2 spike s protein binds ace2 with significantly high affinity in addition the main host protease that suggested to promote the pathogenesis of this coronavirus program httpsportalgdccancergov they compared ace2 expression levels across human tissues between males and females and between younger and older persons in these individual tissues furthermore other reports have analyzed the correlations between ace2 in order to provide insights into the mechanism of sarscov2 infection li analyzed the expression of ace2 in various normal human tissues using the datasets from the genotypetissue expression gtex project and the cancer genome atlas tcga transmembrane serine protease other host proteases such as furin have also been mediates sprotein activation on primary target cells and initial viral entry is the type ii preproofreported by li ace2 expression levels showed no significant difference between have no significant association with sex age or race is the liver a direct target for sarscov2 males and females between younger and older persons or between asian and nonasian races this finding suggests that the infection risk of sarscov2 and sarscov may expression levels and immune signature enrichment levels in individual tissues as as we expected because the systemic manifestations of covid19 it has been reported that sarscov2 has an anotropism beyond the respiratory tract including the kidneys 0cliver heart and brain and possibly that anotropism influences the course of covid19 disease and aggravates preexisting conditions the ace2 protein is found at high levels in the gi tract as the colon biliary system and liver on the other hand it is well documented a sarscov2 rna shedding in the gi tract supporting its tropism for architecture express ace tmprss1 receptors the presence of these two host factors in the liver suggests that a direct viral cytopathic effect occurs also in sars infection the presence of viral rna in liver tissue was documented but not as extensively as the new coronavirus data published by gordon suggest that mitochondrial proteins interact directly with the virus which helps to understand the potential mechanism by which elevated ast the gi tract and liver cells and these may be sites of active viral replication and either direct or indirect tissue injury indeed a large part of the cells distributed in the liver preproofeffect the exacerbated inflammatory response in covid19 may play a central role in profiles are detected in these patients furthermore in addition to this intracellular more recently identified the clinical and laboratory characteristics of covid19 patients with abnormal liver transaminases and they reported that sarscov2 is able to which high levels of il6 have been reported which are involved in both infect liver cells and cause liver impairment by direct cytopathic effect inflammatory and repair responses in liver disease mechanisms of liver pathogenicity 0cif sarscov2 replication has direct adverse effects on liver function it is still unknown findings in liver biopsy of patients killed by covid19 showed moderate micro vesicular steatosis and mild portal and necroinflammatory activity this seems to indicate that a direct injury occurred while the infection that could have been directly caused by sarscov2 another possibility is that a druginduced liver injury occurred to date there are the following possible mechanisms figure infection the massive release of cytokines by the immune system in response to the viral infection can result in a cytokine storm and symptoms of sepsis that are the cause of death in of fatal covid19 cases in these cases uncontrolled inflammation induces multian damage leading including liver failure biomarkers of inflammation such as creactive protein pcr serum ferritin ldh ddimer il6 il2 are have been found to be significantly elevated in immune damage from exacerbated inflammation in response to sarscov2 preproofpathogenesis of sars cov related liver disease more studies should be liver is a potential target for direct infection with this virus to understand the performed for evidence of viral mechanisms of replication in different cell anelles as cytoplasm endoplasmic reticulum golgi apparatus and lipidrafts cov2 enters cells through the ace2 molecule which is abundantly expressed in the liver and in particular in bile epithelial cells based on this expression the direct cytopathic effect due to active viral replication in various liver cells sarscritically ill patients with covid19 into hepatocytes and liver histology characterization it is also important to know in cells their capacity to efficiently produce both infectious and defective non 0cinfective whole virions there are not yet enough data to know the viral dynamics in the different tissues and the associated pathogenesis anoxia respiratory failure is one of the main characteristics of covid19 anoxic hypoxic hepatitis is common in patients with severe symptoms reactivation of preexisting liver disease patients with preexisting chronic liver medications such as tocilizumab and baricitinib used to combat the adverse immune cholestatic liver disease various studies such as lopinavir ritonavir remdesivir chloroquine tocilizumab uminefovir traditional chinese medicine so it is important to consider that they could be potentially hepatotoxic in some patients druginduced liver damage dili initial clinical guidelines recommended antiviral agents for covid19 so a variety of drugs have been administered in disease may be more susceptible to liver damage from sarscov2 biological preproof genetic factors genetics may well be one of the determining factors in some reaction may also cause hbv reactivation and induce eventual impairment of liver function in those patients with hbv on the other hand it is still unknown whether sarscov2 infection exacerbates cholestasis in people with underlying patients who become seriously ill with covid19 but until now we cannot be sure it is possible for example that the genetic variation that makes an individual more susceptible to high blood pressure or diabetes also makes him more vulnerable to the virus it will be important to find out what role genetic factors predisposing to liver steatosis have and their sensitivity to severe symptoms of covid19 ji and 0ccolleagues showed that subjects with metabolicassociated fatty liver disease mafld have a higher risk of covid19 severity disease and abnormal liver blood tests than patients without mafld in contrast louise biquard demonstrated that mafld is not associated with changes in liver expression blood test abnormalities reported by ji and colleagues is thus likely not explained by concluding remarks the scenario is not yet complete which does not allow us to establish or understand the natural history of the disease and the participation or commitment of the liver in this disease certainly the application of new technological platforms such as singlecell increased hepatic sarscov2 uptake still several contradictory reports will help of genes implicated in sarscov2 infection the observed persistence of liver to find the real role of genetic factors in the evolution of this disease preprooftranscriptomic assays will allow us to quickly know the commitment of each cell type in affected ans and the meaning of viral dynamics in the various affected systems including the liver however as we have already learned from the old hepatotropic viruses history still is ongoing and we have much to learn and understand about the virologic characteristics of this emerging rna virus that allow us to develop specific antivirals such as the case of hcv and the vaccine to decrease the impact of this acute infection declarations of interest none ethical approval not required 0c references chen n zhou m dong x qu j gong f han y epidemiological and clinical characteristics of cases of novel coronavirus pneumonia in wuhan china httpsdoi101002path1570 wang d eraslan b wieland t hallström b hopf t zolg dp a deep proteome and transcriptome abundance atlas of healthy human tissues mol syst hamming i timens w bulthuis mlc lely at navis gj van goor h tissue distribution of ace2 protein the functional receptor for sars coronavirus a first step in understanding sars pathogenesis j pathol a descriptive study lancet httpsdoi101016s0140 preproofa manifestation of covid19 rev gastroenterol méxico english ed biol 201915e8503 httpsdoi1015252msb20188503 patients with covid19 j am coll cardiol httpsdoi101016jjacc202005001 paranjpe i fuster v lala a russak a glicksberg bs levin ma association of treatment dose anticoagulation with inhospital survival among hospitalized schmulson m dávalos mf berumen j beware gastrointestinal symptoms can be httpsdoi101016jrgmxen202004001 cai q huang d yu h zhu z xia z su y covid19 abnormal liver function tests j hepatol httpsdoi101016jjhep202004006 0c siddiqi hk mehra mr covid19 illness in native and immunosuppressed states a clinicaltherapeutic staging proposal j hear lung transplant off publ int soc hear transplant httpsdoi101016jhealun202003012 feng g zheng ki yan qq rios rs targher g byrne cd covid19 and between markers of liver injury and mortality in covid19 in china hepatology httpsdoi101002hep31301 de la rica r bes m aranda m del castillo a socias a payeras a low transl hepatol httpsdoi1014218jcth202000018 albumin levels are associated with poorer outcomes in a case series of covid19 patients in spain a retrospective cohort study medrxiv liver dysfunction current insights and emergent therapeutic strategies j clin lei f liu ym zhou f qin jj zhang p zhu l longitudinal association preproofmortality of adult inpatients with covid19 in wuhan china a retrospective cohort study lancet httpsdoi101016s0140liver directly contributes to hepatic impairment in patients with covid19 j hepatol httpsdoi101016jjhep202005002 httpsdoi1011012020050720094987 zhou f yu t du r fan g liu y liu z clinical course and risk factors for wang y liu s liu h li w lin f jiang l sarscov2 infection of the gerussi a rigamonti c elia c cazzagon n floreani a pozzi r coronavirus disease covid19 in autoimmune hepatitis a lesson from 0cimmunosuppressed patients hepatol commun 2020na httpsdoi101002hep41557 richman d whitley r hayden f clinical virology 4th ed asm press ksiazek tg erdman d goldsmith cs zaki sr peret t emery s a novel httpsdoi101056nejmoa030781 kuiken t fouchier ram schutten m rimmelzwaan gf van amerongen g van respiratory syndrome lancet httpsdoi101016s0140 drosten c günther s preiser w van der werf s brodt hr becker s identification of a novel coronavirus in patients with severe acute respiratory riel d newly discovered coronavirus as the primary cause of severe acute coronavirus associated with severe acute respiratory syndrome n engl j med preproofsyndrome n engl j med httpsdoi101056nejmoa030747 outbreak associated with a new coronavirus of probable bat origin nature httpsdoi101038s4158602020127 mortola e roy p efficient assembly and release of sars coronaviruslike ps by a heterologous expression system febs lett httpsdoi101016jfebslet200409009 fehr a perlman s coronaviruses methods and protocols methods in molecular biology chapter coronaviruses an overview of their replication and zhou p yang xl lou wang xgg hu b zhang l zhang w a pneumonia 0cpathogenesis springer berlin heidelberg belouzard s millet jk licitra bn whittaker gr mechanisms of coronavirus cell entry mediated by the viral spike protein viruses httpsdoi103390v4061011 vennema h godeke gj rossen jw voorhout wf horzinek mc opstelten dj et snijder ej decroly e ziebuhr j the nonstructural proteins directing coronavirus neuman bw buchmeier mj supramolecular architecture of the coronavirus p adv virus res httpsdoi101016bsaivir201608005 van der hoeven b oudshoorn d koster aj snijder ej kikkert m barcena m neuman bw kiss g kunding ah bhella d baksh mf connelly s a structural analysis of m protein in coronavirus assembly and morphology j struct biol httpsdoi101016jjsb201011021 expression of viral envelope protein genes embo j al nucleocapsidindependent assembly of coronaviruslike ps by co preproofbiogenesis and architecture of arterivirus replication anelles virus res httpsdoi101016jvirusres201604001 rna synthesis and processing vol 1st ed elsevier inc httpsdoi101016bsaivir201608008 rabi f al zoubi m kasasbeh g salameh d alnasser a sarscov2 and coronavirus disease what we know so far pathogens masters p the molecular biology of coronaviruses adv virus res 0c shang j ye g shi k wan y luo c aihara h structural basis of receptor recognition by sarscov2 nature httpsdoi101038s415860202179y rabaan aa alahmed sh haque s sah r tiwari r malik ys sarscov li w moore mj vasilieva n sui j wong sk berne ma angiotensin httpsdoi101038nature02145 hoffmann m kleineweber h pöhlmann s a multibasic cleavage site in the cohen fs how viruses invade cells biophys j httpsdoi101016jbpj201602006 sarscov and merscov a comparative overview le infez med converting enzyme is a functional receptor for the sars coronavirus nature preproofem structure of the 2019ncov spike in the prefusion conformation science httpsdoi101126scienceabb2507 spike protein of sarscov2 is essential for infection of human lung cells mol cell 202078779784e5 httpsdoi101016jmolcel202004022 ziegler cgk allon sj nyquist sk mbano im miao vn tzouanas cn sarscov2 receptor ace2 is an interferonstimulated gene in human airway epithelial cells and is detected in specific cell subsets across tissues cell httpsdoi101016jcell202004035 wrapp d wang n corbett ks goldsmith ja hsieh cl abiona o cryo 0c follis k york j nunberg j furin cleavage of the sars coronavirus spike glycoprotein enhances cellcell fusion but does not affect virion entry virology httpsdoi101016jvirol200602003 millet jk whittaker gr host cell proteases critical determinants of coronavirus li myy li l zhang y wang xss expression of the sarscov2 cell receptor httpsdoi101186s4024902000662x xu h zhong l deng j peng j dan h zeng x high expression of ace2 receptor of 2019ncov on the epithelial cells of oral mucosa int j oral sci httpsdoi101038s413680200074x tropism and pathogenesis virus res httpsdoi101016jvirusres201411021 gene ace2 in a wide variety of human tissues infect dis poverty 20209na preproofcov2 protein interaction map reveals targets for drug repurposing nature infection of sarscov2 gastroenterology 202015818311833e3 httpsdoi101053jgastro202002055 httpsdoi101038s4158602022869 xu l liu j lu m yang d zheng x liver injury during highly pathogenic human coronavirus infections liver int off j int assoc study liver httpsdoi101111liv14435 coomes ea haghbayan h interleukin6 in covid19 a systematic review and xiao f tang m zheng x liu y li x shan h evidence for gastrointestinal gordon de jang gm bouhaddou m xu j obernier k white km a sars 0cmetaanalysis medrxiv httpsdoi1011012020033020048058 xu z shi l wang y zhang j huang l zhang c pathological findings of covid19 associated with acute respiratory distress syndrome lancet respir med httpsdoi101016s221326002030076x zhang b zhou x qiu y feng f feng j jia y clinical characteristics of chen g wu d guo w cao y huang d wang h clinical and chai x hu l zhang y han w lu z ke a specific ace2 expression in invest httpsdoi101172jci137244 death cases with covid19 medrxiv httpsdoi1011012020022620028191 immunological features of severe and moderate coronavirus disease j clin preproofcholangiocytes may cause liver damage after 2019ncov infection biorxiv patients medrxiv httpsdoi1011012020040120047381 httpsdoi10110120200203931766 herold t jurinovic v arnreich c hellmuth jc von bergweltbaildon m klein m level of il6 predicts respiratory failure in hospitalized symptomatic covid grein j ohmagari n shin d diaz g asperges e castagna a compassionate use of remdesivir for patients with severe covid19 n engl j med httpsdoi101056nejmoa2007016 u s food and drug administration fact sheet for health care providers emergency 0cuse authorization eua of hydroxychloroquine sulfate supplied from the strategic national stockpile for treatment of covid19 in certain hospitalized patients varona pérez j rodriguez chinesta jm riesgo de reactivación de la hepatitis b asociado al tratamiento con corticoides frente a sarscov2 covid19 rev clÃnica española httpsdoi101016jrce202004012 ji d qin e xu j zhang d cheng g wang y nonalcoholic fatty liver httpsdoi101016jjhep202003044 sarscov2 in metabolicassociated fatty liver disease j hepatol diseases in patients with covid19 a retrospective study j hepatol preproof biquard l valla d rautou pe no evidence for an increased liver uptake of httpsdoi101016jjhep202004035 0cfigure legends preprooffig1 proposed mechanisms of liver pathogenicity of sarscov2 in infected cells sars cov2 infection2 cytokinestorm3 drugeffects4 hypoxia5 previousliverdamagebiochemicallabmarkerswhite bloodcellsgenomereleasereplicationtranslationvirionassemblyviral proteinsmaturevirus release\uf0e9aado2mitochondrialproteinshypoxicisquemicliverinjuryliver damagelopinavirritonavirremdesivirchloroquinetocilizumaboxidativeimbalancesteatosisace2s proteincytopathiceffect\uf0e9gmcsf\uf0e9il6\uf0e9il1β\uf0e9il2\uf0e9il8\uf0e9ccl2\uf0e9ccl3\uf0e9ccl5\uf0e9cxcl \uf0e9alt\uf0e9ast\uf0eaalbumin\uf0e9pcr\uf0e9ldh\uf0e9ddimer\uf0e9ferritin\uf0e9bilirubin 0c' | Colon_Cancer |
preoperative heart rate variability a prognosticindicator for overall survival and cancer recurrencein patients with primary colorectal cancer one e0237244 101371 pone0237244editor louise emilsson university of oslonorwayreceived february accepted july published august copyright strous this is an openaccess distributed under the terms of thecreative commons attribution license whichpermits unrestricted use distribution andreproduction in any medium provided the originalauthor and source are crediteddata availability statement data cannot beshared publicly because of ethical concernspatients were included on a no objection base toconduct retrospective data studies and publishfindings but were not asked for permission topublish full encrypted data data are available fromthe viecuri institutional data access contact viawetenschapsbureauviecurinl for researcherswho meet the criteria for access to confidentialdata heart rate variability hrv represents efferent vagus nerve activity which is suggested tobe inversely related to fundamental mechanisms of tumorigenesis and to be a predictor ofprognosis in various types of cancer hrv is also believed to predict the occurrence andseverity of postoperative complications we aimed to determine the role of preoperativehrv as a prognostic factor in overall and cancer free survival in patients with colorectalcancermethodsretrospective analysis was performed in a detailed dataset of patients diagnosed with primary colorectal cancer between january and december who underwent curative surgical treatment hrv was measured as timedomain parameters sdnn standarddeviation of nnintervals and rmssd root mean square of successive differencesbased on preoperative second ecgs groups were created by baseline hrv low hrvsdnn 20ms or rmssd 19ms and normal hrv sdnn �20ms or rmssd �19msprimary endpoints were overall and cancer free survivalresultsa total of patients were included in this study hrv was not significantly associated withoverall survival sdnn 20ms vs sdnn �20ms244 vs adjusted hr p rmssd 19ms vs rmssd �19ms270 vs adjustedhr p or cancer recurrence sdnn 20ms vs�20ms201 vs adjusted hr p rmssd 19ms vs�19ms vs adjusted hr p there was no one 101371 pone0237244 august one 0chrv and prognosis in colorectal cancerfunding the authors received no specific fundingfor this workcompeting interests the authors have declaredthat no competing interests existsignificant association between hrv and cealevel at one year followup or between hrvand occurrence of a postoperative complication or the severity of postoperativecomplicationssheart rate variability was not associated with overall or cancer free survival in patients withprimary colorectal cancer who underwent curative surgical treatment these results do notalign with results found in studies including only patients with advanced cancer which suggests that there is only an association in the other direction cancer causing low hrvintroductionin there were over million newly diagnosed colorectal cancer patients worldwide andover in the netherlands alone it is the fourth most common cause of death worldwide to improve survival it is of importance to get a better insight into modifiable prognosticfactors emerging evidence suggests that vagal nerve activity indexed by heart rate variabilityhrv could be one of these prognostic factors []hrv is the physiological phenomenon of the fluctuation in time intervals between adjacentheartbeats and represents efferent vagus nerve activity to the heart [] it has been suggestedthat efferent vagal activity is inversely related with fundamental mechanisms of tumorigenesisas inflammation oxidative stress and excessive sympathetic activity these mechanisms arebelieved to be controlled by the vagus nerve via a bidirectional braintoimmune pathwaysthrough the release of neurotransmitters via the cholinergic antiinflammatory pathway a higher vagal tone may reflect a more flexible topdown regulation of the immunesystem and physiological activity moderated by the brain absence of vagus activity due tovagotomy has been shown to increase the risk of developing colorectal cancer in addition to influencing development of cancer vagus nerve activity seems to be a predictor of prognosis in various types of cancer recent studies show an association betweendecreased activity of the vagus nerve and worse survival in patients with cancer of the gastrointestinal tract liver pancreas lung prostate and breast among others also patientswith normal hrv seem to live longer in different sorts of metastatic cancer independent ofconfounders in patients with colorectal cancer a low hrv at baseline has shown to beassociated with higher cea levels at months after diagnosis which predicts a poorer prognosis in patients undergoing curative treatment for colorectal cancer hrv does not only seemto influence cancer prognosis a recent study showed that patients with lower hrv have moreintraoperative blood loss and more and more severe postoperative complications identifying patients with low hrv is easy and noninvasive when its predictive value forthe prognosis of cancer patients is of satisfactory significance vagus nerve activation prior toor during cancer treatment could theoretically be beneficial in improving prognosis alsoif we could predict the occurrence and severity of postoperative complications based on hrvimproving hrv before surgery could possibly accelerate postoperative recovery and indirectlyaffect patients prognosis recent studies focussing on improving hrv by improving physicalfitness by means of physical exercise show promising results in both older men and woman however the only previous study on colon cancer and hrv including patients receiving curative treatment included a small sample and did not examine whether hrv predicts one 101371 pone0237244 august one 0chrv and prognosis in colorectal cancersurvival in these patients to clarify the predictive value of hrv in prognosis of patientswith colorectal cancer further exploration is needed current studies identifying hrv as aprognostic factor did not specifically focus on colorectal cancer have small study populationsdid not correct for confounders and mainly focused on metastatic disease []the aim of this study was to determine the role of preoperative hrv as a prognostic factorin overall and cancer free survival in patients with primary colorectal cancer who underwentcurative surgical treatmentmethodsdata collectiondata from the netherlands cancer registry ncr were used the ncr collects data on allnewly diagnosed cancer patients in the netherlands information on patient and tumour characteristics diagnosis and treatment is routinely collected from the medical records by trainedadministrators of the cancer registry anatomical site of the tumour is registered according tothe international classification of diseases oncology the tumournodemetastasis tnmclassification is used for stage notification of the primary tumour according to the editionvalid at time of cancer diagnosis quality of the data is high due to thorough training of theregistration team and consistency checks for the study population additional data were collected from the medical records of thepatients this encompassed information on hrv cealevels asa classification comorbidities identified at admission divided into groups cardiac disease hypertension diabetes mellitus thyroid disease pulmonary disease vascular disease neurological disease and otheroccurrence and severity of postoperative complications and cancer recurrence groups ofcomorbidities were chosen based on matching features within these groups and their potentialinfluence on hrv or the endpoint being analysed severity of the postoperative complicationsaccording to the claviendindo classification was also documented medical records wereassessed between january and july and reevaluated for revision of this between the 20th and 25th of april this study was approved by the research committee and the board of directors of viecurimedical centre data was obtained under the law scientific research and statistics in the interest of public health where asking for permission is not possible or appropriate for several reasons in the netherlands unless patients objected to use of their personal medical record forscientific research data was encrypted with an encryption key provided by the ncr encryption was shortly lifted to access the patients number for accessing hisher medical record following extraction data were encrypted againstudy populationthe study population included all consecutive patients diagnosed with primary colorectal adenocarcinoma between january and january at viecuri medical centre who underwent curative surgical treatment patients with metastatic disease at time of surgery orcarcinoma in situ were excluded as their treatment and prognosis differs from those receivingcurative treatment for colorectal cancer metastasis found within months after surgery wereconsidered present at time of surgery and therefore excluded other excluded patients werepatients with neuroendocrine tumours because of different tumour characteristics and prognosis patients with cardiac arrhythmias including atrial and ventricular extrasystole pacemakers patients taking betablockers as this enhances hrv indexes or patients withbradycardia heart rate bpm or tachycardia heart rate bpm as this precluded reliable calculation of hrv we did not exclude patients taking alphablockers calcium one 101371 pone0237244 august one 0chrv and prognosis in colorectal cancerinhibitors diuretics amiodarone aceinhibitors or arbs as these types of medicationreduce central sympathetic functioning rather than peripheral and their influence on hrv istherefore less univocal and sometimes completely absent heart rate variabilityheart rate variability was analysed using a 12lead 10second ecg 150hz used for preoperative screening in case of multiple ecgs per patient the most recent ecg before date of surgery was used for hrvanalysis in case of multiple ecgs per patient on the same date theecg with the best quality was chosen meaning an ecg without motion artefacts in case ofmotion artefacts there was always an ecg without motion artefacts available recorded on thesame date time between two consecutive rpeaks was measured in lead ii with an accuracy of02ms using museecg hrv was presented using the timedomain hrv parameters sdnnstandard deviation of nnintervals and rmssd root mean square of successive differences in milliseconds calculated using the following calculations rsdnn ¼ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffii¼1 ðrri 00 rrmeanþ2pnn 00 rmssd ¼rffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffii¼1 ðrriþ1 00 rriþ2pn 00 n 00 ð1þð2þsdnn and rmssd obtained from 10s ecgs were found to correlate with results of ecgsof longer durations power spectral analysis hrv parameters as lf and hf can only beobtained in longer recording ecgs and were therefore not implementable in this study sdnn and rmssd were both analysed as continuous variables as well as binary variablesusing cutoffs of 20ms versus �20ms and 19ms versus �19ms respectively in case of ansdnn 20ms or rmssd 19ms hrv was classified as low and in case of sdnn �20ms orrmssd �19ms as normal these cutoff values were based on cutoff values used in otherstudies showing an association between lowhrv as sdnn 20ms and rmssd 19ms andcolorectal cancer as there is no standardised definition of low and normal hrv endpoints and definitionsthe primary endpoints of this study were overall and cancer free survival overall survival wasdefined as the time between the date of surgery to the date of death or last followup date inmonths cancer free survival was defined as the time in months from the date of surgery untilthe date of cancer recurrence defined as the first date of either radiologic or pathologic diagnosis of metastases or tumour recurrence of colorectal cancer patients dying without cancerrecurrence were censored on day of death secondary endpoints were elevated cealevel ugl at oneyear followup occurrence of postoperative complications within daysafter surgery and severity of postoperative complications according to the claviendindoclassificationstatistical analysisin this retrospective observational cohort study we utilized descriptive statistics to provide anoverview of control variables of the study population patient characteristics as age sex bmicomorbidities and asaclassification heart rate and tumour characteristics as tnmstagetumour localisation and tumour differentiation and their association with hrv and one 101371 pone0237244 august one 0chrv and prognosis in colorectal cancerprognosis normal distribution of the continuous variables heart rate age and bmi as well assdnn and rmssd were tested with a kolmogorovsmirnov test because of normal distribution heart rate age and bmi were compared between hrvgroups using unpaired ttest allother variables were categorical and were compared between hrvgroups using chisquarestatistics as groups were all of sufficient powerdifferences in overall survival and cancer free survival in months according to sdnn andrmssd were visualized by means of kaplanmeier curves and statistically tested using the logrank test multivariate coxregression analyses were conducted to calculate the prognosticassociation between hrv and overall and cancer free survival while adjusting for other prognostic variables multivariate logistic regression was used to assess the independent effect ofsdnn and rmssd on cealevels and the occurrence and severity of postoperative complications variables included for adjustment were chosen by means of forward stepwise selectionbased on clinical judgment differences at baseline eg differences on any predictor betweenpatients who later died or not and database availability and depended on the analysed endpoint those included patient demographics age sex bodymassindex comorbidities identified at admission divided into groups cardiac disease hypertension diabetes mellitus thyroiddisease pulmonary disease vascular disease neurological disease other including crohnsdisease hepatitis kidney failure disorders anaemia depression arthritis tumour characteristics localisation stage differentiation and the occurrence of postoperative complicationswhen the later was not an outcome differences in cealevel at baseline and one year checkup between and within groups of low hrv and normal hrv were assessed with a repeatedmeasures linear model and tested using the tukey test to test the implication of a longer timebetween ecg and treatment all analyses were repeated after excluding patients with an ecgolder than months a twotailed pvalue � was considered significant in all analysesdata were analysed using ibm spss statistics version ibm corp ny armonk usaresultsof colorectal cancer patients that underwent a surgical resection a total of patientswere included in this study reasons for exclusion are presented in fig median sdnn andrmssd were 204ms interquartile range iqr 115ms351ms and 175ms iqr 99ms299ms respectively table shows descriptive data of the included patients by hrv groupsbaseline heart rate and age were negatively associated with hrv the group of patients withlow hrv contains more patients with a history of cardiac disease regardless of the hrv defining parameter when defining low hrv by rmssd 19ms more patients in this group havehypertension as comorbidity this group also contains more patients with an asa classification greater than oneduring a median followup of months iqr all causedeath occurred in patients cancer recurrence occurred in patients during a median followup of months iqr to rule out any distort in results caused by a delay between ecg and treatment all analyseswere repeated after exclusion of ecgs older than months this did not lead to any new significant results therefore these results were not displayed in detail in this papersurvivalin low hrv groups slightly more patients died compared to normal hrv groups sdnn20ms versus �20ms versus respectively rmssd 19ms versus�19ms versus respectively these observed differences between hrvgroups in overall survival were not significant fig a sdnn p b rmssd one 101371 pone0237244 august one 0chrv and prognosis in colorectal cancerfig flowchart of the study101371 pone0237244g001p after adjustment for some potential confounders these associations remained notsignificant sdnn 20ms versus �20ms hr p and rmssd19ms versus �19ms hr p tables and age cardiac disease tumour stage and postoperative complications had a significant influence on overall survival age also differed at baseline and was identified as a confounder when defining low andnormal hrvgroups by sdnn cardiac disease was identified as confounder when conducting sensitivity analyses with sdnn and rmss as continuous variables results remained thesame there was no association between hrv and overall survival sdnn hr p and rmssd hr p in low hrv groups slightly more patients had recurrence of cancer compared to normalhrv groups sdnn 20ms versus �20ms versus respectivelyrmssd 19ms versus �19ms versus respectively these observed differences between hrv groups in cancer free survival were not significant fig a sdnnp b rmssd p as in overall survival after adjustment for some potentialconfounders these associations remained not significant sdnn 20ms versus �20mshr p and rmssd 19ms versus �19ms hr p tables and in sdnnbased groups baseline heart rate was identified asa confounding variable sensitivity analyses with sdnn and rmssd as continuous variables one 101371 pone0237244 august one 0ctable descriptive data of included patients according to prespecified hrv groupssdnn 20ms n sdnn �20ms n prmssd 19ms n rmssd �19ms n phrv and prognosis in colorectal cancerheart rate�age�age n year� yearsex nmalefemale [] [] [] [] [] [] [] [] mean bmi sd [] [] [] []comorbidities ncardiac diseasehypertensiondiabetes mellitusthyroid diseasepulmonary diseasevascular diseaseneurological diseaseotherasa nasa1asa2asa34localisation tumour nright colonleft colonrectumtumour stage niiiiii differentiation grade nwellmoderate poorundifferentiated � non normaldistributed data presented as median with interquartile range101371 pone0237244t001did not alter these results there was no association between hrv and cancer free survivalsdnn hr p and rmssd hr p cealevelcealevel at baseline and one year checkup was registered in patients and elevated in of these patients this was elevated at one year checkup in only patients low hrv was notsignificantly associated with elevated cealevels at one year checkup as shown in table sensitivity analyses with sdnn and rmssd as continuous variables did not alter these resultssdnn hr p and rmssd hr one 101371 pone0237244 august one 0chrv and prognosis in colorectal cancerfig kaplanmeier curves for overall survival in groups of low hrv and normal hrv presented as a sdnn andb rmssd101371 pone0237244g002 one 101371 pone0237244 august one 0ctable multivariate analyses of associations of sdnn and covariates with overall and cancer free survivalhrv and prognosis in colorectal cancersdnn20ms�20msheart rateagebmicardiac diseasenoyeshypertensionnoyesasaclassificationasa1asa2asa34localisationright colonleft colonrectumtumour stageiiiiiioverall survivalhr cipcancer free survivalhr ci reference reference p reference reference reference reference reference reference reference reference reference postoperative complicationnoyes reference reference 101371 pone0237244t002p adjusting for covariates was not possible because of the small number of patientswith an elevated cealeveldifferences between cealevel at baseline and one year checkup were compared betweenand within hrvgroups results were displayed in fig there were no significant differencesin cealevel at baseline and one year checkup between the low hrv group and normal hrvgroup defined by sdnn and rmssd p and p respectively also there were nosignificant differences in cealevel at baseline and at one year checkup within the low hrvgroup and normal hrv group defined by sdnn and rmssd p and p respectivelypostoperative complicationsin patients one or more postoperative complications occurred within days after surgerythe occurrence of a postoperative complication was not significantly associated with lowhrv defined as sdnn 20ms or rmssd 19ms even after adjustment for some potentialconfounders table heart rate age cardiac disease and hypertension were identified asconfounding covariates when conducting sensitivity analyses with sdnn and rmss as continuous variables the association between occurrence of a postoperative complication with one 101371 pone0237244 august one 0ctable multivariate analyses of associations of rmssd and covariates with overall and cancer free survivalhrv and prognosis in colorectal cancerrmssd19ms�19msheart rateagebmicardiac diseasenoyeshypertensionnoyesasaclassificationasa1asa2asa34localisationright colonleft colonrectumtumour stageiiiiiioverall survivalhr cipcancer free survivalhr ci reference reference p reference reference reference reference reference reference reference reference reference reference postoperative complicationnoyes reference reference 101371 pone0237244t003baseline hrv remained not significant sdnn hr p andrmssd p the same applied when postoperative complicationswere graded according to the claviendindo classification and the complication that is gradedthe highest for each patient is compared to the absence of postoperative complicationstable discussionin this observational cohortstudy we determined the heart rate variability in preoperativeecgs of patients with primary colorectal cancer who underwent curative surgical treatment hrv refers to physiological variations in beattobeat intervals it was presented intimedomain parameters sdnn and rmssd hrv was not significantly associated with overall survival or cancer free survival independent of some risk factors also this study showedno significant differences in cealevels at one year checkup between patients with low hrvand patients with normal hrv patients with low hrv did not have more or more severepostoperative complications compared to patients with normal hrvtumorigenesis has three fundamental mechanisms inflammation promoting oxidativestress and stimulating tumour growth oxidative stress causing dnadamage and one 101371 pone0237244 august one 0chrv and prognosis in colorectal cancerfig kaplanmeier curves for cancer free survival in groups of low hrv and normal hrv presented as a sdnnand b rmssd101371 pone0237244g003 one 101371 pone0237244 august one 0chrv and prognosis in colorectal cancertable univariate analyses of low hrv and risk of elevated cealevel at one year checkupcea μglor cipcea μglor ciindependent of baseline ceaindependent of baseline ceasdnn 20ms n rmssd 19ms n sdnn �20ms n normal baseline cea � μglsdnn 20ms n sdnn �20ms n elevated baseline cea μglsdnn 20ms n reference reference rmssd �19ms n normal baseline cea � μglrmssd 19ms n rmssd �19ms n elevated baseline cea μglrmssd 19ms n reference reference sdnn �20ms n referencermssd �19ms n referencep101371 pone0237244t004interfering with subsequent repair mechanisms and sympathetic neurotransmitters stimulating tumour metastasis and progression it has been suggested that afferent fibres of thevagus nerve inform the brain about peripheral inflammation this is followed by a braintoimmune response via the efferent route of the vagus nerve that modulates the function ofimmuneregulatory cells through the release of neurotransmitters via the cholinergic antiinflammatory pathway malfunctioning of this route may play part in the onset of cancer this theory has been supported by studies of patients with gastric ulcers who underwent avagotomy who then showed an increased risk of developing lung and colorectal cancer [] the vagus nerve is also believed to modulate tumour progression an active vagus nervecan inhibit inflammation oxidative stress and the release of sympathetic neurotransmittersthat stimulate tumour metastasis and progression [] absence of this inhibitory effect inturn results in metastasis and tumour progression as shown in a study of erin showingthat mice who underwent chemical vagotomy developed more metastasis of breastcancer cellsthan controls epidemiologically low hrv has been associated with worse overall survivalover time in patients with recurrent or metastatic cancer of various types even after correctionfor confounders [] however the results of the present study do not support thesefindingsto our knowledge this is the first study including only patients with colorectal cancer whoare eligible for curative treatment by partial bowel resection and not those receiving palliativetreatment de couck studied the relationship between hrv and tumour burden in bothcurative and palliative patients with prostate cancer or nonsmall cell lung cancer independent of confounders the hypothesised inverse relationship of hrv and the tumour markerpsa at and months after diagnosis was only significant in patients with stage iv prostatecancer not stage ii and iii in colorectal cancer mouton found that low hrv definedas sdnn ms predicted significantly higher levels of the tumour marker cea at months after diagnosis again these results were only found in patients receiving palliativetreatment not curative only one previous study showed a significant inverse relationshipbetween hrv and mortality in cancer in general independent of stage this study of guo had a large study population of patients with various types of cancer low hrv wasdefined as sdnn 70ms and was significantly associated with shorter survival times thissuggests that hrv is a predictive indicator of survival time not only in palliative but also incurative patients however results were not controlled for cancer type which could affect bothhrv and survival and should therefore be interpreted with caution the fact that thepatients recruited in the present sample were only with less advanced cancer may partlyexplain the lack of prognostic role in hrv in this sample one 101371 pone0237244 august one 0chrv and prognosis in colorectal cancerfig bar chart for cealevel at baseline and one year checkup in groups of low hrv and normal hrv presented as asdnn and b rmssd101371 pone0237244g004 one 101371 pone0237244 august one 0chrv and prognosis in colorectal cancertable low hrv and risk of occurrence of a postoperative complication within dayspostoperative complicationor ciunadjustedsdnn 20ms n sdnn �20ms n referenceadjusted�sdnn 20ms n sdnn �20ms n referenceunadjustedrmssd 19ms n rmssd �19ms n referenceadjusted�rmssd 19ms n rmssd �19ms n reference�adjusted for heart rate age cardiac disease and hypertension101371 pone0237244t005psome of these previous studies suggest a bidirectional relationship between vagus nerveactivity and cancer however based on current evidence on this subject we cannot supportthis hypothesis the positive association between low hrv and worse prognosis found inpatients with colorectal cancer receiving palliative treatment but not in patients receivingcurative treatment suggest that this relation is not bidirectional but that advanced cancer isassociated with low hrv midlatestage tumours are often accompanied by damage to autonomic nerves resulting in decreased hrv a study of de couck showed that cancerpatients in general have a significantly lower hrv than healthy people the same resultswere found in studies of bijoor where rmssd was found to be significantly lower inpatients with early and advanced stage cancer compared to a healthy control group when comparing patients with advanced stage cancer tnm iii and iv to patients with anearly stage of cancer tnm i and ii rmssd was found to be significantly lower in patientswith advanced stages of cancer thus though experimental studies in animals showthat vagal nerve functioning can causally slow tumorigenesis the human data suggests that themalignant tumour causes vagal nerve dysfunction and therewith decreased hrv besides the proposed influence of low hrv on survival of colorectal cancer patientsthrough development and increased progression of cancer reimer suggested that lowhrv could influence survival of those undergoing surgical treatment due to more and moresevere postoperative complications however the results found in this study were notconcurrent with those of reimer who included patients with asa undergoingelective surgery their analysis of hrv was through powerspectrum parameters based on longer ecgrecordings instead of the timedomain parameters based on 10s ecgs used in thisstudy but the difference in used parameters used in both studies is probably not the explanation for the differences in results between both studies since it has been demonstrated thatrmssd and sdnn based on ecg recordings of 10s are in substantial agreement with those of45min and a 10s ecg therefore suffices to determine time domain hrv parameters howeverreimer did not correct for possible confounders in their study patients with low hrvwere more likely to have diabetes a known risk factor for postoperative ileus and wound infections both found to be common postoperative complications in their low hrv group correcting for this comorbidity may change the significance of their findings a study of one 101371 pone0237244 august one 0chrv and prognosis in colorectal cancertable low hrv and severity of postoperative complications according to claviendindo classificationunadjustedminor grade i and iior cipmajor grade iii iv vor cisdnn 20msn n sdnn �20msn referencen referenceadjusted�sdnn 20msn sdnn �20msn referenceunadjustedrmssd 19msn rmssd �19msn referenceadjusted�n n referencen n referencermssd 19msn n rmssd �19msn referencen referencep�adjusted for heart rate age categories vs � and hypertension101371 pone0237244t006scheffler did not show any significant preoperative differences in hrv between patientswith or without postoperative complications or between patients with postoperative complications of different severity levels thus also in relation to predicting postoperative complications results are not uniformthis study was subject to a number of limitations due to its retrospective character confounding may have occurred we attempted to correct for all possible confounders withindatabase availability with regression analyses also not all possible confounding factors werewithin database availability reasons for diagnostic procedure smoking alcohol abuse are presumed to have an effect on hrv and outcomes but were not documented in enough patientsto be taken into account when conducting multivariate analyses analysed ecgs were thoseperformed before or at time of preoperative screening in this cohort ecgs were madewithin the year before surgery only ecgs were older than five years at time of surgery theprecise effects of these differences in time from ecg to cancer treatment on hrv areunknown to test the implication o | Colon_Cancer |
in a novel coronavirus sarscov2 was found to cause a highly contagious disease characterized by pneumonia the disease covid19 quickly spread around the globe escalating to a global pandemic in this review we discuss the virological immunological and imaging approaches harnessed for covid19 diagnosis and research covid19 shares many clinical characteristics with other respiratory illnessesaccurate and early detection of the infection is pivotal to controlling the outbreak as this enables case identification isolation and contact tracing we summarize the available literature on current laboratory and pointofcare diagnostics highlight their strengths and limitations and describe the emerging diagnostic approaches on the horizonwe also discuss the various research techniques that are being used to evaluate host immunity in laboratoryconfirmed patients additionally pathological imaging of tissue samples from affected patients has a critical role in guiding investigations on this disease conventional techniques such as immunohistochemistry and immunofluorescence have been frequently used to characterize the immune microenvironment in covid19 we also outline the emerging imaging techniques such as the rnascope which might also aid in our understanding of the significance of covid19specific biomarkers such as the angiotensinconverting enzyme ace2 cellular receptoroverall great progress has been made in covid19 research in a short period extensive global collation of our current knowledge of sarscov2 will provide insights into novel treatment modalities such as monoclonal antibodies and support the development of a sarscov2 vaccinekeywordscovid19 immunology pathology diagnostics specific t cellsintroductionin december a novel respiratory disease named coronavirus disease covid19 was detected by physicians in wuhan china the disease was found to be caused by the severe acute respiratory syndrome sarscov2 rna virus12 within a matter of weeks covid19 had spread rapidly and escalated to a global pandemic at the time of writing june million cases had been reported and patients had succumbed to the disease worldwide3 indeed patients with covid19 are at high risk of developing a severe and critical disease4 therefore rapid and accurate diagnostic tests are urgently needed to effectively isolate identify and treat infected individuals and to contain the spread of the virus failure to do so will inevitably lead to spikes in cases and the resultant overcrowding and collapse of healthcare services5 moreover research into this novel virus is also critical to understand its pathogenesis and its interaction with the human immune system insights from such research will guide the design of public health policies and protocols to 1lee kong chian school of medicine nanyang technological university singapore singapore2yong loo lin school of medicine national university of singapore singapore singapore3institute of molecular cell biology imcb agency of science technology and research astar singapore singapore4department of anatomical pathology singapore general hospital singapore singaporethese authors contributed equallyreceived june and in revised form july accepted for publication july corresponding authorjoe poh sheng yeong institute of molecular cell biology imcb agency of science technology and research astar college road academia level diagnostics tower singapore singapore email yeongpsimcbastaredusg 0c slas technology identify susceptible individuals and diagnostic prognostic and treatment approaches for patientscurrent diagnostic approaches predominantly involve established virological procedures such as nucleic acid hybridization techniques reversetranscriptase pcr [rtpcr] and recombinase polymerase amplification rpa as well as immunologic approaches like antibody assays each approach boasts unique strengths and weaknesses for instance while rtpcr demonstrates high sensitivity and specificity its capabilities have been severely limited for practical reasons during this current pandemic due to global shortages of skilled personnel reagents and equipment and a processing time of up to days by contrast immunologic tests such as antibody assays are rapid and require minimal equipment but they have limited utility in the context of acute diagnosis of sarscov2 infections this is because it can take several days to weeks following symptom onset for a patient to mount a detectable antibody response6immunological tools in research include enzymelinked immunosorbent assays elisas flow cytometry and mass cytometry cytof imaging techniques for pathological analyses include conventional approaches such as hematoxylineosin he staining immunohistochemical ihc staining or transmission electron microscopy tem and rnascope each of these methods is used to examine the pathophysiology underlying covid19 from a different perspective each with their own advantages and disadvantages for example it has been established that the entry of sarscov2 intro cells depends on the binding of viral proteins with the human receptor angiotensinconverting enzyme ace2 receptors7 additionally evidence shows that the type ii transmembrane protease tmprss2 is also essential for viral entry by priming the viral spike protein for binding to ace28 therefore considerable research efforts employing different techniques have been directed at mapping the distribution of ace2 and tmprss2 in tissues and their relationship to the observed manifestations of disease together the combination of these approaches has advanced our understanding of covid19in this review we discuss the current approaches in covid19 diagnosis and research with a focus on findings from virological and pathological imaging methods we also discuss immunological methods which are increasingly recognized as an integral component of the disease processdiagnosticsthe most common symptoms of covid19 at initial presentation are nonspecific and include a high fever a new and persistent cough and fatigue910 due to similarities between the clinical characteristics of covid19 and many other respiratory illnesses the accurate and early detection of infection is pivotal for outbreak control any delays in diagnosis are increasingly measured in lives lostaccording to the world health anization who the immediate goal for research into covid19 diagnostics is the development of rna assays antibody and antigen assays and pointofcare detection11 the intermediateterm priority would be their integration into multiplex diagnostic platforms while the longterm goal would be the investigation of prognostic markersin this section we summarize the current and emerging diagnostic tools for sarscov2 through the lens of immunologylabbased testsrtpcr molecular testing the detection of viral nucleic acids by rtpcr is the primary method used to confirm a suspected case of covid19 rtpcr and other nucleic acid hybridization techniques are an integral part of virology and are applied in a broad range of settings including screening diagnosis informing medical and therapeutic decisions and assessing cure rates from therapy12 chinese officials released the genomic sequence of sarscov2 to public databases early in the course of the outbreak13 and the who has since published seven protocols for rtpcrbased diagnostics because of the high sensitivity and specificity of rtpcr it is regarded as the gold standard for virus detection14 there are two essential steps in the process viral rna extraction and pcr amplification and probebased detection multiple largescale highthroughput instruments are available for automating both steps such as the roche cobas system which has an advertised throughput of tests per hours15however rtpcrbased testing is costly and timeconsuming requiring up to days using centralized laboratory equipment and skilled personnel furthermore global supply chain challenges have led to significant shortages of essential reagents lastly falsenegative results due to low sample volumes variable sampling techniques and sampling locations sample degradation during transportation andor improper nucleic acid extraction are a concern16 in addition the differences in detectable viral material in different sampling locations eg nasopharyngeal vs bronchoalveolar lavage fluid [balf] vs rectal samples might also explain the falsepositive rtpcr results on repeat testing in recovered covid19 patients indeed one postmortem case study revealed residual virus in lung tissue despite consecutive negative results on pcr testing from nasopharyngeal swabs19 separately winichakoon et al outlined a case of repeatedly negative nasopharyngeal and oropharyngeal swabs in a clinically deteriorating patient where only a balf pcr test returned positive20given the high expression of ace2 on alveolar epithelial cells and negative expression on nasal oral and nasopharynx 0ctan et al cells21 it would be prudent to perform bronchoalveolar lavage on patients to rule out falsenegative results from swabs of upper respiratory tract samples20labbased immunological assays in contrast to rtpcr techniques that detect viral nucleic acids serological and immunological assays aim to detect antibodies against sarscov2 or antigenic proteins in infected individuals neutralization assays are considered the gold standard for assessing neutralizing protective antibodies22 however these assays require specialized biosafety level bsl3 facilities and still take several days to complete another type of labbased antibody assay is the traditional elisa which detects all binding antibodies the four principal types of elisa are direct indirect competitive and sandwich elisa the indirect elisa is the most common method for determining antibody concentrations elisas have good concordance with neutralization assays for the detection of antibody responses in sarscov223 unfortunately both methods require skilled operators and are limited by low throughput due to the absence of fully automated systemsserological diagnostics offer several advantages re quirements for specimen quality are comparatively less stringent than for nucleic acid tests as the antibodies are uniformly distributed in the serum24 consequently sampling location concerns do not apply here furthermore good correlation between igg elisas performed on both conventional serum samples and plasma samples have been reported25 of which the latter may be conveniently obtained from residual blood submitted for other routine laboratory testsone pitfall of antibody assays is their limited utility early in the course of any infection sparse data are available with regard to the antibody responses produced by patients with covid19 it seems that sarscov2 igm is detectable at a median of days after symptom onset while igg is detectable after days26 with the seroconversion rate approaching by day an italian research group noted that the performance of a commercial vivadiag covid19 igmigg test was very poor with a sensitivity of only and a negative predictive value of in a cohort of suspected covid19 patients in the emergency room setting27 as such we believe that for now rtpcr testing is likely more appropriate for diagnosing acute covid19notably a longitudinal study examining the iggigm profiles of patients found that seroconversion for igg and igm occurred in no specific chronological order with a median of days after symptom onset28 all patients achieved seroconversion by day consequently the detection of both igg and igm simultaneously rather than one antibody alone would be idealanother concern surrounding serologic diagnostics is the production of falsepositive results from crossreactivity due to the high prevalence of the four endemic human coronaviruses in the human population in sarscov2 the spike s protein which includes two regions s1 and s2 and the nucleocapsid n protein np are the major immunogens29 and therefore most diagnostics rely on the detection of antibodies specific for these antigens one work suggests that of the possible targets the s1 subunit antigen is more specific than either the whole s antigen or the n antigen for detecting sarscov2 antibodies with no crossreactivity to other coronaviruses except for sarscov23 given that only sarscov infections were recorded worldwide30 the risk of false positives from this crossreactivity is miniscule however np elisas are more sensitive than s1 in detecting antibodies in those with a mild infection23 importantly as in sarscov most of the neutralizing antibodies are directed against the s protein31 of which s1 contains a receptorbinding domain rbd responsible for making contact with ace2 to facilitate viral entry7 thus theoretically only diagnostics that detect s1specific antibodies are suitable to infer immunity to covid19 this fact is corroborated by evidence that antis rbd but not antinp igg levels correlated with neutralizing antibody titers in sera from a cohort of recovered patients32 the number of commercial antibody assays is growing detecting either antinp antibodies antis1s antibodies or both there is also large variation in their claimed sensitivities and specificities33 based on the available evidence an ideal serological assay would be a combined test that simultaneously detects both antibodies to np and s1 antigens assessment of antinp antibodies has good sensitivity and would be best suited for supporting the diagnosis of infection while the additional antis1 antibody assay would allow for the determination of immunityrapid testspointofcare rtpcr tests a small number of commercial pointofcare tests utilizing rtpcr have been developed these typically involve the same methodology as conventional rtpcr but implemented with automated and portable benchtopsized instruments that can be operated closer to patient care settings than a centralized laboratory a prominent example is cepheids xpert xpress sarscov2 run on the gene xpert platform this apparatus can provide a result within min others include the mesabiotech accula test and microsensdx rapiprep covid19 despite displaying good sensitivity and specificity these instruments are generally limited by a very low throughput of only one to four tests per run per machine34 and as such are only suited to small laboratories or clinics 0c slas technology figure loopmediated isothermal amplification lamp a lamp begins when the forward inner primer fip binds to the a2c region while the forward primer a1 binds to a1c which displaces the fip complementary strand b the backward inner primer bip binds b2c while the backward primer b3 binds b3c and displaces the bip complementary strand c a complementary sequence that initiates loop formation is produced d loop structures are formed that allow for lamp with the use of loop primersfigure crispr technique viral rna is converted to dsdna using rtrpa recombinase polymerase amplification a the cas12a nuclease enzyme is activated upon complex binding to the target sequence resulting in cleavage of the target sequence and the fluorescent rna reporter b t7 transcription converts dna to complementary rna cas13 nuclease enzyme activity is activated upon complex binding to the target sequence resulting in a similar cleavage of the target sequence and the fluorescent rna reporterimmunological assaysrapid antibody assays compared with labbased antibody assays rapid assays such as lateral flow immunoassays lfias fig and chemiluminescent immunoassays clias fig offer the benefits of rapid diagnostic testing at a low cost these assays do not require specialized equipment or expertise35 and are thus excellent candidates for pointofcare testing or deployment on a large scale this an area of intense interest with governments worldwide aiming to order millions of tests to inform policy makers about attack rates in their populations36 lfias are predominantly singleuse kits designed for pointofcare use while clias are fully automated analyzers that permit very high testing throughputunfortunately these tests do not quantify the antibody titers and the performance of lfias has been called into question one evaluation of nine commercial lfias reported a sensitivity ranging from only to versus rtpcr and to versus elisa37 meanwhile the performance of clias is superior with good sensitivity and specificity levels similar to those of elisa38 otherwise these tests share the same advantages and drawbacks as the 0ctan et al table summary of diagnostic approaches for covid19categorytype of testtypical test result timecharacteristicsexamplesvirologicmolecular rtpcrdaysgold standard high sensitivity who rtpcr protocolstests pointofcare rtpcr minlamp crispr himmunologic testslfia for antibodies minantigensand specificity high throughput but lab basedrapid good sensitivity and specificity pointofcare testing but low throughputrapid good sensitivity and specificity pointofcare testing but low throughputrapid pointofcare testing but not quantitative poor sensitivitycepheid xpert xpress sarscov2sherlock biosciences sherlockvivadiag covid19 igmigg rapid testcorisbio covid19 ag respistripepitope diagnostics kt1033 edi novel coronavirus covid19 elisa kitroche elecsys antisarscov2 traditional elisa hgood sensitivity and specificity but lab based not automatedclia minrapid good sensitivity and specificity high throughput but lab basedneutralization assaydaysgold standard high sensitivity not commercially and specificity able to quantify neutralizing antibodies but requires bsl3 lab facilityavailablelabbased antibody assays discussed above the characteristics and unique advantages and disadvantages of these different methodologies are outlined in table antigen assays an alternative approach to immunological assays is to directly detect sarscov2 viral antigens several commercial pointofcare antigen tests are available but their performance remains to be evaluated these tests may be suitable for making an early diagnosis and are deployable as pointofcare assays however they face the same sampling limitations as rtpcr and are hypothetically hampered by limited sensitivity due to the omission of an amplification process unlike nucleic acid testing for example one multicenter study evaluating the corisbio covid19 ag respistrip a lateral flow assay for the sarscov2 np reported a test sensitivity of only rapid nonpcr molecular testing nucleic acid testing using nonpcr methods is an emerging approach for rapid diagnostics and several assays have received food and drug administration fda emergency use authorization which facilitates the distribution of unapproved medical products or the offlabel use of approved medical products when certain criteria are met these methods share high sensitivity and specificity on par with rtpcr but with the principal advantages of more rapid testing at a lower cost40lamp fig is one such novel isothermal nucleic acid amplification method that does not require a thermal cycler one example is the id now covid19 test from abbott diagnostics which can deliver results in just min43 and uses a lightweight portable instrument allowing onsite testing of swab samples however it has a limited throughput of only one sample per runthe crispr enzymes cas12 and cas13 have also been adapted for rapid nucleic acid sensing fig the detectr assay by mammoth biosciences45 as well as the sherlock assay by sherlock biosciences46 potentially offers sensitivity and specificity comparable to those of rtpcr but can be completed in h however these approaches are still in the early stages of commercialization and current applications are available only as test kits to be run in labs while pointofcare versions exist as proofofconcept demonstrations47 nonetheless their inherent characteristics hold great potential for diagnosis in the futureprognostication of diseaseprofiling of genetic susceptibility work is in progress to ascertain the possible genetic basis for the apparent variations in covid19 susceptibility and disease severity cao et al compared expression quantitative trait loci eqtl for ace2 in different populations finding significantly greater eqtl variants associated with higher ace2 expression in 0c slas technology figure lateral flow immunoassay lfia a serum sample deposited on the sample pad b antisarscov2 antibodies in the sample will bind to the target antigen with a labeled tag c immobilized antihuman igm antibodies will capture the sarscov2 antibodyantigen complex d control antibodies are captured by immobilized antibodies in the control lineserum prognostic markers another application of immunological methods would be to measure markers that enable prognostication in covid19 higher titers of antibodies against sarscov2 have been associated with more severe disease2350 similar to previous studies in middle east respiratory syndrome merscov51 elisa has been used to provide a quantitative measurement of serum and plasma igm and igg antibodies by monitoring the kinetics of igm and igg antibodies specific to the n and s proteins on sarscov2 it was found that intensive care unit icu patients had a significantly lower level of sigg within weeks of symptom onset but a higher level of nigg antibodies compared with nonicu patients52 this finding highlights the possible utility of sigg and nigg as a prognostic tool for covid19 patientsthe ddimer level which consists of crosslinked fibrin degradation products that reflect ongoing blood clot formation and breakdown activity in the body is another proposed prognostic marker modern commercial assays for ddimers are based on monoclonal antibodies employing either elisa or microlatex agglutination assays53 reports have emerged that elevated ddimer levels suggestive of a hypercoagulable state are associated with drastically worse outcomes a chinese group reported that ddimer levels of ¥ µgml on admission were associated with a times increased mortality relative to ddimer levels of µgml in a cohort of covid19 patients54 this finding of ddimer levels as a negative prognostic marker was also noted in other studies conducted in china455 and the netherlands56similarly interleukin il6 a key component of the cytokine release syndrome is another marker measured by elisa and has been described to independently predict adverse outcomes in covid195758 tumor necrosis factor alpha tnfα another important proinflammatory cytokine has also been found to be strongly correlated with figure chemiluminescence enzyme immunoassay clia sarscov2 antigens will capture igm and igg antibodies from the sample serum secondary antibodies that are conjugated with horseradish peroxidase hrp bind to the captured primary igm and igg antibodies and react with a chemiluminescent substrate to generate a strong chemiluminescent signal that is measured in terms of relative light units rlueast asian populations but reported no direct evidence supporting the existence of s proteinbindingresistant ace2 mutants48 out of identified protein altering variants separately stawiski et al analyzed ace2 polymorphisms within a much larger population dataset spanning more than population groups across the world and performed structural predictions to identify variants that might confer protection or rather increase susceptibility to sarscov2 s protein binding49 out of a total of identified proteinaltering ace2 variants variants were predicted to increase susceptibility while variants were speculated to confer protection however the degree of changes in receptorvirus binding interactions for each structural variant was not quantified these findings represent significant developments in our understanding of population risk profiles for covid19 and future coronavirus infections 0ctan et al endan damage and mortality even after adjusting for disease severity scores59 gao et al examined both il6 and ddimer levels they proposed a panel comprising tandem testing of these two markers which produced a sensitivity of and specificity of in early prediction of severe covid1958 elevated troponin levels a marker of myocardial injury measured with elisa immunoassays also strongly predict progression to death in patients with severe illness60 these results suggest that multiplex cytokine and serum marker profiling will be a powerful tool in stratifying patients that may guide clinical decisions and resource allocationsummary in sum rapid progress has been made in diagnostics for covid19 yet the race against time continues for researchers and biotechnology firms to develop rapid costeffective and reliable test kits that can be deployed on a large scale at the time of writing labbased rtpcr testing has been the dominant diagnostic approach but alternative molecular approaches like isothermal amplification and crispr which have clear advantages are on the horizon immunological tests such as clia and lfia will become increasingly important because of the urgent need for pointofcare diagnostics for mass testing of infected asymptomatic individuals and their close contacts and will be valuable in complementing molecular approaches for confirming infection furthermore immunological assays will be in great demand by policy makers worldwide for the assessment of immunity to covid19 however the performance of these serological tests varies significantly particularly their degree of sensitivity and specificity we believe that caution must be taken in the interpretation of these tests detailed evaluation of the reliability of serological tests will be a key area for future research lastly given the importance of techniques like elisa in prognosticating covid19 immunological methods will undoubtedly occupy a crucial role in achieving all levels of the whos short medium and longterm diagnostic goalscovid19 research toolsimmunological approachescovid19 infection has a poor prognosis in individuals with comorbidities and abnormal immune functions although research surrounding covid19 is still in its infancy several studies have revealed lymphopenia and the cytokine storm as underlying mechanisms correlating to disease progression here we discuss the various immunological techniques involved in assessing host immunity in covid19 patientselisa as discussed elisa has also been used to detect the inflammatory cytokines implicated in the cytokine storm seen in patients with severe respiratory failure due to covid19 one study found that the immune dysregulation in patients with severe respiratory failure was due to a significantly increased production of il6 and defective lymphoid function because of an il6mediated decrease in hladr expression on cd14 monocytes interestingly interferongamma ifnγ levels were below the detection level in these patients suggesting that t helper th cells are unlikely to be major players in the overinflammatory response of severe patients61 a similar observation was made in a separate study whereby inflammatory cytokines that mediate major immune responses such as tnfα and il1β were not significantly elevated in icu patients62 these findings demonstrate that the immunophenotype of patients with covid19 can vary depending on presently unclear host immune factors and the severity of their condition this relationship between disease severity and cytokine storm has also been highlighted in other studies that found a significantly elevated plasma concentration of granulocyte colonystimulating factor gcsf ip10 ccl2 and ccl3 in icu patients compared with nonicu patients63elisa is also being used as a companion diagnostic tool for therapeutic purposes in a study that explored the use of convalescent plasma therapy from donors as a form of treatment in severe covid19 elisa was used to assess the neutralizing activity of the rbdspecific igm and igg antibodies found in the donor convalescent plasma64 after the transfusion was complete elisa was also used to detect igg igm and neutralizing antibody titers in the sera of patients to assess the response to treatment65enzymelinked immunosorbent spot enzymelinked immunosorbent spot elispot is a sensitive immunoassay that quantitatively measures cytokinesecreting cells at the singlecell level providing insight into immunerelated cellular activities66 hence it is a promising tool for characterizing specific tcell immunity in covid19 patients by ifnγ elispot analysis it was revealed that convalescent covid19 patients had significantly increased levels of ifnγsecreting t cells when compared with healthy donors a significant correlation between neutralizing antibody titers and npspecific t cells was identified in these patients suggesting that a combination of humoral and cellular immunity is integral to clearing sarscov2 interestingly it was noted that in convalescent patients weeks ifnγsecreting tcell numbers had postdischarge decreased suggesting that they may not be maintained for a prolonged period of time even in recovered patients67elispot is also serving a vital role in vaccine development through the detection of potential tcell epitopes in the s protein rbd of sarscov268 one study was able to harness elispot assays to identify three tcell epitopes that induced a strong adaptive immune response 0c slas technology postimmunization demonstrating the promise of elispot assays in the area of vaccine development32 recently elispot has also been applied to assess the immunogenicity of newly developed vaccines one such study successfully utilized an ifnγ elispot assay to evaluate tcell responses to a new sarscov2 vaccine in murine splenocytes and rhesus macaque peripheral blood mononuclear cells pbmcs the promising findings from this animal study informed the start of a phase i clinical trial with the same vaccine highlighting the usefulness of elispot in assessing immune responses to new vaccines and promoting vaccine development69flow cytometry unlike elisa and elispot flow cytometry determines the number of cytokinesecreting cells and has the capacity to immunophenotype based on surface and intracellular markers70 in relation to the current pandemic this technique enables the detection sorting and analysis of multiple subpopulations of immune cells specific to covid using flow cytometry researchers detected a cytotoxic immune environment in patient blood samples despite a reduction in the overall lymphocyte population71 as part of the sarscov2 antiviral response peripheral lymphocytes retain the capacity to activate and differentiate into subpopulations such as antibodysecreting cells cd3cd19cd27hicd38hi follicular t cells cd4cxcr5icospd1 cd4 th cells cd38hladrcd4 cytotoxic t tc cells cd38hladrcd8 and regulatory t treg cells cd3cd4cd25cd127 these tc cells harbor large amounts of cytotoxic granules while cd4 th cells skewed toward a proinflammatory th1 and th17 phenotype727375 the overall hyperinflammation and cytotoxic environment supports the notion that a cytokine storm could be liable for the multisystemic insults in patients with severe covid19elicitation of antiviral tcell responses specific to sarscov2 is of utmost importance to establishing viral control many studies have demonstrated robust antiviral responses however there is no known set of markers reported to identify sarscov2specific t cells collectively most groups have characterized sarscov2specific t cells based on hladr cd38 cd69 cd25 cd44 and ki67 expression th | Colon_Cancer |
during the covid19 pandemic emergency departments have noted a significant decrease in strokepatients we performed a timely analysis of the bavarian telestroke tempis working diagnosis databasemethods twelve hospitals from the tempis network were selected data collected for january through april in years through were extracted and analyzed for presumed and definite ischemic stroke is amongst otherdisorders in addition recommendations for intravenous thrombolysis rtpa and endovascular thrombectomy evtwere noted and mobility data of the region analyzed if statistically valid groupcomparison was tested with fishersexact test considering unpaired observations and apvalue was considered significantresults upon lockdown in midmarch we observed a significant reduction in recommendations for rtpa compared to the preceding three years [] vs p¼ recommendations for evt werep¼ reflecting its increasing importance following the covid19 lockdown midmarch the number ofevt decreased back to levels in [] vs p¼ absolute numbers of issignificantly higher in january to midmarch compared to [] vs decreased in parallel to mobility datas the reduced stroke incidence during the covid19 pandemic may in part be explained by patientavoidance to seek emergency stroke care and may have an association to population mobility increasing mobilitymay induce a rebound effect and may conflict with a potential second covid19 wave telemedical networks maybe ideal databases to study such effects in nearreal timekeywordstelestroke covid19 lockdown stroke thrombolysisdate received may date accepted june introductionimplementation of social distancing to combat theimpact of corona virus pandemic sequelae has emergedas the major strategy to contain the spread of infectiongiven the lack of speciï¬c treatments for covid19 andlimited intensive care resources1 major concerns forstroke neurologistsin this extraordinary scenarioinclude the following a rapid speciï¬c managementof cases of acute stroke with possible covid19from initiation of the stroke call in the preclinical setting through the ambulance system emergency department and hospital stroke department and in the1department of neurology university of regensburg bezirksklinikumregensburg tempis telemedical stroke center regensburg germany2cts herdecke germany3department of neurology tempis telemedical stroke centeracademic teaching hospital of the university of munich mu¨nchen klinikharlaching munich germanycorresponding authorfelix schlachetzki md department of neurology university ofregensburg center for vascular neurology and intensive care tempistelemedical stroke center bezirksklinikum regensburguniversit¬atsstr84 regensburg germanyemails felixschlachetzkiklinikuniregensburgde 0c of telemedicine and telecare bthe factneuroradiological department when needed to aid instroke diagnosis and treatmentthatpatients with mild stroke symptoms or transient ischemic attacks tias may be reluctant to request hospitaladmission for acute stroke23 and c that covid19itself is associated with severe stroke syndromes this issuggested in a recent case series of covid19 patientsfrom wuhan china focusing on neurological symptoms that described cerebrovascular events in of cases especially in elderly patients and in thosewith more severe infections also authors of a secondcase series reported unusual cases of young covid19patients yrs with large vessel stroke and otherauthors reported three stroke patients with coagulopathy and antiphospholipid antibodies in the context ofsevere covid infections4the number ofin contrast several stroke departments in germanyincluding our own the usa and china have noted asignificant drop in the number of stroke patient admissions during the corona pandemic7 data on this phenomenon are still scarce howeverin a descriptivereport by morelli from piacenza lombardyitaly covering the period february appearance ofthe ï¬rst sarscov2 patient recorded in italy to march stroke admissionsdecreased from an average of with largevessel occlusions lvos to two tias one lvoand three lacunar strokes8 using a commercial neuroimaging database with the rapid software platform kasangra and hamilton observed a decrease in stroke imaging procedures with the nadirfollowing the ï¬rst statewide stayathome order in theusa9 the decrease was observed in all age sex andstroke severity subgroups within all participatinghospitals which processed overall patientsbetween july and april cardiologistsin france observed a similar significant drop in admissions to nine intensive cardiac care units after initiationof social distancing and selfquarantine in midmarch overall there are scarce data available on theimpact of the covid19 infection itself on cardiovascular morbidity including cerebral stroke11aims and hypothesisthe primary aim of this study was to evaluate the effectof the covid19 pandemic lockdown on stroke consultations and treatment recommendations using theacute consultant database of the telestroke networktempis12 we focused on data collected during theï¬rst four months of which included the emergence of the corona virus pandemic in southeasternbavaria through the ï¬rst two months of social distancingregion shutdown we compared these data withcomparable data collected during the same months inthe years methodsdata from daily consultations at clinics withoutneurology departmentsin the telestroke networktempis form the basis of this study the consultationstook place between january and april in the years all data were pseudonymized weextracted the actual working diagnoses based on telemedical consultation and neuroimaging results mainlycerebral computed tomography two major databaseswere used to calculate the population within these districts wwwdestatisde and experiencearcgiscomexperience478220a4c454480e823b17327b2bf1d4pagepage_1 this retrospective study was approvedby the local ethics committee of the university ofregensburg and performed in accordance with guidelines of the declaration of helsinkimobility data available at wwwapplecomcovid19mobility were extracted these data were generated from the relative request volume for directionsin munich germany compared with a base volumeon january to observe the relationship ofmobility and the reported stroke decline in piacenzawe also extracted mobility data from milan close topiacenza italy8the major working diagnostic groups were asfollows a ischemic stroke b tia c intracranialhaemorrhage d epileptic seizure e migraine andf other disorder including facial palsy headacheand brain tumour also included were cases in whichthere were recommendations for iv thrombolysis ivrtpa or endovascular therapy evt thrombectomyfor lvoexploratory descriptive summary statistics withmean values and standard deviations were appliedin an analysis of data covering january through aprilin years in comparison with data coveringthe same period in counts are presented as agraphic display showing incidences standardized to15day periods if statistically valid especially percentage of recommendations for iv thrombolysis andthrombectomy groupcomparison was tested withfishers exact test considering unpaired observationsa pvalue was considered significantresultsthere were telemedical consultations during thespeciï¬c time frames investigated and the population inthe geographical areas covered by these rural hospitals is most hospitals reside in areas with ahigh number of covid19 cases figure 1a the 0cschlachetzki number of covid19positive cases in the whole ofbavaria rose from ï¬ve at the end of february to cases on april the public lockdownwas initiated on march however the recommendation of personal quarantine for people who hadtravelled to northern italy was broadcast earlier on march in munich applevr mobility trends demonstrated a decrease in walking activity in midmarch to to of the baseline levelin milan lombardy italy on march walking activity began to decrease soon reaching of baselineactivity and remaining fairly constantthereafterfigure 1boverall consultations were analysed and excluded being nonacute consultations within thenetwork ie followup examinations statistically significant changes in the number of recommendations foriv thrombolysis were observed in figure 1cwhile in iv thrombolysis was recommendedin of consultations with suspected ischemicstroke of the frequency of this recommendation decreased to of in p¼ no differences in the number of ivthrombolysis recommendations were observed duringthe time period covering january to march in vs in notfigure a incidence of new covid19 infections in bavaria on april red dots indicate network hospitals and green andyellow squares depict the two academic stroke centres that alternate weekly for the tempis consult service modified with permission from the bavarian state office for health and food safety httpwwwlglbayerndegesundheitinfektionsschutzinfektionskrankheiten_a_zcoronaviruskarte_coronavirus b mobility data according to covid19 mobility trends reports apple thedata reflect requests for routing in apple maps for munich which resides in the centre of the tempis network and for milan nearpiacenza where the first decline in the number of strokes was reported morelli 8 horizontal dotted line indicates reportedreduced stroke activity in piacenza c recommendations absolute numbers for application of iv thrombolysis and thrombectomyvertical dashed line indicates the official beginning of lockdown in bavaria time and patient numbers on yaxis are standardized to15day periods xaxis in each month to compensate for shorter february and longer january and march months j1¼ january first half j2 january second half f¼ february m¼ march a¼ april d working diagnoses of the telestrokeconsultations vertical dashed line indicates the official beginning of lockdown in bavaria time and patient numbers on yaxis arestandardized to 15day periods xaxis in each month to compensate for shorter february and longer january and march months j1¼ january first half j2 january second half f¼ february m¼ march a¼ april 0c of telemedicine and telecare significant no trend in fewer recommendations forevt was observed between march and april in compared with the same time periods in of vs of however in the preceding time frame january to march significantly more recommendations for thrombectomy were made comparedwith of vs of in p¼ the data reï¬ect the development of consultationsand treatment recommendations for lvo in the network from onward the number of recommendations for evt steadily rose with increasing evidencefor recanalization even in later time windows andincreasing employment of computed tomography angiography in the tempis network table shows thedevelopment of consultations in up to the end ofthe study including the lockdown period it shows adrop in the number of consultations and more importantly fewer recommendations for iv thrombolysisand evt which suggests fewer incidences of ischemicstroke severities table figure 1dalthough bavaria is the state with the highestnumber of covid19 cases in germany especially inour region we only performed ï¬ve telestroke consultations for the network hospitals in which possiblecovid19 infection was discussed including a singlepatient with stroke symptoms and feverdiscussionthe tempis telestroke working data conï¬rm thecurrent observation of a low stroke incidence insoutheastern bavaria with relative proportions of theworking diagnosis remaining similar the number ofcases of disabling stroke from intracranial haemorrhage and ischemic stroke requiring iv rtpa or evtalso diminished challenging the theory that onlypatient avoidance to call for emergency treatment isresponsible for this phenomenon this study also demonstrates the potential and importance of telestrokenetworks in the current covid19 pandemic313the observation of fewer stroke cases during thecovid19 pandemic seems to contradict two essentialassumptions with regard to stroke risk a sarcov is a strong risk factor for stroke and b physicalinactivity in a lockdown setting may increase the riskof stroke especially among elderly persons firstsarcov2 may induce hypercoagulability and highlevels of creactive protein ddimer and interleukin6placing patients at risk to develop thrombotic complications14 in a series of intensive care unit patientsin the netherlands reported by klok only threestrokes complicated the course of covid19 whereasthe majority of complications included pulmonarythrombosiscatheterassociatedembolism n¼ and peripheral venous thrombosisn¼ andobservations in case series that concurrent covid infection complicates or triggers unusual ischemicstroke may well prevail but case control studies focusing on this phenomenon are urgently needed to afï¬rmor deny the assertion5 second physical inactivity has aprofound effect on atrial ï¬brillation obesity diabetesmellitus management and hypertension among othersand contradicts current recommendations on mid andlongterm stroke prevention16 a recent study in consecutive patients with nonstsegment elevationacute coronary syndromes acss and optical coherence tomography of the culprit lesion reported bykato found that the combination of greaterphysical activity outdoor acs onset and high bodymass index had a significant effect on the incidence ofcoronary plaque erosion17 interestingly mobility datasuch as those provided by the apple mobility databasevr demonstrated a parallel reduction in incidencesof stroke and acs in three published papers8 inaddition to oursour data conï¬rm the observation from morelli who termed the phrase bafï¬ing case of ischemicstroke disappearance these authors also discuss thatthis effect cannot be totally explained merely by thereluctance of patients to call for help in a stroke emergency because the number of cases presenting withsevere stroke requiring evt and the number of generalconsultations in tempis also decreased an analysisbased on a large database associated with the application of rapid software in acute stroke by kansagra is in line with our observation that also severestroke patients diminished during the early lockdownphase9 the number of ischemic core volumes ml and greater than ml were observed to decreaseby and respectively core volumes ml decreased by and and very smallcore infarct volumes measuring ml decreased the decrease in the number of very small infarctvolumes may well be explained by the generally proposed hesitation to seek emergency care while thereduction in large ischemic core volumes is morelikely due to fewer lvos as observed in our studywith a sharp decline in iv thrombolysis and thrombectomy recommendationsanother explanation may be a concurrent low infection rate with other viruses that can trigger atherosclerosis and plaque rupture resulting in neuro andcardiovascular morbidity18 the lockdown not onlyreduces physical activity strict social distancing anduse of facial masks should also lead to low rates ofexposure to and transmission of other common virusesand allergens that by themselves appear to triggerstroke19 additional studies with detailed analyses of 0cschlachetzki stekcarberauqsniatadhtnomehtfoshtgneltnereffidrofstnemtsudajtuohtiwtubsdoirepkeewotnidedvdiilirpayraunajrofsnoitatlusnocforebmunlatotlebatrparparamrambefbefnajnajsopdvocilatotairavabnisesacnoitaiveddradnatsdnaseulavnaemnimorfatadwohssipmetkrowtenekortsnoitatlusnoceetl] 06 06 06 06 06 06 06 06 06 06 06 06 06 06 06 06[snp p¡ 8a 06[¡snp§p§§§§ 8a 06[ 8a 06[ 8a 06[ 8a 06[ 8a 06[ 8a 06[§ 8a 06[ 8a 06[ 8a 06[ 8a 06[ 8a 06[ 8a 06[ 8a 06[ 8a 06[ 8a 06[lsisyobmorhtvi 8a 06[ 8a 06[ 8a 06[ 8a 06[ 8a 06[ 8a 06[ 8a 06[ 8a 06[ 8a 06[ 8a 06[ 8a 06[ 8a 06[ 8a 06[ 8a 06[ 8a 06[ 8a 06[ 8a 06[ 8a 06[ 8a 06[ 8a 06 06[ 8a 06[ 8a 06 06[ytilibadaerrettebrofldoblynoerasrebmunldobskcattaicmehcsitneisnartatiamotamehlarudbushdsegahrromehidonhcarabushasegahrromeahlainarcartnihciigndeebllainarcartnsnsn¼¼¼¼bp§pcpp§§ ¡iids 06naemsnoitatlusnocekortsicmehcsids 06naemds 06naemymotcebmorhtds 06naemds 06naema tids 06naemh cids 06naemhashdsbcieniargimeruziessrehto 0c of telemedicine and telecare symptom onsettodoor times stroke severity neuroimaging and inï¬ammatory markers are needed tounderstand the reason for the reduced number of revascularization therapies requested during the covid19pandemiclimitations of the studyanalysis of daily working diagnoses in the tempistelestroke network has the advantage of being highlytimely yet it lacks speciï¬city because the ï¬nal diagnosismay differ from the initial one this may be compensated by the creation of a large common database fortelestroke networks that incorporates corrections forthe actual population covered analyses of otherstrokerelated databases such as the one associatedwith rapid software healthcare provider databasesand common stroke registries for quality control thedecrease in the number of thrombectomy recommendations in our cohort midmarch did not reach statistical signiï¬cance when compared with the sameperiod in years through because rates forthis procedure increased according with levels of evidence2021 in agreement with this development thrombectomy recommendations by tempis neurologists in prior to the covid19 pandemic occurred morefrequently than in previous yearssour study using the tempis telestroke database conï¬rms lower incidences of ischemic stroke and otheracute neurological disorders requiring consultationsuch as intracerebral haemorrhage seizure disorderand migraine next to a reluctance within the population to seek immediate medical assistance for acutestroke the covid19 lockdown which resulted inless physical activity and fewer other common infections may also be responsible for the fewer numberof patients with severe stroke especially those withintracranial haemorrhage and those eligible for recanalization therapies if lockdownassociated factors areindeed responsible for a lower stroke incidence we mayexpect a rebound effect following the lockdown periodwith an increased incidence of stroke as well as ofmyocardial infarcts and traumatic brain injuries aspatients frailty may have increased during the lockdown analyses of large stroke databases may revealfurther insights into this phenomenon however telestroke networks such as tempis may be ideal tools tomonitor stroke occurrence in real timeacknowledgmentsthe authors acknowledge all consulting neurologists intempis and colleaguesin badin partner hospitalsebersbergburglengenfeldreichenhalleggenfeldenerding freising kelheim mu¨ hldorf rotthalmu¨ nstervilsbiburg dingolï¬ng and zwiesel the authors like tothank jo ann elison ma elsdfor editing thispaper for english grammar and languagedeclaration of conflicting intereststhe authors declared no potential conï¬icts of interest withrespect to the research authorship andor publication of thisarticleanonymized data are available on requestfundingthe authors received no ï¬nancial support for the researchauthorship andor publication of this articleorcid idfelix schlachetzkiorcidorg0000000161672597references jawaid a protecting older adults during social distancing science khosravani h rajendram p notario l protectedcode stroke hyperacute stroke management during thecoronavirus disease covid19 pandemic stroke markus hs and brainin m covid19 and stroke a global world stroke organization perspective int jstroke mao l jin h wang m neurologic manifestationsof hospitalized patients with coronavirus disease inwuhan china jama neurol oxley tj mocco j majidi s largevessel stroke asa presenting feature of covid19 in the young n engl jmed e60 zhang y xiao m zhang s coagulopathy andantiphospholipid antibodies in patients with covid19n engl j med e38 zhao j rudd a and liu r challenges and potentialsolutions of stroke care during the coronavirus disease covid19 outbreak stroke morelli n rota e terracciano c the bafï¬ing caseofischemic stroke disappearance from the casualtydepartment in the covid19 era eur neurol kansagra ap goyal ms hamilton s collateraleffect of covid19 on stroke evaluation in the unitedstates n englnejmc2014816 online ahead of printj meddoi huet f prieur c schurtz g one train may hideanother acute cardiovascular diseases could be neglectedbecause of the covid19 pandemic arch cardiovasc dis bansal m cardiovascular disease and covid19diabetes metab syndr audebert hj schenkel j heuschmann pu effectsof the implementation of a telemedical stroke networkthe telemedic pilot project for integrative stroke care 0cschlachetzki tempis in bavaria germany lancet neurol patel uk malik p demasi m multidisciplinaryapproach and outcomes of teleneurology a reviewcureus e4410 terpos e ntanasisstathopoulos i elalamy i hematological ï¬ndings and complications of covid am j hematol klok fa kruip m van der meer njm incidenceof thrombotic complications in critically ill icu patientswith covid19 thromb res kyu hh bachman vf alexander lt physicalactivity and risk of breast cancer colon cancer diabetesischemic heart disease and ischemic stroke eventsaystematic review and doseresponse metaanalysis forthe global burden of disease study bmj i3857 kato a minami y katsura a physical exertion asa trigger of acute coronary syndrome caused by plaqueerosion j thromb thrombolysis grau aj urbanek c and palm f common infectionsand the risk of stroke nat rev neurol pagliano p spera am ascione t infections causing stroke or strokelike syndromes infection campbell bcv donnan ga lees kr endovascular stent thrombectomy the new standardof care for large vessel ischaemic stroke lancet neurol vinny pw vishnu vy and padma srivastava mvthrombectomy to hours after stroke n engl jmed 0c' | Colon_Cancer |
" the ampactivated protein kinase ampk is an evolutionarily conserved regulator of cellular energyhomeostasis as a nexus for transducing metabolic signals ampk cooperates with other energysensing pathwaysto modulate cellular responses to metabolic stressors with metabolic reprogramming being a hallmark of cancerthe utility of agents targeting ampk has received continued scrutiny and results have demonstrated conflictingeffects of ampk activation in tumorigenesis harnessing multiomics datasets from human tumors we seek toevaluate the seemingly pleiotropic tissuespecific dependencies of ampk signaling dysregulationmethods we interrogated copy number variation and differential transcript expression of ampk pathway genesacross diverse cancers involving over patients cox proportional hazards regression and receiver operatingcharacteristic analyses were used to evaluate the prognostic significance of ampk dysregulation on patientoutcomesresults a total of and seven ampk pathway genes were identified as having loss or gainoffunction featuresthese genes exhibited tissuetype dependencies where survival outcomes in glioma patients were most influencedby ampk inactivation cox regression and logrank tests revealed that the 24ampkgene set could successfullystratify patients into high and lowrisk groups in glioma sarcoma breast and stomach cancers the 24ampkgeneset could not only discriminate tumor from nontumor samples as confirmed by multidimensional scaling analysesbut is also independent of tumor node and metastasis staging ampk inactivation is accompanied by the activationof multiple oncogenic pathways associated with cell adhesion calcium signaling and extracellular matrixanization anomalous ampk signaling converged on similar groups of transcriptional targets where a commonset of transcription factors were identified to regulate these targets we also demonstrated crosstalk between procatabolic ampk signaling and two proanabolic pathways mammalian target of rapamycin and peroxisomeproliferatoractivated receptors where they act synergistically to influence tumor progression significantlycontinued on next page correspondence alvinalaiuclacukinstitute of health informatics university college london euston roadlondon nw1 2da uk the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cchang and lai bmc cancer page of continued from previous page genetic and transcriptional aberrations in ampk signaling have tissuedependent pro or antitumorimpacts pancancer investigations on molecular changes of this pathway could uncover novel therapeutic targetsand support risk stratification of patients in prospective trialskeywords ampk glioma lossoffunction tumor metabolism pancancer the ampactivated protein kinase ampk is an evolutionary conserved key player responsible for energy sensing and homeostasis orthologous copies of ampkprevail universally as heterotrimeric complexes wherethe human genome encodes two genes for the α catalyticsubunit two regulatory subunit genes and three Πsubunit genes historically ampk was discovered as a crucial regulator of lipid metabolism since then ampkis implicated in a wide variety of fundamental metabolicprocesses as well as in metabolic diseases such as cancerand diabetes the first link between ampk and cancer was identified through the tumorsuppressive function of lkb1 which is upstream of the mtor pathway theroles of ampk werepharmacologically demonstrated by the application ofmetabolic inhibitors such as the antidiabetic metforminand the mimetic of amp aicar [] numerousstudies have since compellingly established the promiscuous nature of these pharmacological agents wherebythe inhibition of cancer cell proliferation occurs throughnonspecific ampkindependent avenues [ ]tumorsuppressivestressorssuchasin contrastof metabolicto the tumorsuppressive results frompharmacological studies genetic experiments on cancercells have credibly demonstrated that ampk activationis crucial for tumor progression and survival [] amyriadoxygendeprivation nutrient starvation and oxidative stress exists within the tumor microenvironment metabolic reprogramming during carcinogenesis would thus triggerampk activation to enable cells to survive under conditions of stress typically found in the tumor microenvironment hence conferring an overall tumorpromotingeffect ampk is also shown to support cancer growthand migration through crosstalk with other prooncogenicpathways for instance overexpression of oncogenes mycand src or the loss of the tumor suppressor folliculincould lead to ampk activation []genetic and pharmacological studies have paved theway for our understanding of the function of ampk incancer however anti and proneoplastic features ofampk remain controversial potentially due to the oversimplification of ampkmodulated processes in in vitroand nonhuman invivo models the genetic and clinicallandscape of ampk signaling has not been systematically investigated thus our study aims to address anunmet need to rigorously investigate the role of ampkin diverse cellular context using multiomics data fromactual tumors where we examined somatic copy numberalterations transcriptional and clinical profiles of tumorsfrom cancer types our analyses of clinical samples atscale would complement evidence from pharmacologicaland genetic studies to better elucidate the multifacetedand cellspecific nature of ampk signaling on tumorprogressionmethodsampk pathway genes and cancer cohortsninetytwo ampk pathway genes were retrieved fromthe kyoto encyclopedia of genes and genomes keggdatabase additional file clinical genomic and transcriptomic datasets of cancers involving patients were downloaded from the cancer genome atlastcga copy number variation differential expressionmultidimensional scaling and survival analysesdetailed methods of the above analyses were previouslypublished and thus will not be repeated here as per the guidelines [] to summarize discrete amplification and deletion indicators for copy number variation analyses were obtained from gistic geneleveltables gistic values of and were annotatedas shallow amplification and shallow deletion heterozygous events respectively gistic values of and were annotated as deep amplification and deep homozygous deletion events respectively multidimensionalscaling analyses and permutational multivariate analysisof variance permanova were performed using the rvegan package survival analyses were performed usingcox proportional hazards regression and the logranktest sensitivity and specificity of the 24ampkgene setwere assessed using receiver operating characteristicanalyses differential expression analyses were performed on patients stratified into high 4th quartileand low 1st quartile expressing groups using the genesetto determine the transcriptional effects ofanomalous ampk signalingpathway and transcription factor analysesgenes that were differentially expressed degs betweenthe 4th and 1st quartile patient groups were mapped to 0cchang and lai bmc cancer page of kegg gene ontology and reactome databases using gprofiler to ascertain biological processes and signaling pathways that were enriched the enrichr tool was used to map degs to the chea and encodetranscription factor tf databases to identify tfs thatwere significantly enriched as regulators of the degsirs2pik3r1sirt1 tbc1d1calculating the 24ampkgene score peroxisomeproliferatoractivated receptors ppar score andmammalian target of rapamycin mtor scoreampk scores were calculated from the mean expressionthe following genes slc2a4 foxo3 ppp2cbofpik3cd cab39l ccna1 fbp1 fbp2 foxo1 hmgcrppargc1appp2r2c mlycd pfkfb3 ppp2r2b prkaa2 leprcab39 irs1 and pfkfb1 ppar scores for each patientwere calculated by taking the mean expression of pparsignature genes plin5 pparg acadm gk cpt2scp2 acaa1 apoa1 ppara acox2 angptl4fabp3 plin2 aqp7 acsl1 fabp5 acadl andpck2 mtorpi3kakt scores for each patientwere calculated using the following equation mtorpi3kakt score akt mtor gsk3 s6k s6 pten all figures were generated using r version andadobe illustrator version cs6resultspancancer genomic and transcriptional alterations ofampk pathway genesfocusing on the genomic and transcriptomic landscapeof genes associated with ampk signaling retrievedfrom kegg across cancer types involving patients additional file we interrogated somatic copynumber alterations scna and mrna expression seeadditional file for a flowchart illustrating the study design to determine the effects of genomic alterations inampk pathway genes we classified genes as havinghighlevel amplifications gains lowlevel amplificationsdeep homozygous deletions and shallow heterozygousdeletions to evaluate pancancer patterns of scnaswe considered genes that were gained or lost in at least of samples within a cancer type and in at least onethird of cancer types ie at least seven cancer types atotal of genes were recurrently amplified while genes were recurrently lost fig additional file ampk is the central regulator of cellular energy levelswhich controls a number of downstream targets an example being the nuclear receptor hnf4a remarkablyhnf4a was found to be the most amplified gene identified as being recurrently amplified in of samplesin all cancers fig additionalfile this isfollowed by cftr cancer types and four other genesthat were amplified in cancer types adipor2 lepfile fig additionalprkag2 and rhebincontrast ppp2r2a was the most deleted gene found in of samples across cancers followed by the deletion of slc2a4 in cancers and five additional genesfoxo3 ppp2cb ppp2r2d ppp2r5c and ppp2r5ein cancer types fig additional file among allcancer types the highest number of amplified ampkpathway genes was observed in esophageal carcinomaesca genes followed by bladder cancer blca genes and lung cancer genes in both lung squamouscell carcinoma [lusc] and adenocarcinoma [luad]fig glioma tumors gbmlggin contrast hadonly five genes that were recurrently amplified fig in terms of somatic deletions lusc and esca both had genes deleted while no recurrent deletions were observed in papillary renal cell carcinoma kirp fig lossoffunctionevents differentialwe reasoned that scnas associated with transcriptional alterations could be considered as putative gainorexpressionanalyses between tumor and nontumor samples in eachcancer revealed that and genes were significantlyupregulated and downregulated in at least seven cancertypes respectively additional file of these differentially expressed genes seven and genes were alsorecurrently amplified and deleted respectively venn diagram in fig both gene sets were mutually exclusiveie the genes either had gainorfunction or lossoffunction features but not bothmolecular underpinnings of patient survival involvingputative lossoffunction ampk pathway componentswe next investigated the impact of transcriptional dysregulation ofthe putative gain and lossoffunctiongenes identified previously on patient survival outcomesacross all cancer types employing cox proportional hazards regression we observed that all genes sevengainoffunction and lossoffunction genes wereprognostic in at least one cancer type fig 2a thehighest number of prognostic genes was observed inglioma gbmlgg tumors genes while none ofthe genes were significantly associated with overallsurvival outcomes in esca and cholangiocarcinomachol fig 2a intriguingly although esca had thehighest number of scnas fig none of the genesharbored prognostic information suggesting that alterations in ampk signaling components have minimalroles in driving tumor progression and patient outcomes fbp1 was significantly associated with overallsurvival outcomes in cancers while ppp2r2c andppp2r2b in cancers fig 2a fbp2 is the least prognostic gene in only one cancer type cervical squamouscell carcinoma and endocervical adenocarcinoma cescfig 2a 0cchang and lai bmc cancer page of fig the landscape of somatic copy number alterations of ampk pathway genes heatmaps depict a fraction of samples within each cancertype that harbor somatic deletions and b somatic amplifications fortynine genes are recurrently deleted in at least of tumors within eachcancer and in at least seven cancer types fortysix genes are recurrently amplified in at least of tumors within each cancer and in at leastseven cancer types stacked bar charts on the yaxes illustrate the fraction of samples that possess copy number variation of a gene underconsideration grouped by shallow and deep deletions or amplifications stacked bar charts on the xaxes illustrate the fraction of samples withineach cancer type that contain shallow and deep deletions or amplifications the bar charts on the right of each heatmap depict the number ofcancer types with at least of samples affected by gene deletions and amplifications the venn diagrams demonstrate the identification of putative loss and seven gainoffunction genes from gene sets that are somatically altered and differentially expressed cancer cohorts analyzedwith corresponding tcga abbreviations are listed in parentheses bladder urothelial carcinoma blca breast invasive carcinoma brca cervicalsquamous cell carcinoma and endocervical adenocarcinoma cesc cholangiocarcinoma chol colon adenocarcinoma coad esophagealcarcinoma esca glioblastoma multiforme gbm glioma gbmlgg head and neck squamous cell carcinoma hnsc kidney chromophobekich pankidney cohort kipan kidney renal clear cell carcinoma kirc kidney renal papillary cell carcinoma kirp liver hepatocellularcarcinoma lihc lung adenocarcinoma luad lung squamous cell carcinoma lusc pancreatic adenocarcinoma paad sarcoma sarcstomach adenocarcinoma stad stomach and esophageal carcinoma stes and uterine corpus endometrial carcinoma ucec number ofsamples for each cancer type are indicated in parentheses blca brca cesc chol coad esca gbm gbmlgg hnsc kich kipan kirc kirp lihc luad lusc paad sarc stad stes and ucec given the prevalence of lossoffunction phenotypes indetermining clinical outcomes fig 2a we proceeded toexamine the combined impact of all lossoffunctiongenes on patient survival and oncogenic dysregulationto determine the extent of ampk pathway variationacross the cancers we calculated pathway scores foreach of the tumor samples by taking the meantranscript expression values of the genes slc2a4foxo3 ppp2cb pik3cd cab39l ccna1 fbp1fbp2 foxo1 hmgcr irs2 pik3r1 sirt1 tbc1d1ppargc1a ppp2r2c mlycd pfkfb3 ppp2r2bprkaa2 lepr cab39 irs1 and pfkfb1 we observedinteresting patterns when cancers were ranked from lowto high based on their median pathway scores fig 2bgbmlgg had the highest median pathway score whileblca and cesc were found at the lower end of thespectrum fig 2b as expected kaplanmeier analysisrevealed a significant difference in overall survival between glioma patients p stratified by low andhigh 24gene pathway scores fig 2c interestingly thecontribution of ampk signaling in cancer prognostication is cancertype dependent as in gliomalogranktests revealed that patients with high 24gene scores hadsignificantly improved survival outcomes in breast cancer p and sarcoma p fig 2c incontrast high expression of the genes was associated 0cchang and lai bmc cancer page of fig prognostic significance of ampk loss and gainoffunction genes a heatmap illustrates significant hazard ratio values from coxproportional hazards regression analyses on the lossoffunction and seven gainoffunction genes across all cancers b the distributions of ampkgene scores in each cancer are illustrated in the boxplot cancers are sorted from low to high median scores refer to fig legend forcancer abbreviations c kaplanmeier analyses and logrank tests revealed the prognostic significance of the 24ampkgene set in four cancertypes patients are stratified into q1 1st quartile and q4 4th quartile groups based on their 24gene scores for logrank tests dmultidimensional scaling analyses of the 24gene set depicted in 2dimensional space significance differences in the distribution between tumorand nontumor samples are confirmed by permanovawith increased mortality rates in stomach adenocarcinoma p fig 2c these results were in agreement when independently validated using the coxregression approach breast hazard ratio [hr] p glioma hr p sarcomahr p and stomach hr p cancers additionalfile since the 24genescore could be used to stratify patients into high andlowrisk groups we predict that when considered together gene expression values could discriminate tumorfrom nontumor samples although analysis could notbe performed on sarcoma this dataset only had twonontumor samples multidimensional scaling analysesand permanova tests of breast p gliomap and stomach p cancers revealed significantseparation between tumor and nontumor 0cchang and lai bmc cancer page of samples in twodimensional space fig 2d overall thissuggests that the 24gene set could be harnessed as adiagnostic biomarker for early cancer detectionconsistent with our previous observation that highpathway scores were associated with good prognosis insarcoma fig 2cto determine the independence of the 24gene setfrom other clinicopathologicalfeatures we employedmultivariate cox regression and observed that the gene set is independent of tumor node and metastasistnm staging where available in breast hr p and stomach cancers hr p additional file similarly kaplanmeier analyses andlogrank tests confirmed that the 24gene set allowedfurther risk stratification of patients with tumors of thesame tnm stage breast p and stomach p fig 3a furthermore we observed that within ahistological subtype of sarcomaleiomyosarcoma patients with elevated ampk signaling had significantlybetter3asurvival outcomesp figexploredthepredictivewe nextperformancesensitivity versus specificity of the 24gene set in allfour cancer types using receiver operating characteristicanalysis the area under the roc curve auc is an indication of how well the gene set could predict patientsurvival which ranges from to we found that thecombined model uniting both 24gene set and tnm staging outperformed the 24gene set when considered onits own in breast cancer patients auc vs fig 3b for stomach cancer the 24gene set onlycontributed to a marginally higher auc when used incombination with tnm staging when compared to the24gene set alone auc vs fig 3baucs of the 24gene set in glioma and sarcoma werefig the 24ampkgene set is independent of tumor stage and histological subtype a kaplanmeier analyses of patients grouped by tumornode and metastasis tnm stage breast and stomach cancers or by the histological subtype of leiomyosarcoma and the 24gene score forleiomyosarcoma the logrank test reveals a significant difference in survival rates between 1st and 4th quartile patients b receiveroperatingcharacteristic roc analyses on the 5year predictive performance of the 24gene set roc curves generated by the 24gene set are compared tocurves generated from both 24gene set and tnm staging where available or histological subtype auc area under the curve 0cchang and lai bmc cancer page of and respectively fig 3b within the leiomyosarcoma histological subtype auc was even higherat fig 3boncogenic transcriptional alterations associated withampk pathway inactivationampk pathway inactivation was associated with alteredsurvival outcomes in patients figs and we predictthat this could be due to broad transcriptional dysregulation arising from abnormal ampk signaling to investigate this phenomenon we performed differentialexpression analyses between patients stratified by the24gene set into high 4th quartile and low 1st quartileexpression groups and found that an outstanding number of common genes that were significantly differentially expressed in all four cancer types fig 4a thehighest number of differentially expressed genes degswas observed in stomach cancer genes followedby sarcoma genes glioma genes and breastcancer genes fig 4a additionalfile thedegs were mapped to kegg gene ontology andreactome databases to determine whether they were associated with any functionally enriched pathways intriguingly all four cancer types share similar patterns offunctional enrichments fig 4b and c for instance biological processes associated with cell communicationsignal transduction cell differentiation cell signalingcell adhesion and cell morphogenesis were enriched inall four cancers fig 4c in terms of specific signalingpathways calcium signaling camp signaling and processes associated with extracellular matrix anizationwere among the most enriched fig 4cto further identify potential transcriptional regulatorsof the degs we mapped the degs to encode andchea transcription factor tf binding databases remarkably we identified common tfs shared across allfour cancers that were implicated as direct binding partners of the degs fig 4c five tfs suz12 smad4rest ezh2 and nfe2l2 were found to be enriched inall four cancers suggesting that transcriptional dysregulation of tumors with aberrant ampk signaling involveddirect physical associations of these tfs with targetdegs fig 4c curiously foxm1 and e2f4 wereenriched only in glioma tumors which deserves furtherexploration in the next section overall our analysesdemonstrated that impaired ampk signaling resulted incommon patterns of oncogenesis which affect the severity of cancer and consequently mortality ratesinpatientsdownstream targets of ezh2 nfe2l2 rest smad4 andsuz12 were associated with survival outcomespathways modulating energy homeostatic may transducesignals to influence other cognate signaling modulesezh2 nfe2l2 rest smad4 and suz12 were all implicated as common transcriptional regulators of degsin glioma sarcoma breast and stomach cancers suggesting that altered ampk signaling converged on similargroups of transcriptional targets of all the target degsof the aforementioned tfs and geneswere found to be common targets of ezh2 nfe2l2rest smad4 and suz12 respectively in all four cancers fig 5a concatenating all five gene sets yielded unique genesie genes that were binding targets ofmore than one tf were considered only once to gainfurther insights into how ampk inactivation influencestumor progression we performed cox regression analyses to determine the association between each of the genes and survival outcomes the highest number ofprognostic genes was observed in glioma genes good prognoses and five adverse prognoses fig 5b incontrast out of genes were associated with adverseprognosis in stomach cancer fig 5b these observations were consistent with the 24ampkgene set beingpositive and negative prognostic factors in glioma andstomach cancer respectively fig which mirrored thebehavior of degs identified as a result of aberrantampk signaling fig 4c of the genes only andten were significantly associated with survival outcomesin sarcoma and breast cancer respectively fig 5b collectively our results suggest that the ampk pathwayand its interaction with other signaling modules are keydeterminants of patient outcomes in multiple cancertypesprognostic significance of joint ampk pathway activityand transcriptional levels of five oncogenic tfs inpatients with gliomahaving discovered the importance of the 24ampk geneset we sought to explore the crosstalk between ampksignaling and tf activity in glioma as previously mentioned glioma had the highest 24ampkgene scorefig 2b with a vast majority of the genes conferringprognostic information fig 2a moreover of the transcriptional targets of the five common tfs identifiedin patients with altered ampk signaling were significantly associated with survival outcomes in glioma fig5b additionally tfs foxm1 and e2f4 were identifiedto be enriched only in glioma tumors fig 4c thus wepredict that a joint model uniting ampk and tf expression profiles would allow further delineation of patientsinto additional risk groups and if so allowing combinedtargeting of ampk and candidate tfs for therapeuticaction as done previously we calculated ampk scoresfor each patient based on the mean expression of the genes interestingly we found that ampk scores weresignificantly negatively correlated with tf expressionlevels in glioma e2f4 rho p ezh2 0cchang and lai bmc cancer page of fig see legend on next page 0cchang and lai bmc cancer page of see figure on previous pagefig ampk inactivation drives oncogenic transcriptional alterations in diverse biological processes and signaling modules a venn diagramillustrates the number of differentially expressed genes degs between 1st and 4th quartile patients as stratified using the 24ampkgene set infour cancer types a total of degs were common in all four cancers b dot plots depict the number of significantly enriched pathways andbiological processes upon the mapping of degs to kegg gene ontology and reactome databases each dot represents an enriched event contologies that exhibit similar patterns of enrichment across four cancers are shown degs are also mapped to encode and chea transcriptionfactor tf databases to determine enriched tf binding associated with degsrho p foxm1 rho p smad4 rho p and suz12rho p fig 6a we subsequentlycategorized patients into four groups using the mediancutoff of the ampk scores and tf expression values lowlow highhigh low ampk score and high tfexpression and high ampk score and low tf expression logrank tests revealed that patients stratified intothe four groups had survival rates that were significantlydifferent e2f4 p ezh2 p foxm1p smad4 p and suz12 p fig 6b for e2f4 ezh2 foxm1 and suz12patients with low ampk scores and high tf expressionperformed the worst e2f4 hr p ezh2 hr p foxm1 hr p and suz12 hr p fig6c for smad4 patients within the lowlow categoryhad the highest mortality rates hr p fig 6ccrosstalk between ampk and other anabolicrelatedpathways ppar and mtorampks antianabolic and procatabolic activities maywork in concert with other metabolic pathways toinvestigate the synergistic effects of ampk and two proanabolic pathways peroxisome proliferatoractivated receptors ppar and mammalian target of rapamycinmtor signaling in tumor progression we calculatedppar and mtor pathway scores detailed in themethods section for each glioma tumor low ampkscores were associated with poor outcomes in gliomafig to evaluate ampk and ppar or mtor ascombined models patients were separated into fourgroups using the median cutoff as mentioned previously interestingly when ampk and ppar scores werecollectively used for patient stratification we found thatpatients with low ampk and high ppar scores had thehighest death rates hr p confirmingthat ppar hyperactivation is associated with poor outcomes in glioma tumors with low ampk activity fig in contrast when considering mtor activitypatients with low ampk and low mtor scores performed the worst hr p fig theresults overall suggest that the ampk pathway could actsynergistically with ppar and mtor signaling to influence cancer progression significantlydiscussionwhile the role of ampk in energysensing is wellunderstood its full potential in metabolic diseases suchas cancer remains an open topic of debate despite extensive efforts spent on elucidating the role of ampksignaling [ ] there remains no consensus onwhether ampk promotes or suppresses tumor progression exploiting a rich reservoir of pancancer datasetsafforded to us by tcga we performed a thoroughexamination of genomic and transcriptomic profiles of ampk pathway genes in diverse cancer types ourcurrent understanding of ampk signaling is fueled bygenetic studies in cell lines and animal models although useful in determining causal relationships resultsfrom in vitro cell lines and animal models may have limited translational relevance as they do not accurately reflect human pathology animal models may offeradditional mechanistic insights but limitations in ethicsand costs remain moreover the complexity of humancancers is not accurately modeled in animals less than of results from animal models are translated to clinical trials despite analyses on tumor genetic datasets providing mostly correlative outcomes they remainvaluable in understanding diseasespecific molecularpathology when interrogated at scale on large patientgroups [] and when results are considered in relation to those obtained from celllines and animalmodelsemploying pancancer population data our studyidentified conserved and unique patterns of ampk signaling across diverse cancer types analyses at two molecular levels genetic and transcriptional yielded amore comprehensive depiction of ampk signalingwhere we identified genes that were both somatically altered and differentially expressed these putative lossor gainoffunction genes are more likely to impacttumor progression as they are altered at both macromolecular levels as reported in other studies we confirmedthat ampk signaling could either be oncogenic ortumor suppressive depending on the cellular context intuitively since ampk is antianabolic its function maynot be fitting for tumor growth and proliferation this isconsistent with reports demonstrating ampks tumorsuppressive activity [ ] a study on lymphoma demonstratesthewarburg effect and hypoxia signaling in mice that ampk downregulation induces 0cchang and lai bmc cancer page of fig see legend on next page 0cchang and lai bmc cancer page of see figure on previous pagefig prognostic significance of degs targeted by enriched tfs a venn diagrams illustrate the extent of overlap between degs targeted byezh2 nfe2l2 rest smad4 and suz12 across four cancers b forest plots depict degs that are significantly associated with overall survivaloutcomes hazard ratios are denoted as purple squares while pink bars represent the confidence intervals significant wald test p values areindicated in blueits loss ofampk is proposed to act as a metabolic gatekeeper tolimit cancer cell division hencefunctionwould contribute to tumor aggression because of theloss in metabolic checkpoints [ ] ampk regulatesthe tumorsuppressive function of the serinethreoninekinase lkb1 ablation of lkb1 results in enhanced riskof developing gastrointestinal lung and skin squamouscell cancers [ ] moreover ampk is shown to inhibit pi3kaktmtor signaling which is activated inmany cancers [ ] also metabolic inhibitors such asmetformin which indirectly activates ampk could suppress tumor growth via autophagy induction and mtorinhibition [ ] metformin is shown to inhibit theproliferation of estrogen receptor α erα negative andpositive breast cancer cell lines through ampk stimulation however when tested in mice models metformin contributes to enhanced tumor progression andincreased angiogenesis providing us with a glimpse ofpotential proneoplastic effects of ampk activation in our study we observed that high levels of ampkpathway activity were associated with better outcomes inglioma breast cancer and sarcoma fig corroboratingprevious results on the tumorsuppressive function ofthe opposite is true in stomachampk converselyfig prognostic relevance of candidate tfs and the 24ampkgene set in glioma a scatter plots illustrate significant negative correlationsbetween ampk scores and tf expression levels in glioma patients are separated and colorcoded into four categories based on median ampkand tf scores density plots appended to the y and xaxes demonstrate the distribution of ampk and tf scores b logrank tests are performedon the four patient grou | Colon_Cancer |
"adipogenesis is the process through which mesenchymalstem cells mscs commit to the adipose lineage and diï¬erentiate into adipocytes during this process preadipocytescease to proliferate begin to accumulate lipid droplets anddevelop morphologic and biochemical characteristics ofmature adipocytes such as hormoneresponsive lipogenesisand lipolytic programs currently there are mainly twomodels of benign adipocyte diï¬erentiation in vitro one isï¬broid pluripotent stem cells which can diï¬erentiate intonot only adipocytes but also muscle cartilage and othercells there are two kinds of ï¬broid pluripotent stem cellsbone marrow and adipose mesenchymal stem cells anothergroup is ï¬broblastic preadipocytes which have a single direction of diï¬erentiation namely lipid diï¬erentiation including3t3l1 and 3t3f422a cells cancer cells with tumorinitiation ability designated as cancer stem cells cscshave the characteristics of tumorigenesis and the expressionof speciï¬c stem cell markers as well as the longterm selfrenewal proliferation capacity and adipose diï¬erentiationpotential in addition to cscs cancer cells undergoing epithelialmesenchymaltransformation emt havebeen reported to be induced to diï¬erentiate into adipocytes[] lung cancer ncih446 cells can be induced to differentiate into neurons adipocytes and bone cells in vitro the adipogenesis diï¬erentiation treatment is promisingin the p53 gene deletion type of ï¬broblastderived cancer cancer cells with homologous recombination defectssuch as ovarian and breast cancer cells with breast cancersusceptibility genes brca mutations can be inducedto diï¬erentiate by poly adpribose polymerase parp 0cstem cells internationalinhibitors the nuclear receptor peroxisome proliferatoractivated receptor Î pparÎ agonist antidiabetic thiazolidinedione drug can induce growth arrest and adipogenicdiï¬erentiation in human mouse and dog osteosarcoma cells thyroid cancer cells expressing the pparÎ fusion proteinppfp can be induced to diï¬erentiate into adipocytes bypioglitazone adipogenesis can be induced in welldiï¬erentiated liposarcoma wdlps and dediï¬erentiatedliposarcoma ddlps cells by dexamethasone indomethacininsulin and 3isobutyl1methyl xanthine ibmx in this review we highlight some of the crucial transcription factors that induce adipogenesis both in mscs and inincluding the wellstudied pparÎ and ccaatcscsenhancerbinding proteins cebps as well as othercell factors that have been recently shown to have an important role in adipocyte diï¬erentiation we focus on understanding the complex regulatory mechanism of adipocytediï¬erentiation that can contribute to the clinical treatmentof human diseases including those caused by obesity andadipocytes dysfunction especially for the malignant tumorwhich can be transdiï¬erentiated into mature adipocytes adipocyte differentiationcell proliferation and diï¬erentiation are two opposingprocesses and there is a transition between these two processes in the early stages of adipocyte diï¬erentiation theinteraction of cell cycle regulators and diï¬erentiation factors produces a cascade of events which ultimately resultsin the expression of adipocyte phenotype adipogenesishas diï¬erent stages each stage has a speciï¬c gene expression pattern in general adipocyte diï¬erentiation ofpluripotent stem cells is divided into two phases the ï¬rstphase known as determination involves the commitment ofpluripotent stem cells to preadipocytes the preadipocytescannot be distinguished morphologically from their precursor cells but also have lost the potential to diï¬erentiate intoother cell types in the second phase which is known asterminal diï¬erentiation the preadipocytes gradually acquirethe characteristics of mature adipocytes and acquire physiological functions including lipid transport and synthesisinsulin sensitivity and the secretion of adipocytespeciï¬cproteins the diï¬erentiation of precursor adipocytes is also dividedinto four stages proliferation mitotic cloning early diï¬erentiation and terminal diï¬erentiation after the precursors are inoculated into the cell culture plates the cellsgrow exponentially until they converge after reaching contact inhibition the growth rate slows and gradually stagnatesand the proliferation of precursor adipocytes stops which isvery necessary for initiating the diï¬erentiation of precursoradipocytes adipocyte precursors exhibit transient mitosiscalled clonal expansion a process that relies on the actionof induced diï¬erentiation factors some preadipocyte cellsmouse cell lines 3t3l1 3t3f442a undergo one or tworounds of cell division prior to diï¬erentiation whereasother cell lines mouse c3h10t12 diï¬erentiating into adipocyte do not undergo mitosis clonal expansion whether mitotic clonal expansion is required for adiposediï¬erentiation remains controversial however it is certainthat some of the checkpoint proteins for mitosis regulateaspects of adipogenesis [ ] when cells enter the terminaldiï¬erentiation stage the de novo synthesis of fatty acidsincreases significantly the transcription factors and adipocyterelated genes work cooperatively to maintain precursor adipocyte diï¬erentiation into mature adipocytes containing largelipid droplets regulatory pathways inpreadipocytes commitmentadipocyte diï¬erentiation is a complex process in which geneexpression is ï¬nely regulated the most basic regulatory network of adipose diï¬erentiation has not been updated inrecent years but some factors and signaling pathways thatdo aï¬ect adipose diï¬erentiation have been continuouslyreported adipocyte diï¬erentiation is the result of the geneexpression that determines the phenotype of adipocyteswhich is a complex and delicate regulatory process figure wnt signal pathway in adipogenesis wnt signaling isimportant for adipocytes proliferation and diï¬erentiationboth in vitro and in vivo the wnt family of secretedglycoproteins functions through paracrine and autocrinemechanisms to uence cell fate and development wntprotein binding to frizzled receptors initiates signalingthrough catenindependent and independent pathways wnt signaling inhibits adipocyte diï¬erentiation in vitroby blocking the expression of pparÎ and cebpα constitutive wnt10b expression inhibits adipogenesis wnt10b isexpressed in preadipocytes and stromal vascular cells butnot in adipocytes in vivo transgenic expression of wnt10bin adipocytes results in a reduction in white adipose tissuemass and absent brown adipose tissue development wnt10a and wnt6 have also been identiï¬ed as determinantsof brown adipocyte development [ ] wnt5b is transiently induced during adipogenesis and promotes diï¬erentiation indicating that preadipocytes integrate inputs fromseveral competing wnt signals the hedgehog hh signaling pathway mechanismthree vertebrate hh ligands including sonic hedgehogshhindian hedgehog ihh and desert hedgehogdhh have been identiï¬ed and initiated a signaling cascademediated by patched ptch1 and ptch2 receptors [ ]hh signaling had an inhibitory eï¬ect on adipogenesis inmurine cells such as c3h10t12 ks483 calvaria mscslines and mouse adiposederived stromal cells thesecells were visualized by decreased cytoplasmic fat accumulation and the expression of adipocyte marker genes after hhsignaling was inhibited although it is generally agreedthat hh expression has an inhibitory eï¬ect on preadipocytediï¬erentiation the mechanisms linking hh signaling andadipogenesis remain poorly deï¬ned erkmapkppar signal pathway extracellularregulated protein kinase erk is required in the proliferativephase of diï¬erentiation erk activity blockade in 3t3l1 0cstem cells internationaldex insulin demxwnt 10band othersshhpbc smotgfð½p smad3 smad3testosteroneð½catentinarirspi3kaktcrebpkapcrebfoxo1a2tcflef gata23cebpð½mapkg3k3ð½p2cebpð½cebpαppará½»bmpssmad1srebpadipocytegenesfigure regulation pathways in preadipocytes commitment bmp and wnt families are mediators of mscs commitment to producepreadipocytes exposure of growtharrested preadipocytes to diï¬erentiation inducers igf1 glucocorticoid and camp triggers dnareplication leading to adipocyte gene expression due to a transcription factor cascade the dotted line indicates an uncertain molecularregulatory mechanismcells and embryonic stem cells can inhibit adipogenesis inthe terminal diï¬erentiation phase erk1 activity leads topparÎ phosphorylation which inhibits adipocyte diï¬erentiation this implies that erk1 activity must be reduced afteradipocyte proliferation so that diï¬erentiation can proceedthis reduction is mediated in part by mitogenactivatedprotein kinase mapk phosphatase1 mkp1 [ ]these extracellular and intracellular regulation factors causeadipocytespeciï¬c gene expression and eventually lead toadipocyte formation adipocyte differentiationregulatory proteins pparÎ and adipocyte diï¬erentiation pparÎ is a member of the nuclearreceptor superfamily and is both necessaryand suï¬cient for adipogenesis forced expression ofpparÎ is suï¬cient to induce adipocyte diï¬erentiation broblasts indeedthe proadipogenic cebps andkrüppellike factors klfs have all been shown to induceat least one of the two pparÎ promoters in contrast antiadipogenic transcription factor gata functioned in part byrepressing pparÎ expression pparÎ itself has twoisomers the relative roles of pparÎ1 and pparÎ2 in adipogenesis remain an open question pparÎ2 is mainlyexpressed in adipose tissue while pparÎ1 is expressed inmany other tissues although both can promote adipocytediï¬erentiation pparÎ2 could do so eï¬ectively at very lowligand concentration compared with pparÎ1 the twoprotein isoforms are generated by alternative splicing andpromoter usage and both are induced during adipogenesispparÎ1 can also be expressed in cell types other than adipocytes ren et al used engineered zincï¬nger proteins tothe expression ofthe endogenous pparÎ1 andinhibitpparÎ2 promoters in 3t3l1 cells ectopic expression ofpparÎ2 promotes adipogenesis whereas that of pparÎ1does not zhang et al reported that pparÎ2 deï¬ciencyimpairs the development of adipose tissue and insulin sensitivity there are transcriptional cascades between adipocytesgenes including pparÎ and cebpα which are the coreadipocyte diï¬erentiation regulators in the early stage of adipocyte diï¬erentiation the expression of cebp and cebpδincrease which upregulates cebpα expressionfurtheractivate pparÎ pparÎ activating cebpα in turn resultsin a positive feedback pparÎ binding with retinoic acid xreceptor rxr forms diï¬erent heterodimers the variousdimmers can combine with the pparÎ response elementppre and initiate the transcription of downstream genesfor diï¬erentiation into adipocytes cebps participate in adipogenesis and several cebpfamily members are expressed in adipocytesincludingcebpα cebp cebpÎ cebpδ and cebphomologous protein chop the temporal expression of thesefactors during adipocyte diï¬erentiation triggers a cascadewhereby early induction of cebp and cebpδ leads tocebpα expression this notion is further supported by thesequential binding of these transcription factors to severaladipocyte promoters duringadipocyte diï¬erentiationcebp is crucial for adipogenesis in immortalized preadipocyte lines cebp and cebpδ promote adipogenesis atleast in part by inducing cebpα and pparÎ cebpαinduces many adipocyte genes directly and plays an important role in adipose tissue development once cebpα isexpressed its expression is maintained through autoactivation despite the importance of cebps in adipogenesis 0cstem cells internationalthese transcription factors clearly cannot function eï¬cientlyin the absence of pparÎ cebp cannot induce cebpαexpression in the absence of pparÎ which is required torelease histone deacetylase1 hdac1 from the cebpαpromoter furthermore ectopic cebpα expressioncannot induce adipogenesis in pparÎï¬broblasts however cebpα also plays an important role in diï¬erentiated adipocytes overexpression of exogenous pparÎ incebpαdeï¬cient cells showed that although cebpα isnot required for lipid accumulation and the expression ofmany adipocyte genes it is necessary for the acquisition ofinsulin sensitivity [ ] figure human ï¬broblasts withthe ability to diï¬erentiate into adipocytes also do not undergomitotic cloning ampliï¬cation however pparÎ exogenousligands need to be added to promote adipocyte diï¬erentiation therefore it can be inferred that mitotic cloning expansion can produce endogenous ligands of pparÎ bmp and transforming growth factor tgf inadipocyte diï¬erentiation a variety of extracellular factorsaï¬ect the preadipocyte commitment of stem cells includingbone morphogenetic protein bmp transforminggrowth factor tgf insulininsulinlike growthfactor igf1 tumor necrosis factor α and interleukin matrix metalloproteinase ï¬broblast growthfactor fgf and fgf2 bmp and tgf have variedeï¬ects on the diï¬erentiation fate of mesenchymal cells the tgf superfamily members bmps and myostatinregulate the diï¬erentiation of many cell types includingadipocytes tgf inhibitor can promote adipose diï¬erentiation of cancer cells with a mesenchymal phenotypein vitro and transgenic overexpression of tgf impairsadipocyte development inhibition of adipogenesis couldbe obtained through blocking of endogenous tgf with adominantnegative tgf receptor or drosophila mothersagainst decapentaplegic protein smad inhibitionsmad3 binds to cebps and inhibits their transcriptionalactivity including their ability to transactivate the pparÎ2promoter [ ] exposure of multipotent mesenchymalcells to bmp4 commits these cells to the adipocyte lineageallowing them to undergo adipose conversion theeï¬ects of bmp2 are more complex and depend on the presence of other signaling molecules bmp2 alone has little eï¬ecton adipogenesis and it interacts with other factors such astgf and insulin to stimulate adipogenesis of embryonicstem cells bmp2 stimulates adipogenesis of multipotentc3h10t12 cells at low concentrations and can contribute tochondrocyte and osteoblast development at higher concentrations klfs in adipocyte diï¬erentiation during adipocyte differentiation some klf family members are overexpressedsuch as klf4 klf5 klf9 and klf15 while klf16 expression is reduced [ ] klf15 is the ï¬rst klf family members which were identiï¬ed to be involved in adipocytediï¬erentiation its expression increased significantly on thesixth day of 3t3l1 adipocyte diï¬erentiation and peakedon the second day of adipocyte induction in mscs andmouse embryonic ï¬broblasts inhibition of klf15 by sirnaor mutation led to a decrease in pparÎ cebpα fatty acidbinding protein fabp4 and glucose transporter glut4 however overexpression of klf15 in nih3t3cells was found to be associated with lipid accumulation aswell as increases in pparÎ and fabp4 mice with complete absence of klf5 showed embryonal lethality and micewith singlechromosome klf5 knockout showed a significant reduction in white fat in adulthood suggesting thatklf5 plays an important role in adipocyte diï¬erentiationklf5 can be activated by cebp or cebpδ which isinvolved in early adipocyte diï¬erentiation klf5 can beactivated by cebp or cebpδ which is involved in earlyadipocyte diï¬erentiation direct binding of klf5 to thepparÎ2 promoter in combination with cebps inducespparÎ2 expression transfection of klf5 dominantnegative mutants in 3t3l1 cells reduced lipid droplet accumulation and inhibited pparÎ and cebpα expressionwhereas overexpression of wild klf5 significantly promotedadipocyte diï¬erentiation even without exogenous hormonestimulation similar to klf5 klf9 knockdown can inhibitthe expression of a series of adipocyte diï¬erentiation genessuch as pparÎ cebpα and fabp4 hence inhibitingadipocyte diï¬erentiation however klf9 overexpressiondid not upregulate the expression of pparÎ and cebpα in addition klf4 can transactivate cebp by bindingto the region of kb upstream of the cebp promoter and promote lipid diï¬erentiation klf6 can forma complex with histone deacetylase3 hdac3 inhibitingpreadipocyte factor1 pref1 expression and promotinglipid diï¬erentiation klf2 is highly expressed in adiposeprogenitors and its expression decreases during the processof lipid diï¬erentiation overexpressed klf2 can bind to thecaccc region of pparÎ2 proximal promoter and inhibitlipid diï¬erentiation as well as the expression of pparÎcebpα and sterolregulated elementbinding proteinssrebp by inhibiting the promoter activity rnasequence analysisshowed that klfl6 expression wasdecreased on the ï¬rst day of adipocyte diï¬erentiation of3t3l1 cells adipocyte diï¬erentiation was promoted byklf16 knockdown but was inhibited by klf16 overexpression via inhibition of pparÎ promoter activity in addition klf3 and klf7 were also found to play a negativeregulatory role in adipocyte diï¬erentiation [ ] signal transducers and activators of transcriptionstats and adipocyte diï¬erentiation the activated statprotein enters the nucleus as a dimer and binds to the targetgene to regulate gene transcription in the adipocyte diï¬erentiation of mouse 3t3l1 cells the expression of stat1 andstat5 was significantly increased while that of stat3and stat6 was not significantly changed in the adipocyte diï¬erentiation of human subcutaneous adipose precursor cells stat1 expression was significantly decreased while the expression of stat3 and stat5 wasincreased and stat6 expression was unchanged therole of stat1 in adipocyte diï¬erentiation is not clearbecause its expression trend in humans and mice diï¬ersduring the adipocyte diï¬erentiation process early adipocytediï¬erentiation of 3t3l1 cells was inhibited by stat1 0cstem cells internationalklf5srebp1cklf15klf2chopcebpá½»krox20ligandcebpð½cebpð¿gata23ppará½»cebpð¼proadipogenicantiadipogenicgenes of terminaladipocytedifferentiationfigure a cascade of transcription factors that regulate adipogenesis pparÎ is one of the key transcription factors in adipogenesis and thecore of the transcriptional cascade that regulates adipogenesis pparÎ expression is regulated by several proadipogenic blue andantiadipogenic red factors cebpα is regulated through a series of inhibitory proteinprotein interactions some transcription factorfamilies include several members that participate in adipogenesis such as the klfs black lines indicate eï¬ects on gene expression violetlines represent eï¬ects on protein activityagonist interferon Î loss of stat1 in 3t3l1 cells can rescue the inhibition of adipocyte diï¬erentiation caused byprostaglandin factor 2α other studies have found thatstat1 is required for adipose diï¬erentiation and stat1overexpression in c3h10t12 cells can prevent the inhibition of lipid diï¬erentiation caused by bcell lymphoma6knockdown there was no abnormal adipose tissuein stat1 knockout mice stat3 not only aï¬ectsthe proliferation of 3t3l1 cells but also coregulates theiradipocyte diï¬erentiation with high mobility group protein the fabp4 promoter was used to speciï¬callyknock out stat3 in the adipose tissue of mice and theresults showed that mice weight significantly increasedand the adipocyte quantity increased compared with thewildtype mice stat5a and stat5b have diï¬erenteï¬ects on adipocyte diï¬erentiation abnormal adipose tissuewas found in the mice with stat5a or stat5b knockout ordouble knockout and the amount of adipose tissue was onlyoneï¬fth of the original adipose tissue in mice withoutknockdown histone modiï¬cation in adipocyte diï¬erentiation histone deacetylase sirtuin sirt plays an important rolein biological processes such as stress tolerance energymetabolism and cell diï¬erentiation during the adipocyte diï¬erentiation of c3h1012 cells sirt1 expressiondecreased overexpression of sirt1 activated thewnt signal which caused the deacetylation of cateninthe accumulation of catenin in the nucleus could inhibitadipocyte diï¬erentiation sirt1 knockdown resulted inincreased acetylation of the histones h3k9 and h4k16 inthe secreted frizzledrelated protein sfrp and sfrp2 promoters thereby promoting transcription of these genes andpromoting lipid diï¬erentiation forkhead box proteino foxo is a member of the transcription factor foxofamily it can recruit cyclic amp response elementbindingprotein cbphistone acetyltransferase p300 to initiate anacetylation the acetylated foxo1 can be phosphorylatedby phosphorylated protein kinase b pkbakt the phosphorylation of foxo1 by akt inhibits the transcriptionalactivation of foxo1 the acetylation of foxo1 lost the ability of dnabinding aï¬nity and promoted its shuttling fromnuclei to cytoplasm sirt1 and sirt2 can deacetylateand active foxo1 activated foxo1 nonphosphorylatednuclear foxo1 in the nucleus binds to the promoters of target genes encoding p21 p27 and pparÎ and initiates subsequent transcriptions sirt2 inhibits the acetylation andphosphorylation of foxo1 thereby induces the accumulation of activated foxo1 in the nucleus activated foxo1could inhibit adipogenesis via pparÎ [] lysinespeciï¬c histone demethylase lsd1 expression increasedduring the adipocyte diï¬erentiation of 3t3l1 cells lsd1could reduce the dimethylation levels of histone h3k9 andh3k4 in the cebpα promoter region thereby promotingadipocyte diï¬erentiation set domaincontaining setd8 catalyzed the monomethylation of h4k20 andpromoted pparÎ expression the activation of pparÎ transcriptional activity leads to the induction of monomethylatedh4k20 and modiï¬cation of pparÎ and its targets therebypromoting adipogenesis enhancer of zeste homolog ezh2 is a methyltransferase and can bind methyl groupsto histone h3k27 which is also necessary for lipid diï¬erentiation the absence of ezh2 in brown fat precursors results inreduced levels of the wnt promoter histone h3k27me3which is also saved by the ectopic ezh2 expression or theuse of a wntcatenin signal inhibitor in addition histone demethylases such as lysinespeciï¬c histone demethylase lsdkdm kdm6 and histone lysine demethylasephf2 are also involved in adipose diï¬erentiation andkdm2b inhibits transcription factor activator protein 2αpromoter via h3k4me3 and h3k36me2 role of microrna and long noncodingrna in adipogenesismicrorna mir can bind and cut target genes or inhibittarget gene translation endogenous sirna can be producedby the action of dicer enzyme and bind to a speciï¬c proteinto change its cellular location many kinds of mirsare involved in regulating adipocyte diï¬erentiation the 0cstem cells internationalexpression of mir143 increased during the diï¬erentiationof adipose progenitor cells overexpression of mir143promoted gene expression involved in adipose diï¬erentiationand triglyceride accumulation inhibition of mir143 prevented the adipose diï¬erentiation of human fat progenitorcells [ ] additionally mir8 promotes adipocyte diï¬erentiation by inhibiting wnt signaling moreover mir mir103 mir21 mir519d mir210 mir30mir204211 and mir375 also play a certain role in promoting adipocyte diï¬erentiation while mir130 mir448and let7y inhibit lipid diï¬erentiation [ ] in additionto mirs long noncoding rna lncrna is a type of noncoding rna and is important during epigenetic regulationand can form a doublestranded rna complex with mrnacauses protein transcription lncu90926 inhibits adipocytediï¬erentiation by inhibiting the transactivation of pparÎ2 as a novel lncrna hoxaas3 expression increasedduring the adipose diï¬erentiation of mscs and hoxaas3 silencing reduced the marker gene of adipose diï¬erentiation and inhibited the adipose diï¬erentiation zhu et al reported that hoxaas3 interacted with ezh2 toregulate lineage commitment of mscs hoxa as3 canregulate the trimethylation level of h3k27 in the runx2promoter region by binding to ezh2 therefore hoxaas3 is considered to be an epigenetic switch regulating mscslineage speciï¬city adipocyte diï¬erentiationassociatedlncrna can act as a competitive endogenous rna of mir in the process of lipid diï¬erentiation thereby promotingthe expression of sirt1 the target gene of mir204 and thusinhibiting lipid diï¬erentiation the lncrna neat1can also regulate adipocyte diï¬erentiation under the uence of mirna140 other lncrna including lncrnablnc1 and plnc are also involved in regulating adipocytediï¬erentiation [ ] other biochemical response involved inadipocyte differentiation unfolded protein responses in adipocyte diï¬erentiationin the endoplasmic reticulum of eukaryotes unfolded protein response involves three proteinsinositolrequiringenzyme 1α doublestranded rnadependent proteinkinaselike er kinase and activating transcription factoratf 6α knockdown of atf6α aï¬ects the expressionof adipocytes genes and inhibits c3h10t12 adipocyte differentiation the inhibitory eï¬ect of berberine on adipocyte diï¬erentiation of 3t3l1 cells is also due to inducedchop and decorin expressions and this inhibitory eï¬ectis ameliorated by chop knockout in the adipocytediï¬erentiation process of 3t3l1 cells increases in pparÎand cebpα as markers of adipocyte diï¬erentiation wereaccompanied by an increase in the corresponding proteinexpressions of phosphorylated eukaryotic translation initiaeif 2α phosphorylated endoribonucleasetion factorire1α atf4 chop and other unfolded protein responsesendoplasmic reticulum stress inducer or hypoxic endoplasmic reticulum stress can inhibit adipocyte diï¬erentiationadditionally eif2α mutation results in continuous activation or overexpression of chop which also inhibits adipocyte diï¬erentiation after the initiation of adiposediï¬erentiation numerous diï¬erentiationassociated proteinsare synthesized exogenous endoplasmic reticulum stressinducers can lead to excessive endoplasmic reticulumresponse which in turn aï¬ects the synthesis of proteinsrelated to diï¬erentiation and inhibits adipocyte formationfigure role of oxidative stress in adipogenesis during thedirectional diï¬erentiation of mscs mitochondrial complexi and iii and nadph oxidase nox4 are the main sourcesof oxygen species ros production currently it is believedthat ros aï¬ects not only the cell cycle and apoptosis but alsodiï¬erentiation through uencing the signaling pathwaysincluding the wnt hh and foxo signaling cascade duringmscs diï¬erentiation the diï¬erentiation ability ofstem cells is determined by the arrangement of perinuclearmitochondria which speciï¬cally manifests as low atpcellcontents and a high rate of oxygen consumption the lackof these characteristics indicates stem cell diï¬erentiation adipocyte diï¬erentiation is a highly dependent rosactivation factor related to mitosis and cell maturation schroder et al found that exogenous h2o2 could stimulate adipocyte diï¬erentiation of mouse 3t3l1 cells andhuman adipocyte progenitor cells in the absence of insulinh2o2 regulates adipocyte diï¬erentiation of 3t3l1 cells ina dosedependent manner high doses of h2o2 and μm promote adipocyte diï¬erentiation [ ] tormos et al found that ros synthesis increased in humanmscs at the early stage of adipose diï¬erentiation and targeted antioxidants could inhibit lipid diï¬erentiation byknocking down rieske ironsulfur protein and ubiquinonebinding protein ros produced by mitochondrial complexiii was found to be necessary in initiating adipose diï¬erentiation however other studies have shown that theexpression levels of adiponectin and pparÎ were decreasedby using h2o2 mm in 3t3l1 cells free radical nitric oxide no also promotes lipid diï¬erentiationbecause treatment with no inducer hydroxylamine or nosynthase nos substrate arginine can significantly induceadipose diï¬erentiation of rat adipose progenitor cells nosinduced adipose diï¬erentiation mainly via enos rather thaninos ros can induce adipose diï¬erentiation primarily by inhibiting wnt foxo and hh signaling pathwaysthat inhibit lipid diï¬erentiation autophagy in adipocyte diï¬erentiation the increase inautophagosomes during lipid diï¬erentiation indicates thatautophagy may play an important role in lipid diï¬erentiation baerga et al conï¬rmed that the adipocyte diï¬erentiation eï¬ciency was significantly inhibited in mouse embryonic ï¬broblasts lacking autophagyrelated gene atg agene encoding an essential protein required for autophagy knockdown of atg5 in 3t3l1 cells promotesproteasomedependent degradation of pparÎ2therebyinhibiting adipocyte diï¬erentiation zhang reportedthat autophagyrelated gene 7atg7 is also crucial for adipose development atg7deï¬cient mice were slim and onlyhad of white fat compared to wildtype mice and the 0cstem cells internationalcebpð½ geneebf1 geneklf4egr2cebpð½cytosolcebpð¿ genecebpð¿klf5geneppará½» geneklf5nr2f2nfkb11433relasrebf1a2rxrappará½»ppará½»rxra heterodimerppará½»rxracorepressor complexfabp4ligands of ppará½»fam120bthrap3ep300ncoa2ncoa3helz2ncoa1crebbpebf1adipoq geneaidrfcebpð¼ geneznf638znf467cebpð¼ncor1hdac3ncor2 slc2a4 geneglut4 genelep genefabp4 genecdk4ccnd3plin1 genepck1 genefabp4cd36 geneppararxracoactivator complexppará½»fatty acidrxramediatorcoactivator complexangptlgeneppargc1amediator complex consensuslpl genenucleoplasmproteins bind to gene promoterstranscription of genes into proteinsacting on proteins compoundingtgfð½1wnt1wnt10btnf77233adipoqglut4slc2a4 tetramerlepfabp4lipid dropletplin1pck1papa pa4xpalmccd36paangptl4lplfigure regulation of adipocyte diï¬erentiation a regulatory loop exists between pparÎ and cebp activation transcription factor coeebf activates cebpα cebpα activates ebf1 and ebf1 activates pparÎ cebp and cebpδ act directly on the pparÎ gene bybinding its promoter and activating transcription cebpα cebp and cebpδ can activate the ebf1 gene and klf5 the ebf1 and klf5proteins in turn bind the promoter of pparÎ which becomes activated other hormones such as insulin can aï¬ect the expression ofpparÎ and other transcription factors such as srebp1c pparÎ can form a heterodimer with the rxrα in the absence of activatingligands the pparÎrxrα complex recruits transcription repressors such as nuclear receptor corepressor ncor ncor1 andhdac3 upon binding with activating ligands pparÎ causes a rearrangement of adjacent factors corepressors such as ncor2 are lostand coactivators such as transcription intermediary factor tif2 cbp and p300 are recruited which can result in the expression of cyclicampresponsive elementbinding protein creb followed by pparÎ pparÎ expression initiates the expression of downstream genesincluding angiopoietinrelated protein pgar perilipin fabp4 cebpα fatty acid transportrelated proteins carbohydrate metabolismrelated proteins and energy homeostasisrelated proteinslipid metabolism and hormoneinduced lipolysis in the adipocytes were altered autophagy related gene atg4b isactivated by cebp in the process of lipid diï¬erentiationand autophagy activation is necessary for the degradationof klf2 and klf3 two negative regulators of lipid diï¬erentiation these results showed that adipose diï¬erentiation andautophagy are mutually complementary in 3t3l1cells autophagy was inhibited by aspartate ammonia or 0cstem cells internationalmethyladenine at diï¬erent lipid induction periods and days and only autophagy inhibition at days hindered the formation of lipid droplets and the expression of lipid marker genes indicating that autophagy wasvery important in the early stage of lipid diï¬erentiation recent studies showed that lc3 is overexpressed in3t3l1 cells further demonstrating the important role ofautophagy in lipid diï¬erentiation role of alternative splicing in adipogenesis selectivesplicing is uenced by splicing regulators which regulateadipocyte diï¬erentiation by regulating the selective splicingof genes speciï¬c to this process lipin1 is an important regulator in the process of adipocyte diï¬erentiation and includestwo isomers lipin1α and lipin1 which have diï¬erenteï¬ects high expression of lipin1α promotes adipocyte differentiation while that of lipin1 promotes lipid droplet formation in sam68deï¬cient mice the ï¬fth intron ofserinethreonineprotein kinase mtor was retained resulting in unstable and rapid mtor degradation and inhibitionof adipocyte diï¬erentiation furthermore there arefour isomers of pref1 pref1a and pr | Colon_Cancer |
endometrial cancer ec is a common malignancy of the female reproductive system circular rnas circrnas were demonstrated to exert critical roles in cancers including ec this study aimed to investigate the effects of hsa_circrna_0001776 circ_0001776 on ecmethods realtime quantitative pcr rtqpcr was used to measure circ_0001776 microrna182 mir182 and leucinerich repeats and immunoglobulinlike domains lrig2 expression the diagnostic and prognostic values of circ_0001776 were identified by receiver operating characteristic roc curve analysis and survival analysis respectively rnase r digestion was used to characterize circ_0001776 and the localization of circ_0001776 was evaluated by cell fractionation assay then cell counting kit8 cck8 colony formation and flow cytometry analysis were used to detect cell proliferation and apoptosis respectively the realtime glycolytic rate ecar and lactate production were measured by extracellular flux analysis and a lactate assay kit respectively bioinformatics analysis and dualluciferase reporter assay were used to determine the interaction among circ_0001776 mir182 and lrig2 the protein expression of lrig2 was determined by western blot moreover circ_0001776 overexpression vector was used to upregulate circ_0001776 expression in an animal tumor modelresults circ_0001776 and lrig2 were downregulated while mir182 was upregulated in ec tissues and cells low expression of circ_0001776 was correlated with the 5year survival rate of ec patients upregulated circ_0001776 markedly attenuated cell proliferation and glycolysis and enhanced cell apoptosis besides circ_0001776 sponged mir182 to regulate lrig2 expression circ_0001776 could suppress ec progression by mir182lrig2 axis furthermore we also found that circ_0001776 significantly inhibited tumor growth in vivo our results confirmed that circ_0001776 inhibited ec tumorigenesis and progression via mir182lrig2 axis providing a potential therapeutic target for eckeywords endometrial cancer hsa_circrna_0001776 mir182 lrig2correspondence xgiaee163com department of obstetrics weifang peoples hospital weifang shandong chinafull list of author information is available at the end of the the authors this is licensed under a creative commons attribution international license which permits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the creative commons licence and indicate if changes were made the images or other third party material in this are included in the s creative commons licence unless indicated otherwise in a credit line to the material if material is not included in the s creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtain permission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40 the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to the data made available in this unless otherwise stated in a credit line to the data 0cjia a0et a0al cancer cell int page of highlights circ_0001776 was lowly expressed in endometrial cancer tissues and cells circ_0001776 overexpression inhibited endometrial cancer cell proliferation and glycolysis and promoted cell apoptosis circ_0001776 upregulated expression lrig2 through targeting mir182 circ_0001776 suppressed endometrial cancer progression via mir182lrig2 axis endometrial cancer ec mainly occurred in postmenopausal women is one of the most common malignancies of the female reproductive system with the incidence of about in worldwide ec is conventionally classified into type i estrogendependent ec and type ii estrogennondependent ec according to molecular genetic features and clinicopathological features and grades and are regarded as type i while grade is regarded as type ii [ ] the prognosis of type i ec patients was relatively favorable while type ii ec was always accompanied by the poor outcomes however the early diagnosis of ec was extremely difficult due to the complex uterus endocrine function and the unadvanced technology of anatomy thus it is meaningful to explore the potential targets for ec diagnosis and treatmentcircular rnas circrnas are a class of abundant endogenous conserved noncoding rnas which have a circular structure lacking ² poly a tails and ² caps [] altered expression of circrnas was found in numerous cancers such as hsa_circ_0001313 in colon cancer circbanp in lung cancer hsa_circ_0072995 in breast cancer several circrnas were confirmed to be the ideal biomarkers for the diagnosis treatment and prognosis of various human cancers [ ] for example hsa_circ_0052112 could regulate breast cancer tumorigenesis through facilitating cell metastasis additionally hsa_circrna_0001776 circ_0001776 was downregulated in ec tissues however the regulatory effects of circ_0001776 in ec remain largely unknown and the underlying mechanisms need further understandingmicrornas mirnas are a group of small singlestranded noncoding rnas with the length of nucleotides which can be involved in the pathological and physical processes including cell survival proliferation and metastasis in various tumors [] recent studies showed that circrnas could sponge mirnas to exert the regulatory effects for instance repeats circ_0044516 could target mir29a3p to facilitate cell metastasis in prostate cancer microrna182 mir was confirmed to aberrantly express in ec in this research circinteractome showed that mir182 might be a target of circ_0001776 we aimed to explore the functional effects of circ_0001776 and mir182 on ec tumorigenesisleucinerich immunoglobulinlike domains lrig2 is a member of lrig protein family which harbored a single transmembrane domain [ ] lrig2 was demonstrated to play a suppressive role in ec the molecular mechanism of lrig2 in ec remains unclear in this study we predicted that lrig2 contained the potential binding site of mir182 thus we aimed to explore the functional role of lrig2 in ec mechanically we investigated the relationship among circ_0001776 mir182 and lrig2 and their effects on regulating the tumorigenesis and progression of ecand material and a0methodstissues sampleshuman normal endometrial tissue samples were collected from healthy volunteers while the tumorous tissues were obtained from endometrial cancer patients who underwent surgery at weifang peoples hospital the clinicopathologic features of these patients were presented in table a0 all participants signed written table correlation between a0 circ_0001776 expression with a0clinicopathologic features of a0ecparametersncirc_0001776 expressionhigh n low n page years histological stage g1 g2 g3lymph node metastasis yes nofigo stage i ii iii iver expression positive negativepr expression positive negative p p 0cjia a0et a0al cancer cell int page of informed consent all experiments in this study were approved by the human research ethics committee of weifang peoples hospitalcell linesrl952 ishikawa hec1a hec1b and an3ca cells were human endometrial cancer cell line while heec cells were normal human endometrial epithelial cells rl952 hec1a and hec1b cells purchased from american type culture collection manassas va usa and heec ishikawa and an3ca cells obtained from shanghai zeye biological technology co ltd shanghai china were used in this study dulbeccos modified eagles medium dmem gibco rockville md usa containing streptomycinpenicillin uml invitrogen carlsbad ca usa and fetal bovine serum fbs invitrogen was used to incubate the cells in a humidified chamber while the cell culture condition was co2 at Ëccell transfectionrl952 and ishikawa cells were selected for the following experiments circ_0001776 overexpression vector circ_0001776 and empty vector vector mir182 mimics mir182 and the control mirnc mir182 inhibitor antimir182 and the corresponding control antinc and small interfering rna sirna targeting lrig2 silrig2 and the control group sinc genepharma shanghai china were transfected into rl952 and ishikawa cells by lipofectamine reagent invitrogenrealtime quantitative polymerase chain reaction rtqpcrtotal rna was extracted from nontumorous tissues and tumor tissues as well as endometrial cancer cells and heec cells using trizol reagent invitrogen and a0µg of total rna was reversetranscribed using superscript iii rt invitrogen the specific gene amplified transcript level was measured by a realtime quantitative pcr system u6 and glyceraldehyde 3phosphate dehydrogenase gapdh were used to normalize the expression of hk2 mir182 circ_0001776 and lrig2 respectively the specific primer sequences were as follows circ_0001776 tca aac ctc gac aag gtg ct sense and cct tag aac acc cgg aag gt antisense mir182 gag aac agc agg tcc agc at sense and ctt cct cag cac aga ccg ag antisense lrig2 cag tgc ata gct gga ggg agtc sense and tac aat gat gag aag ctg att ggc tgca antisense hk2 caa agt gac agt ggg tgt gg sense and gcc agg tcc ttc act gtc tcantisense u6 ctc gct tcg gca gcaca sense and aac gct tca cga att tgc gt antisense gapdh aag gct gag aat ggg aaa c sense and ttc agg gac ttg tca tac ttc antisense the relative expression of circ_0001776 mir182 and lrig2 was assessed by the ct methodrnase r digestion ug total rna isolated from rl952 and ishikawa cells were incubated with units of rnase r epicenter biotechnologies shanghai china for a0min at a0°c after the incubation of total rna and rnase r the expression of circ_0001776 was determined through rtqpcrsubcellular localizationthe localization of circ_0001776 was evaluated by cytoplasmic nuclear rna purification kit nen biotek corp belmont ma usa briefly cells were lysed by lysis buffer j and then centrifuged subsequently the cytoplasmic rna and nuclear rna were treated with buffer sk and anhydrous ethanol respectively then the nuclear rna and cytoplasmic rna were eluted using the spin column finally the expression of circ_0001776 in cytoplasmic and nucleus fractions was detected by rtqpcrcell counting kit cck assaythe proliferation ability of rl952 and ishikawa cells was assessed by cck8 assay à a0cells in a0μl cell suspension were seeded into the 96well plates and cultured for a0h then cells were treated with a0μl cck8 solution dojindo tokyo japan and the absorbance was detected at and a0h after transfection at a0nm respectivelycolony formation assaythe selected rl952 and ishikawa cells were plated into the 6well plates after incubation for a0weeks cells were fixed with paraformaldehyde stained with crystal violet and counted using a microscope biorad laboratories inc hercules ca usacell apoptosis assaythe apoptotic cells were detected by flow cytometry analysis using an annexin vfluorescein isothiocyanate fitc apoptosis detection kit bd pharmingen san diego ca usa in brief cells were treated with trypsin gibco and then centrifuged and removed the supernatant subsequently cells were washed and resuspended with the binding buffer then fitc and propidium iodide pi were added in dark for a0min finally cell apoptosis was assessed by a facscalibur flow cytometer bd pharmingen 0cjia a0et a0al cancer cell int page of determination of a0the a0realtime glycolytic rate ecarin this study we used the seahorse extracellular flux analyzer xf96 seahorse bioscience north billerica ma usa to monitor cell metabolic alternation in a0vitro after cell transfection cells with a density of à per well were added into the xf96well plate and incubated overnight then cells were starved with the nonserum medium for a0h for measurement of ecar cells were cultured with unbuffered medium following sequentially injected glucose oligomycin om and 2deoxyglucose 2dg ecar detection was noted as mphmindetermination of a0lactateafter the transfection of rl952 and ishikawa cells cells were treated with nonserum medium and starved for a0h then the cell culture medium was collected to detect the lactate production using a lactate assay kit keygen nanjing china lactate production was normalized on the basis of the total protein concentrationcarrying the widetype dualluciferase reporter assayplasmids circ_0001776 circ_0001776 wt or mutanttype circ_0001776 circ_0001776 mut were cotransfected with mir182 mimics or mirnc in ec cells while wildtype sequence of lrig2 ²utr lrig2 wt or mutant sequence of lrig2 ²utr lrig2 mut plus mir182 mimics or mirnc were cotransfected into rl952 and ishikawa cells after transfection the relative luciferase activity was detected by the dualluciferase reporter assay system promega madison wi usawestern blottotal protein was isolated from the ec tissues and normal tissues as well as rl952 and ishikawa cells by ripa lysis buffer beyotime shanghai china briefly the total protein was separated using odium dodecyl sulfatepolyacrylamide gel electrophoresis and then the proteins were transferred onto the polyvinylidene difluoride membranes sigma billerica ma usa subsequently the membranes were blocked using nonfat milk and incubated with the primary antibodies against lrig2 ab157492 abcam cambridge uk hk2 ab104836 abcam and gapdh ab8245 abcam overnight at a0°c then the blots were incubated with goat antirabbit ab6721 abcam or antimouse ab205719 abcam for a0h finally the protein signals were calculated by an electrochemiluminescent system perkinelmer life science waltham ma usamouse xenotransplantation a0 weeks old balbc mice were purchased from the beijing laboratory animal center beijing china mice were randomly divided into two groups vector and circ_0001776 rl952 cells stably transfected with circ_0001776 overexpression vector and empty vector were injected into the right flank of the mice after a0days tumor volume was detected every a0days after the mice euthanasia on day tumor weight was measured all the in a0 vivo experiments were approved by weifang peoples hospital experimental animal ethics committeestatistical analysisall statistical analyses were performed using spss software data were expressed as the mean ± the standard deviations sd the interaction between variables was analyzed by pearson correlation analysis students ttest and oneway anova analysis were performed for comparisons the diagnosed value was evaluated by the receiver operating characteristic roc curve analysis and the area under the curve auc meant no diagnostic value a value of p was considered as a statistically significant differencein ec resultscirc_0001776 was a0downregulated in a0endometrial cancertissues and cells circ_0001776 expression was measured using rtqpcr as shown in fig a0 1a circ_0001776 was lowly expressed in ec tissues in comparison to that in normal tissues compared with the grades in and g1 g2 ec tissues circ_0001776 had a lower expression level in grade g3 ec tissues fig a01b besides we constructed the roc curve to explore the potential value of circ_0001776 as described in fig a01c circ_0001776 was confirmed to have the diagnosis value of auc p more importantly we observed that the low expression of circ_0008285 was closely related to the poor survival of ec patients after surgery fig a01d furthermore circ_0001776 expression was decreased in rl952 ishikawa hec1a hec1b and an3ca cells relative to that in heec cells fig a01e all these data demonstrated that circ_0001776 was decreased in ec which might be a biomarker in ec progressioncirc_0001776 overexpression suppressed proliferation and a0facilitated apoptosis of a0ec cellsto elucidate whether circ_0001776 played a key role in ec tumorigenesis the function assays were performed firstly we confirmed that circ_0001776 was indeed circrna which was resistant to rnase r digestion in both 0cjia a0et a0al cancer cell int page of fig circ_0001776 was downregulated in endometrial cancer a rtqpcr was used to detect the expression of circ_0001776 in ec tissues b the expression of circ_0001776 in g1 g2 and g3 ec tissues was measured by rtqpcr c the roc of circ_0001776 as a biomarker was evaluated d survival was analyzed and compared between patients with high and low levels of circ_0001776 using kaplanmeier analysis e the expression of circ_0001776 in ec cells was assessed by rtqpcr p rl952 and ishikawa cells fig a02a b then we detected the subcellular localization of circ_0001776 in ec cells the results showed that circ_0001776 was mostly located in the cytoplasm of ec cells fig a02c d as expected the expression of circ_0001776 was significantly increased in rl952 ishikawa and heec cells transfected with circ_0001776 fig a0 2e f and additional file a0 fig s1a cck8 assay indicated that circ_0001776 overexpression markedly inhibited cell proliferation in rl952 and ishikawa cells compared with the control group fig a02g h similarly we observed the reduction of cell colonies number in rl952 and ishikawa cells transfected with circ_0001776 fig a0 2i additionally we measured the effects of circ_0001776 overexpression on cell apoptosis the data suggested that overexpression of circ_0001776 significantly induced cell apoptosis in ec cells fig a02j moreover we detected the protein levels of cleavedcasp3total casp3 and cleavedcasp9total casp9 in ec cells transfected with vector or circ_0001776 the data showed that circ_0001776 overexpression increased the protein expression of cleavedcasp3 and cleavedcasp9 in ec cells additional file a0 fig s2a b however circ_0001776 overexpression showed no significant effects on cell proliferation the number of colonies and apoptosis in heec cells additional file a0 fig s1bd these results demonstrated that circ_0001776 was a suppressive circrna in ec tumorigenesiscirc_0001776 overexpression inhibited the a0glycolytic metabolism in a0ec cellsnext we explored whether overexpression of circ_0001776 could directly affect glycolytic metabolism in ec cells through detecting extracellular acidification rate ecar and lactate2 production circ_0001776 upregulation significantly reduced ecar in rl952 and ishikawa cells fig a03a b overexpression of circ_0001776 did not change ecar level in heec cells additional file a0 fig s1e moreover lactic acid production was markedly decreased by circ_0001776 overexpression in rl952 and ishikawa cells fig a0 3c we further detected the mrna and protein expression of hk2 and the data showed that circ_0001776 upregulation significantly suppressed hk expression fig a03d e overall our data suggested that circ_0001776 could regulate glycolytic metabolism in ec cellslevel circ_0001776 sponged mircircinteractome showed that mir182 contained the binding sites of circ_0001776 fig a0 4a moreover the relative luciferase activity was significantly decreased in rl952 and ishikawa cells cotransfected with circ_0001776 wt and mir182 mimic compared with the control group while there was no significant change of the luciferase activity in ec cells cotransfected with 0cjia a0et a0al cancer cell int page of fig circ_0001776 overexpression suppressed proliferation and facilitated apoptosis of ec cells a b circ_0001776 resistance to rnase r was detected by rtqpcr c d rtqpcr was used to assess the levels of cytoplasmic control transcript gapdh nuclear control transcript u6 and circ_0001776 in nuclear and cytoplasmic fractions e f the overexpression efficiency of circ_0001776 was evaluated by rtqpcr gi cell proliferation in ec cells transfected with circ_0001776 or vector was detected by cck8 assay and colony formation assays j cell apoptosis in ec cells transfected with circ_0001776 or vector was measured by flow cytometry analysis p circ_0001776 mut and mir182 mimics fig a04b c as shown in fig a0 4d mir182 was highly expressed in ec tumor tissues relative to that in normal tissues besides the expression of mir182 was inversely correlated with circ_0001776 level in ec tissues fig a0 4e consistently mir182 level was also facilitated in rl952 and ishikawa cells compared with heec cells fig a04f moreover overexpression of circ_0001776 could significantly repress mir182 expression in ec cells fig a0 4g these data revealed that circ_0001776 could serve as a competing endogenous rna to sponge mir182circ_0001776 overexpression inhibited ec progression by a0sponging mirto further investigate whether circ_0001776 exerted its suppressive functions on tumor through mir182 rescue experiments were performed using mir182 mimics in ec cells with circ_0001776 overexpression rtqpcr demonstrated that mir182 expression was dramatically increased in ec cells transfected with mir182 mimics fig a0 5a b subsequently we performed the functional experiments in ec cells cck8 assay indicated that the inhibitory effect of circ_0001776 overexpression on cell proliferation was reversed by upregulating mir182 in both rl952 and ishikawa cells fig a05c d we also discovered that mir182 overexpression significantly rescued the reduction of colonies induced by circ_0001776 overexpression in ec cells fig a0 5e f flow cytometry analysis indicated that mir182 upregulation markedly reduced circ_0001776 overexpressioninduced cell apoptosis in both rl952 and ishikawa cells fig a05g h furthermore we evaluated the glycolytic metabolism in ec cells the data suggested that circ_0001776 upregulation triggered a decrease of glycolytic metabolism which was reversed by mir182 overexpression fig a05i j consistent with the results in glycolytic metabolism the decrease of lactate production in rl952 and ishikawa cells transfected with circ_0001776 was attenuated by upregulating mir182 fig a05k l taken together circ_0001776 could inhibit cell proliferation and glycolytic metabolism and promote cell apoptosis by targeting mir182 in ec cells 0cjia a0et a0al cancer cell int page of fig circ_0001776 overexpression inhibited the glycolytic metabolism in ec cells a b the change of ecar level with different treatment in rl952 and ishikawa cells was determined after transfecting with control or circ_0001776 overexpression plasmid c the relative lactic acid level in rl952 and ishikawa cells transfected with control or circ_0001776 overexpression plasmid was examined d e the mrna and protein expression of hk2 were detected by rtqpcr and western blot p fig circ_0001776 sponged mir182 a circinteractome predicted that mir182 harbored the binding sites of circ_0001776 b c the interaction between circ_0001776 and mir182 was evaluated by the dualluciferase reporter assay d the expression of mir182 in ec tissues was detected by rtqpcr e the relationship between circ_0001776 and mir182 was analyzed by kaplanmeier analysis r p f the expression of mir182 in ec cells and heec cells was examined by rtqpcr g the expression of mir182 in ec cells transfected with vector or circ_0001776 was detected by rtqpcr p 0cjia a0et a0al cancer cell int page of fig circ_0001776 overexpression inhibited ec progression by sponging mir182 a b rtqpcr was used to measure mir182 expression in rl952 and ishikawa cells transfected with mirnc or mir142 c d cell proliferation was measured by cck8 assay after transfection e f cell colony formation assay was used to detect the number of colonies after transfection in ec cells g h flow cytometry was performed to examine cell apoptosis in rl952 and ishikawa cells after transfection i j ecar was determined in ec cells after transfection k l the lactic acid level was measured in ec cells after transfection p lrig2 was a0a a0direct target of a0mirto investigate the potential molecular mechanism of mir182 in ec we screened the potential target gene of mir182 via targetscan as shown in fig a06a lrig2 harbored the binding sites of mir182 the luciferase activity was significantly decreased in ec cells cotransfected with lrig2 wt and mir182 mimics compared with the control group while the luciferase activity had no significant change in rl952 and ishikawa cells cotransfected with lrig2 mut and mir182 mimics relative to the corresponding control group fig a06b c moreover rtqpcr and western bolt assays showed that the mrna and protein expression of lrig2 were dramatically attenuated in tumor tissues relative to that in normal tissues fig a0 6d e furthermore lrig2 expression was negatively related to mir182 level fig a06f and positively correlated with circ_0001776 expression fig a06gmoreover the expression of mir182 was significantly decreased in ec cells transfected with antimir182 fig a06h we found that the protein expression of lrig2 was downregulated in ec cells compared with that in heec cells fig a06i as described in fig a06j downregulation of mir182 could increase lrig2 protein expression in ec cells besides overexpression of circ_0001776 facilitated the protein expression of lrig2 while mir182 overexpression reversed the promotion effect in both rl952 and ishikawa cells fig a06k l overall mir182 directly targeted lrig2 and circ_0001776 could sponge mir182 to regulate lrig2 expression in ec cellslrig2 knockdown reversed the a0effects of a0mir downregulation on a0ec cellswe detected the knockdown efficiency of lrig2 in ec cells and observed that transfection of silrig2 could significantly decrease the protein expression of lrig2 in ec cells fig a0 7a as shown in fig a0 7b c the suppressive effect of mir182 downregulation on cell proliferation was blocked by lrig2 deletion in ec cells analogously the colonies were decreased by downregulating mir182 while lrig2 deletion significantly increased the number of colonies in both rl952 and ishikawa cells fig a0 7d e mir182 deletion induced 0cjia a0et a0al cancer cell int page of fig lrig2 was a direct target of mir182 a the binding site of lrig2 and mir182 was predicted by targetscan b c the relative luciferase activity in ec cells was detected by dualluciferase reporter assay d the mrna expression of lrig2 in ec tissues was examined by rtqpcr e western blot was used to detect the protein expression of lrig2 in ec tissues f the negative relationship between mir182 and lrig2 was discovered in ec tissues r p g the positive relationship between circ_0001776 and lrig2 was observed in ec tissues r p h lrig2 expression was determined after ec cells transfected with antinc or antimir182 i the protein expression of lrig2 in ec cells was detected by western blot j lrig2 expression in ec cells transfected with antinc or antimir182 was measured by western blot k l lrig2 expression in ec cells transfected with vector circ_0001776 circ_0001776 mirnc or circ_0001776 mir182 was examined by western blot p cell apoptosis which was restrained by lrig2 downregulation in ec cells fig a07f g moreover the glycolytic metabolism was markedly suppressed in ec cells transfected with antimir182 while lrig2 deletion could reverse the inhibitory effect of mir182 deletion on glycolytic metabolism fig a07h i consistently mir182 deletion reduced the lactate production in ec cells while lirg2 knockdown significantly rescued the reduction of lactate production fig a0 7j k these results indicated that lrig2 downregulation could block the effects of mir182 deletion on cell proliferation apoptosis and glycolytic metabolism in ec cellscirc_0001776 inhibited tumor growth in a0vivoto determine the effects of circ_0001776 on tumor growth in a0 vivo rl952 cells stably transfected with circ_0001776 or vector were injected into the right flank of the mice the result suggested that circ_0001776 overexpression suppressed tumor growth in a0vivo fig a08a b furthermore tumor weight in the circ_0001776 group was significantly lower than that in the control group 0cjia a0et a0al cancer cell int page of fig lrig2 knockdown reversed the effects of mir182 downregulation on ec progression a the expression of lrig2 in ec cells transfected with sinc or silrig2 was determined by western blot b c cell proliferation was measured in ec cells transfected with antinc antimir182 antimir182 sinc and antimir182 silrig2 by cck8 assay d e the number of colonies was assessed in ec cells after transfection f g the apoptotic rate of ec cells after transfection was detected by flow cytometry h i the change of ecra level in ec cells after transfection was determined j k the lactic acid level in ec cells was examined p fig circ_0001776 inhibited tumor growth in vivo ac the tumor volume and weight were measured in vivo d e the expression of circ_0001776 mir182 and lrig2 in the tumor tissues from balbc mice was detected using rtqpcr and western blot respectively p 0cjia a0et a0al cancer cell int page of fig a0 8c besides we discovered that circ_0001776 expression was upregulated while mir182 was downregulated by circ_0001776 overexpression fig a0 8d moreover the protein expression of lrig2 was significantly facilitated in the tissues extracted from the mice in circ_0001776 group compared with the control group fig a0 8e these data confirmed the suppressive role of circ_0001776 in ec in a0vivodiscussionthe malignant endometrial tumor is the most common female cancer among all reproductive system diseases accompanying with increasingly younger women diagnosed with ec thus it is urgent to develop the novel methods for the diagnosis treatment and prognosis for ec and find out the regulators in the tumorigenesis of ec recently accumulating evidence indicated that circrnas were closely involved in the regulation of the biological processes in human cancers including ec [ ] for instance hsa_circ_0000190 was demonstrated as a novel biomarker for gastric cancer diagnosis which was significantly associated with lymphatic metastasis tumor diameter and distal metastasis in gastric cancer besides circ8073 was confirmed to be a crucial regulator in cell proliferation of endometrial epithelial cells and further targeted mir449a to regulate the pi3kaktmtor pathway in endometrial receptivity development in this research we found a reduction of circ_0001776 expression in ec tissues and cells low expression of circ_0001776 was demonstrated to be related to the grade of ec and the g3 ec existed lower expression than that in g1 g2 ec patients the data in our study were consistent with the results from ye and his colleagues besides we also observed that ec patients with low circ_0001776 expression had a poor survival rate additionally the functional effects of circ_0001776 were explored by performing functional assays as expected overexpression of circ_0001776 significantly inhibited cell growth and glycolytic metabolism and facilitated cell apoptosis in ecseveral researches reported that glycolytic metabolism was a key hallmark in human cancers the activation of glycolysis and the increased lactic acid production were observed in multiple cancer cells which led to the energy metabolism alteration and were correlated with the prognosis of cancer patients a recent study confirmed that the glycolytic activity was positively related to ec cell proliferation through activating the mapk and ampkmtors6 pathways thus we detected the effect of circ_0001776 on glycolytic metabolism in ec cells our data suggested that circ_0001776 overexp | Colon_Cancer |
the european commission asked efsa for a scientiï¬c opinion on the risks for animal and humanhealth related to the presence of glycoalkaloids gas in feed and food this risk assessment coversedible parts of potato plants and other food plants containing gastomato andaubergine in humans acute toxic effects of potato gas asolanine and achaconine includegastrointestinal symptoms such as nausea vomiting and diarrhoea for these effects the contampanel identiï¬ed a lowestobservedadverseeffect level of mg total potato gaskg body weight bwper day as a reference point for the risk characterisation following acute exposure in humans noevidence of health problems associated with repeated or longterm intake of gas via potatoes hasbeen identiï¬ed no reference point for chronic exposure could be identiï¬ed from the experimentalanimal studies occurrence data were available only for asolanine and achaconine mostly forpotatoes the acute dietary exposure to potato gas was estimated using a probabilistic approach andapplying processing factors for food due to the limited data available a margin of exposure moeapproach was applied the moes for the younger age groups indicate a health concern for the foodconsumption surveys with the highest mean exposure as well as for the p95 exposure in all surveysfor adult age groups the moes indicate a health concern only for the food consumption surveys withthe highest p95 exposures for tomato and aubergine gas the risk to human health could not becharacterised due to the lack of occurrence data and the limited toxicity data for horses farm andcompanion animals no risk characterisation for potato gas could be performed due to insufï¬cient dataon occurrence in feed and on potential adverse effects of gas in these species european food safety authority efsa published by john wiley and sons ltd on behalfof european food safety authoritykeywords glycoalkaloids gas solanine chaconine potato margin of exposure moe food feedrequestor european commissionquestion number efsaq201600811correspondence contamefsaeuropaeu leon brimer was a member of the working group on glycoalkaloids in food and feed until august wwwefsaeuropaeuefsa efsa 0cglycoalkaloids in feed and foodpanel members margherita bignami laurent bodin james kevin chipman jes 13us del mazo bettinagraslkraupp christer hogstrand laurentius ron hoogenboom jeancharles leblanc carlo stefanonebbia elsa nielsen evangelia ntzani annette petersen salomon sand dieter schrenk tanjaschwerdtle christiane vleminckx and heather wallaceacknowledgements the panel wishes to thank the following for the support provided to thisscientiï¬c output kelly niermans the panel wishes to acknowledge all european competentinstitutions member state bodies and other anisations that provided consumption and occurrencedata for this scientiï¬c outputsuggested citation efsa contam panel efsa panel on contaminants in the food chain schrenk dbignami m bodin l chipman jk del mazo j hogstrand c hoogenboom lr leblanc jc nebbia csnielsen e ntzani e petersen a sand s schwerdtle t vleminckx c wallace h brimer l cottrill bdusemund b mulder p vollmer g binaglia m ramos bordajandi l riolo f rold 13antorres r and graslkraupp b scientiï¬c opinion risk assessment of glycoalkaloids in feed and food in particular inpotatoes and potatoderived products efsa pp 102903jefsa20206222issn european food safety authority efsa published by john wiley and sons ltd on behalfof european food safety authoritythis is an open access under the terms of the creative commons attributionnoderivs licensewhich permits use and distribution in any medium provided the original work is properly cited and nomodiï¬cations or adaptations are madereproduction of the images listed below is prohibited and permission must be sought directly from thecopyright holderfigure elsevier figure springer figure american chemical society springerthe efsa is a publication of the european foodsafety authority an agency of the european unionwwwefsaeuropaeuefsa efsa 0cglycoalkaloids in feed and foodsummarythe european commission asked efsa for a scientiï¬c opinion on the risks for animal and humanhealth related to the presence of glycoalkaloids gas in feed and food in particular in potatoes andpotatoderived products this risk assessment covers edible parts of potato plants and other foodplants containing gas in particular tomato and aubergine nonedible parts of ga containing plantshave not been considered with the exception of potato sprouts the panel developed the draftscientiï¬c opinion which underwent a public consultation from february to april thecomments received and how they were taken into account when ï¬nalising the scientiï¬c opinion werepublished in an efsa technical report efsa gas are present in many plants of the family of solanaceae and contribute to plant resistanceagainst pests and pathogens gas are composed of a steroidal aglycone and an oligosaccharide sidechain in commercial potato cultivars s tuberosum the main gas are achaconine and asolanineconsisting of the aglycone solanidine and chacotriose and solatriose as oligosaccharide side chainsrespectively the aubergine fruit s melongena contains primarily the gas asolamargine and asolasonine composed of the aglycone solasodine and chacotriose and solatriose respectively inlycopersicum atomatine and adehydrotomatine are the major gas withtomato fruitlycotetraose coupled to the aglycones tomatidine and tomatidenol respectivelyshuman risk assessmentin experimental animals the potato gas asolanine and achaconine show a relatively low oralbioavailability with differences between species hamsters exhibit higher absorption and slowerexcretion rates for both substances when compared to rats due to the limited information themetabolic proï¬les of potato gas in experimental animals could not be characterisedin humans asolanine and achaconine are systemically absorbed following ingestion for bothsubstances relatively long serum halflives were reported suggesting a possible accumulation the bloodclearance of the respective aglycone solanidine appears to be slow accordingly levels of solanidine wereregularly detected in the blood of human volunteers in several studies suggesting hydrolysis of gas nofurther information is available on metabolism and excretion of potato gas in humansthere are no toxicokinetic data on tomato and aubergine gas and their aglycones in experimentalanimals and humansin acute oral toxicity studies no adverse effects of asolanine were observed at doses of mgkgbody weight bw per day in rats and mgkg bw per day in mice reliable data on other potatogas or tomato and aubergine gas and their aglycones are missingin repeated oral dose studies on potato gas rodents showed nonspeciï¬c effects such as reducedbody weight and relative liver weight with indication of similar potencies of asolanine and achaconine hamsters exhibited these symptoms after a 5day treatment with mg of asolanine ora chaconinekg bw per day while mice showed these effects after one week of daily treatments with mg of asolanine or mg of achaconinekg bw solanidine however increased the absoluteand relative liver weight at mgkg bw per day in mice suggesting a different effect of theaglycone compared to the gasthe tomato ga atomatine and its aglycone tomatidine exerted no effects in rats when applied at mgkg bw per day for a period of day at higher doses atomatine reduced the cholesterol uptakeand increased fecal sterol and coprostanol excretion in hamsters and rats in mice a to 2weektreatment with the aubergine ga asolasonine increased the body weight gain at mgkg bw perday while its aglycone solasodine decreased body weight gain and caused gastric gland degenerationand liver toxicity at mgkg bw per daydevelopmental studies have been performed mainly in hamsters treated with potato gas and theiraglycones for only one day or for a short very restricted time period during gestation outcomes weremainly analysed in late gestational embryos and comprised effects in the central nervous systempredominantly exencephaly encephalocele and anophthalmia these malformations occurred at dosesof mgkg bw per day and above for gas and of mgkg bw per day and above for theaglycones no noobservedadverseeffectlevelloael could be identiï¬ed from these studies reduced postnatal survival of pups due to insufï¬cientmilk production was reported when pregnant holtzman rats had been exposed to mg of asolaninekg bw per day studies on the male fertility in dogs have been performed only with theaubergine aglycone solasodine decreased epididymal weight and cauda epididymal epithelial heightand also an epididymal lumen depleted of sperm occurred in dogs after mgkg bw per day givenlowestobservedadverseeffectnoael orlevelwwwefsaeuropaeuefsa efsa 0cglycoalkaloids in feed and foodfor month similar effects were observed in rhesus monkeys exposed to mgkg bw per day for monthsfrom the limited number of studies available there was no evidence for genotoxicity of the potatogas asolanine and achaconine and the aglycone solanidine as well as for the aubergine ga asolamargine however there is not sufï¬cient information to conclude on the genotoxic potential ofthese gasno longterm chronic toxicitycarcinogencity study for potato tomato or aubergine gas or for therespective aglycones could be identiï¬edin humans acute toxic effects following ingestion of potato gas include gastrointestinal symptomsof varying severity such as vomiting diarrhoea and abdominal pain which may occur from a totalpotato gas potato tga intake of mgkg bw or more further symptoms including drowsinessapathy confusion weakness vision disturbances rapid and weak pulse and low blood pressure maybe the consequence of dehydration following vomiting and diarrhoeain severe cases paralysis respiratory insufï¬ciency cardiac failure coma and death have beenreported doses in the range of mg potato tgaskg bw are considered to be potentially lethal forhumans results from limited volunteer studies suggest possible differences in the human populationwith respect to the individual susceptibility towards adverse effects associated with the intake ofpotato gasregarding the mode of action adverse effects of gas may be due to their ability to complex withmembrane 3bhydroxy sterols thereby causing disruption and loss of integrity of cell membranesafter oral exposure these effects may affect the mucosa of the gastrointestinal tract and cause thesymptoms observed in intoxicated humans such as nausea vomiting and diarrhoeagas inhibit acetylcholinesterase ache and serum butyrylcholinesterase buche by a reversiblecompetitive mode of action the relative potency of inhibition of asolanine and achaconine appearsto be similar the aglycones exert weak or no inhibitory effects the excess of acetylcholine at theneuronal and neuromuscular junctions upon inhibition of the enzymes might also contribute to thesymptoms described for intoxications with gasat high doses atomatine may form a nonabsorbable complex with cholesterol and other sterols inthe enteral lumen which may impair the absorption of cholesterol as a consequence blood cholesterollevels were lowered in rodentsthe contam panel considered that the use of rodent data on acute toxicity was not appropriate toestablish a reference point for acute exposure to potato gas in humans the contam panel selectedthe loael of mg potato tgakg bw per day as the reference point for acute risk characterisationbased on human data from case reports outbreaks and studies in volunteers the available data onacute toxicity were considered insufï¬cient to establish a healthbased guidance value instead thepanel used the margin of exposure moe approach to assess a possible health concern from acuteexposure to potato tgas via foodassuming the main symptoms to be mainly due to localirritation of the gastrointestinal mucosarather than inhibition of ache activity the panel considered that the possible interindividual variabilityin toxicodynamics is more relevant than the interindividual variability in toxicokinetics accordingly anmoe higher than indicates that there is no health concern this moe of takes into account theextrapolation from a loael to a noael a factor of and the interindividual variability intoxicodynamics a factor of the experimental data available for repeated dose toxicity are not sufï¬cient to identify a referencepoint for chronic exposure to potato gas in humans no evidence of health problems associated withrepeated or longterm intake of gas via potatoes has been identiï¬edregarding gas or aglycones occurring in edible parts of food plants other than s tuberosum nosuitable study for determining a reference point for tomato or aubergine gas or aglycones wasidentiï¬edoccurrence data were only available for asolanine and achaconine and mostly for maincroppotatoes and new potatoes few data were available for processed food no data on the occurrenceof tomato and aubergine gas and their aglycones were submitted to efsasince the occurrence data on potato gas did not cover all the food categories containing potatoesin the consumption database it was decided that the best approach for the exposure assessmentwould be to use the occurrence data in the raw primary commodities rpc maincrop potatoes andnew potatoes and the rpc consumption database the panel decided to combine the occurrence ofnew potatoes with that of maincrop potatoes and the mean upper bound ub occurrence sum ofwwwefsaeuropaeuefsa efsa 0cglycoalkaloids in feed and foodasolanine and achaconine for these two groups was mgkg and the p95 occurrence was mgkg the minimum and maximum reported concentrations were and mgkg respectivelythe acute dietary exposure to potato tgas was estimated using a probabilistic approach includingonly days in which there was consumption of maincrop potatoes as no occurrence data wereavailable for gas in tomato and aubergine these foods were not included in the exposure assessmentprocessing of potatoes has been reported to reduce the content of gas in the ï¬nal processedproduct in general and according to the literature the peeling of potatoes reduced the ga contentby boiling in water and blanching of peeled potatoes by and frying in oil of peeledpotatoes by microwave and oven baking of unpeeled potatoes may cause a reduction in thega content by and by respectively no information has been found about thechemical nature of the ga degradation products for the exposure assessment processing factors forthe major food processing steps comprising peeling and heat processing boiling frying bakingwere applied to the occurrence data as follows processing factors between and wereattributed to the peeling of potatoes between and for frying and deep frying and between and for all other cooking methodsinformation about the peeling of potatoes was not available in the consumption database but itwas assumed that of the potatoes are consumed as peeled where information of the cookingmethod was not available a cooking method was randomly attributed to the eating event based onthe relative frequency of cooking methods reportedthe mean ub exposure to potato tgas across surveys ranged from lgkg bw per day inadults to lgkg bw per day in toddlers the 95th percentile exposure ranged from lgkgbw per day in adults to lgkg bw per day in toddlers up to lgkg bw per day in theupper limit of the conï¬dence intervalcomparing the loael for potato tgas of mgkg bw per day with the acute exposure estimatesthe moes for the younger age groups indicate a health concern for the food consumption surveys withthe highest mean exposure as well as for the p95 exposure in all surveys for adult age groups themoes indicate a health concern only for the food consumption surveys with the highest p95exposuresthe contam panel calculated the mean percentage of days with potato consumption acrosssurveys per age group on which the potato tga intake may be below the moe of the highestnumber of survey days with intake of potatoes below the moe of was estimated for toddlers followed by children for the other age groups the estimated tga intake was below the moeof in up to of the survey daysfor tomato and aubergine gas the risk to human health could not be characterised due to the lackof occurrence data in food and the limited information on the adverse effects in experimental animalsand humansthe contam panel considered that the impact of the uncertainties on the risk assessment of acuteexposure to potato gas in food is moderate and that overall the identiï¬ed uncertainties may eithercause an over or underestimation of the riskfarm animals horses and companion animals risk assessmentinformation on the toxicokinetics of gas was limited to ruminants for which the data suggest anextensive conversion of asolanine and achaconine to aglycones in rumen and a low potential ofsolanidine to transfer into cows milkno data on the potential adverse effects of potato gas in horses companion animals cats anddogs or fur animals were identiï¬ed due to an insufï¬cient database on the adverse effects of gas inruminants pigs poultry rabbits and ï¬sh an acute reference dose could not be derivedpotatoes are not grown speciï¬cally as feed for livestock but when supply exceeds marketrequirements for human consumption whole raw potatoes may be used as feed for ruminants andpigs some byproducts of potato processing and starch extraction are used as feeds for farmedlivestock principally nonruminants and for companion animalsdata on potato gas in feed were insufï¬cient to perform an exposure assessmentthus no risk characterisation could be performed due to insufï¬cient occurrence data of gas forfeed and the lack of or limited data on the adverse effects of gas in farm animals horses orcompanion animalswwwefsaeuropaeuefsa efsa 0cglycoalkaloids in feed and foodrecommendationsthe following needs have been identiï¬ed to improve the risk assessment for humans and reducethe uncertaintiescid129research on the occurrence of gas and their aglycones and other potentially toxicologicallyrelevant secondary plant metabolites in the potato cultivars available on the market and onnew potato cultivars resulting from breeding experimentscid129 occurrence data on gas and their aglycones in potato processed products including foods forinfantscid129 occurrence data on gas and their aglycones in tomato and aubergine and products thereofcid129 data on the toxicokinetics of potato tomato and aubergine gas and aglycones in experimentalanimals and humanscid129 data on repeated dose toxicity including reproductive and developmental toxicity of potatotomato and aubergine gas and aglycones in experimental animalsstudies in humans linking dietary exposure biomarkers of exposure and adverse effectscid129the following needs have been identiï¬ed to improve the risk assessment for farm animals horsesand companion animals and reduce the uncertaintiescid129 occurrence data on potato gas and their aglycones in feedcid129studies on the kinetics and the potential adverse effects from feed material containing gas ofpotato gas in farm animals horses and companion animalswwwefsaeuropaeuefsa efsa 0cglycoalkaloids in feed and foodtable of contentsabstractsummaryintroduction background and terms of reference as provided by the requestor interpretation of the terms of reference supporting information for the assessment chemistry analytical methods sources potatoes tomatoes aubergine previous risk assessments legislation and other standards data and methodologies methodology for data collection selection of evidence and study appraisal food and feed occurrence data submitted to efsa data collection and validation data analysis food and feed consumption data food consumption data feed consumption data food classiï¬cation methodology for exposure assessment methodology for risk characterisation assessment hazard identiï¬cation and characterisation toxicokinetics experimental animals asolanine achaconine humans mixtures of asolanine and achaconine solanidine biomarkers of exposure farm animals horses and companion animals summary on toxicokinetics toxicity in experimental animals acute toxicity studies gas from edible parts of s tuberosum gas from edible parts of food plants other than s tuberosum summary on acute toxicity studies repeated dose toxicity studies gas and aglycones from edible parts of s tuberosum gas and aglycones from edible parts of food plants other than s tuberosum developmental and reproductive toxicity studies developmental effects reproductive effects immunotoxicity studies studies on cardiovascular effects neurotoxicity studies genotoxicity gas from edible parts of s tuberosum gas from edible parts of food plants other than s tuberosum carcinogenicity studies studies on metabolic effects gas from edible parts of s tuberosum gas from edible parts of food plants other than s tuberosum observations in humans wwwefsaeuropaeuefsa efsa 0cglycoalkaloids in feed and food gas from s tuberosum reports on intoxications studies in human volunteers epidemiological studies summary gas from food plants other than s tuberosum case reports adverse effects in farm animals horses and companion animals ruminants pigs poultry rabbits fish horses companion animals cats and dogs fur animals reports on intoxications mode of action membrane effects with implications for the gastrointestinal tract inhibition of cholinesterases ches comparative determination of inhibition of ches in vitro determination of inhibitory constants ki for gas on inhibition of ches in vitro inhibition of ches in vivo developmental and reproductive effects of gas and their aglycones inhibition of cholinesterases and effects in the immune system interference with metabolism considerations of critical effects and doseresponse analysis for the human risk assessment gas from edible parts of s tuberosum considerations of critical effects and doseresponse analysis derivation of a healthbased guidance value hbgv or margin of exposure moe approach gas from edible parts of food plants other than s tuberosum considerations of critical effects and doseresponse analysis consideration of critical effects and doseresponse analysis for the farm animal horses andcompanion animals risk assessment occurrence data occurrence data submitted to efsa previously reported occurrence data in the open literature literature on occurrence data on food occurrence data on gas in potatoes occurrence data on gas in tomatoes occurrence data on gas in aubergines occurrence data on gas in other food products literature occurrence data in feed inï¬uence of storage and processing on the content of gas gas from s tuberosum storage of potatoes processing of potatoes for food consumption processing of potatoes for feed gas from food plants other than s tuberosum summary on the inï¬uence of storage and processing on the levels of gas exposure assessment current acute dietary exposure assessment for humans previously reported dietary exposure assessments current dietary exposure assessment for farm animals horses and companion animals risk characterisation human health risk characterisation ga from edible parts of s tuberosum gas from edible parts of food plants other than s tuberosum farm animals horses and companion animal risk characterisation uncertainty analysis assessment objectives exposure scenarioexposure model wwwefsaeuropaeuefsa efsa 0cglycoalkaloids in feed and foodhazard identiï¬cation and characterisation summary of uncertainties conclusions hazard identiï¬cation and characterisation toxicokinetics toxicity in experimental animals observations in humans adverse effects in farm animals horses and companion animals mode of action margin of exposure moe approach occurrence and exposure food feed risk characterisation human health risk characterisation farm animals horses and companion animal health risk characterisation recommendations documentation provided to efsa references abbreviations appendix a major glycoalkaloids and their aglycones present in solanum species appendix b identiï¬cation and selection of evidence relevant for the risk assessment of glycoalkaloids infeed and food appendix c details of the study design of the toxicokinetic studies appendix d comparison of developmental toxicity of single dose studies appendix e inhibition of cholinesterases by gas appendix f rapid alert system for food and feed rasff reports on the presence of solanum nigrum infood products appendix g studies on the toxicity of glycoalkaloids not considered in the risk assessment appendix h additional scenario for the human risk characterisation annex a occurrence data in food and feed submitted to efsa and dietary exposure assessment forhumans wwwefsaeuropaeuefsa efsa 0cglycoalkaloids in feed and foodintroductionbackground and terms of reference as provided by the requestorbackgroundmany plants in the family solanaceae contain glycoalkaloids and they are considered to be naturaltoxins the plant glycoalkaloids are toxic steroidal glycosides and the commonest types found in foodplants are asolanine and achaconine their natural function is probably to serve as stress metabolitesor phytoalexins for the protection of the plant when attacked by insects fungi etcamongst the most widely cultivated food crops aubergines tomatoes and potatoes are in thesolanaceae family but the levels of glycoalkaloids in tomatoes and aubergines are generally quite lowthe glycoalkaloids of most relevance to food safety are those occurring in the potato thepredominant toxic steroidal glycosides in potato are asolanine and achaconine they occur in potatotubers peel sprouts berries leaves and blossoms and their concentration in tubers depends on anumber offactors concentrations ofglycoalkaloids are times greater in the peel than in the ï¬esh there is considerable variation inglycoalkaloid content among potato cultivars storage conditions especially light and temperature aremainly responsible for increases in solanine although the glycoalkaloid content can increase in thedark the rate of formation is only about the rate of formation in light increases of solanine inthe potato peel are closely associated with greening synthesis of chlorophyll of the peel thesebiochemical processes are independent of each other but are both activated by lightsuch as cultivar maturity and environmentalfactorsbitter or burning sensation in the mouth are sensory impressions which may accompanyglycoalkaloid poisoning symptoms from potatoes that include ï¬ulike symptoms such as nauseavomiting stomach and abdominal cramps and diarrhoea more severe cases of glycoalkaloid poisoningmay be accompanied by a variety of neurological effects ie drowsiness apathy restlessnessshaking confusion weakness and disturbed vision there are a few reports of deaths beingattributed to glycoalkaloid exposure from the consumption of potatoes potato leaves and potatoberriespotatoes and potatoderived products are listed in the catalogue of feed materials1terms of referencein accordance with art of regulation ec no the european commission asks theeuropean food safety authority for a scientiï¬c opinion on the risks for animal and human healthrelated to the presence of glycoalkaloids in feed and food in particular in potatoes and potatoderivedproductsinterpretation of the terms of referencethe contam panel considered that the opinion should cover edible parts of potato plants and alsoof other food plants containing glycoalkaloids gas eg tomato and aubergine nonedible parts ofga containing plants have not been considered with the exception of potato sprouts in particular thecontam panel concluded this opinion should comprise thea evaluation of the toxicity of gas in feed and food in particular in potatoes and potatoderivedproducts for farm and companion animals and humans considering all relevant toxicologicalend pointsb evaluation of the alkaloid proï¬le ie composition of the alkaloids and their concentration ofthe food and feed samples submitted to efsac estimation of the dietary exposure of the european population to gas in food in particular inpotatoes and potatoderived products including the consumption patterns of speciï¬c groupsof the population if appropriated estimation of the dietary exposure offarm and companion animals to gas in feedinparticular in potatoes and potatoderived productse assessment of the human health risks for the european population including speciï¬c groupsof the population if appropriate as the consequence of the estimated dietary exposure commission regulation eu no of january on the catalogue of feed materials ojl p wwwefsaeuropaeuefsa efsa 0cglycoalkaloids in feed and foodf assessment of the farm and companion animal health risks in europe as the consequence ofthe estimated dietary exposure exposure to gas from weeds containing ga is only addressedin this opinion in the context of accidental intake by farm animalswhen referring to gas in potatoes the term total gas tga refers to a material comprising asolanineand achaconine as major fraction with no speciï¬cation on the occurrence of minor gas as well as band cforms of solanine and chaconine similarly when referring to tomato and aubergine the termtga refers to the gas from the corresponding species and forms thereofsupporting information for the assessment chemistrysolanine is one of the ï¬rst alkaloids that has been isolated from nature by desfosses in friedman et al in zwenger and kind reported that solanine contains a glycoside sidechain zwenger and kind only in it was shown that solanine extracted from potato is infact a mixture of two glycoalkaloids gas asolanine and achaconine that share the same solanidineaglycone kuhn and l¬ow since then at least different gas have been isolated and fullystructurally elucidated from over species of the solanaceae family s 13anchezmata et al alsinani and eltayeb the chemical structures and some physical properties of the most importantones are listed in appendix agas are composed of a steroidal aglycone and an oligosaccharide sidechain attached to the 3bhydroxy group of the aglycone see figure friedman et al friedman milner et al the gas of relevance can be divided into the i solanidane group with solanidine as thesteroid backbone and the ii spirosolane group with either the solasodine or the tomatidenoltomatidine backbone gas often contain a double bond between c5 and c6 but the corresponding 5a6hydrogenated forms are also common and in some species eg tomato they constitute the majorcomponents the stereochemistry at carbons c22 and c25 is well deï¬nedtheconï¬guration is 22r 25stheitconï¬guration is 22s 25s friedman et al in solanidineis 22r 25r and in tomatidenoltomatidinein solasodinefurther diversiï¬cation is generated by the composition of the glycoside sidechain most gascontain either a trisaccharide chacotriose or solatriose or a tetrasaccharide lycotetraose ascarbohydrate in commercial potato cultivars solanum tuberosum mostly achaconine and asolaninecomposed ofthe solanidine aglycone and chacotriose and solatriose respectively are presentfigure wild s tuberosum varieties may contain a much wider range of gas friedman et al distl and wink the aubergine fruit derived from s melongena contains primarily asolamargine and asolasonine composed of the solasodine aglycone and chacotriose and solatrioselycopersicum varieties atomatine and arespectivelydehydrotomatine are the major compounds composed of the aglycones tomatidine and tomatidenolrespectively coupled to lycotetraose friedman derived from sin tomato fruitthe preï¬x alpha a refers to the intact glycoside while the preï¬xes beta b gamma c anddelta d refer to the corresponding gas with progressively truncated carbohydrate sidechains due tothe action of enzymatic or acidic hydrolysis friedman milner et al wwwefsaeuropaeuefsa efsa 0cglycoalkaloids in feed and foodohoooohohoohohooohohohsolatrioseohohooohohohsolatrioseohoohoohhh22rnhsolanidine25shhhohsolanine22r 25rnhohsolasodinehhhhooohsolasonineohohoohohohoooohoohoohohoohchacotrioseohohohchacotrioseoohoohohohoooohohohoooohohohohlycotetraoseoohohhhhoohoohoohohhohohoooohoh25sohoh22snhohtomatidinelycotetraoseoohohoohoohooohhhhhhnhsolanidinechaconinehnhohsolasodinehhhsolamarginehhhhnhohtomatidenoltomatinedehydrotomatinefigure s | Colon_Cancer |
gastroenteropancreatic neuroendocrine neoplasms gep nens as well as neuroendocrine tumors gep nets are heterogeneous tumors that originate from peptidergic neurons and neuroendocrine cells previously described as carcinoid tumors in most nets are indolent tumors compared with other epithelial malignancies however they are reported to have the potential to metastasize even in well differentiated tumors and are resistant to therapies1 data from the surveillance epidemiology and end results seer database indicate that the incidence of nets has increased significantly approximately times reaching casesyear of which gep nets account for approximately to of all nets and the correspondence qiang feng department of colorectal surgery national cancer centernational clinical research center for cancercancer hospital chinese academy of medical sciences and peking union medical college no panjiayuan south road chaoyang district beijing peoples republic of china email fengqiang2008vipsinacomsubmit your manuscript wwwdovepresscomdovepresshttp102147cmars256723 cancer management and research wu this work is published and licensed by dove medical press limited the full terms of this license are available at wwwdovepresscomtermsphp and incorporate the creative commons attribution non commercial unported v30 license httpcreativecommonslicensesbync30 by accessing the work you hereby accept the terms noncommercial uses of the work are permitted without any further permission from dove medical press limited provided the work is properly attributed for permission for commercial use of this work please see paragraphs and of our terms wwwdovepresscomtermsphp 0cwu dovepressincidence and prevalence of colorectal nets are inferior only to those of colorectal adenocarcinoma146 in addition the tumor sites varied markedly by race with the incidence of rectal nets among asian populations increasing from per in to per in which was among white populations147 similarly the incidence of rectal neuroendocrine tumors rnets grew fastest among all nets by approximately times compared with the incidence in accounting for of all nets which makes it the second most common net in china8significantly higher than that for localized colorectal nets endoscopic resection including endoscopic mucosal resection emr and endoscopic submucosal dissection esd and surgery including transanal excisions as well as surgical resections are both effective methods for metastatic tumors somatostatin analogs ssas radiation radiofrequency ablation rfa chemotherapy targeted therapy and peptide receptor radionuclide therapy prrt are all alternatives however the 5year survival rate of nets with lymph node metastasis lnm or distant metastasis is still disappointing with fiveyear overall survival rates of approximately and respectively6911the prognostic factors of colorectal nets have been explored by numerous studies tumor stage location size grade lymphovascular invasion and the status of resection margins are major factors that have been reported to be associated with lnm and poor prognosis12 however these studies were highly heterogeneous which affected the accuracy of the metaanalysis and the effectiveness of these scurrently controversies remain in the treatment of colorectal nets experts from the chinese society of clinical oncology csco agreed that colonic nets greater than cm and less than cm could be completely resected endoscopically when the t stage was less than t2 but the national comprehensive cancer network nccn recommends that these tumors be treated by surgery in accordance with the guidelines for colon adenocarcinoma13the aim of this study was to evaluate the outcomes of colorectal nets explore the risk factors for lymph node metastasis in colorectal nets and identify the prognostic factors for survival outcomesmethodsclinical data collectionbetween and a total of consecutive patients received treatments for colorectal nets in our center we constructed a database of retrospectively collected data from patients medical records including clinical characteristics pathological reports recurrence and survival during the followup periodfor radical resection with lymph node dissection lymph node metastasis was detected by pathological evaluation for local excision such as endoscopic mucosal resection emr endoscopic submucosal dissection esd or transanal excision tae lnm was evaluated through computed tomography ct or magnetic resonance imaging mri before the treatment and during the followup periods the diagnosis of a metastatic lymph node was based on the following criteria size criteria short axis diameter of lymph nodes was greater than mm for round lymph nodes and greater than mm for ovoid lymph nodes morphological abnormalities irregular contour and margin unclear border heterogeneous internal echoes or signal intensity1617 the tumor diameter refers to the longest diameter of the tumor according to pathology reports for patients with distant metastases tumor diameter was determined by endoscopic findings before treatmentpathological diagnosisthe tumor stage was classified according to the american joint committee on cancer ajcc cancer staging manual 7th edition and 8th edition1819 and the tumor grade was classified according to the classification20 for patients before we revised their pathology results and found that they were all neuroendocrine carcinomas necs therefore we classified them as having g3 grade tumors the mitosis count n23 was expressed as the number of mitotic cells in ten highpower fields hpfs from hematoxylin and eosin hestained slides examined with microscopy according to enetswho guidelines g1 grade mitotic image hpfs g2 grade mitotic image hpfs and g3 grade mitotic images2010 hpfs the ki index was calculated as the percentage of cells labeled by immunohistochemistry according to enetswho guidelines g1 grade ki67 positive index g2 grade ki67 positive index to and g3 grade ki67 positive index20 the expression levels of chromogranin a cga n101 and synaptophysin syn n109 were scored according to the percentage of positive cells and the intensity of cell staining the positive cell percentage score was based on the following system points no positive cells point positive cells accounting for to points positive cells accounting for to points submit your manuscript wwwdovepresscom dovepress cancer management and research 0cdovepress wu positive cells accounting for to and points positive cells accounting for to the positive cell staining intensity score was based on the following system points negative point weakly positive points moderate positive and points strong positive the two scores were multiplied together points for negative to points for weak positive to points for moderate positive and to points for strong positive inclusion criteriapatients who were treated in our center for localized and metastatic colorectal nets from to exclusion criteria patients who had colorectal nets combined with other malignancies patients for whom the pathological diagnosis was mixed adenoneuroendocrine carcinoma patients for whom there was insufficient clinical inappropriate pathology reports information or from outside hospitalsrisk factors for lymph node metastasis and prognostic factors related to survival were investigated in all patientsthe primary endpoint was progressionfree survival pfs which was defined as the interval between initial treatment and the first documentation of disease progression or deathstatistical analysisstatistical analysis was performed using spss for mac version spss chicago il usa continuous data are described as means±sds in this study the risk factors for lnm were assessed using pearsons Ï2 test in univariable analysis and logistic regression analyses in multivariable analysis the 5year overall survival os and progression free survival pfs were analyzed with the kaplanmeier method variables were compared with the log rank test and the multivariable analysis for survival outcomes was conducted using the cox proportional hazards model statistical significance was accepted for p values female ratio was the frequencies of grade g1 g2 and g3 nets were and respectively of the patients patients were resected locally by emr by esd and by transanal excision in addition nets were surgically resected including radical resections multivisceral resections and palliative resections due to distant metastasis the remaining patients were treated by systemic treatment due to distant metastasis the most commonly used chemotherapeutic regimen in our center was the ep regimen etoposide and cisplatin as the firstline chemotherapy and the secondline chemotherapy was variable and included the xelox regimen oxaliplatin and capecitabine the folfox regimen oxaliplatin calcium folate and 5fu and everolimus temozolomide and tegafurgimeraciloteracil and combinations between them the tumor diameter was less than cm in patients and the distance from the anal verge was less than cm in patients lnm was found in cases and distant metastasis occurred in patients two patients had radiologically determined lnm after tae in the followup period and one of them went on to undergo radical surgery the other patient was also found to have liver metastasis therefore he was treated with chemotherapy the clinical and histopathological characteristics are summarized in table risk factors for lnmthe risk factors for lnm through univariate analysis were tumor location in the colon p0001 tumor diameter ¥ cm p0001 t stage p0001 tumor grade p0001 and the positive degree of syn p0012 and cga p0049 table in multivariable analyses tumor diameter ¥ cm or ci p0040 and tumor grade g3 or ci p0001 were independent risk factors for lnm in colorectal ntes tumor location in the colon or ci p0083 and tumor grade g2 or ci p0066 might be independent risk factors for lnm even though the p value was greater than table resultsclinical and histopathological characteristics figure a total of patients were included in our study figure the age of the patients was ± years and the male risk factors for survival outcomesthe median followup period was months range months a total of patients died in this cohort in patients with distant metastasis before treatment patients died during chemotherapy cancer management and research submit your manuscript wwwdovepresscom dovepress 0cwu dovepressfigure flowchart of the selection of patientspatients died after multivisceral resections and patients died after palliative resections due to tumor progression in patients without distant metastasis before treatment patients died due to the recurrence of distant metastasis at the liver peritoneum lung pleura and brain and patient died of severe lung infections the 5year progressionfree survival pfs and overall survival os rates of all patients were and respectively the prognostic factors for the 5year pfs and os rate in all patients were age neoadjuvant chemotherapy tumor diameter tumor location tumor grade lnm cga level and treatment method table in the multivariable analysis age ¥ hr ci p00020001 and lnm yes hr ci p00180025 were independent risk factors for 5year pfs and os the cga level [moderate positive hr ci p0010 and strong positive hr ci p0007] were independent risk factors for 5year pfs tumor diameter ¥ cm hr ci p0063 and tumor grade g3 hr ci p0090 were independent risk factors for 5year pfs even though the p value was greater than table comparison of two age groupsin univariable analyses patients were in the 65year group and patients were in the ¥65year group the proportion of tumors with a diameter ¥ cm was significantly higher in the ¥65year group than in the 65year group vs p0016 there were also significantly more patients with lnm in the ¥65year group vs p0041 for t stage the proportion of earlystage t1 patients in the ¥65year group was significantly less than that in the 65year group although p was vs p0086 for treatments there were significantly more patients who were treated with systematic chemotherapy in the ¥65year group vs p0040different operative methods for t1n0m0 colorectal netsthere was no significant difference in tumor grade tumor location surgical margin relapse or 5year os except for tumor diameter p0012 the diameters of tumors resected by emr esd and transanal excision were ± cm ± cm and ± cm respectively table submit your manuscript wwwdovepresscom dovepress cancer management and research 0cdovepress wu table clinical and histopathological characteristics of colorectal nets n135table continued variablesage¥sexmalefemalebmi¥smoking historydrinking historyfamily cancer historyyesnoneoadjuvant chemotherapyyesnoneoadjuvant radiotherapyyesnodistance from the anal verge cm¥tumor locationrectumcolonappendixtreatmentemresdtransanal excisionradical resectionmultivisceral resectionpalliative resectionsystemic treatmenttumor diameter cm¥t stage after chemotherapyxn variableslnmnegativepositivedistant metastasisnegativepositivetumor gradeg1g2g3cga n101negativesyn n109negativen kaplanmeier survival curveskaplanmeier survival curves for os and pfs according to tumor grade diameter location and cga level are shown in figures discussionnets have a relatively good prognosis with a median os time reported to be years months and a 5year os rate of however the survival outcomes varied significantly at different stages of nets the 5year os rate of stage i and ii tumors was reported to be as high as and but dropped to and for stage iii and iv tumors respectively22 according to epidemiological data from the seer database and gkr joint cancer registry the 5year os rates of lymph node metastases stage iiib and distant metastases stage iv are and respectively1610 the 5year os rates of all patients and patients without distant metastasis were and respectively and the 5year pfs rates were and respectively lymph node metastasis is the most important factor that determines the prognosis of nets and the prediction of lymph node continuedcancer management and research submit your manuscript wwwdovepresscom dovepress 0cwu dovepresstable univariable analysis for risk factors for lymph node metastasis n table multivariable analysis for risk factors for lymph node metastasis n135lnm n Ï2 valuep valuen hr cip valueage¥tumor diameter¥ tumor locationrectumcolonappendix tumor gradeg1g2g3t staget0t1t2t3t4cganegativesynnegative notes by logistic regression analyses p values abbreviation hr hazard ratiomost nets are at the g1 phase and g2 or g3 phases account for only to of all nets and have been reported to be risk factors for lnm by numerous studies1223 a multicenter clinical study in china showed that pathological type g3 nec is an independent risk factor affecting the prognosis of patients with rectal nets p similarly sohn et al3 found that the lnm rate of g1 phase rectal nets was only but it increased remarkably to at the g2 phase in our study our results showed that histological tumor grades g2 p and g3 p0001 were independent risk factors for lymph node metastasis lymph node metastasis occurred in of patients with g3 tumors and with g2 tumors but only with g1 tumors the 5year variablessexmalefemaleage¥bmi¥smoking historyyesnodrinking historyyesnotumor locationrectumcolonappendixtumor diameter¥t staget0t1t2t3t4 tumor gradeg1g2g3cga n101negativesyn n109negative notes by pearsons Ï test p values metastasis is necessary for clinicians to choose a suitable treatment the aim of our research was to explore the predictive factors for lymph node metastasis of colorectal nets and assess the current therapeutic algorithmsubmit your manuscript wwwdovepresscom dovepress cancer management and research 0cdovepress wu table prognostic factors for survival outcomestable continued variables5year pfs 5year os Ï2 valuep valueg3lnmyesnocga negativesyn negativetreatment variablessexmalefemalebmi¥age¥smoking historyyesnodrinking historyyesnoneoadjuvant radiotherapyyesnoneoadjuvant chemotherapyyesnotumor locationrectumcolontumor diameter cm¥t stagextumor gradeg1g25year pfs 5year os Ï2 value p value continuedemresdtransanal excisionradical or palliativesurgerysystemic treatmentnotes kaplanmeier method and log rank test p values os rate decreased sharply from to when the tumor grade increased from g3 to g1 however tumor grade was not significant in survival outcomes possibly because tumor grade affects survival outcomes indirectly by directly affecting lymph node statustumor size has been reported to be a strong predictive factor for lymph node metastasis previous studies have shown that a tumor less than mm is usually limited to the submucosa with a low metastasis risk of less than and the 5year survival rate can reach approximately to according to the enets guidelines surgical treatment is recommended if rnets are greater than cm g1g3 or are g3 phase with or without metastasis endoscopic resection is feasible when the tumors are less than cm g12 phase and t1 stage15 the treatment of rnets in western countries and in china is similar but there have been controversies regarding the treatment of 2cmsized cnets chinese experts agree that endoscopic resection can be considered for cnets less than cm however there is cancer management and research submit your manuscript wwwdovepresscom dovepress 0cwu dovepresstable multivariable analysis for survival outcomes5year pfsos hr cip valueage ¥tumor diameter¥tumor locationrectumcolonappendixlnmnoyescganegativesynnegativetumor gradeg1g2g3neoadjuvant chemotherapynoyestreatmentemresdtaesurgerysystemic treatmentnotes by the cox proportional hazards model p values abbreviation hr hazard ratio0019987800003633e1700003066e29200001826e4500343994900007412e29010015970800001686e30no explicit mention of treatment in the enets guidelines and experts from the nccn recommended surgical resection instead of endoscopic resection1315 in our study survival curves were significantly better p0001 among patients with tumors less than cm figure a tumor diameter greater than cm was an independent risk factor for lnm p0040 table and we believe this was due to the small sample size however patients with tumors less than cm had lnm and patients with tumors less than cm also developed lnm the lnm in small nets might be due to the tumor cells extending to the submucosal layer which has abundant lymphatic vessels for them to spread through previous studies have reported that small nets also have malignant potential26 therefore even if tumor size was submit your manuscript wwwdovepresscom dovepress cancer management and research 0cdovepress wu table comparison of different treatment methods for t1n0m0 colorectal netsemresdtransanal excisiong gradeg1g2g3tumor diameter±±±tumor locationrectumcolonappendixsurgical marginpositivenegativerelapse cases5year os rate notes by anova p values Ï2f valuep valuea strong predictive factor for lnm lnm should not be predicted only by tumor size and further examinations such as eus or ct can help us to evaluate the status of lymph nodes more specificallychromogranin a cga and synaptophysin syn are two neuroendocrine differentiation ned immunohistochemistry markers frequently used in nets in univariable analysis there was a significant difference for colorectal nets with different expression levels of cga in terms of both risk factors for lnm and survival outcomes both p005 in multivariable analysis moderate positive p0010 and strong positive cga p0007 were independent risk factors for 5year pfs which has been proven in a wide variety of retrospective analyses27 in addition prospective clinical trials radiant1 and have been performed to assess the prognostic value of cga in advanced nets and the results showed shorter os for patients with elevated cga31 however the increase in the expression level of cga was not proportional to the increase in the lnm rate or 5year os rate and cga was negative in patients with lnm which may affect the accuracy of the prognostic value of cga lindholm et al35 also found that a relevant portion of nets do not show elevated cga levels the major problem is that several confounding factors can including gastrointestinal and affect cga levels figure a pfs curves according to tumor grade b os curves according to tumor gradecancer management and research submit your manuscript wwwdovepresscom dovepress ab050100150200020406080100progressionfree survivalg1g2g3 0cwu dovepressfigure a pfs curves according to tumor diameter b os curves according to tumor diameterfigure a pfs curves according to tumor location b os curves according to tumor locationfigure a pfs curves according to age b os curves according to agesubmit your manuscript wwwdovepresscom dovepress cancer management and research ababab 0cdovepress wu figure a pfs curves according to cga level b os curves according to cga levelcardiovascular disorders or proton pump inhibitor ppi consumption and a variety of other nontumor reasons36 regarding syn there was only a significant difference in the univariable analysis for lnm previous studies have shown that patients with a low level of synaptophysin had a better os rate than those with a high level however the small sample size limited the accuracy of the results37 based on the findings of this research and previous studies38 it can be suggested that ned might affect the survival outcomes of colorectal net patients and markers especially cga might be suitable for clinicians to predict the prognosis of patientsthe location of the tumor is also an important factor affecting the prognosis and treatment of nets tumors in the colon are more common in necs and generally have a worse prognosis with higher metastatic potential than tumors in the rectum the outcomes of neuroendocrine tumors from the right hemicolon of the midgut and from the left hemicolon from the hindgut are not the same the welldifferentiated biological behavior of the left hemicolon is closer to that of the rectum a recent chinese multicenter study found that more than of colonic nets of midgut origin are necs or mixed adenoneuroendocrine carcinomas manecs39 according to statistics from the seer database the 5year survival rate of patients with rnets is which is significantly higher than that of colonic nets in our study the lymph node metastasis rate in the colon was which was significantly higher than that in the rectum the 5year os rate and pfs rate of individuals with lnm in the appendix and in the rectum were significantly better than those of individuals with lnm in the colon p0005 and p0003 respectively which was consistent with previous studies1 based on the findings of this research and previous studies it can be suggested that colonic nets should be completely resectedin our research the survival outcomes of patients years and older in our study were worse than those of patients younger than years p0001 we also found that tumors from elderly patients ¥ years were larger and more advanced than those from younger patients years both p0001 the reason for the poor prognosis in elderly patients may be that elderly patients have lower tolerance to surgical trauma and side effects of chemotherapy because of their weakened an physiological functions which leads to multiple complications4142 therefore when we encounter elderly patients minimally invasive therapies such as laparoscopic surgery could help reduce surgical trauma and chinese herbs can relieve and reduce the adverse events of chemotherapy43for nets in the colon the recommended treatment varies among different guidelines but surgical resection is generally recommended because of the greater likelihood of malignant behavior than with rectal nets endoscopic resection may be considered if the tumor diameter is less than cm and does not reach the muscularis propria for nets in the rectum eus is required before surgery surgical resection is recommended when the tumor diameter is more than cm g3 grade t3 to t4 stage or when there is peripheral lymph node metastasis when the tumor diameter is less than cm g1 or grade and t1 stage endoscopic resection is feasible in other cases the treatment method is determined according to the depth of tumor invasion assessed by eus21cancer management and research submit your manuscript wwwdovepresscom dovepress 050100150200020406080100months progressionfree survivalnegativeweak positivemoderate positivestrong positivep0023050100150200020406080100months overall survivalnegativeweak positivemoderate positivestrong positivep0038ab 0cwu dovepressthis study has some limitations including its retrospective design and the relatively small number of patients included although lnm should be evaluated after radical resection with lymph node dissection we analyzed the risk factors for lnm by ct or mri in those who underwent local excision before the treatment and during the follow up periods and we believe the results are reliable because this study lasted more than years we could investigate the longterm survival outcomes and prognostic factors after different treatments even with the small number of patients finally further studies should be performed to validate our main sthe clinical and pathological characteristics of rectal and colon neuroendocrine tumors are different | Colon_Cancer |
"ammatory bowel disease ibd is a long life disease with remission and relapse periods ibd arises as a result of inappropriateimmune response to intestinal commensal anisms in individualswith genetic predisposition and consequently causes ammation andintestinal ulcers in addition ibd has a complex pathogenesis andmany factors such as dysbiosis oxidative stress and epigenetics thatmay also be involved in disease pathogenesis [] ulcerative colitisuc and crohn's diseases cd are known as two main forms of ibdaccordingly these diseases cause intestinal ulcers and some annoyingsymptoms such as diarrhea abdominal pain and rectal bleeding occasionally the severity of these symptoms is very high which can leadpatients to be hospitalized in this regard therapeutic approaches totreat these diseases mainly focus on prolonging remission and are almost similar however diï¬erential diagnosis can also help to treat thedisease in a more eï¬ective way for example 5asa which is acommon drug in the treatment of ibd is less eï¬ective on maintainingremission in cd patients on the other hand antibiotic therapy is notrecommended for the treatment uc but it can be eï¬ective on cd patients diï¬erential diagnosis is a serious challenge because cdand uc have significant similarities in terms of their clinical endoscopic and histological features however there are some diï¬erencesbetween uc and cd which are summarized in table1 in addition tointestinal complications uc and cd also have significant extraintestinal manifestations for example it was shown that uc is significantly associated with primary sclerosing cholangitis and cd is alsoassociated with cholelithiasis especially in cases that the ileum is involved furthermore cd can cause ï¬stulas to the urinary systemwhich leads intestinal bacteria to enter the urethra and recurrent urinary tract infections both cd and uc can cause several disorderssuch as arthritis erythema nodosum pyoderma gangrenosum andanemia which are known as the most important extraintestinal manifestations of ibd the latest statistics showed that the global corresponding author at department of clinical biochemistry and laboratory medicine faculty of medicine tabriz university of medical sciences daneshgahstreet po box tabriz iranemail address vagharimtbzmedacir m vagharitabari101016jcca202008025received july received in revised form august accepted august available online august elsevier bv all rights reserved 0cf khakikhatibi table1clinical endoscopic and histological features of cd and ucclinical featuresfeaturesrectal bleedingabdominal painfevermucus defectionintestinal obstructionperineal diseasepostoperative recurrenceasca positiveanca positiveendoscopic featurescdoccasionallyfrequentlyfrequentlyoccasionallyyesyesyesfrequentlynot commonucfrequentlyoccasionallynot commonfrequentlynonononot commonfrequentlyfeaturescduclocationmucosal involvementdepth of ulcerationï¬stulacobblestone appearanceaphthous ulcerationmucosal friabilityhistological featuresfeaturesgranulomascrypt abscessespatchinessany part of gi tractdiscontinuousdeepyesyesfrequentlynot commoncdfrequentlynot commonfrequentlycolon and rectumcontinuoussuperï¬cialnonooccasionallyfrequentlyucrarefrequentlynot commonprevalence of ibd currently is on the rise and it is not an exaggerationif we consider it as a global serious health problem according to areport published in ibd has the highest prevalence rate ineurope and its prevalence in the newly industrialized countries of asiaafrica and south america also appears to be increased over the pastthree decades unfortunately the peak of the disease is at the young age of years old therefore in addition to the suï¬ering from icts on the patients it also has many negative eï¬ects on societymoreover many ï¬nancial burdens are annually imposed on countriesfor controlling and treating this chronic disease the invasive diagnosticand therapeutic measures are currently undertaken to diagnose andmanage ibd which are unpleasant for patients as well as having thehigh associated costs now the gold standard method for diagnosingibd and monitoring patient status is performing colonoscopy examination and histopathologic evaluation which are invasive measures therefore in recent years many studies have been conducted toï¬nd a suitable laboratory marker with suï¬cient sensitivity and speciï¬city for the purpose of diagnosing and noninvasive management ofibd a high proportion of these studies have investigated the eï¬cacy offecal calprotectin in diagnosing and monitoring patients althoughsome of these studies reported auspicious results there are still somedoubts on the eï¬ectiveness of fecal calprotectin on diagnosing andmonitoring ibd patients so in this review we addressed the advantages and limitations of fecal calprotectin for the diagnosis andmanagement of ibd the role of fecal calprotectin in diagnosis and management ofibdthe eï¬cacy of fecal calprotectin as an laboratory marker in various areas of ibd diagnosis and management have been studied including ibd diï¬erentiation from irritable bowel syndrome ibs evaluation of endoscopic activity of the disease evaluation of histologicalactivity of the disease and prediction of disease recurrence andclinica chimica acta response to treatment in following after a brief introduction andmentioning the important points regarding laboratory measurement offecal calprotectin we reviewed the most interesting ï¬ndings in all ofthe abovementioned areas calprotectin a clinically valuable proteincalprotectin is an antimicrobial protein mainly secreted by neutrophils this protein competes with bacteria over zinc thus kills thebacteria however this is not the only contribution that it has to antimicrobial activity moreover this protein has many potential clinicalapplications such as the elevated serum levels that have been observedunder various immunological and immunopathological conditionsserum calprotectin levels rapidly increase in response to bacterial infections in the kidney and heart or during transplant rejection at theearly stages of ammation of the lung serum calprotectin can also beconsidered as a reliable marker besides plasma levels of calprotectinappear to be useful in reï¬ecting disease activity in ammation of thejoints in addition it has been demonstrated that serum calprotectin levels are increased in patients with bacterial sepsis so it can beconsidered as a reliable biomarker in neonatal sepsis the serumlevel of calprotectin increases as well as a sensitivity of and aspeciï¬city of that have been reported for serum calprotectin indiagnosis of neonatal sepsis it has been recently shown thatserum calprotectin levels increase in patients with aneurysmal subarachnoid hemorrhage and higher levels in the ï¬rst of onset areassociated with a poor prognosis at the ï¬rst three months serumcalprotectin levels also increase in patients with rheumatoid arthritisand even in patients with a moderate to high disease activity who havenormal or low crp levels so they appear to be more eï¬cient at reï¬ecting disease activity some studies have also investigated the eï¬cacy of serum calprotectin in the diagnosis of cancers correspondingly in one of thesestudies it was shown that serum calprotectin levels significantly increased in patients with laryngeal carcinoma compared with healthyindividuals and those with benign laryngeal pathologies moreover inthis study a direct relationship was also observed between serum levelsof calprotectin and stage of cancer another study showed that theserum level of calprotectin increased in patients with papillary thyroidcarcinoma but it significantly decreased after operation alsoregarding the eï¬cacy of serum and saliva calprotectin for the diagnosisof ibd impressive results have been reported a study onpatients with ibd both uc and cd have shown that serum calprotectinlevels were directly correlated with fecal calprotectin levels and weremore potent in ibd diagnosis compared to crp and albumin this studyalso indicated that the combination of serum calprotectin with crp oralbumin can be helpfulin the prediction of treatment escalationespecially in patients with cd however no significant correlationwas observed between serum calprotectin and fecal calprotectin levelsin patients with cd and uc as well as a slight correlation betweenserum calprotectin level and crp that was observed only in patientswith uc another study showed that the serum level of calprotectin was significantly higher in patients with cd compared to healthyindividuals in addition although a significant correlation was observedwith the clinical activity of the disease no significant correlation wasfound between the level serum calprotectin and endoscopic activity ofthe disease the eï¬cacy of salivary calprotectin in the diagnosisof ibd has also been studied which showed that salivary calprotectinsignificantly increased in patients with ibd compared to healthy individuals in this study auc values for unstimulated saliva and stimulated saliva to distinguish ibd patients from healthy individualswere reported to be and respectively however thepopularity of calprotectin is mainly due to the use of fecal calprotectinin the diagnosis and management of ibd that is discussed in the following 0cf khakikhatibi clinica chimica acta laboratory measurement and reference intervalfecal calprotectin is a stable protein that remains stable for daysat room temperature this property is an excellent advantage for alaboratory marker also it seems that keeping the specimen at refrigerated temperature °c can increase the stability of fecal calprotectin however evidence has been obtained regarding thatthe stability of this protein decreases after staying for three days atroom temperature on the other hand it is not also recommended tokeep samples in the refrigerator for more than days it seemsthat fecal calprotectin remains stable up to one year at °c measurement of fecal calprotectin can be done both qualitatively andquantitatively accordingly in the qualitative measurement monoclonal antibodies are used to detect fecal calprotectin and the positiveresults are characterized by the appearance of colored lines on the testcassette however in the qualitative one only positive or negative results are reported and despite of sensitivity test speciï¬city in theevaluation of disease activity was reported to be only it seems thatthe main application of this test is to diï¬erentiate healthy individualsfrom ibd patients rapidly however some studies have shown that it isnot accurate enough in this case as well nevertheless asignificant concordance has been reported between home test resultsibdoc and fecal calprotectin laboratory measurement results whenquantum blue calprotectin elisa kit was used notably the agreements between results were and depending on the selectedcutoï¬s several commercial kits are also available for fecal calprotectin qualitative test known as rapid calprotectin these tests reportpositive results ranged from to µgg there are also severalcommercial kits that can be used for the quantitative measurement offecal calprotectin these kits are usually designed in terms of the elisamethod and some have a measurement range between and µgg moreover the chemiluminescence immunoassays cliamethod can also detect values between and µgg fluoro enzyme immunoassays feia and particle enhanced turbidimetric immunoassays petia can also be used for the measurement of fecalcalprotectin in this regard one of the most serious challenges to thelaboratory evaluation of fecal calprotectin is the determination of theupper limit in healthy individuals among healthy adults there is asignificant agreement on µgg as an upper limit one study suggested values up to µgg in people over years old and up to µgg in children aged between and years old as referenceranges of fecal calprotectin in healthy individuals fecal calprotectin levels appear to be higher in healthy infants andchildren under four years old than in adults and further studies areneeded in this regard to determine the acceptable upper limit for diagnosis of pediatric ibd table lists the median levels of fecalcalprotectin in healthy individuals with diï¬erent ages reported in somestudies according to these reports age can aï¬ect fecal calprotectinlevels fecal calprotectin and ibd diagnosisonly a small percentage of patients complaining of abdominal painand diarrhea have ibd in many cases ibs as a functional gastrointestinal disorder is known as the cause of such clinical symptomspatients with ibs have normal colonoscopy results while ibd patientsindicate abnormal colonoscopy results and have intestinal ulcersunfortunately the significant prevalence of ibs and the overlap between clinical symptoms and ibd can increase the colonoscopy ratetherefore a noninvasive diagnostic marker can be very helpful in thisregard notably the ï¬rst evidence of the eï¬cacy of fecal calprotectin inthe diagnosis of ibd was obtained in the 1990s røseth in proposed a method for measuring calprotectin in stool specimens one of the ï¬rst and most interesting studies regarding fecal calprotectinutility in ibd diagnosis was the study by røseth published in in this study patients with ulcerative colitis were studied and according to their results fecal calprotectin levels are higher in patientswith ulcerative colitis compared to healthy controls this study havealso shown that even patients with low disease activities had higherlevels of fecal calprotectin compared to healthy individuals subsequent studies somehow conï¬rmed and complemented the ï¬ndings of this study in another study published in auc values of ci were reported for fecal calprotectin in thediagnosis of colorectal ammation moreover in a study onchildren with ibd it was shown that the level of fecal calprotectin washigher in these patients compared to healthy children so it can beconcluded that it is also directly correlated with esr levels in astudy published in kolho reported auc values of ci for fecal calprotectin in the diagnosis of pediatric ibd in a study on patients with crohn disease a sensitivity of and a speciï¬city of at cutoï¬ of μgg have been reportedfor fecal calprotectin in diagnosis of the disease the results of ourrecent study along with other studies showed that fecal calprotectin ispreferred over traditional ammatory biomarkers such as crp andesr in the diagnosis of ibd diamanti reported a sensitivity of and a speciï¬city of for fecal calprotectin at a cutoï¬ of μgg in ibd diagnosis in our recent study a sensitivityof and a speciï¬city of at a cutoï¬ of μgg were observed for fecal calprotectin in the diagnosis of ibd however oursample size was and the majority of patients were in the active phaseof the disease in another study conducted on patients with ulcerative colitis asensitivity of and a speciï¬city of at cutoï¬ of μgg havebeen reported in this regard in one study it was shown that fecalcalprotectin in cutoï¬ of μgg is able to distinguish patients withibd from patients without ibd patients with diseases other than ibdpatients with ibs and healthy persons with sensitivity and speciï¬city caviglia in their study reported a sensitivity of and a speciï¬city of at a cutoï¬ of μgg for fecalcalprotectin in diï¬erentiating between ibs and ibd howeversome studies have reported significantly lower values accordingly in astudy on patients with ulcerative colitis kalantari reported asensitivity of and a speciï¬city of at a cutoï¬ of μgg besides there is a considerable agreement between fecal calprotectinand capsule endoscopy ï¬ndings in patients with crohn's disease asensitivity of and a speciï¬city of have also been reported at acutoï¬ of mgkg for fecal calprotectin in predicting ce ï¬ndings anddiagnosis of crohn's disease in another study lower sensitivityand speciï¬city rates sensitivity speciï¬city were reportedfor fecal calprotectin in this regard furthermore in one studythat examined the eï¬cacy of fecal calprotectin in predicting wirelesscapsule endoscopy ï¬ndings a sensitivity of and a speciï¬city oftable reported median levels of fecal calprotectin in healthy individuals of diï¬erent agesagesmedian levels of fecal calprotectin range µggnumber of subjectsused kitup to monthchildren yearschildren yearsadultsover years bühlmann elisabühlmann elisacalpro® calprotectin elisa test alpphicalphicalreference 0cf khakikhatibi were reported for this biomarker at μgg in the diagnosis ofsmall bowel ammation in crohn's disease given these ï¬ndings it seems that fecal calprotectin has no ideal sensitivity and speciï¬city for the diagnosis of ibd where the small intestine is involvedbesides there are some preanalytical limitations which are explainedin the next sections therefore optimistically speaking fecal calprotectin measurement can eliminate the need for colonoscopy howeverin a metaanalysis performed to evaluate the eï¬cacy of fecal calprotectin and some other ammatory markers to diï¬erentiate betweenibd and ibs the probability of ibd was less than at fecal calprotectin values lower than µgg or crp values lower than mgdl therefore it seems that fecal calprotectin can be helpful at leastin ruling out the possibility of ibd in patients with ibslike symptoms aswell as reducing the rate of colonoscopy moreover it should be notedthat although a systematic review has reported pooled sensitivity andspeciï¬city above for fecal calprotectin to diï¬erentiate between ibdand ibs it emphasized more on the possibility of falsepositive resultsin low cutoï¬ points hence performing extensive studies indiï¬erent countries on the healthy population and the ibd patient is beneeded to determine a suitable cutoï¬ with maximum sensitivity andspeciï¬city and minimum falsepositive resultstable summarizes the results of various clinical investigationsregarding fecal calprotectin utility in the diï¬erential diagnosis of ibdfrom ibs and table4 summarizes some metaanalysis results in thisregard as shown in table the most important limitation of the majority of clinical studies conducted to date is the small sample size alarge global study may be helpful in providing a more precise evaluation of fecal calprotectin clinical value in discrimination between ibdand nonibd diseases fecal calprotectin and endoscopic and histologic activity evaluationundoubtedly one of the most serious challenges in the managementof ibd is evaluating the endoscopic and histologic activities of thedisease nowadays colonoscopy and histopathologic examinations arethe routine tools for the assessment of mucosal healing in patients withibd as noted earlier several scoring systems have been devised toscore disease activity based on the ï¬ndings of colonoscopy and histopathologic examinations in recent years many promising results havebeen reported regarding the correlation between these scores and fecalcalprotectin levels in addition many studies have been performed inthe last decade all of which cannot be reviewed in this article the ï¬rstevidence of a link between fecal calprotectin and disease endoscopicactivity was obtained in the late 1990s in one of the ï¬rst studiesroseth found a significant correlation between fecal calprotectinlevels and endoscopic and histologic activities in patients with ulcerative colitis furthermore in another study they observed that ibdpatients who were in remission clinically and had normal fecal calprotectin levels less than mgl had normal colonoscopy results these interesting ï¬ndings indicate that fecal calprotectin can beconsidered as a biomarker in the evaluation of endoscopic activity andclinica chimica acta table4summarized results of some metaanalysis regarding the utility of fecal calprotectin in discrimination between patients with ibd and without ibdsample sizepooled sensitivitypooled speciï¬cityreferences mucosal healing in ibd patients also these studies were the startingpoint of extensive studies that have been conducted up to now in astudy conducted on patients with crohn's disease sipponen investigated the sensitivity and speciï¬city of fecal calprotectin in predicting endoscopic activity of crohn's disease correspondinglythe researchers used the crohn's disease endoscopic index of severitycdeis scoring system in their study to evaluate the endoscopic activity of crohn's disease as a result they found that there was a significant correlation between the endoscopic activity of the disease andthe level of fecal calprotectin besides the ï¬ndings of this study demonstrated that fecal calprotectin at µgg cutoï¬ can predict theendoscopic activity of crohn's disease with sensitivity and speciï¬city in another study cdeis and mayo disease activity indexmdai were used to evaluate the endoscopic activity of crohn's diseaseand ulcerative colitis respectively according to the results of thatstudy on ibd patients there was a significant correlation between fecalcalprotectin levels and disease endoscopic activity another studyshowed that fecal calprotectin is more strongly correlated with theendoscopic activity of the disease in ulcerative colitis compared to therachmilewitz clinical activity index in addition in this study theoverall accuracy of fecal calprotectin for endoscopically active diseaseidentiï¬cation was obtained as some studies have also shown the superiority of fecal calprotectinover traditional ammatory markers like crp besides one studyfound that fecal calprotectin was more strongly correlated with thesimple endoscopic score for crohn's disease sescd compared to thecrp and even crohn's disease activity index cdai the modiï¬edbaron index was also used in another study to evaluate the endoscopicactivity of ulcerative colitis as a result it was shown that calprotectinis more strongly correlated with the endoscopic activity of ulcerativecolitis compared to crp and clinical activity of the disease in thisregard similar results were also observed in our recent study in whichthe ulcerative colitis endoscopic index of severity uceis and sescdwere used therefore fecal calprotectin appears to be superior totraditional ammatory markers in the prediction of ibd endoscopicactivity the high values of sensitivity and speciï¬city that were mentioned earlier have raised the hope that using fecal calprotectin canreduce colonoscopy rate for patients monitoring however severalrecent studies have reported some significantly lower values accordingly in a recent study in which mayo endoscopic score [mes] wasused to evaluate the endoscopic activity of ulcerative colitis atable summary of the results of some studies regarding the utility of fecal calprotectin in discrimination between patients with ibd and without ibdnumber of ibd patientsage grouplocationcut oï¬sensitivityspeciï¬city cd and uc cd and uc cd and uc and unclassiï¬ed68cd and uc cd and uc and unclassiï¬ed cd and uc cd and uc uc cd ucadultsadultsadultsboth adult and pediatricpediatricadultspediatricadultsadultsboth adult and pediatrictaiwanchinaitalyspainfinlandiranitalyirandenmarkindia48µgg µgg150µgg150µgg595µgg784µgg160µgg164µgg150µgg188µggaucreferences spsreï¬dbib60 0cf khakikhatibi clinica chimica acta table summary of the results of some studies regarding the correlation of fecal calprotectin with endoscopic activity in ibd patientsage groupstudylocationusedendoscopicactivity indexcorrelationcoeï¬cientrreferencenumberof ibdpatients cd uc uc cd ucadultsadultsadultsadultsadultsfinlandiranswitzerlandswitzerlandswitzerland modiï¬edcdeisuceisrachmilewitzsescd uc cdadultsadults uc cd uc cd cd uc ucadultsadultsadultsadultsadultsadultsadultsbaron scorerachmilewitzsescdgermanyusa andcanadajapanitalyitalybrazilfrancefrancesouth korea uceismattssescdmayo scoresescdcdeismayo score sensitivity of and a speciï¬city of were reported for fecalcalprotectin at µgg to diï¬erentiate active endoscopic from inactivemes or from mes or in another study the sensitivityand speciï¬city of fecal calprotectin at a cutoï¬ of µgg for diï¬erentiating mes ¤ in patients with ulcerative colitis were and respectively overall as presented in table several studiesperformed in diï¬erent countries reported the correlation between fecalcalprotectin and ibd endoscopic activity although some of these studies reported a strong correlation some others reported a relativelyweak correlation as noted earlier there are significant diï¬erencesbetween the reports on the sensitivity and speciï¬city of fecal calprotectin to predict the endoscopic activity of ibd undoubtedly a widerange of factors from sample size and the inclusionexclusion criteriato preanalysis variables and indexes used to evaluate the endoscopicactivity may also contribute to these diï¬erences however fecal calprotectin does not appear to be a very reliable marker for the predictionof ibd endoscopic activity so currently it seems a bit optimistic toconsider fecal calprotectin as a reliable alternative for colonoscopy inthis regard further studies are still needed however under some certain circumstances such as pregnancy or pandemics the use of fecalcalprotectin to evaluate ibd endoscopic activity can be helpfulpregnant patients with ibd have serious limitations for colonoscopyexamination and it has been recommended that colonoscopy should beonly performed in the second trimester of pregnancy and where there isa strong indication therefore noninvasive markers such as fecalcalprotectin can be helpful during pregnancy in one study physicianglobal assessment [pga] which is a clinical symptombased criterionwas used to evaluate ibd activity and subsequently the associationbetween fecal calprotectin and this criterion was investigated in pregnant women with ibd the results of this study showed a significantcorrelation between fecal calprotectin and pga levels at prepregnancyduring pregnancy and postpartum stages in another study asignificant association was reported between fecal calprotectin levelsand clinical activity of ibd in pregnant women moreover it was shownthat stool calprotectin at a cutoï¬ of mgkg had a sensitivity between and as well as a speciï¬city between and in the assessment of ibd clinical activity at diï¬erent stages ofpregnancy a recently published systematic review has also conï¬rmed the conclusions obtained from these studies according tothese results it seems that fecal calprotectin is not aï¬ected by physiological changes during pregnancy however it is significantly correlatedwith ibd clinical activity during pregnancy therefore from the viewpoint of relatively acceptable sensitivity and speciï¬city in predictingthe endoscopic activity of ibd fecal calprotectin may be considered as anoninvasive biomarker for the evaluation of ibd endoscopic activity inpregnant women in addition under pandemic conditions fecal calprotectin can be very helpful following the covid19 pandemicwhich began in late and is still ongoing healthcare systems indiï¬erent countries were forced to impose significant limitations oncolonoscopy therefore noninvasive ibd management and fecal calprotectin as a noninvasive laboratory marker have become moreimportant than before the combination between disease clinical activity and fecal calprotectin has been recommended as a noninvasiveapproach that can help in making decisions on treatment duringcovid19 pandemic therefore it seems that fecal calprotectincan be considered as an alternative for colonoscopy used for ibd endoscopic activity evaluation during pandemic fecal calprotectin appears to be associated with ibd histologic activity as well given thediï¬culty in the evaluation of the histologic activity of crohn's disease some studies have been focused on the ulcerative colitis andmany scoring systems have been devised so far correspondingly thesesystems score the disease's histologic activity based on histologic observationstherefore for this purpose a biopsy of the intestinal tissue is required which can be prepared by colonoscopy and then sent to thelaboratory in this regard one of these histologic scoring systems isroberts score that was used in one of our recent studies where weobserved a significant correlation between the level of fecal calprotectinand the histologic activity of ulcerative colitis which was calculatedbased on the roberts scoring system theede also used themodiï¬ed harpaz index and performed some interesting studies in thisregard in one of their studies fecal calprotectin was found to be significantly associated with the histologic activity of the ulcerative colitisand it was shown that it could predict histological mucosal healingauc ci95 sensitivity speciï¬city andcutoï¬ mgkg in another study on patients with endoscopically inactive ulcerative colitis mayo endoscopic score the researchers showed that patients with ulcerative colitis who were inendoscopic remission but had histologically active disease had higherlevels of fecal calprotectin compared to patients with no histologicallyactive disease versus mgkg p also despite thehigh speciï¬city the sensitivity of fecal calprotectin in theprediction of score of histological activity was achieved as at mgkg in a recent study the geboes index has been used toevaluate histologic activity in patients with clinically quiescent ulcerative colitis as a result this study reported relatively low sensitivityand speciï¬city for fecal calprotectin in the prediction of geboesscore sensitivity speciï¬city and cut oï¬ µgg in another recent study the nancy index has been used toevaluate the histologic activity of ulcerative colitis and a high sensitivity and a low speciï¬city were ï¬nally reported for fecalcalprotectin at a cutoï¬ of µgg in the prediction of histologic activity however some studies have reported both high sensitivityand speciï¬city for fecal calprotectin in the prediction of histologicalremission for example one study reported a sensitivity of aswell as a speciï¬city of for fecal calprotectin at a cutoï¬ of µggin the prediction of gs it seems that the cause of theseconï¬icts should be explored in the endoscopic and clinical activity ofthe disease the inclusion and exclusion criteria of these studies andpossibly in the diï¬erent indexes used for the evaluation of the histologicactivity of the disease another notable issue is that all of these studieshave been conducted on a relatively low number of patients with ulcerative colitis so the need for a study with a large sample size is stillstrongly felt moreover a large global study may be helpful in this regard prediction of relapse and response to treatmentas mentioned previously ibd has recurrence and relief periods sopredicting response to treatment and relapse is one of the significant 0cf khakikhatibi clinica chimica acta challenges in ibd management the ï¬rst evidence of the eï¬cacy offecal calprotectin to predict recurrence dates back to the early 2000saccordingly a study published in by tibble is one of the ï¬rststudies in this regard in this study ibd patients in clinical remissionwere followed up for one year for the assessment of recurrence afterpreparing a stool sample to measure calprotectin this study has alsoshown that fecal calprotectin levels were higher in ibd patients withrecurrent disease and it was found that fecal calprotectin had a sensitivity of and a speciï¬city of at a cutoï¬ of mgl to predictibd recurrence in a study published in costa showedthat the sensitivity and speciï¬city of fecal calprotectin to predict ulcerative colitis recurrence are more than that of crohn's disease asensitivity of and a speciï¬city of versus a sensitivity of and a speciï¬city at a cutoï¬ of μgg another studyconducted on patients with ulcerative colitis has also reported appropriate sensitivity and speciï¬city rates for fecal calprotectin in the prediction of relapse a sensitivity of and a speciï¬city at a cutoï¬of μgg however another study was conducted on patientswith ulcerative colitis who have been followedup for year and ï¬nally a lower sensitivity was reported this study have shown that fecalcalprotectin at cutoï¬ of μgg could predict diseas | Colon_Cancer |
ulcerative colitis is a type of ammatory bowel disease thatcan potentially lead to cancer e age of onset of colitis istypically years old and it can seriously threaten thequality of life of patients e immunopathogenesis andimmunosuppressive treatment of colitis are currently theresearch topics of significant interest e research goals areto diagnose and treat colitis in order to prevent exacerbationof the disease e drugs used to treat colitis in the clinicoften have adverse eï¬ects after their longterm applicationanother crucial area of colitis research is focused on thediscovery of functional foods that can prevent colitis withoutside eï¬ects natural plants including aegle marmeloslinn also have the intervention eï¬ect on colitis a recentstudy has shown that the intestinal ï¬ora is closely related tocolitis and that the intestinal ï¬ora participates in the mucosalimmune response bacteria are an important promoter ofammatory bowel disease e symptoms of colitis can bealleviated by regulating the intestinal ï¬ora preventing ï¬oralimbalance and increasing the number of probiotics yak yoghurt is a natural fermented food that is rich innutrients and is common in the minority areas of theqinghai tibet plateau previous research has suggested thatyak yoghurt exerts various physiological activities such asantioxidationimmunitycholesterolreductionand 0cevidencebased complementary and alternative medicineenhancement e qinghai tibet plateau has a speciï¬cclimate and unique environment for the fermentation of yakyoghurt additionally the availability of yak milk and specialtibetan fermentation utensils eg certain fermentationmicroanisms can make the ï¬avor and quality of yakyoghurt diï¬er greatly from ordinary fermented milk aprior study on the intestinal physiological activity of lacticacid bacterial species in yak yoghurt showed that the lacticacidproducing bacteria isolated from yak yoghurt hadantioxidant and constipation preventing eï¬ects in this studythe potential eï¬ects of lactobacillusplantarum ys4 lpys4 on oxazolidoneinduced colitiswere investigated for the ï¬rst time e ï¬ndings provide apossible foundation for further development of lpys4especially its application in functional food or medicineratio � solution massnormal group were daub treated with ml of ethanolwith those in the remaining four groups being daub treated with ml of oxazolidonesolvent � after treatment for days the mice wereanesthetized en the blunt head of a silicone tube wasinserted into the intestinal tract from the anus of the mouse at adepth of cm e mice in normal group were administeredwith ml of ethanol solution while those in theremaining four groups were administered with ml of oxazolidone solution mass ratio � solvent � ethanoltwenty seconds later the catheters were removed and the micewere lifted up by their tails for half a minute on the last dayof treatment day all the mice were sacriï¬ced by decapitation and their plasma samples and colon tissues were collected e length and weight of the colon were documentedexperiment animal materials materials and methodsand reagentsexperimental strain the strain was isolated from yak yoghurt in the yushu area of qinghai province china by ourteam it was named lpys4 and stored in china center fortype culture collection cctcc wuhan china nom2016750 e negative control strain lb was purchasedfrom the cctcc no ab fifty male balbc mice weeks old were purchasedfrom the experimental animal center of chongqingmedical university certiï¬cate no syxk yu oxazolone was purchased from sigmaaldrich co llcusa il2 il10 et1 sp ss and vip serum cytokine kitswere purchased from biolegend inc usa gsh sodmpo and mda kits were purchased from nanjing jiancheng bioengineering institute nanjing china trizol reagent oligodt18 rnase dntp mlv primer bca proteinquantitative kit aps temed sdspage pvdf membrane ï¬rst antibody and second antibody were purchasedfrom ermo fisher scientiï¬c inc usa instruments and equipment imark microplate readerwas purchased from biorad usa steponeplus pcrinstrument was purchased from ermo fisher scientiï¬cinc usa tanon chemiluminescence imager waspurchased from tanon science and technology co ltdchina sas v91 statistical software package was purchasedfrom sas institute inc usalb animal grouping and intervention a total of balbcmale mice were assigned to ï¬ve groups model groupnormal group lactobacillus bulgaricustreatmentgroup and highdose lpys4h and lowdoses lpys4l treatment groups and there were mice in each groupe mice in lb lpys4h and lpys4l groups were fedwith and cfukg ml livingbacteria physiological saline of each corresponding straindaily by oral gavage for consecutive days and normal andmodel groups were fed with ml physiological salineafter days of treatment the abdomens of all mice wereshaved with an area of cm cm e mouse abdomens in detection of endothelin1 et1 substance p spsomatostatin ss and vasoactive intestinal peptide vipconcentrations in serum samples e whole blood samplesof mice were allowed to clot at room temperature for h andthen centrifuged at rpmmin for min after collecting the serum samples the concentrations of et1 sssp and vip were detected using commercial kits determination of interleukin2 il2 and interleukin10il10 levels in serum samples e mouse serum sampleswere prepared according to section en the serumlevels of il2 and il10 cytokines were assessed usingcommercial kits determination of glutathione gsh malondialdehydemda myeloperoxidase mpo and superoxide dismutasesod activities in colon tissues a mixture of g colontissue and ml normal saline was prepared at weightratio after homogenizing the mixture the activities of gshmda mpo and sod in colon tissues were evaluated usingcommercial kits pathological observation of he staining e lesionsite cm of colon was cut with a scalpel etrimmed tissue and corresponding label were placed in neutral formalin solution for h e colon tissue wasdehydrated embedded sliced dewaxed stained and thendehydrated transparent and sealed finally the pathologicalstate of colon tissue was observed under a microscopebx43 olympus tokyo japan qpcr assay total rna was isolated using rnazol andthen diluted to the ï¬nal concentration of μgμl for cnasynthesis μl of the diluted rna extract was taken andcdna was prepared using a reverse transcriptase kit enthe cdna template μl was added into μl of sybrgreen pcr master mix and μl of forward primer andreverse primer each table qpcr ampliï¬cation wascarried out for cycles under the reaction conditions of95oc s °c s °c s and °c s followed by 0cevidencebased complementary and alternative medicineckitinosenosnnossequencegene nametable sequences of primers used in this studyforward ²agagagatcgggttcaca3²reverse ²cacagaactgagggtaca3²forward ²tcgtccaacttctgggctctt3²reverse ²ccttctcttcctcccctctcttc3²forward ²tcagccatcacagtgttccc3²reverse ²atagcccgcatagcgtatcag3²forward ²catagcccaggtaaagcacaat3²reverse ²gaacactccagaatcgtcaactcforward ²tcagggactacgctgcgaaag3²reverse ²aagagctggcagaccgactca3²forward ²tgcaccaccaactgcttag3²reverse ²gatgcagggatgatgttc3²Î´ctdetection gene δctgapdh measured according to the following equation δct �cycle under the reaction conditions of °c s and°c s gapdh was used as internal control for thisdetermination and the relative mrna expression was²gapdhscf western blotting mg of tissue samples was mixedwith μl of pmsf and ml of ripa and then homogenized at rmin 4oc for min protein quantiï¬cationwas conducted using the bca protein quantitative kit andthe protein samples were diluted to μgml en thediluted protein and sample buï¬er were mixed at heatedat 100oc for min and icebathed for min subsequentlyacrylamide starting buï¬er resolving buï¬er temed aps and diï¬erentiated water were mixed in speciï¬c proportions in order to prepare sdspage separation glue andconcentration glue e prestained samples and proteinladder were placed into the sample hole of the rubber sheetrespectively and then the proteincontaining sdspageglue was subjected to vertical gel electrophoresis for minafter activation with methanol for min the pvdf wereblocked with skimmed milk in tbst solution for hen the blocked pvdf membranes were rinsed with tbst followed by incubation with the primary antibodyat °c for h after washing with tbst for times thesecondary antibody was incubated at °c for h lastly theprotein bands were visualized by supersignal west picoplus chemiluminescent substrate and the images werecaptured using a chemiluminescence imager multiple comparisons were conducted using onewayanova followed by tukeys test statistical analysis e average value of three experimental results was determined and the statistical softwaresas was used to analyze whether there was a significantdiï¬erence between each group at the level of p longest in normal group ± cm while being the results eï¬ect of lpys4 on colon parameters e experimentalresults demonstrated that the length of mouse colon was thepared to those in the remaining four groups in contrast thethe ratio of colon weightlength was the highest in normalpgml and the lowest concentrations were found for sslpys4h group and these eï¬ects were better than those of eï¬ect of lpys4 on the serum contents of et1 sp ss andvip in mice it can be seen from figure that the serumml was decreased in normal mice compared to that of theremaining four groups e colitis model mice exhibited theopposite results in which the highest concentrations wereserum concentrations of ss and vip in colitis mice andmarkedly decrease those of et1 and sp more importantlythe eï¬ects were better after treatment with lpys4hshortest in the colitis model group ± cm similarlygroup ± while being the lowest in the colitis modelgroup ± figure it was found that lpys4 couldsignificantly p attenuate the decline in colon lengthand weightlength ratio induced by colitis ± cm and± for lpys4l group ± cm and ± forlb ± cm and ± concentrations of ss ± pgml and vip± pgml in normal mice were increased comlevel of et1 ± pgml and sp ± pgobserved for et1 ± pgml and sp ± ± pgml and vip ± pgml interestingly lpys4 could significantly p improve the± ± ± and ± pglb ± ± ± and ±± pgml in colitis model mice was signiï¬groups p following the treatment with lpys4 anhigher in lpys4h ± pgml and lpys4ltreated mice ± pgml than in lbtreated mice± pgml and the serum levels of il10 werelower in lpys4h ± pgml and lpys4ltreated mice ± pgml than in lbtreated mice± pgml p gsh ± μmolmg and sod ± μmolhighest while those of mpo ± mumg and mda± nmolmg were the lowest among the ï¬ve± μmolmg and sod ± μmolgprotwere significantly reduced p while those of mpo± mumg and mda ± nmolmg wereremarkably increased p in the model group mice eï¬ect of lpys4 on the serum concentrations of il2 andil10 in mice it can be seen from figure that the serumcontentand il10± pgml eï¬ect of lpys4 on the activities of mpo sod gsh andmda in mouse colon as shown in figure the activities ofincrease in il2 and a decrease in il10 cytokine level wereobserved notably the serum levels of il2 were markedlyml and the eï¬ects of lpys4 were better than those ofgroups after induced by oxazolidone the levels of gshcantly reduced and raised compared to the remaining fourgprot in the colon tissue of the normal group were the pgmlofil2 0cevidencebased complementary and alternative medicinebacmice treated with a low concentration of lactobacillus plantarum ys4 cfukg lpys4h mice treated with a high concentrationof lactobacillus plantarum ys4 cfukg and lb mice treated with lactobacillus bulgaricus cfukgfigure ac colon length and colon weightcolon length of each group of mice data are presented as the mean± standard deviationad diï¬erent letters mean values are significantly diï¬erent p according to tukeys honestly significantly diï¬erent test lpys4land mda levels p such eï¬ect was stronger comparedto lbtreated mice ± μmolmg ± μmolgprot ± mumg and ± nmolmg more± μmolgprot in colon tissue and markedly decreased those of mpo ± mumg and mda± nmolmg when compared to colitis model groupand the goblet cells were increased compared with the modelgroup among them lpys4h had the most obvious eï¬ecton improving colon tissue which indicated that lpys4 couldreduce the colon injury caused by dss and the high eï¬ciencyeï¬ect was also enhanced with the increase of concentrationlpys4 could markedly attenuate the decline in gsh andsod levels and prevented colitisinduced increase in mpo± μmolmgspeciï¬cally treatment with lpys4h significantly increasedthesodlevelsandof pathological observation as shown in figure in thenormal group the epithelial cells of colon mucosa were intactthe ammatory cells were normal without ltration andthe goblet cells were arranged orderly without congestionand edema in the model group the epithelial cells of colontissue were obviously damagedthe intestinal wall wasthickened and edema ammatory cell ltration andgoblet cells were reduced after treatment with lb and lpys4 congestion edema and cell ltration were alleviated eï¬ect of lpys4 on mrna and protein expression inmouse colon as shown in figures and colitis inductioncould lead to the upregulated mrna and protein expressionof inos in mouse colon but downregulated the relativeexpression of ckit enos nnos and scf treatment withlpys4h could increase the relative expression of nnosenos ckit and scf and decrease that of inos in the colontissues of colitis mice such eï¬ects were stronger than thoseof lpys4l or lb treatment group discussione ratio of colon weightlength is employed as a vitalstandard for assessing colitis in vivo e colon length ofnormalmodellblpys4llpys4hadccb0020406080100normalmodellblpys4llpys4hcolon length cmcolon lengthadccb00100200300400500normalmodellblpys4llpys4hcolon weightcolon lengthcolon weightcolon length 0cevidencebased complementary and alternative medicineabfigure a et1 b ss c sp and d vip serum levels of each group of mice data are presented as the mean± standard deviationad diï¬erent letters mean values are significantly diï¬erent p according to tukeys honestly significantly diï¬erent test lpys4lmice treated with a low concentration of lactobacillus plantarum ys4 cfukg lpys4h mice treated with a high concentrationof lactobacillus plantarum ys4 cfukg and lb mice treated with lactobacillus bulgaricus cfukgdcfigure a il2 and b il10 serum levels of each group of mice data are presented as the mean± standard deviation ae diï¬erentletters mean values are significantly diï¬erent p according to tukeys honestly significantly diï¬erent test lpys4l mice treatedwith a low concentration of lactobacillus plantarum ys4 cfukg lpys4h mice treated with a high concentration oflactobacillus plantarum ys4 cfukg and lb mice treated with lactobacillus bulgaricus cfukgbadabbc0050100150200250normalmodellblpys4llpys4het1 level pgmlet1aedcb00100200300400500600700normalmodellblpys4llpys4hss level pgmlssvipdabbc00100200300400500600700normalmodellblpys4llpys4hsp level pgmlspaedcb00100200300400500600700normalmodellblpys4llpys4hvip level pgmlvipadccb050100150200250300normalmodellblpys4llpys4hil2 level pgmlil2eabcd02004006008001000normalmodellblpys4llpys4hil10 level pgmlil10 0cevidencebased complementary and alternative medicineabfigure a mpo b no c gsh and d mda colon tissue levels of each group of mice data are presented as the mean± standarddeviation ae diï¬erent letters mean values are significantly diï¬erent p according to tukeys honestly significantly diï¬erent testlpys4l mice treated with a low concentration of lactobacillus plantarum ys4 cfukg lpys4h mice treated with a highconcentration of lactobacillus plantarum ys4 cfukg and lb mice treated with lactobacillus bulgaricus cfukgcdcolitis mice was shorter on average than that of control miceand the ratio of colon weightlength was lower in colitis micethan in control mice howeverit appeared thattreatment with lpys4 could attenuate the decline in colonlength and weightlength ratio induced by colitisa prior study has shown that vasoconstriction of theendothelin can lead to colonic mucosa erosion and ulceration which in turn can exacerbate the progression of colitis ss however can reduce gastrointestinal ammationby suppressing the production of gastric acid and othergastrointestinal ï¬uids us a decrease in ss level can inducethe secretion of gastrointestinal ï¬uids thus aggravatingcolitis excessive accumulation of sp can induce colitisbut after antagonizing it has been shown to relieve colitis invivo vip inhibits no production by regulating thetranscriptional activity of inos in the colon tissue thusprotecting the intestinal mucosa besides vip can also uence certain immune aspects of colitis in this studylpys4 inhibited colitis by downregulating the levels of etand sp and upregulating those of ss and vipil2 is an eï¬ector cytokine produced by cells whichhas been closely associated with colitis cells regulate theammatory response that causes colitis and il2 is involved in the suppression of ammatory process andseverity reduction of colitis by uencing cells il is another cytokine produced by treg cells with immunoinhibitory eï¬ects which plays a significant role in thedevelopment of colitis ammatory bowel diseaseibd is a chronic refractory intestinal ammatory diseasemainly including ulcerative colitis uc and crohns diseasecd although the etiology of ibd is still unclear theimbalance between and has been recognized as themain cause of mucosal damage in ibd under the action ofdiï¬erentiation factor il10 regulatory t cells derived fromintestinal mucosa associated lymphoid tissue can correct deviation by secreting high level of il10 andmedium level of tgfβ so as to achieve the purpose oftreating ammatory bowel disease to a certain extent it was observed that lpys4 could increase the level of il2thus regulating immunity and alleviating colitis and lpeabcd00100200300400normalmodellblpys4llpys4hmpo level mumgmpoadccb00100200300400normalmodellblpys4llpys4hsod level μmolgprotsodaedcb0020406080100120normalmodellblpys4llpys4hsgh level μmolmggshdabbc000510152025normalmodellblpys4llpys4hmda level nmolmgmda 0cevidencebased complementary and alternative medicinefigure observation of colon pathology in mice by he stainingys4 could inhibit the colitis and reduce the secretion of ile aggregation of neutrophils began to decline afterintestinal ammation and a large amount of them enteredinto the circulation and migrated to tissues at the sametime free radicals such as reactive oxygen species and reactive nitrogen species gathered in large quantities which inturn led to damage and toxicity of colon tissue and furtheraggravated colitis after colitis the levels of sod andgsh were reduced in colon tissue while those of mda andmpo were elevated our ï¬ndings also indicated that colitiscould lead to decrease in gsh and sod levels as well asincrease in mda and mpo levels [ ] in addition lpys4 attenuated colitis by inhibiting the transcriptional responses to oxidative stressnos can be divided into nnos enos and inos it hasbeen reported that no produced by enos plays a key rolein response to colonic tissue damage and excessive nogenerated by inos promotes colitis damage enoscontrols the production of no to keep the colonic tissue ina normal state which plays an important role in reducingcolitisinduced colonic injury e presence of excessiveno aggravates colon damage nnos can also controlthe level of no in tissue and protect the tissue from beingdamaged by excessive no in this study lpys4upregulated the expression of enos and nnos in the colonand downregulated that ofthereby attenuatingcolitisinosulcerative colitis not only shows hematochezia anddiarrhea but also presents colonic motility disorders it hasbeen proved that interstitial cells of cajal icc are related tocolonic motility dysfunction and directly participate in theprogression of colitis as a speciï¬c marker of gastrointestinalicc ckit is a transmembrane glycoprotein speciï¬callyexpressed on icc cell membrane ckit gene located onchromosome 4q1213 belongs to protooncogene and itsproduct is tyrosine kinase type iii as a receptor of scfckit can regulate the proliferation and diï¬erentiation ofhematopoietic stem cells through a series of signalingpathways [ ] scf exerts a direct eï¬ect on ammatorybowel disease by regulating the function and number oficc scf can interact with its ligand ckit and the dysregulation of scfkit signaling pathway may decrease theproliferation and diï¬erentiation of icc thus exacerbatingcolitis [ ] e abnormal expression of scfckitsignaling pathway can also change the physiologicalfunction of icc weaken gastrointestinal motility andaggravate intestinal dysfunction in the present studylpys4 could inhibit colitis by regulating the expressionlevels of scf and ckiticc autophagy regulation has become a new target forthe treatment of intestinal motility disorder in ulcerativecolitis because the drug treatment of colitis often hasside eï¬ects and once stopped it is easy to relapse ereforethe use of natural harmless substances through the regulation of icc prevention and treatment of colitis canmaintain longterm health a study has shown thataurantii fructusimmaturus and atractylodis macrocephalae rhizoma can inhibit the autophagy of cajal stromalcells induced by glutamate which may play an inhibitoryrole in colitis meanwhile there is also a study showingthat lactic acid bacteria can regulate icc thus regulatingnormallblpys4llpys4hmodel 0cevidencebased complementary and alternative medicineacbdthe mean± standard deviation ae diï¬erent letters mean values are significantly diï¬erent p according to tukeys honestlyfigure eï¬ect of lpys4 on a nnos b enos c inos d ckit and e scf mrna expression in mouse colon data are presented asesignificantly diï¬erent test lpys4l mice treated with a low concentration of lactobacillus plantarum ys4 cfukg lpys4hmice treated with a high concentration of lactobacillus plantarum ys4 cfukg and lb mice treated with lactobacillusbulgaricus cfukgintestinal function and protecting the intestine isstudy also conï¬rmed that the lpys4 can regulate the scfckitsignaling pathway and the scfckit signalingpathway is an important icc regulatory pathway erefore the lpys4 may also inhibit the colon by regulating iccaedcb001020304050normalmodellblpys4llpys4hrelative to multiple of model groupnnosadccb001020304050normalmodellblpys4llpys4hrelative to multiple of model groupenoseabcd00020406081012normalmodellblpys4llpys4hrelative to multiple of model groupinosaedcb0010203040normalmodellblpys4llpys4hrelative to multiple of model groupscfaedcb0010203040normalmodellblpys4llpys4hrelative to multiple of model groupckit 0cevidencebased complementary and alternative medicineacbdmean± standard deviation ae diï¬erent letters mean values are significantly diï¬erent p according to tukeys honestly sigfigure af eï¬ect of lpys4 on nnos enos inos ckit and scf protein expression in mouse colon data are presented as theniï¬cantly diï¬erent test lpys4l mice treated with a low concentration of lactobacillus plantarum ys4 cfukg lpys4hmice treated with a high concentration of lactobacillus plantarum ys4 cfukg and lb mice treated with lactobacillusbulgaricus cfukgef conclusionin this study oxazolone was used to induce colitis in balbcmice and the inhibitory eï¬ects of lpys4 on colitis weredetected rough the observation of colon tissues and serum samples of mice it was found that lpys4 treatmentcould alleviate colitis by restoring the levels of ammatoryindicators closer to those measured in healthy control miceis work suggests that lpys4 is superiorquality lactic acidbacteria with a potential role in colitis treatment and provides a foundation for further research and developmentabbreviationslpys4 lactobacillus plantarum ys4lbqpcr quantitative polymerase chain reactionhelactobacillus bulgaricushematoxylineosinnormalmodellblpys4llpys4hckitenosinosnnosscfβactinadcbcb0010203040normalmodellblpys4llpys4hrelative to multiple of model groupnnosaedcb00102030405060normalmodellblpys4llpys4hrelative to multiple of model groupenoseabcd00020406081012normalmodellblpys4llpys4hrelative to multiple of model groupinosadccb0010203040506070normalmodellblpys4llpys4hrelative to multiple of model groupscfadccb00102030405060normalmodellblpys4llpys4hrelative to multiple of model groupckit 0cevidencebased complementary and alternative medicineendothelin1substance psomatostatinvasoactive intestinal peptideinterleukin2interleukin10et1spssvipil2il10mpo myeloperoxidasesodgshmda malondialdehydeenosnnos neuronal nitric oxide synthaseinducible nitric oxide synthaseinosscfstem cell factorsuperoxide dismutaseglutathioneendothelial nitric oxide synthasedata availabilityno data were used to support this studyconflicts of intereste authors of this manuscript state that they do not haveconï¬icts of interest to declareauthors contributionsruokun yi and fang tan contributed equally to this workruokun yi and fang tan performed the majority of theexperiments and wrote the manuscript huayi suo wenfengli xianrong zhou and jianfei mu contributed to the dataanalysis xin zhao and peng xie designed and supervised thestudy and read the ï¬nal manuscriptacknowledgmentsis research was funded by national key rd program ofchina 2018yfd0502300 childrens research institute ofnational center for schooling development programmeand chongqing university of education csdp19fs01103theand technology research program ofchongqing municipal education commission kjzdk201901601 and research project of chongqing universityof education ky2015tbzc chinasciencereferences r k yi f tan w liao et al isolation and identiï¬cation oflactobacillus plantarum hfy05 from natural fermented yakyogurt and its eï¬ect on alcoholic liver injury in mice microanisms vol no p j liu f tan x h liu et al exploring the antioxidanteï¬ects and periodic regulation of cancer cells by polyphenolsproduced by the fermentation of grape skin by lactobacillusplantarum kfy02 biomolecules vol no p t s olson b k reuter k g e scott et al e primarydefectfrom a nonhematopoietic source journal of experimental medicinevol no pp ileitis originatesin experimental b manandhar k r paudel b sharma and r karkiphytochemical proï¬le and pharmacological activity of aeglemarmelos linn journal of integrative medicine vol no pp x zhou h liu j zhang et al protective eï¬ect of lactobacillus fermentum cqpc04 on dextran sulfate sodiuminduced colitis in mice is associated with modulation of thenuclear factorκb signaling pathway journal of dairy science vol no pp m c arrieta k madsen j doyle and j meddings reducing small intestinal permeability attenuates colitis in theil10 genedeï¬cient mouse gut vol no pp h suo x zhao y qian et al erapeutic eï¬ect of activatedcarboninduced constipation mice with lactobacillus fermentum suo on treatment international journal of molecular sciences vol no pp y qian h suo m du et al preventive eï¬ect of lactobacillus fermentum lee on activated carboninduced constipation in mice experimental and eerapeutic medicinevol no pp x zhao y qian h suo et al preventive eï¬ect of lactobacillus fermentum zhao on activated carboninduced constipation in mice journal of nutritional science andvitaminology vol no pp x long y pan and x zhao prophylactic eï¬ect ofkudingcha polyphenols on oxazolone induced colitis throughits antioxidant capacities food science and human wellnessvol no pp k zhu g huang j xie x zhou j mu and x zhaopreventive eï¬ect of ï¬avonoids from wushan shencha malus doumeri leaves on ccl induced liver injury foodscience nutrition vol no pp x zhao j zhang s yi et al lactobacillus plantarumcqpc02 prevents obesity in mice through the pparα signaling pathway biomolecules vol p w strober i j fuss and r s blumberg e immunologyofmucosalmodels oï¬nï¬ammation annual review of immunology vol no pp jl song y qian gj li and x zhao antiammatoryeï¬ects of kudingcha methanol extract ilex kudingcha cjtseng in dextran sulfate sodiuminduced ulcerative colitismolecular medicine reports vol no pp y qian x zhao jl song et al inhibitory eï¬ects of resistant starch rs3 as a carrier for stachyose on dextransulfate sodiuminduced ulcerative colitis in c57bl6 miceexperimental and eerapeutic medicine vol no pp x y chen x zhao h w wang et al prevent eï¬ects oflactobacillus fermentum hy01 on dextran sulfate sodiuminduced colitis in mice nutrients vol no p y qian a l lei x j liu et al inhibitory eï¬ects oflactobacillus plantarum ys2 in dextran sulfate sodiuminduced c57bl6j mice colitis science and technology of foodindustry vol no pp s chen x zhao p sun j qian y shi and r wangpreventive eï¬ect of gardenia jasminoides on hclethanolinduced gastric injury in mice journal of pharmacologicalsciences vol no pp l xie z h xing r x jiang et al eï¬ect of sanpi decotionon the expression of il2 and il10 of mice with ulcerativecolitis chinese journal of experimental traditional medicalformulae vol no pp l hang s kumar a m blum j f urban m c fantini andj v weinstock heligmosomoides polygyrus bakeri infection decreases smad7 expression in intestinal cd4 t cells 0cevidencebased complementary and alternative medicinewhich allows tgfβ to induce il10producing regulatorytcells that block colitis ee journal of immunology vol no pp j zhang q li y wei et al process design of the antioxidant shuidouchi and its eï¬ect on preventing dextransulfate sodium dssinduced colitis in mice via antioxidantactivity applied sciences vol no p c fiocchi ammatory bowel disease new insights intomechanisms of ammation and increasingly customizedapproaches to diagnosis and therapy current opinion ingastroenterology vol no pp j zhang r yi y qian p sun x zhao and z yanglactobacillus plantarum cqpc06 activity prevents dextransulfate sodiuminduced colitis by regulating the il8 pathway journal of food science vol no pp j zhang x chen jl song et al preventive eï¬ects oflactobacillus plantarum cqpc07 on colitis induced bydextran sodium sulfate in mice food science and technologyresearch vol no pp lh chen jl song y qian x zhao hy suo and j liincreased preventive eï¬ect on colon carcinogenesis by use ofresistant starch rs3 as the carrier for polysaccharide ofinternationallarimichthysjournal of molecular sciences vol no pp x feng j zhang y qian et al preventative eï¬ects oflactobacillus plantarum ys3 on oxazoloneinduced balbccolitis in mice applied biological chemistry vol no pp swimming bladdercrocea j m wang m li r tang et al eï¬ect of yiqi jianpitongbian recipe on icc and scfckit signaling pathway incolon tissue of slow transport type constipation rats chinesearchives of traditional chinese medicine vol no pp j feng j gao s zhou et al role of stem cell factor in theregulation of icc proliferation and detrusor contraction inrats with an underactive bladder molecular medicine reports vol no pp c lu h lu x huang et al colonic transit disordermediated by downregulation of interstitial cells of cajalanoctamin1 in dextran sodium sulfateinduced colitis micejournal of neurogastroenterology and motility vol no pp y c dai l zheng y l zhang et al jianpi qingchangdecoction regulates intestinal motility of dextran sulfate sodiuminduced colitis through reducing autophagy of interstitial cells of cajal world journal of gastroenterologyvol no pp s yan y z yue m m sun et al suppressive eï¬ect ofaurantii fructus immaturus and atractylodis macrocephalaerhizoma on glutamic acidinduced autophagy of interstitialcells of cajal journal of integrative medicine vol no pp c li sp nie kx zhu et al eï¬ect oflactobacillusplantarumncu116 on loperamideinduced constipation inmice international journal of food sciences and nutritionvol no pp r yadak m breur a | Colon_Cancer |
primary colorectal cancer pcrc is a common digestive tract cancer in the elderly the primary lesioncan be seen in the left colon the right colon the upper or lower rectum pcrc is the second mostcommonly diagnosed cancer in women and the third most commonly diagnosed cancer in men and theprevalence of male is higher than that of female in most areas with the social environment lifestyleand dietary structure changes the incidence of pcrc is on the rise and there is a trend of rejuvenationthis is a social issue worthy of attention at present there is controversy about the pathogenesis ofpcrc it is generally believed that smoking drinking greasy diet obesity lack of exercise colorectalinflammation and genetic factors are all involved in the onset of cancer but these factors are also the causeof many other tumors therefore the specific etiological mechanism of pcrc has not yet been elucidated some scholars believe that some genes or molecules are involved in the development of pcrc thesefindings promote the research and treatment of pcrc [] at present the treatment of pcrc includestraditional surgery chemotherapy radiotherapy emerging immunotherapy molecular targeted therapyetc clinically the single or combination therapy that best suits the condition is usually selected accordingto the actual situation of the patient however the treatment effect of pcrc is still limited andreceived march revised august accepted august accepted manuscript online august version of record published august the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commons attributionlicense cc by 0cbioscience reports bsr20200963101042bsr20200963the longterm survival rate is low the early prognosis of patients with early diagnosis is often better thereforefurther exploring the pathogenesis of pcrc searching for specific molecular targets for diagnosis and treatment realizing early diagnosis targeted treatment and individualized treatment are the development trends of precise medicaltreatment which have important clinical significancepersonalized medicine refers to the treatment of existing diseases based on the information of each persons disease genome it is now widely believed that majority of individual differences in drug response are due to geneticfactors personalized medicine is a discipline that emphasizes studying the effect of genetic factors on a drug recently due to the smooth implementation of the human genome project and the rapid development of bioinformaticspersonalized medicine has been strongly promoted and the concept has been gradually developed bioinformatics is a method to process and analyze biological data by combining biological knowledge with information processing technology it is commonly used in highthroughput data analysis such as gene and proteomicsas a frontier interdisciplinary subject bioinformatics analysis technology can realize the biological analysis of thestructure and function of histological data find the genes or proteins most relevant to diseases and further analysis may find the molecules most relevant to diseases and can be used as disease markers at present a largenumber of scholars have applied this technology to tumor research that is processing gene sequence or omics databy bioinformatics analysis technology to find genes or molecular markers most relevant to tumors []therefore the present study aimed to use the bioinformatics to identify the hub genes of pcrc and to verify theirrole on the overall survival of patients with pcrc based on the clinical data and the research would provide novelinsights for the personalized medicine on the treatment of patients with pcrcmaterial and methodslease start with dates and time location of study and the recruitmentsof patientsthe present study recruited a total of pcrc patients between and from the fourth hospital of hebeimedical university shijiazhuang of hebei province clinical and histopathological characteristics and followup andsurvival information were available for all patients and were collected retrospectively from medical records patientsaged years old histologically confirmed as pcrc not received tumor treatment and no history of gastrointestinal surgery will be screened for inclusion criteria exclusion criteria included age years old or yearsold combined with other malignant tumors operation time more than one month after the last examination andsevers heart diseaseethical clearance and informed contentthe research conformed to the declaration of helsinki and was authorized by the human ethics and research ethicscommittees of the fourth hospital of hebei medical university the written informed consents were obtained fromall participatesdownload public datathe gene expression omnibus geo database httpwwwncbinlmnihgovgeo is the largest most comprehensive and publicly available source of gene expression data it contains information about the expression levels ofmultiple genes in different groups of clinical samples such as the differences in gene expression between tumor tissues and normal tissues gse41258 gpl96 [hgu133a] affymetrix human genome u133a array and gse81558gpl15207 [primeview] affymetrix human gene expression array were obtained from the geo database a totalof samples including tumor colorectum tissues from pcrc patients and normal colorectum tissues wereselected from gse41258 a total of samples including tumor colorectum tissues from pcrc patients and normal colorectum tissues were selected from gse81558veriï¬cation for repeatability of intragroup datafirst repeatability of intragroup data were verified by the pearsons correlation test the heatmap was drew via the rlanguage environment and presented the correlation among intragroup data second principal component analysispca was the general method for sample clustering and is commonly performed for diversity analysis resequencinggene expression and other sample clustering based on various variable information the verification for repeatabilityof intragroup data was executed by pca the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commons attributionlicense cc by 0cbioscience reports bsr20200963101042bsr20200963differentially expressed genes degs between normal and pcrcgeo2r httpwwwncbinlmnihgovgeogeo2r could import data of the geo database into the r languageand perform differential analysis essentially through the following two r packages including limma packages andgeoquery therefore through the geo2r tool degs were identified between normal and pcrc group the adjusted pvalues adj p and the fold change fc ¥ or were defined as significance sangerboxshengxinren one open tool was used to draw volcano maps venn diagrams were delineated using anonline venn tool httpbioinformaticspsbugentbewebtoolsvenn which would visualize common degs sharedbetween gse41258 and gse81558proteinprotein interaction ppi networkthe common degs shared between gse41258 and gse81558 were converted into differently expressed proteinsthe string search tool for the retrieval of interacting genes online database tringdb could construct ppi network which was visualized by cytoscape version go and kegg analysis via david toolone online tool david davidncifcrfgovhomejsp version maryland america was applied to carriedout the functional annotation for degs gene ontology go generally perform enrichment analysis of genomesand there are mainly cellular components cc biological processes bp and molecular functions mf in thego analysis kyoto encyclopedia of genes and genomes kegg wwwkeggjp is a comprehensivedatabase of genomic chemical and systemic functional information therefore david was used to make analysisof go and keggsignificant module and hub genesmolecular complex detection tool mcode version an open plugin of cytoscape was performed toidentify tested most significant module from the ppi network and the criteria was that the maximum depth mcode scores cutoff kscore and node score cutoff then cytohubba a free plugin of cytoscape was applied to authorize the hub genes when the degree ¥chiahao chins research introduce a novel cytoscape plugin cytohubba for ranking nodes in a network by theirnetwork features cytohubba provide a userfriendly interface to explore important nodes in biological networkswhen the degree¥ in the cytohubba the hub genes would be obtained and in the former publications []numerous researchers chose hub genes out of the degs therefore the present study chose hub genes out of degsinteraction between the hub genespearsons correlation analysis was also performed to present the interaction between the hub genes the cbioportalhttpwwwcbioportal one online software constructed the coexpression network of these hub genessimultaneously the coexpression network of hub genes in the field of pcrc was also analyzed via coexpedia a freeand open online toolhttpwwwcoexpedia expression analysis of hub genesucsc xena xenaucsceduwelcometoucscxena could integrate the public genomic data sets to analyzeand visualize the expression level of hub genes then the clustering analysis of expression level of hub genes wasperformed using heatmaps based on the gse41258 and gse81558 also the expression profiles of hub genes in thehuman different ans were displayed with gene expression profiling interactive analysis gepia httpgepiacancerpkucn in order to compare the expression of hub genes in the various tumors gepia was used andthe expression profiles of hub genes in the pcrc and normal groups were analyzed using gepiaeffect of hub gene expression for pathological stage and overall survivaleffect of hub gene expression for pathological stage and overall survival was analyzed by the gepia finally the correlation and linear regression analysis between pcrc and hub gene expression were performed and the receiveroperator characteristic roc curve analysis was performed to test the sensitivity and specificity of hub gene expression for diagnose pcrc the spss software version ibm new york america was used to conduct all thestatistical analysis a pvalue was defined as statistical significance the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commons attributionlicense cc by 0cbioscience reports bsr20200963101042bsr20200963table summaries for the function of hub genesnogene symbolfull namefunctionclca4guca2achloride channel accessory guanylate cyclase activator 2agcgsstglucagonsomatostatinmay be involved in mediating calciumactivated chloride conductanceendogenous activator of intestinal guanylate cyclase it stimulates this enzyme through thesame receptor binding region as the heatstable enterotoxinsregulates blood glucose by increasing gluconeogenesis and decreasing glycolysisglp1 is a potent stimulator of glucosedependent insulin release glp2 stimulatesintestinal growth concomitant with increased crypt cell proliferationsomatostatin inhibits the release of somatotropin this hormone is an important regulatorof the endocrine system through its interactions with pituitary growth hormone thyroidstimulating hormone and most hormones of the gastrointestinal tractms4a12membrane spanning 4domainsmay be involved in signal transduction as a component of a multimeric receptor complexa12silencing of this gene in colon cancer cells inhibits the proliferation cell motility andchemotactic invasion of cellsplp1chgapyyvipproteolipid protein this is the major myelin protein from the central nervous system it plays an important rolein the formation or maintenance of the multilamellar structure of myelinchromogranin athis gene product is a precursor to three biologically active peptides vasostatinpancreastatin and parastatinpeptide yythis gut peptide inhibits exocrine pancreatic secretion has a vasoconstrictory action andinhibitis jejunal and colonic mobilityvasoactive intestinal peptidevip causes vasodilation lowers arterial blood pressure stimulates myocardial contractilityincreases glycogenolysis and relaxes the smooth muscle of trachea stomach andgallbladderguca2bguanylate cyclase activator 2b may be a potent physiological regulator of intestinal ï¬uid and electrolyte transport may bean autocrineparacrine regulator of intestinal salt and water transportrtqpcr assaytotal rna was extracted from tumor colorectum tissues from pcrc patients and adjacent normal colorectum tissuesby the rnaiso plus trizol kit thermofisher massachusetts america and reverse transcribed to cdna rtqpcrwas performed using a light cycler® system with specific primers for the ten hub genes table presents theprimer sequences used in the experiments the rq values 01 01ct where ct is the threshold cycle of each samplewere calculated and are presented as fold change in gene expression relative to the control group gapdh was usedas an endogenous control the expression level of clca4 and ms4a12 in pcrc patients was measured by rtqpcroverall survival analysis of the pcrcthe kaplanmeier method was performed to analyze the overall survival all statistical analyses were conductedusing spss software version and p005 was considered statistically significantresultshigh repeatability of datathere exist strong correlations among samples in the pcrc group and also strong correlations among samples in thecontrol group in the gse41258 via the pearsons correlation test supplementary figure s1a and there also existstrong correlations among samples in the pcrc group and also strong correlations among samples in the controlgroup in the gse81558 via the pearsons correlation test supplementary figure s1b furthermore pca was performed to verify the repeatability of data through the pca the repeatability of the data in gse41258 was fine thedistances between per samples in the pcrc group were close and the distances between per samples in the controlgroup were also close in the dimension of pc1 supplementary figure s1c through the pca the repeatability ofthe data in gse81558 was fine the distances between per samples in the pcrc group were close and the distancesbetween per samples in the control group were also close in the dimension of pc1 supplementary figure s1ddegs between control and pcrcthere are plenty of degs on the all chromosomes between pcrc and control samples supplementary figure s1eone volcano plot presents the degs in the gse41258 figure 1a and another volcano plot presents the degs in thegse81558 figure 1b in the volcano plots the green nodes indicate the downregulated degs and the red nodesindicate the upregulated degs the venn diagram manifested that a total of degs were exist in the two datasetsgse41258 and gse81558 simultaneously figure 1c after construction of ppi network for the common degsthere are nodes and edges in the ppi network figure 1d the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commons attributionlicense cc by 0cbioscience reports bsr20200963101042bsr20200963figure the differently expressed genes and ppi networka one volcano plot presents the degs in the gse41258 b another volcano plot presents the degs in the gse81558 in thevolcano plots the green nodes indicate the downregulated degs and the red nodes indicate the upregulated degs c thevenn diagram manifested that a total of degs were exist in the two datasets gse41258 and gse81558 simultaneously dthe ppi network of the common degs the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commons attributionlicense cc by 0cbioscience reports bsr20200963101042bsr20200963the functional enrichment analysis of degs via go and kegggo analysis manifested that variations in degs related with biological processes bp were significantly enrichedin bicarbonate transport onecarbon metabolic process cell surface receptor signaling pathway collagen catabolicprocess transport xenobiotic transport body fluid secretion axon development positive regulation of guanylate cyclase activity drug transmembrane transport response to steroid hormone response to tumor necrosis factor positiveregulation of peptidylthreonine phosphorylation cell proliferation and regulation of intracellular ph figure 2athe variations in degs related with cellular components cc were significantly enriched in extracellular space extracellular region anchored component of membrane proteinaceous extracellular matrix plasma membrane apicalplasma membrane integral component of plasma membrane apical part of cell extracellular exosome and basolateralplasma membrane figure 2b the variations in degs related with molecular functions mf were significantly enriched in hormone activity carbonate dehydratase activity xenobiotictransporting atpase activity arylesterase activity metalloendopeptidase activity neuropeptide hormone activity and hydrolase activity hydrolyzing oglycosylcompounds figure 2c kegg pathway enrichment analysis showed that the top pathways related with degs werenitrogen metabolism bile secretion proximal tubule bicarbonate reclamation and pancreatic secretion figure 2dsignificant module network and identiï¬cation of hub genesa significant module was screened from the ppi network and the module network consisted of nodes and edgesfigure 2e and ten hub genes were identified including clca4 guca2a gcg sst ms4a12 plp1 chgapyy vip and guca2b figure 2f the function of hub genes were summarized in the table strong interaction among the hub genesthrough the pearsons correlation test heat maps manifested that there were strong correlations among hub genes inthe gse41258 supplementary figure s2a and gse81558 supplementary figure s2b datasets pyy sst gcg andvip existed simultaneously in the coexpression network via cbioportal supplementary figure s2c and throughthe analysis of coexpedia there were strong interactions among pyy sst gcg chga clca4 guca2b andms4a12 supplementary figure s2ddifference of expression of hub genes between pcrc and controlsamplesheat map showed that the expressions of all the hub genes were lower in the pcrc samples than the control samplessupplementary figure s2e hierarchical clustering allowed for simple differentiation of pcrc tissues from normalcolorectal tissues via the expression levels of hub genes in the gse41258 supplementary figure s3a and gse81558supplementary figure s3b datasets the expressions of all the hub genes were lower in the pcrc group than thecontrol groupthe analysis of expression level of hub genesthe hub genes in the human different ans were expressed in the supplementary figure s3c the pink presents thetumor individuals and the green presents the normal individuals the expression of hub genes in the colorectum washigher in the normal individuals compared with the tumor samples supplementary figure s3c when we comparedthe expression of hub genes in the various tumors the all hub genes were downregulated in the pcrc samples alsonamed colon adenocarcinoma coad supplementary figure s3d through gepia analysis the expressions ofhub genes in the pcrc patients were lower than the normal individuals supplementary figure s4aassociation between hub gene expression pathological stage andoverall survivalthe results of gepia manifested that the expression of vip was significantly positively related with pathological stagep0027 while the expression of clca4 guca2a gcg sst ms4a12 plp1 chga pyy and guca2b wasnot as supplementary figure s4b kaplanmeier analysis showed that pcrc patients with low expression levelsof clca4 and ms4a12 had poorer overall survival times than those with high expression levels p005 figure3ae pcrc patients with high expression levels of gcg sst plp1 and chga had poorer overall survival timesthan those with low expression levels p005 figure 3cdf supplementary figure s5a however there was nostatistically significant effect on os associated with the expression of guca2a pyy vip and guca2b p005figure 3b supplementary figure s5bd the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commons attributionlicense cc by 0cbioscience reports bsr20200963101042bsr20200963figure the enrichment analysis for degs and the identiï¬cation of hub genesa detailed information relating to changes in the biological processes bp of degs in pcrc and control colorectal samplesb detailed information relating to changes in the cellular components cc of degs in pcrc and control colorectal samples cdetailed information relating to changes in the molecular functions mf of degs in pcrc and control colorectal samples d keggpathway analysis for degs e the significant module of the ppi network f the hub genes identified from the ppi network the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commons attributionlicense cc by 0cbioscience reports bsr20200963101042bsr20200963figure the overall survival kaplanmeier of six hub genesa clca4 b guca2a c gcg d sst e ms4a12 f plp1 the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commons attributionlicense cc by 0cbioscience reports bsr20200963101042bsr20200963table the correlation and linear regression analysis between pcrc and relevant gene expressionpcrcmultiple linear regressionvifodtgene symbolpearsons correlation coefï¬cientpvalueÏaclca4guca2agcgsstms4a12plp1chgapyyvipguca2bpvalueapearsons correlation coefï¬cient between pcrc and relevant characteristics Ï pearsons correlation coefï¬cientbmultiple linear regression analysis pcrc primary colorectal cancer p005 p001 p0001table receiver operator characteristic curve analysis of hub gene expression for pcrcgene symbolpcrcclca4guca2agcgsstms4a12plp1chgapyyvipguca2bauc0987maxpvalue95ciauc area under curve max the maximum of auc significant variables odt optimal diagnostic thresholdpcrc primary colorectal cancer p0001correlation linear regression and roc analysisthe pearsons correlation coefficient was used in the correlation analysis and clca4 Ï p0001guca2a Ï p0001 gcg Ï p0001 sst Ï p0001 ms4a12 Ï p0001 plp1 Ï p0001 chga Ï p0001 pyy Ï p0001 vip Ï p0001 and guca2b Ï p0001 were significantly correlated with pcrc table in themultivariate linear regression model holding all other variables at a fixed value the natural logarithmic dn remainedassociated with clca4 guca2a sst ms4a12 plp1 chga pyy and guca2b p005 table to identify accurate thresholds for hub genes to predict pcrc we constructed roc the expression of all hubgenes was associated with a diagnosis of pcrc auc pvalue0001 table and figure the roccurve of clca4 was shown in figure 4a and the area under curve of clca4 was maximal the roc curve ofguca2a was shown in figure 4b the roc curve of gcg was shown in figure 4c the roc curve of sst wasshown in figure 4d the roc curve of ms4a12 was shown in figure 4e the roc curve of plp1 was shown infigure 4f the roc curve of chga was shown in figure 4g the roc curve of pyy was shown in figure 4h theroc curve of vip was shown in figure 4i the roc curve of guca2b was shown in figure 4j the roc curves ofper hub genes are shown in figure 4k the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commons attributionlicense cc by 0cbioscience reports bsr20200963101042bsr20200963figure roc curves of hub genes for pcrca clca4 b guca2a c gcg d sst e ms4a12 f plp1 g chga h pyy i vip j guca2b k roc curves of allhub genes the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commons attributionlicense cc by 0cbioscience reports bsr20200963101042bsr20200963table clinicopathological variables and the expression status of clca4 and ms4a12clca4pms4a12plow high low high sexagemalefemale years¥ yearsoverall survival months¥ monthspearsons chisquared test was usedp005figure the veriï¬cation of expression and overall survival analysis for clca4 and ms4a12a the relative expression of clca4 based on pcr b the relative expression of ms4a12 based on pcr c the overall survivalof pcrc based the expression of clca4 d the overall survival of pcrc based the expression of ms4a12basic information of pcrc patientspatients basic information were presented in table the mean patient age was years old range yearsand the median os was months range monthsrtqpcr analysis validation of hub genesas presented in the result clca4 p005 figure 5a and ms4a12 p005 figure 5b were markedly the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commons attributionlicense cc by 0cbioscience reports bsr20200963101042bsr20200963downregulated in pcrc samples when compared with the adjacent normal colorectum tissues it should be notedthat the expression situation of clca4 and ms4a12 were consistent in above results of bioinformaticslow expression of clca4 and ms4a12 in pcrc patients wereindependent prognostic factors for the poor overall survivalthe kaplanmeier os curves were analyzed low expression of clca4 was predictive of a shorter os in the pcrc patients p005 figure 5c low expression of ms4a12 was predictive of a shorter os in the pcrc patients p005figure 5ddiscussionpcrc is a common digestive tract cancer which seriously affects the life expectancy and quality of life of patients inrecent years the survey results show that the morbidity and mortality rate are on the rise the clinical manifestations of patients with pcrc are related to the location and pathological type of the tumor the most common typeof pathology is adenocarcinoma the primary lesion located in the colon often causes diarrhea obstruction bleedingin the rectum anemia and cachexia in the later stage of cancer patients the current treatment is mainly surgerycombined with chemotherapy or radiotherapy while advocated exercise to enhance the bodys immunity and preventinfection gavrilas et al found that combination of dietary preparations such as curcumin and resveratrol withchemotherapeutic drugs contributed to the prognosis of pcrc clinical application benefit and safety of epidermal growth factor egfrrelated targeted therapy and pd1pdl1 immunotherapy are still to be further studied the investigation found that the cost of pcrc treatment is high and it takes up a lot of medical resources andthe prognosis of patients is not necessarily proportional to the input the early treatment of early treatment patientshas a relatively low total cost of treatment and a good prognosis therefore to further explore the pathogenesisof pcrc to find possible therapeutic targets to achieve early diagnosis targeted therapy individualized treatmenthas important clinical value and market prospectsbioinformatics has been widely used as a new means of exploring disease mechanism and searching fordiseaserelated genetic molecules zhang et al found genes related to hepatocellular carcinoma by bioinformaticsanalysis further analysis confirmed the correlation between these differential genes and diseases suggesting thatthese molecules may be used as molecular targets for early diagnosis and treatment zhang et al found the mostrelevant molecules of gastric cancer mir19b3p and mir165p by analyzing the genomewide mirna microarraydata of gastric cancer patients which provided a new idea for the diagnosis and treatment of gastric cancer sunfound molecules related to the pathogenesis of colorectal cancer by screening from public databases further analysisshowed that differentially expressed genes such as ppbp ccl28 and cxcl12 are likely to be involved in the development of colorectal cancer and may be potential diagnostic and therapeutic targets we found genes thatwere differentially expressed in patients with pcrc by bioinformatics analysis low expression of plp1 vip sstgcg pyy ms4a12 clca4 guca2a chga and guca2b in tumor patients compared with normal subjectsat the same time we performed survival analysis on patients with pcrc the results showed that clca4 guca2agcg sst ms4a12 and plp1 genes were significantly associated with the survival of patients with pcrcclca4 is the chloride channel accessory clca4 is a member of the calciumsensitive chloridetransportingprotein family involved in intracellular ion channel activity chloride ion transmembrane transport and proteolysis members of the calcium activated chloride channel clca gene family are thought to have multiple functionsincluding cell adhesion and tumor suppression ye et al found that clca4 is low expressed in patients with oraltongue squamous cell carcinoma through genomewide transcriptional mapping which provides ideas for diagnosisand targeted therapy bundela also found multiple differentially expressed genes in oral cancer patients in indiaand suggested that clca4 may be a potential therapeutic target yu et al found that clca4 is low expressedin breast cancer patients further analysis revealed that clca4 is a marker of breast epithelial differentiation andmay be involved in tumor proliferation and metastasis clinical data analysis showed that patients with breast cancerwith low expression of clca4 had lower recurrencefree survival rate suggesting that it may serve as a diagnosticand therapeutic target hu found that clca4 was low expressed in bladder cancer tissues further analysis revealed that clca4 may be involved in the proliferation and invasion of bladder cancer through pi3kakt signaltransduction suggesting that clca4 may be a target for diagnosis and treatment liu found that clca4 mayinhibit epithelialmesenchymal transition emt by affecting pi3katk phosphorylation thereby inhibiting cellmigration and invasion of hepatoma cells yang found that patients with colorectal cancer crc had low expression of clca1 and clca4 and further experiments confirmed that clca1 is involved in tumor proliferation andinvasion zhao found that clca4 was low expressed in colorectal patients chen also found that clca4 the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commons attributionlicense cc by 0cbioscience reports bsr20200963101042bsr20200963was low expressed in patients with colorectal cancer and believed that clca4 inhibited the epithelialmesenchymaltransformation of colorectal cancer through pi3katk signaling pathway thus participating in the proliferation andinvasion of tumors and may be used as a marker for diagnosis and judgment consistent with the above resultswe found that clca4 was lowly expressed in primary colorectal patients by bioinformatics analysis and survivalanalysis of primary colorectal patients found that patients with low expression of clca4 had a worse prognosis insurvival suggesting the protective effect of clca4 on pcrc patients and its inhibitory effect on cancer we speculated that clca4 affects epithelialmesenchymal transition and intercellular communication via pi3katk andparticipates in the development of primary colorectal patients which may be potential diagnostic and therapeutictargetsms4a12 is membrane spanning 4domains a12 as a cell protein ms4a12 participates in cell membrane composition cell differentiation proliferation and cell cycle regulation members of the ms4a family are likely to be part ofthe oligomeric cell surface complex which has different signal transduction functions ms4a12 may promote theproliferation and invasion of colorectal cancer cells by influencing epidermal growth factor receptor further studiesfound that intestinal specific transcription factor cdx2 mediated by rna interference rnai is a transactivatorof growthpromoting gene expressio | Colon_Cancer |
"high mobility group box hmgb1 is a nonhistone chromatinassociated protein widely distributed in eukaryoticcells and is involved in dna damage repair and genomic stability maintenance in response to stimulus like bacteriaor chemoradiotherapy hmgb1 can translocate to extracellular context as a danger alarmin activate the immuneresponse and participate in the regulation of inflammation and cancer progressionkeywords hmgb1 rage tlr damp inflammation cancerchromatinassociated proteinit washigh mobility group box hmgb1 is a highly conservative nucleoprotein and belongs to the group of nonhistonefirstextracted from calfthymus chromatin in andnamed for its high mobility in gel electrophoresis subsequentinvestigations found that hmgb1 couldtranslocate from the nucleus to the cytoplasm after posttranslational modifications including acetylation phosphorylation and methylation hmgb1 can be expressedat the neuron membrane as well in response to chemoradiotherapy or hypoxia hmgb1 could be transferredto the extracellular context mainly through two waysactive secretion from immunocompetent cells or passiverelease from apoptotic or necrotic cells extracellularhmgb1 transmits danger signals to surrounding cellsby interacting with its classical receptors such as thereceptor for advanced glycation end products rageand tolllike receptors tlr249indepth studies implicated that hmgb1 was a multifunctional protein involved in a variety of cellularsubcellularbiological properties depending on itslocalizationandbinding receptors fig posttranscriptional modification correspondence yizhangzzueducn1biotherapy center cancer center the first affiliated hospital ofzhengzhou university zhengzhou china2state key laboratory of esophageal cancer prevention treatmentzhengzhou university zhengzhou chinafull list of author information is available at the end of the in the nucleus hmgb1 plays a key role in the processof dna replication transcription chromatin remodeling and vdj recombinationthus regulating dnadamage repair and the maintenance of genome stabilityas a dna chaperone cytoplasmic hmgb1 is involvedin immune responses by increasing autophagy inhibitingapoptosis and regulating mitochondrial function atthe membrane hmgb1 promotes axonal sprouting andneurite growth activates platelets and induces cellmigration as a typical damage associated molecular pattern damp extracellular hmgb1 is involved in manyimmune responses by promoting immune cell maturation activation and cytokine production extracellular hmgb1 can also interact with chemokines such ascxcl11 to enhance immune responses as a multifunctional protein hmgb1 exerts different biologicaleffects under different stimuli the deregulation ofhmgb1 is associated with many diseases especiallyinflammatory disorders and cancerhmgb1 in inflammationhmgb1 plays a critical role in the regulation of bothinnate and adaptive immune responses to promoteimmune response to sterile or infectious stimulus insterile inflammation during ischemiareperfusion injuryiri hmgb1 becomes disulfidebonded to increasemacrophage production of proinflammation cytokinesin a tlr4 dependent manner indicating that disulfidebonded hmgb1 can be identified as a diagnosis and the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cwang and zhang of hematology oncology page of fig the multifunctions of hmgb1 hmgb1 in the nucleus regulates dna damage repair and genome stability as a nonhistone chromatinassociated protein and dna chaperon in the cytoplasm or mitochondria hmgb1 increases autophagy inhibits apoptosis and regulatesmitochondria functions at the membrane hmgb1 promotes axonal sprouting and neurite growth hmgb1 can be transferred to extracellularcontext by two ways active secretion from immunocompetent cells or passive release from apoptotic or necrotic cells participating inimmune responsesin contrasttreatment biomarker for iri when initially secreted byinnate immune cells like macrophages hmgb1 is proinflammatory during the early stages of sepsis howeverthe extracellular hmgb1 could also induce immune tolerance and immunosuppression when released by othersomatic cellsintracellular hmgb1 caninduce protective autophagy and contribute to cellsurvival by delivering lipopolysaccharide lps andpromoting endocytosis hmgb1 activates the noncanonical inflammasome pathway and induces pyroptosis pyroptotic macrophage death may accelerateundesirable immune hyperactivity and immunosuppression which is a potential mechanism associatedwith late mortality from sepsis in hepatic infectious disease the release and activity of hmgb1 as acytokine could be suppressed by glycyrrhizinic acidga by binding with tlr4 hmgb1 regulatesthe hepatitis virusesinduced immunological axis providing a new therapy strategy for the treatment ofacute viral hepatitis in the clinical practice canbesecretedin severe pulmonary inflammatory diseases including covid19 hmgb1inabundance by necrotic pulmonary epithelial cells andinnate immune cells disulfidehmgb1 triggers proinflammatory cytokine release and further exacerbatessevere inflammation therefore hmgb1 mightbeofinflammationtreatmentpotentialtargetaforthethe dual effects of hmgb1 in canceraccording to the alterations of the subcellular locationsreceptors and expression levels hmgb1 is associatedwith the hallmarks of cancer proposed by hanahan andweinberg hmgb1 appears to play paradoxicalroles during the development and therapy of cancer onthe one hand hmgb1 can contribute to tumorigenesisexcessive hmgb1 production caused by chronic inflammatory response seems to be associated with tumorigenesis for example by combining with rage hmgb1plays an important role in regulating oval cells activationand inflammationassociated liver carcinogenesis in mice in established cancers hmgb1 produced by tumorcells may exacerbate inflammationrelated immunosuppression for instance previous research indicated thatlps induced the release of proinflammatory cytokinessuch as il1 il6 and tnfα in a hmgb1dependentmanner to improve colon cancer progression however the underlying mechanism of hmgb1 in the transformation ofinflammation and cancer needs to befurther studied it has been reported that hmgb1 canbe released to extracellular context by necrotic cellsunder hypoxia in growing solid tumor extracellularhmgb1 promotes the release of cytokines such as il6and il8 by activating mapk and myd88dependentnfκb pathways which in turn stimulates tumor cellsproliferation angiogenesis emt invasion and metastasis nucleusand cytoplasmic hmgb1 promotes 0cwang and zhang of hematology oncology page of autophagy and inhibits apoptosis of tumor cells to induce chemotherapy resistance on the other handhmgb1 plays a protective role in the suppression oftumor and tumor chemoradiotherapy and immunotherapy nucleus hmgb1 assists in the regulation of telomere and maintenance of genome stability loss ofhmgb1 results in the instability of genome and leads totumorigenesis thus the roles of hmgb1 in regulationof dna damage repair and cancer etiology indicate thattargeting chromosomal architectural hmgb1 may provide a new perspective for cancer therapy hmgb1located in the cytosol or mitochondria may bind toautophagy associated genes like beclin to regulate cellautophagy and mitophagy absence of hmgb1 resultsin autophagy deficiency and increased apoptosis leadingto tumorigenesis intracellular hmgb1 functions as atumor suppressor by binding tumor suppressor proteinslike rb but it remains to be studied whether hmgb1interacts with other tumor suppressors or oncoproteinsextracellular hmgb1 enhances chemotherapy efficacyby transforming tumor cells from apoptosis to senescence in addition hmgb1 can mediate immunogenic cell death during chemoradiotherapy and enhanceantitumor immunity in response to chemotherapy likeanthracycline or radiotherapy hmgb1 can be rapidlyreleased from dead cells as an alarming molecule uponrelease from necrotic cells or secreted by activated macrophages hmgb1 can recruit inflammatory cells andmediate interactions between nk cells dendritic cellsdcs and macrophages activated nk cells provide anadditional source of hmgb1 which is released into theimmunological synapse between nk cells and immaturedcs promoting the maturation of dcs and the induction of th1 response in addition hmgb1 produced from nsclc cells induced by docetaxel canstimulate t cells for antitumor immune response andimprove immunotherapy effects like cart cells therefore modulating hmgb1 may provide a potentialcombination strategy for cancer chemoradiotherapy andimmunotherapyconclusionhmgb1 is a multifunctional molecule that plays a majorrole in homeostasis as damp molecule hmgb1 playsthe complex roles in various biological processes and isinvolved in the development of many diseases such asautoimmune diseases and cancers hmgb1targetedagents including antibodies and inhibitors have shownbeneficial results in preclinicalinflammatory modelssuch as sepsis these agents await clinical developmentabbreviationshmgb1 high mobility group box rage the receptor for advancedglycation end products tlr tolllike receptors damp damage associatedmolecular pattern iri ischemiareperfusion injury lps lipopolysaccharidega glycyrrhizinic acid covid19 coronavirus disease19 mapk mitogenactivated protein kinase myd88 myeloid differential protein88 nfκb nuclear factor kappalightchainenhancer of activated b cellsemt epithelialmesenchymal transition dcs dendritic cellsacknowledgementsnot applicableauthorsâ contributionsyz designed directed and revised the manuscript sw drafted themanuscript both of the authors read and approved the final manuscriptfundingthis work was supported by the national key research and developmentprogram of china 2016yfc1303500availability of data and materialsnot applicableethics approval and consent to participatenot applicableconsent for publicationnot applicablecompeting intereststhe authors declare that they have no competing interestsauthor details1biotherapy center cancer center the first affiliated hospital ofzhengzhou university zhengzhou china 2state key laboratory ofesophageal cancer prevention treatment zhengzhou universityzhengzhou china 3henan key laboratory for tumor immunologyand biotherapy zhengzhou chinareceived july accepted august referencesgoodwin gh sanders c johns ew a new group of chromatinassociatedproteins with a high content of acidic and basic amino acids[j] febs jâpaudel yn angelopoulou e piperi c balasubramaniam vrmt othman ishaikh mf enlightening the role of high mobility group box hmgb1 ininflammation updates on receptor signalling[j] eur j pharmacol huebener p gwak gy pradere jp et al highmobility group box isdispensable for autophagy mitochondrial quality control and anfunction in vivo[j] cell metab ârivera vargas t apetoh l danger signals chemotherapy enhancers[j]immunol rev âgao q wang sm chen xf et al cancercellsecreted cxcl11 promotedcd8 t cells infiltration through docetaxelinducedrelease of hmgb1 innsclc[j] for immunotherapy of cancer andersson u tracey kj hmgb1 is a therapeutic target for sterileinflammation and infection[j] annu rev immunol âkim hm kim ym hmgb1 lps delivery vehicle for caspase11mediatedpyroptosis[j] immunity âdeng m tang y li w et al the endotoxin delivery protein hmgb1mediates caspase11dependent lethality in sepsis[j] immunity â753e747shi xd yu lj zhang yl et al glycyrrhetinic acid alleviates hepaticinflammation injury in viral hepatitis disease via a hmgb1tlr4 signalingpathway[j] int immunopharmacol saha b tornai d kodys k et al biomarkers of macrophage activation andimmune danger signals predict clinical outcomes in alcoholic hepatitis[j]hepatology baltimore md â andersson u ottestad w tracey kj extracellular hmgb1 a therapeutictarget in severe pulmonary inflammation including covid19[j] molecularmedicine cambridge mass 0cwang and zhang of hematology oncology page of hanahan d weinberg ra hallmarks of cancer the next generation[j] cellâ pusterla t nèmeth j stein i et al receptor for advanced glycationendproducts rage is a key regulator of oval cell activation andinflammationassociated liver carcinogenesis in mice[j] hepatologybaltimore md â yang y yang l jiang s et al hmgb1 mediates lipopolysaccharideinducedinflammation via interacting with gpx4 in colon cancer cells[j] cancer cellint yuan s liu z xu z et al high mobility group box hmgb1 a pivotalregulator of hematopoietic malignancies[j] j hematol oncol mukherjee a vasquez km targeting chromosomal architectural hmgbproteins could be the next frontier in cancer therapy[j] cancer res liu y dong y kong l et al abscopal effect of radiotherapy combined withimmune checkpoint inhibitors[j] j hematol oncol publisherâs notespringer nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations 0c" | Colon_Cancer |
" immune checkpoint inhibitors that block programmed cell death1 pd1 and programmed cell death ligand1 pd l1 have improved outcomes for many cancer subtypes but do exhibit toxicity in the form of immune related adverse eventsobjective the aim of this study was to investigate the emerging toxicities of pd1 and pd l1 inhibitors including acute or reactivation of tuberculosis tb and atypical mycobacterial infection amimethods this study was completed as a retrospective review using the us food and drug administration adverse events reporting system faers for incidence of tb and ami due to pd1 and pd l1 inhibitors compared with other fda food and drug administration approved drugs the statistical methods included disproportionality signal analysis using the reporting or ror to compare cases the wald ci was reported to assess the precision of the rorresults out of the adverse events aes reported to faers for all drugs between january and march aes were due to the five fda approved pd1pd l1 inhibitors seventy two cases of tb were due to pd1pd l1 inhibitors specifically cases due to nivolumab due to pembrolizumab due to atezolizumab and due to durvalumab there were cases of ami due to nivolumab due to pembrolizumab and each due to durvalumab and atezolizumab avelumab was not attributed to any ae of tb or ami from analysis of the faers database the calculated ror for tb due to pd1pd l1 inhibitors was ci to p00001 and for ami was ci to p00001 pd1pd l1 inhibitors used in the treatment of cancer subtypes is associated with increased tb and ami risk although this complication is rare clinicians using pd1pd l1 inhibitors should be aware of the risksintroductionimmune checkpoint inhibitors icis that block programmed cell death1 pd1 and programmed cell death ligand1 pd l1 have transformed care for many cancer subtypes and have improved outcomes for patients with pd l1 overexpression1 through blockade of the pd1pdl1 axis the t lymphocyte mediated response against tumour cells is enhanced resulting in accelerated immune mediated destruction of key questionswhat is already known about this subject º case reports and case series suggest programmedcell death1programmedcell death ligand1 pd1pd l1 inhibitors are associated with acute tuberculosis tb or reactivation of tbwhat does this study add º this is the first large systemic effort to quantify the risk of tb due to pd1pd l1 inhibitors through retrospective analysis of faers food and drug administration adverse events reporting system a pharmacovigilance database pd1pd l1 inhibitors were not only associated with increased risk of tb compared with other drugs but atypicalmycobacterial infection as wellhow might this impact on clinical practice º although this complication is rare clinicians using pd1pd l1 inhibitors should be aware of thiscancer cells however facilitating immune mediated activation is not benign and patients receiving icis are known to exhibit unique toxicities that result in an damage known as immune related adverse events iraes3 the most common iraes with pd1 and pd l1 inhibitors are fatigue pruritus and diarrhoea4 some iraes can be fatal with pneumonitis hepatitis neurotoxicity and most commonly myocarditis reported5 while counterintuitive when the mechanism of action is considered an emerging and increasingly reported toxicity of pd1 and pd l1 inhibitors is acute tuberculosis tb and reactivation of tb6 the first case of tb due to the pd1 inhibitor was described in a patient with relapsed hodgkins lymphoma who developed pulmonary tb following treatment with pembrolizumab7 since then there have been other case reports of tb following initiation of pd1 or pd l1 inhibitors that make the development of tb a relevant concern8 in a preclinical mouse study pd1 deficient mice were found to be highly susceptible to tb with reduced survival compared with wild type mice12 however anand a0k et a0al esmo open 20205e000866 101136esmoopen2020000866 0copen accesstable adverse events of tb and ami due to pd1pdl1 inhibitors from january to march in faerstotal aes due to all drugs in faerstotal aes due to pd1pdl1 inhibitorstotal aes due to pd1 inhibitorstotal aes of tb in faerstotal aes of tb due to pd1pdl1 inhibitorstotal aes of ami in faerstotal aes of ami due to pd1pdl1 inhibitorsror calculation ror for tb due to pd1pdl1 inhibitors versus full database ror for ami due to pd1pdl1 inhibitors versus full database ci to ci to aes adverse events ami atypical mycobacterial infection faers food and drug administration adverse events reporting system pd1 programmed cell death1 pd l1 programmed cell death ligand1 ror reporting or tb tuberculosisthere is no current risk estimate describing the potential risk of developing tb or atypical mycobacterial infection ami from pd1 and pd l1 inhibitors in this study we retrospectively reviewed the us food and drug administration adverse events reporting system faers a pharmacovigilance database for the risk of tb and ami due to pd1 and pdl1 inhibitors compared with other fda foodand drug administration approved drugsmethodsthis study is a retrospective analysis that used data queries from the faers pharmacovigilance monitoring database faers is a public database that contains nearly million adverse event ae reports medication error reports and product quality complaints reported by healthcare professionals manufacturers and consumers from around the world since these reports are managed by fda and evaluated by clinical reviewers in the center for drug evaluation and research and the center for biologics evaluation and research date in each event report where applicable include individual case identification numbers for reference the suspected pharmaceutical agent treatment indication adverse reactions nature of the event ie serious outcomes eg hospitalised death other outcomes sex male female or unknown age weight event date initial fda receipt date latest fda receipt date pharmaceutical company reporter eg healthcare professional consumer pharmaceutical company unknown concomitant medications latest manufacturer received date country where the event occurred and manufacturer control number individual names and date of birth are excluded from these liststhe present study involved data queries of the faers database between january and march for aes secondary to pd1 inhibitors namely pembrolizumab and nivolumab and pd l1 inhibitors namely atezolizumab durvalumab and avelumab in all aes due to above five drugs we then searched for three aes specifically tuberculosis pulmonary tuberculosis and atypical mycobacterial infection tuberculosis and pulmonary tuberculosis were grouped together for analysis all other events that were reported in patients with tb or ami were characterised into subcategories including pulmonary infectious endocrine gastrointestinal hepatobiliary dermatological cardiac haematological neurological vascular infusion related rheumatological and otherstb and ami cases among patients treated with pd1 and pd l1 inhibitors were compared with all reported tb and ami events in the database due to other drugs by conducting a disproportionality signal analysis based on the reporting or ror the ror is a measure of the magnitude of association between an exposure to a pharmaceutical agent and the odds of a specific outcome occurring in the setting of an elevated ror it can be conferred that there is an elevated risk of an adverse event occurring with a specific medication the wald ci was used to assess the precision of the ror when lower limit of ror and ci did not cross ror was considered significant13 the likelihood of association between pd1pd l1 inhibitors and tbami were investigated using two sided Ï2 or fishers exact tests as warranted all analyses were conducted using sas sas institute inc cary north carolina usa and statistical significance was defined as p005resultsbetween january to march a total of adverse events report cases were generated in faers out of ae there were associated with the approved five pd1pd l1 inhibitors the majority of aes were reported with nivolumab and pembrolizumab at in faers there were reports of tb with any drug of which were reported with pd1pdl1 inhibitors the ror for tb due to pd1pd l1 inhibitors was elevated at ci to p00001 for ami there were reports associated with all drugs of which were due to pd1pd l1 inhibitors the anand a0k et a0al esmo open 20205e000866 101136esmoopen2020000866 0ctable details of tb ae due to pd1pdl1 inhibitorstable continuedopen access serious nivolumab pembrolizumab atezolizumab durvalumab lung cancer gastric cancer head and neck cancer hodgkins lymphoma malignant melanoma colon cancer neuroendocrine carcinoma ovarian carcinoma pancreatic carcinoma plasma cell myeloma renal cell carcinoma transitional cell carcinoma unknowntotal number of tb aespd1pdl1 inhibitor used indication for pd1pdl1 use type of reaction sex median age years range min maxoutcome reporter year initial report received region of origin of ae died hospitalised life threatening other outcome asia europe americas africa australia healthcare professional consumer male female n54 continuedsuspected drug pd1pdl1 inhibitor pd1pdl1 inhibitor ¥ aes adverse events pd1 programmed cell death1 pd l1 programmed cell death ligand1 tb tuberculosisror for ami due to pd1pd l1 inhibitors was elevated at ci to p00001 table out of cases of tb due to pd1pd l1 inhibitors were due to nivolumab followed by due to pembrolizumab and due to atezolizumab and durvalumab respectively there were no cases reported with avelumab the most common indication for which pd1pd l1 inhibitor was used was lung cancer median age of the whole cohort was years eighty per cent of the patients were men and were women out of cases cases had a reported outcome of death the most common region of origin in which tb was reported was asia sixteen cases had pd1pd l1 inhibitors plus one of more non checkpoint inhibitor drug listed as a suspect drug leading to ae table out of cases of ami due to pd1pd l1 inhibitors were due to nivolumab followed by due to pembrolizumab and each due to durvalumab and atezolizumab no report of ami attributable to avelumab was found the most common reason for use of pd1pd l1 inhibitor was lung cancer median age of the entire cohort was years seventy three per cent of patients were men and were women out of cases patient died the most common region in which ami was reported was asia one case had pd1pd l1 inhibitors plus one of more non checkpoint inhibitor drug listed as a suspect drug leading to ae table patients who had tb due to pd1pd l1 inhibitors also had additional reported pulmonary complications in of cases followed by other infectious complications in of cases similarly patients who had ami attributed to use of pd1pd l1 inhibitors had pulmonary complications in of cases followed by endocrine dermatological and others in of the cases table discussionin this retrospective pharmacovigilance database review pd1pd l1 inhibitors had a statistically significant positive signal with tb and ami with a proportion of these events associated with mortality nivolumab had the highest frequency of reported tb and ami whereas avelumab had no reported events most commonly affected patients were receiving treatment for lung cancer and the most commonly reported country of origin was japananand a0k et a0al esmo open 20205e000866 101136esmoopen2020000866 0copen accesstb has a high disease burden worldwide with the highest disease associated mortality of any infectious agent in there were million new cases globally and million reported deaths14 amis are estimated to occur in approximately to per persons with an increasing incidence in developed countries15 there is growing evidence that patients receiving icis can develop tb reaction while on treatment6 to date there are reported cases of tb secondary to icis none of which were attributed cytotoxic t lymphocyte associated protein ctla4 inhibitors median time to diagnosis from ici initiation was months range to months17 the mechanism by which a pd1pd l1 inhibitor results in tb is not clear in a murine study pd1 knockout mice had decreased survival compared with wild type mice following exposure to mycobacterium tuberculosis furthermore pd1 inhibition is needed to prevent cd4 t cells from promoting development of tb18 pd1 inhibition mitigates over production of interferon gamma ifnγ which is important for host resistance to tb19increased risk of tb and ami is also found in patients on tumor necrosis factor tnf alpha inhibitors and janus kinase jak inhibitor ruxolitinib20 patients treated with infliximab a tnf alpha inhibitor were and times more likely to develop tb and ami respectively22 in patients prior to start tnf alpha inhibitors screening for latent tb is recommended20 if the patient is found to have latent tb treatment with isoniazid is recommended as it substantially reduces the risk of developing tb reactivation23 however a recent study suggests that pd1 inhibition induced tb reactivation is actually driven by tnf alpha and use of tnf alpha inhibitor could reverse this complication24 there are currently no recommended screening guidelines for latent tb prior to starting pd1pd l1 inhibitors a single institution study in germany found that of patients had positive test for quantiferon gold tb plus qgt prior to starting icis however none of the patients who had a positive qgt test developed tb while on treatment with icis6 of the cases of tb reported in literature due to icis treatment with icis was stopped in all cases tb treatment was initiated and seven cases had re initiation of icis out of seven who had re initiation of ici five had response to therapy one had progression and in one case response was not available17 as tb reactivation may lead to treatment interruptions or discontinuation standardised recommendations for tb screening in patients with planned ici should be considered with substantiation of results from the current study in prospective studiesthis is the first study using faers to demonstrate the potential risk of developing tb and ami in pd1pd l1 inhibitor treated patients as pd1pd l1 inhibitors use becomes more prevalent on a global scale including regions with an elevated prevalence of latent tb clinicians need to consider the risk benefit and economic impacts of screening for latent tb and treatment initiation if the patient is positive these questions cannot be table details of ami ae due to pd1pdl1 inhibitors serious male female nivolumab pembrolizumab atezolizumab durvalumab lung cancer head and neck cancer malignant melanoma unknowntotal number of ami aespd1pdl1 inhibitor used indication for pd1pdl1 use type of reaction sex median age years range min maxoutcome reporter healthcare professionalyear initial report received region of origin of ae suspected drug died hospitalised life threatening other outcome pd1pdl1 inhibitor pd1pdl1 inhibitor ¥ asia europe americas n10 aes adverse events ami atypical mycobacterial infection pd1 programmed cell death1 pd l1 programmed cell death ligand1answered in this observational signal analysis and future prospective research studies should be conducted if a patient develops tb or ami while on treatment with pd1pd l1 inhibitors permanent discontinuation of therapy should be avoided if there is clear clinical benefit from ici and multidisciplinary discussions regarding treatment delay should be conducted with the treating oncologist and infectious disease specialists a majority anand a0k et a0al esmo open 20205e000866 101136esmoopen2020000866 0copen accesstable other aes grouped into major an systems in patients treated with pd1pdl1 inhibitorstb death events n13tb alive events n59tb totaln72ami death events n1ami alive events n12ami totaln13pulmonaryinfectiousendocrinegastrointestinalhepatobiliarycardiachaematologicaldermatologicalneurologicalvascularinfusion relatedrheumatologicalothers aes adverse events ami atypical mycobacterial infection pd1 programmed cell death1 pd l1 programmed cell death ligand1 tb tuberculosisof patients in whom tb or ami have reported are those with lung cancer it is worth pointing out that especially in patients with lung cancer there is significant difficulty in differentiating immune pneumonitis or radiation pneumonitis true disease progression or infectious causes prospective studies of iraes should include testing for tb or ami in diagnostic work upfrom pseudoprogression this study has limitations this analysis was a retrospective study of reported events in faers and as such baseline characteristics including presence of latent tb was not known moreover the actual incidence of tb or ami due to pd1pd l1 inhibitors cannot be determined because faers reports patients with aes not total number of patients taking the medication furthermore it is likely that not all cases of tb that occur in the clinical setting are reported within faers as such there are similar limitations in ror estimate ae reporting for a drug may be influenced by extent of use publicity and bias25 although the use of disproportionality analysis through pharmacovigilance databases to determine the increased risk of aes secondary to particular drug has been shown in various settings25 it is critical that any hypothesis generated by using pharmacovigilance databases are validated through prospective studiespd1pd l1 inhibitors used in treatment for cancer is associated with increased risk of tb and ami the most common drug in faers attributed to tb and ami is nivolumab in this study lung cancer was the most common indication for which use of pd1pd l1 inhibitor leads to tb or ami although this complication is rare clinicians using icis should be aware of this anand a0k et a0al esmo open 20205e000866 101136esmoopen2020000866possibility currently there is no additional data available to support or refute the need to screen patients for latent tb prior to initiation of icis prospective studies are needed to address these questions as well as indications to initiate prophylactic therapytwitter vivek subbiah viveksubbiahcontributors conceptualisation ka gs and spi data collection and analysis ka gs je and spi statistics je first draft preparation ka gs and spi review and final approval all authorsfunding the authors have not declared a specific grant for this research from any funding agency in the public commercial or not for profit sectorscompeting interests vs and spi coi can be found onlinepatient consent for publication not requiredprovenance and peer review not commissioned externally peer revieweddata availability statement data are available in a public open access repository all data relevant to the study are included in the article or uploaded as supplementary information faers used for this study is public databaseopen access this is an open access article distributed in accordance with the creative commons attribution non commercial cc by nc license which permits others to distribute remix adapt build upon this work non commercially and license their derivative works on different terms provided the original work is properly cited any changes made are indicated and the use is non commercial see a0http creativecommons licenses by nc orcid idskartik a0anand http orcid vivek a0subbiah http orcid references coit dg thompson ja albertini mr et a0al cutaneous melanoma version nccn clinical practice guidelines in oncology j natl compr canc netw ettinger ds wood de aggarwal c et a0al nccn guidelines insights non small cell lung cancer version j natl compr canc netw 0copen access postow ma sidlow r hellmann md immune related adverse events associated with immune checkpoint blockade n engl j med wang y zhou s yang f et a0al treatment related adverse events of pd1 and pd l1 inhibitors in clinical trials a systematic review and meta analysis jama oncol wang dy salem j e cohen jv et a0al fatal toxic effects associated with immune checkpoint inhibitors a systematic review and meta analysis jama oncol langan ea graetz v allerheiligen j et a0al immune checkpoint inhibitors and tuberculosis an old disease in a new context lancet oncol 202021e55 lee jjx chan a tang t tuberculosis reactivation in a patient receiving anti programmed death1 pd1 inhibitor for relapsed hodgkin's lymphoma acta oncol fujita k terashima t mio t anti pd1 antibody treatment and the development of acute pulmonary tuberculosis j thorac oncol chu y c fang k c chen h c et a0al pericardial tamponade caused by a hypersensitivity response to tuberculosis reactivation after anti pd1 treatment in a patient with advanced pulmonary adenocarcinoma j thorac oncol 201712e111 anastasopoulou a ziogas dc samarkos m et a0al reactivation of tuberculosis in cancer patients following administration of immune checkpoint inhibitors current evidence and clinical practice recommendations j immunother cancer picchi h mateus c chouaid c et a0al infectious complications associated with the use of immune checkpoint inhibitors in oncology reactivation of tuberculosis after anti pd1 treatment clin microbiol infect lázár molnár e chen b sweeney ka et a0al programmed death1 pd1 deficient mice are extraordinarily sensitive to tuberculosis proc natl acad sci u s a rothman kj lanes s sacks st the reporting odds ratio and its advantages over the proportional reporting ratio pharmacoepidemiol drug saf anization wh global tuberculosis report cassidy pm hedberg k saulson a et a0al nontuberculous mycobacterial disease prevalence and risk factors a changing epidemiology clin infect dis 200949e124 prevots dr shaw pa strickland d et a0al nontuberculous mycobacterial lung disease prevalence at four integrated health care delivery systems am j respir crit care med zaemes j kim c immune checkpoint inhibitor use and tuberculosis a systematic review of the literature eur j cancer barber dl mayer barber kd feng cg et a0al cd4 t cells promote rather than control tuberculosis in the absence of pd1 mediated inhibition j immunol sakai s kauffman kd sallin ma et a0al cd4 t cell derived ifnγ plays a minimal role in control of pulmonary mycobacterium tuberculosis infection and must be actively repressed by pd1 to prevent lethal disease plos pathog 201612e1005667 solovic i sester m gomez reino jj et a0al the risk of tuberculosis related to tumour necrosis factor antagonist therapies a tbnet consensus statement eur respir j anand k burns ea ensor j et a0al mycobacterial infections with ruxolitinib a retrospective pharmacovigilance review clin lymphoma myeloma leuk winthrop kl baxter r liu l et a0al mycobacterial diseases and antitumour necrosis factor therapy in usa ann rheum dis gómez reino jj carmona l ángel descalzo m et a0al risk of tuberculosis in patients treated with tumor necrosis factor antagonists due to incomplete prevention of reactivation of latent infection arthritis rheum tezera lb bielecka mk ogongo p et a0al anti pd1 immunotherapy leads to tuberculosis reactivation via dysregulation of tnfα elife 20209e52668 anand k ensor j trachtenberg b et a0al osimertinib induced cardiotoxicity a retrospective review of the fda adverse events reporting system faers jacc cardiooncology anand k ensor j pingali sr et a0al t cell lymphoma secondary to checkpoint inhibitor therapy j immunother cancer 20208e000104anand a0k et a0al esmo open 20205e000866 101136esmoopen2020000866 0c" | Colon_Cancer |
coronaviruses covs belong to a family of envelopedviruses with a positive sense singlestranded rna genomecovs cause illness ranging from upper respiratory tractinfections urtis resembling the common cold to lowerrespiratory tract infections lrtis such as bronchitis pneumonia and even severe acute respiratory syndrome sarswith most serious disease outcomes in the elderly immunocompromised patients and infants [ ] hcovoc43oc43 hcov229e 229e hcovnl63 nl63 andhcovhku1 hku1 were the ï¬rst documented humancovs hcovs which usually cause urtis and less frequently are associated with ltri diseases in the lastdecades two human coronaviruses created great concernfor the world medical community due to significant diseaseand mortality [ ] in severe acute respiratorysyndromecoronavirus sarscov was characterized byacute atypical pneumonia and diï¬use alveolar damagedad in roughly patients and with almost deathsrepresenting a nearly mortality rate more recentlyin a new human coronavirus designated as middle eastrespiratory syndromecoronavirus merscov was identiï¬ed and the global ongoing outbreak of mers with over oï¬cial cases and deaths represented approximately case fatality rate to date in humans over the last few months a new strain of human coronavirus sarscov2 also known as 2019ncov hascaught the worlds seven continents attention with its rapidglobal spread aï¬ecting at least countries and territoriesinfecting more than and claiming more than lives worldwide the coronavirus pandemichas promoted isolation and uncertainly fear and panicworldwide in additionlikely lead to changes inpolitical and economic power in ways that can be determined only later it willit is important to note that there are many similaritiesamong diï¬erent coronavirus species but not in all aspects 0coxidative medicine and cellular longevitydepending on the molecular mechanism of viral inhibitionpromoted by an antiviral agent the analysis of the data andcomparison between animal and human covs must be donevery carefully in fact it is important to note that there arediï¬erences between human and animal cov receptorswhich will likely result in diï¬erent aï¬nities or unlikely interactions of an antiviral agent with the diï¬erent cov receptors howeverif the antiviral agent interferes with thereplication andor assembly of the covs there is a higherprobability of obtaining similar antiviral activity results inhuman cov tests [ ] following this line our searchin specialized literature was focused mainly on studies thatinvestigated the anticoronavirus eï¬ects of natural antioxidants by inhibiting proteases for viral replication materials and methodsthe present study was carried out based on a search of the literature of natural antioxidants and coronavirus the searchperformed in the pubmed database included studies published until march and used the following keywordscoronavirusstressmerscov sarscov 229e nl63 oc43 hku1merscov virus infection and middle east respiratorysyndrome virus the scientiï¬c publications were selectedfrom studies published in the english languageantioxidants ï¬avonoids oxidative pathogenic mechanism of coronavirusinduced cell damagethe high mortality rate associated with the three pathogenichcovs has been mainly attributed to the development ofdigestive and respiratory tract injuries observed followinginfection acute atypical pneumonia and diï¬use alveolardamage that progress to deposition of ï¬brous tissue denudedairways haemorrhage and elevated macrophage ltrationare sometimes accompanied by watery diarrhoea dehydration and vomiting [ ]despite the molecular mechanisms of coronavirusinduced intestine and lung pathogenesis not fully elucidatedand still unclear studies have suggested that lateterm diseaseprogression is unrelated to viremia it is now believed morelikely to be associated with the immunopathological mechanism [ ] viral clearance and subsequent recovery frominfection require activation of an eï¬ective host immuneresponse however many immune eï¬ector cells may alsocause injury to host tissues together with ammatoryand immune response signaling the presence of oxidativecompounds such as reactive oxygen species ros playsimportant roles in the pathogenic mechanism of cell damageinduced by covs through oxidative stress oxidative stress is deï¬ned as an interruption andorderegulation of the signaling and redox system that can becaused by an imbalance in the production of oxidant andantioxidant species among the main oxidant agentsros and reactive nitrogen species rns stand out in orderto counterbalance the oxidant species there is an antioxidantsystem formed by enzymes and nonenzymatic molecules however during pathological events such as viral infections there may be an increase in the production of oxidantspecies not neutralized by the antioxidant system resultingin oxidative stress that promotes cellular damage throughprotein denaturation changes in the functions of nucleicacids lipid peroxidation and cell death []in addition during viral infection oxidative stress contributes to viral pathogenesis through stimulating ammation loss of immune function and increased viral replicationthat may occur due to the activation of the nuclear factorkappa b nfκb transcription pathway [] currentevidence suggests that cytokine dysregulationalso calledcytokine stormcontributes to severe disease caused by thepathogenic covs [ ] the exact mechanisms are notclear yet but research on uenza a virus shows that infection causes a rapid ux of ammatory cells this isfollowed by an increase in reactive oxygen species productionand cytokine expression and release which ultimately leadsto acute lung injury in general rna viruses promotechanges in the bodys antioxidant defense system aï¬ectingenzymes such as superoxide dismutase sod and catalasecat in addition to reducing the levels of antioxidantmolecules such as ascorbic acid carotenoids and reducedglutathione gsh [] wu reported that glucose6phosphate dehydrogenasean important antioxidantenzyme that produces nadph knockdown cells were moresusceptible to infection by hcov229e than normal cells interestingly ye and colleagues have reported that theinhibition of ros production alleviates ammation causedby uenza a virus infections in an experimental model of sarsinduced acute lunginjury in mice it was noted that phospholipid oxidationdue to oxidative stress is one of the main triggering factorsof acute lung injury this happens through the activation ofthe innate immune response culminating in the activationof pulmonary macrophages via tlr4triftraf6nfκbsignaling furthermore hypoxia caused by acute lunginjury can cause myocardial injury due to the production ofros aggravating infections caused by coronavirus disease covid19 mitochondria have an essential function in energy generation and for this reason their function and integrity arestrictly regulated in order to respond to varying energyrequirements and environmental conditions mitochondria are known to function as the control point in apoptoticpathways releasing proapoptotic factors mainly ros whichfunction as a signaling molecule that may result in cell death[ ] some studies have shown a relationship betweencoronavirus infection and dysfunctional or damaged mitochondria leading to the release of ros and other proapoptotic substances [ ] in a recent study xu reportedthat ros and p53 play key roles in regulating many kindsof the cell process during coronavirus infection in vero cellsaccording to the authors coronavirus infection appears toinduce a timedependent ros accumulation which in turnis linked to regulatory mechanisms of p53 activation andapoptosis in infected cells antioxidant substances promote improvement in casesof disease caused by coronaviruses such as apolipoproteinda lipocalin that promoted a neuroprotective eï¬ect against 0coxidative medicine and cellular longevityencephalitis induced by human coronavirus oc43in micethis protective eï¬ect occurred through the reduction of oxidative stress cerebral lipid peroxidation and regulation ofammation [ ] also the treatment with antioxidantssuch as pyrrolidine dithiocarbamate or nacetylcysteine significantly inhibits moreover melatonin promotes downregulation of acute lungoxidative injury due to its antiammatory and antioxidantactions making it a possible compound in the treatment ofcovid19 based on these studies compounds thathave antioxidant actions can be helpful in the treatment ofinfections promoted by coronaviruscoronavirusinduced apoptosisin general antioxidant properties of polyphenolic compounds such as some ï¬avonoids have been associated withthe presence of aromatic phenolic rings that promote theelectron donation and hydrogen atom transfer to free radicals acting as free radical scavengers reducing agents andquenchers of single oxygen formation thus the aimof this study was to investigate the antioxidant capacity andantiviral activity of natural antioxidants against coronavirusthe compounds are illustrated in figure occurrence and antioxidant properties ofanticoronavirus compoundsquercetin can be found in plants such as rubus fruticosus land lagerstroemia speciosa l pers [ ] also quercetinshows antioxidant activity at a concentration of μmoll inhepg2 cells inhibiting oxidative stress promoted by h2o2 promotes an increase in sod cat and glutathioneperoxidase gpx and reduces lipid peroxidation in rats withchronic prostatitischronic pelvic pain syndrome moreover quercetin improves sepsisinduced acute lung injury inrats by reducing lipid peroxidation and ammation andincreasing sod and cat levels in addition quercetin glycosides with antioxidant activity such as quercetin 3βglucoside have already been isolated from plants such as passiï¬ora subpeltata ortega andchamomilla suaveolens pursh rydb [ ] the administration of quercetin 3βglucoside mgkg poinstreptozotocininduced diabetic rats promotes an increasein the levels of antioxidant enzymes sod cat andgpx and nonenzymatic antioxidants vitamins c and eand gsh and a reduction of lipid peroxidation quercetin 3βgalactoside hyperoside is found mainly in plantsof the hypericum genus such as hypericum perforatum l[ ] moreover it showed cardioprotective activity inhigh glucoseinduced injury of myocardial cells throughdecreased apoptosis and ros production and increasedsod levels quercetin 7ramnoside is also found inplants of the hypericum genus such as hypericum japonicum thunb ex murray this ï¬avonoid shows hepatoprotective activity against carbon tetrachloride in mice bydecreasing lipid peroxidation and increasing cat andgsh levels in addition to presenting values of μmand22diphenyl1picrylhydrazyldpph and ²azinobis3ethylbenzthiazoline6sulphonic acid abts assays respectively μm intheepigallocatechin gallate is present in parkia roxburghii gdon and is one of the main metabolites found in green teaand liubao tea camellia sinensis lo kuntze also gallocatechin gallate can be found in this plant [] literaturedata reveal that the administration ip of mg100 g ofepigallocatechin gallate in rats with streptozotocininduceddiabetes mellitus promotes a reduction in oxidative stressthrough reductions in parameters such as indirect nitricoxide synthesis and status total oxidative as well as anincrease in levels of cat and total antioxidant capacity ofplasma furthermore it promotes cardioprotection byantioxidant mechanisms green tea has a high antioxidant capacity due to the highlevels of catechins present he and collaborators compared the antioxidant activities of catechins and reported thatepigallocatechin gallate has greater antioxidant activity viaradical scavenging activity μm with values of ± ± and ± compared to its epimergallocatechin gallate with values of ± ± and ± in the dpph abts and ferric reducingantioxidant power frap respectively amentoï¬avone is a biï¬avonoid present in leaves ofginkgo biloba l garcinia brasiliensis l and nandinadomestica l [] this biï¬avonoid has a high antioxidantcapacity demonstrated in scavenging tests ofdpph abts superoxide and hydroxyl radicals moreover amentoï¬avone prevents acute lung injury induced bysepsis in rats by decreasing thiobarbituric acid reactive substance tbars levels and by increasing levels of sod andgsh apigenin is mainly present in ï¬owers and leaves beingabundantly found in apium graveolens l petroselinum crispum mill fuss and matricaria chamomilla l sánchezmarzo and collaborators evaluated the antioxidantcapacity of apigenin using the trolox equivalent antioxidantcapacity teac oxygen radical absorbance capacityorac and frap assays the results show that apigeninhas good antioxidant activity with values of ± μmol teammol ± μmol teammol and ± μmol fe2mmol respectively in addition oraladministration of apigenin mgkgday for days in anexperimental model of cardiotoxicity induced by doxorubicin in rats promoted cardioprotection by reducing levels ofmalondialdehyde mda increasing sod levels and preventing cardiomyocyte apoptosis luteolin is present in foods such as carrot cabbage teaand apple and is found in ugni molinae turcz [ ] datashow that luteolin μgml increases the levels of gsh theexpression of gsh synthetase and the activity of sod andcat in human colon cancer cells ht29 furthermoreluteolin attenuates the sepsisinduced acute lunginjury in mice by reducing lipid peroxidation and increasingsod and cat activity in addition to suppressing the nfκbpathway herbacetin is ubiquitous in plants of the genus rhodiolasuch as rhodiola rosea l herbacetin glycosides are alsopresent in the roots of r sachalinensis a bor and show antioxidant activity veeramani reported that theadministration of herbacetin mgkg po in mice with 0coxidative medicine and cellular longevityohohohohhoohooohohhooohoh oquercetinohohoooohohohquercetin 3ð½galactosideohohooohoohoohohhoohohohoh oquercetin 7ramnosideohooohohoohoohoohohohquercetin 3ð½glucosidehooohooohohohohohohepigallocatechin gallateohgallocatechin gallateohhooohoh ohooluteolinhooohoooohoohohoohrhoifolinhohohoohohohohohohoohoohooh oamentoflavoneohhooohohohoherbacetinhohoooohooohohohoapigeninooohooohhopectolinarinooopsoralidinhooohohohohcatechinohhoohoooh ohelichrysetinohohomyricetinohohohhohoohoohoisobavachalconeohhooohscutellareinohresveratrolfigure chemical structures of bioactive antioxidants against coronavirusobesityassociated insulin resistance promotes an increasein the activity of the enzyme glucose6phosphate dehydrogenase which is directly related to the production ofnadph pectolinarin is present in plants of the genus cirsiumsuch as cirsium setidens nakai and cirsium japonicum dcthe administration of pectolinarin and mgkg pofor two weeks in rats promotes antioxidant eï¬ects in hepatic 0coxidative medicine and cellular longevitytable antioxidant properties of natural inhibitors of coronaviruscompoundtestedassaysexperimentalconcentration doseantioxidant eï¬ectreferencetype of cells μmoll mgkg po mgkg poinhibiting oxidative stress promoted byh2o2promoted an increase in sod cat andgpx and reduced lipid peroxidationreduces lipid peroxidation and increasessod and cat levelsincreases levels of sod cat gpx mgkg povitamins c and e and gsh and reduces nmoll mgkgic50 μm dpphec50 μm abtsrats with streptozotocininduced diabetes mellitus mg100 g iprats with streptozotocinnicotinamideinduceddiabetes mellitus mgkg po μm mgkg μgml ± μmol teammol ± μmol teammol and ± μmol fe2mmolrespectively mgkg pomodelshepg2 cellsrats with chronicprostatitischronic pelvicpain syndromesepsisinduced acute lunginjury in ratsstreptozotocininduceddiabetic ratshigh glucoseinducedinjury of myocardial cellsccl4induced liver damagemodel in micedpphabtsquercetinquercetin 3βglucosidequercetin 3βgalactosidequercetin ramnosideepigallocatechingallateepigallocatechingallatedpphabtsfrapgallocatechingallateamentoï¬avoneacute lung injury inducedby sepsis in ratsdpph abts superoxideand hydroxyl radicalsteacoracfrapcardiotoxicity induced bydoxorubicin in ratshuman colon cancer cellsht29acute lung injury inducedby sepsis in micemice with obesityassociated insulinresistance inductionhepatic injury induced bydgalactosamine in ratsoracapigeninluteolinherbacetinpectolinarinrhoifolincatechinlipid peroxidationdecreases apoptosis and ros productionand increases sod levelsdecreases lipid peroxidation and increasescat and gsh levelsscavenging of free radicalsreduces indirect nitric oxide synthesis andtotal oxidative statusincreased levels of cat and totalantioxidant capacity of plasmaincreased levels of cat sod and gshreduced levels of superoxide and proteincarbonyl pco and prevented dnadamage ± ± and ± ± and ± respectively ± respectivelydecreases tbars levels and increaseslevels of sod and gshscavenging of free radicalsscavenging of free radicalsreduces levels of mda increases sodlevels and prevents cardiomyocyteapoptosis μgmlincreases levels of gsh expression of gshsynthetase and the activity of sod and mgkg ip mgkg po and mgkg poapproximately troloxequivalents μmcatreduces lipid peroxidation increases theactivity of sod and cat and suppressesthe nfκb pathwayincreases the activity of glucose6phosphate dehydrogenaseincreases levels of sod gsh glutathionereductase and glutathione stransferasescavenging of free radicalsscavenging of free radicals 0coxidative medicine and cellular longevitytable continuedtype of cellscompoundtestedassaysexperimentalconcentration doseantioxidant eï¬ectreferencemodelsabtsfrap ± mol troloxequivalentsmol ± mol trolox equivalentsmoldihydrorhodamine oxidation assayandic50 ± μmisobavachalconedpph sc50frapabts sc50psoralidinelectron spin resonancemyricetinhelichrysetinscutellareinresveratroldpphchinese hamster lungï¬broblast cells v794treated with h2o2oracdpphabtssuperoxide radicalsdpph sc50r2rats with obstructive lungdiseasehypercholesterolemicapoeko mouserespectively μm ± mmic50 μmequivalent to feso4 mm respectively·7h2o and μgml and μgml μgml ± trolox equivalents ± μm ± μm and ± μm respectivelyscavenging of free radicalsscavenging of free radicalsscavenging of free radicals and respectivelyprevents dna damage and lipidperoxidationincreases the activity of sod cat andgpxscavenging of free radicalsscavenging of free radicals μmoldmscavenging of free radicals mgkg mgkgincreases sod activity and reduces mdalevelsinhibits the activity and expression ofnadph oxidasesincreases sod gpx and cat levels injury induced by dgalactosamine by increasing levels ofsod gsh glutathione reductase and glutathione stransferase [ ]rhoifolin is found in citrus fruits such as citrus limettarisso studies have indicated that its radical peroxyl scavenging capacity is higher than trolox in orac assays approximately trolox equivalents μm [ ]meanwhile the catechin is a ï¬avonoid present inleaves of green tea wine and fruits [ ] grzesik investigated the antioxidant action of catechinthrough the abts scavenging activity and frap teststhe results show values of ± mol troloxequivalentsmol and ± mol trolox equivalentsmol respectively in addition catechin shows greaterprotective properties in the dihydrorhodamine oxida ± μm than gsh and ascorbiction assay ic50acid and μm respectively psoralidin is a prenylated coumestan which is found inplants of the fabaceae family such as psoralea corylifolia lxiao and collaborators investigating the antioxidant potential of compounds isolated from p corylifolia observed thatpsoralidin shows the best antioxidant activity by the methodof electron spin resonance spectroscopy with an ic50 value of μm the compound isobavachalcone has been isolated fromplants of the fabaceae and moraceae families [ ] isobavachalcone shows a strong antioxidant activity in dpphsc50 frap and abts sc50 assays with values of μm ± mm equivalent to feso4·7h2o and mm respectively in addition the compound has beenreported to inhibit the nfκb pathway in sephadexinducedlung injury in rats [ ]helichrysetin is a chalcone that is found in plants of thehelichrysum genus such as helichrysum odoratissimum l in a study investigating the antioxidant activity of natural and prenylated chalcones vogel found that helichrysetin is the substance that shows the highest antioxidantactivity in the orac test with values of ± troloxequivalents myricetin is widely found in the plant families myricaceae and anacardiaceae and is widely used as health foodsupplement due to its antioxidant properties [ ]bennett demonstrated that myricetin reacts withoxygencentered galvinoxyl radicals more than timeshigher than vitamin e dalphatocopherol furthermoremyricetin was able to scavenge and on the dpphassay μgml and μgml respectively interestinglythe compound prevents dna damage by lipid 0coxidative medicine and cellular longevity2h2o2 2gsh2o2h2h2o2vit evit cgshnadphnonenzymatic antioxidantsgpxsodcatsemyzne tnadixoitna2h2o gssgo2 h2o2o2 h2omdapcotbarsbio m arkers of oxidative stressgeneration of rosoxidative stresscell damagenaturalantioxidants sesadixohpdan2o2 nadphnadp 2o2 hfigure the main antioxidant mechanisms of natural compounds reported in this review dashed line inhibition full line activationperoxidation and increasing the activity of sod cat andgpx in chinese hamster lung ï¬broblast cells v794treated with h2o2 scutellarein is found in scutellaria barbata d don andpolygonum viscosum buchham [ ] liu investigated the antioxidant activity of scutellarein through thedpph abts and superoxide scavenging assays they notedthat the compound shows good antioxidant activity withvalues of ± μm ± μm and ± μmrespectively while the trolox a standard antioxidant compound presented ± μm ± μm and ± μm respectively resveratrol is found in grapes peanuts and blueberriesand can be isolated from veratrum grandiï¬orum o loes literature shows that resveratrol has good antioxidantvalues of μmoldmactivity with dpph sc50r2moreover it is able to reduce the production of ros by inhibiting the activity and expression of nadph oxidases byeliminating oxidant agentsincluding radical hydroxylsuperoxide hydrogen peroxide and peroxynitrite the treatment of resveratrol mgkg po in ratsreduces oxidative stress in obstructive lung disease byincreasing sod activity and reducing mda levels indicatinga decrease in lipid peroxidation table shows themain actions of natural antioxidants discussed in this studyand figure illustrates these activities effect of natural antioxidants incoronavirus infectionsthis review focused on studies reporting on the anticoronavirus activity of natural antioxidants based on exclusioncriteria data from nineteen compounds were discussedthe oxidative stress pathway could potentially be a keyelement in coronavirusinduced apoptosis and pathogenesis for this reason it is interesting to investigate the useof antioxidants as potential therapeutic toolseither as analternative or as an adjuvant to conventional therapiesinthe treatment of coronavirus infections among the antioxidant compounds evaluated as for coronavirus infectionsare the ï¬avonoids which are compounds widely found infruits vegetables and certain beverages in fact researchgroups have reported that antioxidant ï¬avonoids includingcatechin luteolin apigenin quercetin and quercetin rhamnosideinhibit ros accumulation and apoptosis ofcells infected with diï¬erent coronavirus including porcineepidemic diarrhoea coronavirus pedv and transmissiblegastroenteritis coronavirus tgev []as shown with the recent covid19 pandemic thesearch for alternative or new antiviral therapies for theremainstreatment of coronavirus diseasesimportantbased on the literature antioxidanttherapies oï¬er anattractive option 0coxidative medicine and cellular longevitytable natural antioxidants tested in in vitro coronavirus infection models and their main results and mechanism of actionantioxidanttype of cells testedconcentrationic50antiviral eï¬ectmechanism of actionreferencecatechintgevinfected st cellscatechin μminhibition of tgevinduced apoptosissuppression of the tgevinducedbcl2 reduction baxredistribution cytochrome crelease and caspase3 activation resveratrolmersinfected vero e6cellsresveratrol μmquercetinepigallocatechingallategallocatechingallate gcgquercetin rhamnosideq7rrecombinant 3clprowas expressed in pichiapastoris gs115quercetin μmepigallocatechingallate μmgallocatechingallate μmpedvinfectedvero cellsq7r μminhibition ofmersinducedreduction of the expression ofinfectionapoptosis andnucleocapsid n protein essential prolonged cellular survivalfor merscov replicationafter virus infectioninhibition of coronavirusreplicationreduction of the formationof a visible cytopathiceï¬ect cpe without dnafragmentationgcg displayed a binding energyof kcal mol1 to the active siteof 3clpro and the galloyl moietyat 3oh position was required for3clpro inhibition activitynot speciï¬city amentoï¬avoneapigeninluteolinquercetinquercetin3βgalactosideherbacetinrhoifolinpectolinarinsarscov 3clproinhibition usingï¬uorescence resonanceenergy transfer analysismolecular dockingsprfretbasedbioassays andmutagenesistryptophanbasedï¬uorescence methodherbacetinisobavachalconequercetin3βdglucosidehelichrysetintryptophanbasedï¬uorescence methodamentoï¬avone3βgalactoside μmapigenin μmluteolin μmquercetin μmquercetin μmherbacetin μmrhoifolin μmpectolinarin μmherbacetin μmisobavachalcone μmquercetin3βdglucoside μmhelichrysetin μminhibition of sarscovreplicationflavonoids exhibited sarscov3clpro inhibitory activity[ ]inhibition of merscovreplicationflavonoids exhibited merscov3clpro inhibitory activity isobavachalconepsoralidinlineweaverburk anddixon plotsmyricetinscutellareinsprfretbasedbioassaysisobavachalcone μmpsoralidin μmmyricetin μmscutellarein μminhibition of sarscovreplicationisobavachalcone and psoralidinexhibited sarscov papainlikeprotease inhibitory activitymyricetin and scutellareininhibition of sarscovpotently inhibit the sarscovreplicationhelicase protein in vitro byaï¬ecting the atpase activity the high number of deaths and clinical complicationsobserved in sars and merscov epidemics motivatedthe search for eï¬ective therapeutic agents this was necessitated when many of the tested conventional drugs andtherapies proved ineï¬ective in treating sarsantiviralcov infections for exampletreatment ofthe initial 0coxidative medicine and cellular longevitycell culture coronavirusnaturalantioxidants suppress bcl2 reductionsuppress bax redistributionsuppress cytochorome c releasesuppress caspase3 activationreduce formation of a visiblecytopathic effect without dnafragmentationreduce nucleocapsid n protein expressioninhibit 3clike proteaseinhibit 3clike proteaseinhibit papainlike proteaseinhibit helicase protein by affectedatpase activitytgevpedvmerscovsarscovfigure inhibitory actions of natural antioxidants against coronavirussarscov with antiviral agents such as ribavirin and corticosteroids did not achieve very satisfactory resultsmainly because corticosteroids exertimmunosuppressoreï¬ects on the humoral and cellular immune systems[ ] other drugs such as pentoxifylline were considered for the treatment of sars due to its interestingtherapeutic propertiesantiammatoryantiviral immunomodulatory and bronchodilatory eï¬ectshowever it too was not successful in the clinical treatment of sarscov infection includethatinhibition ofmany antioxidant compounds show antiviral activityagainst sarscov the antiviral activity has been mainlyattributed to thethe 3clike protease3clpro of sarscov a vital enzyme for sarscov replication as an example multiples studies havereported that quercetin and quercetinderived compoundssuch as quercetin 3βgalactoside display potent 3clproinhibitory e5ï¬ect and consequent reduction of sarscovreplication other antioxidants such as epigallocatechin gallate gallocatechin gallate amentoï¬avone apigeninluteolin herbacetin rhoifolin and pectolinarin are alsofound to eï¬ciently block the enzymatic activity of sarscov 3clpro [ ]moreover some natural antioxidants exhibit promisingantiviral activity against sarscov infection by interferingwith diï¬erent targets involved in sarscov replication inparticular the sarscov papainlike protease plpro andsarscov helicase protein kim reported that isobavachalcone and psoralidin inhibit plpro in a dosedependentmanner with ic50 ranging between and μm previously yu reported that myricetin and scutellareinpotently inhibit the sarscov helicase protein in vitro byaï¬ecting the atpase activity merscov is another zoonotic coronavirus transmitted between animals and human beings that causes severemorbidity and mortality no antiviral medicines with satisfactory eï¬cacy for the treatment of merscovinfectedpatients have been identiï¬ed to date similar to sarscov natural antioxidant libraries have been probed forpotentialinhibitory compounds against merscov 3clike protease jo showed that herbacetin isobavachalcone quercetin 3βdglucoside and helichrysetinfourcompounds with recognized antioxidant activity can blockthe enzymatic activity of merscov 3clpro using atryptophanbased ï¬uorescence method furthermore theexperimental and computational studies show that ï¬avonoland chalcone are favourite scaï¬olds to bind with the catalytic site of merscov 3clpro in a study performed by lin the antiviral activitiesof resveratrol were investigated in mersinfected vero e6cells the authors reported a significantinhibition ofmerscov infection and prolonged host cell survival aftervirus infection which they speculate was promoted by resveratrol in addition they also found that the expression ofthe nucleocapsid n protein which is essential for merscov replicationis decreased after resveratrol treatment it is important to mention that in vitro models of coronavirus infection also show antiviral activity of ï¬avonoidsextracted from ï¬owering cherry cultivars and black tea[ ] finally antioxidants such as resveratrol alsoare able to block infection produced by herpesvirus the discovery of antiviral compounds from a bioactivecompound against other viruses is an interesting strategy forobtaining new antiviral drugs table shows the mainactions of the natural antioxidants against the coronavirusand figure summarizes these activities 0c conclusionsin conclusion this review shows that antioxidant compounds prominently ï¬avonoids exhibit antiviral action inmodels of coronavirus infections in general the antiviralactivity might be attributed at least in part to the inhibitoryeï¬ect on the enzymatic activity of targets involved in coronavirus replication including sarscov 3clpro sarscovpapainlike protease plpro sarscov helicase proteinand merscov 3clpro in addition some studies provideevidence that the reduction of ros accumulation retardsthe coronavirusactivated apoptotic signaling thereforethe mechanisms of oxidative stress could be the key elementto be studied in coronavirus infectionsincluding thoserelated to ammatory processes arising from the action ofthis virus obviously further investigations are needed toelucidate other pharmacological mechanisms by which natural antioxidants play an antiviral eï¬ect despite the ï¬ndingsreported in this reviewthey cannot be generalized tocovid19 however the data provided support to the investigation of natural antioxidants as a potential therapeuticapproach in the treatment for covid19 and its severe clinical complications either as an alternative or as an adjuvantto conventional therapies and contribute to the search fornew prototypes in the development of drugs against coronavirus infectionsabbreviations229e3clproabtshuman coronavirus229e3clike protease²azinobis3ethylbenzthiazoline6sulphonic acidcoronavirusescatalasediï¬use alveolar damage22diphenyl1picrylhydrazylferric reducing antioxidant powerreduced glutathioneglutathione peroxidasehuman coronaviruseshuman coronavirushku1lower respiratory tract infectionsmalondialdehydecovscovid19 coronavirus disease catdaddpphfrapgshgpxhcovshku1lrtismdamerscov middle east respiratory syndromecoronavirusnfκbnuclear factor kappa bhuman coronavirusnl63nl63human coronavirusoc43oc43oxygen radical absorbance capacityoracprotein carbonylpcoporcine epidemic diarrhoea coronaviruspedvplpropapainlike proteasereactive nitrogen speciesrnsreactive oxygenated speciesrossarssevere acute respiratory syndromesarscov severe acute respirator | Colon_Cancer |
" the practice of clinical radiology has become more sedentary in the era of the picture archiving andcommunication system physical inactivity is a wellknown risk factor for various chronic diseases this study aimedto determine the frequency and pattern of physical exercises among radiologists in the eastern province of saudiarabia and the association between physical exercises and the prevalence of workrelated musculoskeletalsymptomsmethods an online survey was sent to radiologists in all hospitals academic public and private in the majorcities of the eastern province of saudi arabia it covered information about demographic characteristics and thefrequency and pattern of physical exercises it also included an evaluation of workrelated musculoskeletalsymptoms using the nordic musculoskeletal questionnaire this survey of radiologists was conducted in april the study outcome was the presence of disabling musculoskeletal symptoms in any body region whichrestricted the performance of normal activities within the last months the study results were analyzeddescriptively using the chisquare testresults the survey was completed by participants men and women with a response rate of most participants were less than years eightythree men did a physical exercise at least weeklycompared to women men were more likely to engage in various physical exercises than womenoverall of participants who did not do any physical exercise regularly less than monthly reported havingdisabling neck pain this figure was found lower among participants who did physical exercises monthly orat least weekly a similar pattern was observed with shoulder pain with found in participants whodid not exercise and only in those engaging in physical activities at least weeklys physical inactivity is common among radiologists especially female ones in the eastern province ofsaudi arabia the physical inactivity was significantly associated with workrelated musculoskeletal symptomsgenderspecific health promotion programs are needed to mitigate the negative health outcomes due to thesedentary nature of the radiology current practicekeywords physical exercises radiologist musculoskeletal symptoms correspondence mbaqirijhotmailcom mohammed al gadeeb and ali hassan contributed equally to this work1department of radiology king fahd hospital of the university imamabdulrahman bin faisal university alkhobar saudi arabiafull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cgadeeb archives of public health page of innovations in science and technology have affected allaspects of modern life and have remarkably transformedseveralthese developmentshave also contributed to a significant increase in sedentary behaviors across fields and medicine is no exception to this pervasive trendfields of work howeverin the last few decades clinical radiology has evolveddramatically owing to the introduction of electronicmedical records and the picture archiving and communication system pacs the transition from hardcopyfilm to the pacs has enabled clinicians to readily accessradiological images and reports at any time or placewhile this development has increased productivity andefficiency [ ] it also has some inherent drawbackstoday radiologists spend long hours in front of computer screens to examine and analyze medical imageswhich increasesand physicalinactivitysedentarybehaviorthere is strong evidence that regular physical exercisehas positive effects on health these include the preventionand management of cardiovascular diseases diabetes mellitus obesity and certain cancers [] in contrast physicalinactivity which is prevalent worldwide and is observedin approximately of all adults is associated with awide range of negative health outcomes such as increasedmortality [ ] prolonged sitting time in particular is anindependent risk factor for mortality a metaanalysis conducted in involving over million individuals showedthat a daily sitting time of over h is associated withincreased allcause mortality interestingly howeverother studies revealed that this risk was attenuated amongindividuals engaging in physical exercises [ ]musculoskeletal symptoms are common worldwide musculoskeletal complaints similar to physicalinactivity are associated with several negative determinants of health [ ] previous crosssectional studiesexamining the relationship between sedentary behaviorsand musculoskeletal symptoms have identified associations between physical inactivity and a higher prevalenceof musculoskeletal symptoms []sedentary behavior has increased among radiologistssince the advent of the pacs putting this group atpotential risk of musculoskeletal complaints thus ourstudy aimed to investigate the prevalence of physicalinactivity among radiologists practicing in the easternprovince of saudi arabia and to examine the associationbetween the frequency of physical exercises and workrelated musculoskeletal symptomsmethodsstudy designthe survey was designed using the questionpro surveysoftware seattle wa usa in our study we used anonline survey format because it is easily accessible timesaving and costeffective besidesthe survey wasdesigned to be taken anonymously without personalidentification data requested or stored and lastedwithin approximately mina cover letter was provided along with the surveyquestion it stated the purpose of the study informedparticipants about the voluntary nature of their participation and assured their anonymity participants wereencouraged to contact the research investigator for anyqueries aboutthe study using the provided contactinformationstudy participantsthis crosssectional study was used to assess the patternand frequency of physical exercises and their effects onworkrelated musculoskeletal symptoms among clinicalradiologists including residents specialists junior staffand consultants staff across all hospitals academicpublic and private in the major cities of the easternprovince of saudi arabiarecruitment of participantswe sent a personalized message with a link to the onlinesurvey to each of radiology residents practicing in theeastern province n who were members of awhatsapp facebook menlo park ca usa groupthe link to the survey was also sent to radiology specialists and consultants whose contactinformation wasavailable to the investigators a reminder message wassent days latereach invited radiologist received a unique link for theonline survey so that the survey could not be filled morethan once from the same link this ensured that the survey would not be compromised by duplicate responsesfrom the participants or responses from individuals notincluded in the target population we used the questionpro respondent anonymity assurance feature to keepthe identities of the participants anonymousadditionally paperbased survey questionnaire formswere distributed to the radiology departments of hospitals in the surveyed region they were intended for radiologists of whom the investigators did not have contactinformation investigators visited those departments aweek later to collect the completed forms the surveybegan on april and lasted days overall thesurvey forms whether online and paperbased weredistributed to a total of radiologistsstructure of the questionnairethis survey of the frequency and pattern of physicalexercises was part of a series of surveys on the generalhealth and wellbeing of radiologists in the eastern province of saudi arabia [ ] the survey comprised 0cgadeeb archives of public health page of multiplechoice questions covering the following areas demographic information pattern and frequency ofphysical exercises and identification of workrelatedmusculoskeletal symptomsthe face and content validity of the survey questionnaire was verified by an expert panel of academiciansthe relevance and appropriateness of each item werediscussed we conducted a pilot study with a group of radiologists to assess the clarity of the questions andthe time needed to complete the survey after the pilotstudy no major changes were made to the questionsexposure variablesthe proposed risk factors were determined based on theliterature focusing on demographic characteristics andworkrelated information demographic characteristics included age group sex years of practice current institutionof practice and type of practice participants were askedhow often they took part in different types of physical exercises walking running group sports swimming stretchingstrength training functional fitness and other aerobic exercises a likerttype scale daily weekly monthly less thanmonthly and never was used to record data about thefrequency of physical exercises in our present study aphysical exercise was defined to be more than minoutcome variablesin this study the outcome was the presence of workrelated musculoskeletal symptoms which prevented theparticipants from doing normal physical activities withinthe last months these symptoms could be found inany of the nine body regions ie neck shoulder elbowwristhand upper back lower back hipthighbuttockknee and ankle the standard nordic musculoskeletalquestionnaire was used as it is a valid and reliablescreening tool the responses of the outcome variables were dichotomized responses of left right orbilateral in any body region were coded as a yeswhereas a respondent who indicated no for all bodyregions was coded as a nosample size estimationthe calculated sample size was radiologists in orderto detect an effect size of with a twosided alpha levelbelow and with a power of however since theanticipated response rate was not expected to be highthe survey was distributed to almost twice the estimatedsample sizestatistical analysisthe obtained data were compiled using the questionproplatform and analyzed using ibm spss for windows version ibm corp armonk ny usa two questionnaire forms were excluded from the analysis because ofmissing data all variables used in the study were categorical descriptive statistics such as percentages and frequency distribution of different characteristics were usedas appropriate for questions based on a likerttype scalefive responses were dichotomized as yes and no thechisquare test was used to examine the bivariate relationship between the exposure and outcome variablesthe level of statistical significance was set at p resultscharacteristics of the participantsa total of participants an overall response rate of including residents specialists andconsultants completed the survey besides maleparticipants outnumbered their female counterparts vs most participants were in the or year age groups accounting for and respectively in addition of the participants had been working in the field of radiology for years whereas had more than years of work experience most participants were righthanded and reportedspending to h daily at computer workstation interpreting and reporting radiological images table table characteristics of the participantsvariableage yearsn¥gendermalefemaleprofessional rankresidentsenior registrarconsultantyears in practiceinstitutionacademicpublicprivaten number of respondents 0cgadeeb archives of public health page of physical exercises among the participantsfrequency of physical exercisesseventyfive participants performed at least onetype of physical exercises on a daily basis compared to and on a weekly or monthly basisrespectively meanwhile participants did notregularly perform any physical exercisesthere was a positive association nonsignificant relationship between age and the frequency of physical exercises where olderindividuals were more likely toexercises frequently compared to younger individualsoverall and of participantsin the 30to 40to 50to and ¥ year age groups exercised at least once a week respectively fig men were statistically more likely to exercise dailythan women p overall male participants did a physical activity at least weekly comparedto female participants there were no statistically significant association between the frequency ofphysical exercises and the professional rank the institution of practice and the workload in terms of the timespent at a computer workstation reviewing medicalimagesrunning and other aerobic exercises were found in and of the participantsrespectively however group sports and swimming werethe least popular type of physical exercise reported byonly participants figure demonstrates the patterns of physical exercises the rank order of popular physical exercises wasgenerally the same for age groups and genders a notable exception is that stretching exercise is the mostcommonly found among individuals aged ¥ yearscompared to walkingage was significantly associated with swimming witholder individuals being more likely to perform swimmingthan younger individuals p for instance of participants aged years and above do swimmingregularly compared to only of those aged years no similar significant associations were foundbetween age groups and other physical exercisesgender was significantly associated with a number ofphysical exercisesincluding walking running groupsports swimming and strength training p menwere more likely to engage in these physical exercisesthan women table pattern of physical exercisesthe most frequently performed physical exercise waswalking as participants reported walkingregularly common physical exercises such as stretchingassociation between physical exercises andmusculoskeletal symptomsoverall participants reported having symptoms within the last months prior to the study thesefig frequency of physical exercises among genders and different age groups 0cgadeeb archives of public health page of fig the pattern of physical exercises among genders and different age groupstable frequency of physical exercise according to the participants characteristicsvariableat least one exercise monthlynat least one exercise weeklyntotalexercise less than monthly or nevernage years¥gendermalefemaleyears in practiceprofessional rankresidentsenior registrarconsultantoveralln number of respondentsapvalue in bold when significantpvalue a 0cgadeeb archives of public health page of symptoms prevented participants from doingusual physical activities due to these symptomsthere was a statistically significant association between the frequency of physical exercises and the prevalence of disabling musculoskeletal symptoms within the months prior to the survey overall participants whodid physical exercises regularly were less likely to havedisabling musculoskeletal symptoms in any body regionincluding in the neck and shoulders for instance of participants who did not do any physical exercise regularly reported having disabling neck pain meanwhile suchsymptoms were lower among those who exercisedmonthly or at least weekly exercises asimilar pattern was observed when it came to shoulderpain more specifically of participants who did notdo physical exercises regularly suffered from disablingshoulder pain whereas the figure for those who exercisedat least once a week was only discussionregular physical exercise is probably the single mostimportant intervention for preventing chronic diseasesmaking it one of the primary determinants of health the world health anization recommends atleast min of moderateintensity physical exercisesper week ie min a day days a week for all adults our study demonstrated that a high proportion of radiologists did not engage in regular physicalactivity and did not exercise even once a week this isaggravated by the fact that the practice of clinical radiology has become more sedentary in the pacs era potentially leading to negative effects on the general healthof radiologists in our study there was a significant difference in thefrequency of physical exercise between men and womenwith men being more likely to engage in most types ofphysical exercises this might be due to traditional cultural practices and the differences in gender roles whichmay contribute to the unavailability of exercise facilitiesand lead to time constraints due to child care requirements previous studies [] have reported similarfindings with one study of adults in malaysia demonstrating differences in the motives of exercise betweenmen and women according to this study intrinsic factors eg gaining strength were more likely to motivatemen to engage in physical exercise whereas extrinsicfactorseg weight management and attaining anattractive appearance were larger motivators amongwomen counterintuitively our study found a positive association between age and the frequency of physical exercises a possible explanation for this finding is thehealthy worker effect phenomenon owing to which individuals who are physically inactive and have medicalcomorbidities are more likely to leave the workforceearlier unsurprisingly walking was the most commonphysical exercise in our study as it can be performed atany time and placeour findings demonstrated that participants performing regular physical exercises had a lower likelihood ofexperiencing disabling musculoskeletal symptoms especially neck and shoulder pain which is consistent withprevious findings [] for example a prospectivestudy by sitthipornvorakul involving workersrevealed a negative association between the number ofsteps per day and the onset of neck pain this studyshowed that for every extra steps walked in adaythe risk of neck pain decreased by moreover in a systematic review of studiesconducted by kelly showed that exercise therapyis effective in reducing symptoms and improvingfunction among sedentary workers with workrelatedupper limb disorders a large proportion of our studys participants didnot engage in regular physical exercises during theirleisure time indicating the importance of incorporating exercises into the hours spent at the workstationin johnson conducted an interestingstudy to investigate the effects of treadmill workstation use on the interpretation of computed tomography findings by radiologists their study revealedthat the use of such dynamic workstations did notsignificantlypulmonarynodules on ct scans but shortened the durationrequired for interpretation moreover the use ofstanding radiology workstations is recommended toprevent the negative health effects of prolonged sitting time because it causes more energy expenditurethan sitting [] to our knowledge these radiology workstations are rarely used in the easternprovince of saudi arabia therefore given the magnitude of physicalinactivity among radiologists theuse of such workstations should be promoted whichmay mitigate the health risks associated with sedentary workdetection ofaffectthethe present study has certain limitations the frequency of physical exercises and the prevalence of workrelated musculoskeletal symptoms were selfreportedalthough selfreporting is timesaving and convenient itmay have led to some bias in addition the data on thefrequency and duration of physical exercises collected inthe study may not have been highly accurate preventingthe calculation of metabolic equivalent values the preferred unit of measurement for physical activity finallythe crosssectional nature of the study made it difficult to assess the causal relationship between physicalinactivityand the occurrence of musculoskeletalsymptoms 0cgadeeb archives of public health page of our study showed that radiologists who performed regular physical exercises had a lower likelihood of experiencing workrelated musculoskeletal symptoms howevera significant number of radiologists particularly thosewho were women did not engage in regular exercisesfurthermore interventional strategies aimed at promoting physical exercise among radiologists such asgenderspecific health promotion programs should bedevelopedabbreviationsci confidence interval ct computed tomography or odds ratiopacs picture archiving and communication systemsacknowledgementsnot applicableauthors contributionsconceptualization oa data analysis mq and ms methodology mk and nzwritingoriginal draft ah writingreview editing af and dg supervisionhs all authors read and approved the final manuscriptfundingthis research received no specific grant from any funding agency in thepublic commercial or notforprofit sectorsavailability of data and materialsthe datasets used andor analyzed during the current study are availablefrom the corresponding author on reasonable requestethics approval and consent to participatethe study was approved by the institutional review board at the imamabdulrahman bin faisal university iau irb no informedconsent of the participants in the study was implied when the participantseither completed the survey electronically or returned the completed paperbased survey all information pertaining to the survey was provided in thecovering letter therefore their acceptance and completion of the surveywas considered as acknowledgement of informed consentconsent for publicationnot applicablecompeting intereststhe authors declare that they have no competing interestsauthor details1department of radiology king fahd hospital of the university imamabdulrahman bin faisal university alkhobar saudi arabia 2department offamily and community medicine college of medicine imam abdulrahmanbin faisal university dammam saudi arabiareceived april accepted july referencestowbin aj perry la larson db improving efficiency in the radiologydepartment pediatr radiol mcenery kw coordinating patient care within radiology and across theenterprise j am coll radiol pt b1217kyu hh bachman vf alexander lt mumford je afshin a estep k veermanjl delwiche k iannarone ml moyer ml physical activity and risk of breastcancer colon cancer diabetes ischemic heart disease and ischemic strokeevents systematic review and doseresponse metaanalysis for the globalburden of disease study bmj 2016354i3857kushi lh doyle c mccullough m rock cl demarkwahnefried w banderaev gapstur s patel av andrews k gansler t american cancer societyguidelines on nutrition and physical activity for cancer prevention reducingthe risk of cancer with healthy food choices and physical activity ca cancerj clin proper ki singh as van mechelen w chinapaw mj sedentary behaviorsand health outcomes among adults a systematic review of prospectivestudies am j prev med dumith sc hallal pc reis rs kohl hw iii worldwide prevalence of physicalinactivity and its association with human development index in countries prev med ekelund u steenejohannessen j brown wj fagerland mw owen npowell ke bauman a lee im does physical activity attenuate or eveneliminate the detrimental association of sitting time with mortality aharmonised metaanalysis of data from more than million men andwomen lancet matthews ce moore sc sampson j blair a xiao q keadle sk hollenbecka park y mortality benefits for replacing sitting time with different physicalactivities med sci sports exerc stamatakis e gale j bauman a ekelund u hamer m ding d sitting timephysical activity and risk of mortality in adults j am coll cardiol vos t abajobir aa abate kh abbafati c abbas km abdallah fabdulkader rs abdulle am abebo ta abera sf global regional andnational incidence prevalence and years lived with disability for diseases and injuries for countries a systematic analysis forthe global burden of disease study lancet andersson hi the course of nonmalignant chronic pain a 12year followup of a cohort from the general population eur j pain mcbeth j silman aj macfarlane gj association of widespread body painwith an increased risk of cancer and reduced cancer survival a prospectivepopulationbased study arthritis rheum morken t magerøy n moen be physical activity is associated with a lowprevalence of musculoskeletal disorders in the royal norwegian navy across sectional study bmc musculoskelet disord nabeel i baker ba mcgrail jm flottemesch tj correlation betweenphysical activity fitness and musculoskeletal injuries in police officers minnmed holth hs werpen hkb zwart ja hagen k physical inactivity is associatedwith chronic musculoskeletal complaints years later results from thenordtrøndelag health study bmc musculoskelet disord balagué f bibbo e mélot c szpalski m gunzburg r keller ts theassociation between isoinertial trunk muscle performance and low backpain in male adolescents eur spine j harisinghani mg blake ma saksena m hahn pf gervais d zalis m dasilva dias fernandes l mueller pr importance and effects of alteredworkplace ergonomics in modern radiology suites radiographics al shammari m hassan a al dandan o al gadeeb m bubshait dmusculoskeletal symptoms among radiologists in saudi arabia a multicenter crosssectional study bmc musculoskelet disord al dandan o hassan a al shammari m al jawad m alsaif hs alarfaj kdigital eye strain among radiologists a surveybased crosssectional studyacad radiol crawford jo the nordic musculoskeletal questionnaire occup med ferguson b acsms guidelines for exercise testing and prescription 9th ed j can chiropr assoc who global recommendations on physical activity for health genevaworld health anization cheah yk poh bk the determinants of participation in physical activity inmalaysia osong public health res perspect chen yj huang yh lu fh wu js lin ll chang cj yang yc the correlatesof leisure time physical activity among an adults population from southerntaiwan bmc public health mao hy hsu hc lee sd gender differences in related influential factorsof regular exercise behavior among people in taiwan in a crosssectional study plos one 2020151e0228191 michaud ds giovannucci e willett wc colditz ga stampfer mj fuchs cs molanorouzi k khoo s morris t motives for adult participation inphysical activity obesity height and the risk of pancreatic cancer jamaphysical activity type of activity age and gender bmc public health 0cgadeeb archives of public health page of gerdle b brulin c elert j eliasson p granlund b effect of a general fitnessprogram on musculoskeletal symptoms clinical status physiologicalcapacity and perceived work environment among home care servicepersonnel j occup rehabil sitthipornvorakul e janwantanakul p lohsoonthorn v the effect of dailywalking steps on preventing neck and low back pain in sedentary workersa 1year prospective cohort study eur spine j kelly d shorthouse f roffi v tack c exercise therapy and workrelatedmusculoskeletal disorders in sedentary workers occup med johnson cr besachio da delonga d kuzniewski c mudge cs effect ofdynamic workstation use on radiologist detection of pulmonary noduleson ct j am coll radiol pt a451 hoffmann jc mittal s hoffmann ch fadl a baadh a katz ds flug jcombating the health risks of sedentary behavior in the contemporaryradiology reading room am j roentgenol levine ja schleusner sj jensen md energy expenditure of nonexerciseactivity am j clin nutr richardson ml wellness in the radiology reading room making yourworkstation a workout station am j roentgenol publishers notespringer nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations 0c" | Colon_Cancer |
" clinical trials have been conducted to clarify the beneficial effects of vd3 1α25dihydroxy vitamind3 also known as calcitriol treatment in prostate cancer however the molecular mechanisms underlying theseeffects are not fully understood recent studies on igfbp3 have indicated its intracellular functions in cell growthand apoptosis the aim of this study was to confirm the benefits of lowdose vd3 treatment and clarify themolecular mechanisms underlying these beneficial effects in prostate cancer cellsmethods the molecular effects of simultaneous treatment of lncap cells and their genetically modified cell lineswith low concentration of docetaxel and vd3 were biologically and biochemically analyzed to further determinethe effects of vd3 treatment on igfbp3 induction system cells were temporarily treated with vd3 in combinationwith a transcriptional inhibitor or protein synthesis inhibitor bcl2 protein and its mrna behavior were also observed inigfbp3 expressionmodified lncap cells to determine the involvement of igfbp3 in the suppression of bcl2 by vd3treatmentresults changes in igfbp3 expression levels in lncap cells indicated that it mediated the inhibition of cell growthinduced by vd3 treatment igfbp3 was also found to be a mediator of the enhanced cytotoxicity of prostate cancercells to vd3 in combination with the anticancer drug we further identified the distinct property of the igfbp3induction system wherein temporal vd3 stimulationinduced prolonged igfbp3 expression and vd3 treatmentinduced increase in igfbp3 expression were optimized based on the protein concentration rather than the mrnaconcentration meanwhile bcl2 expression was downregulated by vd3 treatment in an igfbp3independent manner these findings indicate the molecular mechanisms of igfbp3 induction stimulated by vd3 and igfbp3independent bcl2 suppression by vd3 treatment in prostate cancer cells the results could prompt a reevaluation ofvd3 usage in therapy for patients with prostate cancerkeywords vitamin d nonlinear igfbp3 induction bcl2 suppression prostate cancer treatment correspondence satorusasagawatokushukaijp1molecular biology laboratory research institute nozaki tokushukai hospitaltanigawa daito osaka japanfull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cigarashi bmc cancer page of vitamin d has a central role in calcium and skeletalhomeostasis [ ] its pleiotropic role both in physiological and pathological phenomena such as cell growthimmune function and tumorigenesis has also been examined [] which revealed that exposure of cancercells to vitamin d significantly reduces the cell growthrate in multiple cancer types [] indeed recent epidemiologicalinvestigations have reported that highervitamin d concentration could prevent multiple types oftumorigenesis consistent with such finding for example an increase in colon cancer incidence with lowervitamin d dietary habits has been reported [ ]however suppressive effect on prostate cancer is stillunder discussion [ ]in turntraditional methodscurrently prostate cancer is one of the most commonthecancers in men worldwide clinical use ofprostatespecific antigen psatest dramatically improved the screening sensitivity of prostate cancer compared to that ofthenumber of patients with earlystage prostate cancer hasrapidly increased since the mid1990s [ ] unlikeother cancer types most cases of prostate cancer haveslow progression or have nonprogressive indolentsymptom and are localized in the prostate thus they areunlikely to cause poor physical condition or death therefore patients with prostate cancer need a less burdensome treatment in order to avoid potential harmfrom excessive treatmentalthough the impact of vitamin d as a single agent onprostate cancer has been investigated its significance remains under discussion [ ] meanwhile the synergistic or additive effects of vitamin d and its derivativeswith anticancer drugs on prostate cancer have beenclinically studied and encouraging results have been reported [ ] however the results of larger trials thatevaluated the synergistic effect of vitamin d in combination with docetaxel one of the firstline anticancerdrugs in prostate cancer chemotherapy showed limitedor nonsignificant benefit of vitamin d efficacy in castration or androgen deprivation therapyresistant prostatecancer [ ] furthermore overconsumption of 1α25dihydroxy vitamin d3 vd3 also known as calcitriolthe biologically active form of vitamin d3 from food orprolonged treatment with vd3 derivatives could triggerhypercalcemia resulting in physiological side effects therefore to date vd3 has not been proactivelyused in the treatment of patients with prostate cancerthe biological function of vitamin d is mainly mediatedby vitamin d receptor vdr which acts as a transcriptional factor vitamin d receptor elements vdreon the promoter region of target genes are recognizedand transcriptionally activated by vitamin dcoupledvdr consistent with the diverse physiological functionof vd3 vdre was identified not only in the gene related to calcium and skeletal homeostasis but also in thegene related to fundamental cellular functions includingcell growth igfbp3 is one of the families of sixhigh affinity igfbps and was originally found in plasmaas a stabilizer and transporter of igfs in the bloodstream interestingly vdre was found on the prothe igfbp3 gene and recent studies havemoter ofrevealed that igfbp3 functions inside the cell as wellregulating cell growth and apoptosis [ ]methodsthis study aimed to investigate igfbp3 induction byvitamin d treatment and determine its role in prostatecancer treatment with vitamin d in combination withanticancer drugs in order to provide molecular biologicalevidence of benefit of vitamin d and to suggest effectivevitamin d usage in prostate cancer treatmentchemicals and reagentsdihydrotestosterone dht and calcitriol vd3 purchased from tokyo chemical industry tokyo japanwere resolved in ethanol as a stock solution pei maxmolecular weight was purchased from polysciences pa usa the other chemicals and reagentswere purchased from wako pure chemicalosakajapan and sigmaaldrich st louis mo usaincluding testosteronecharcoal stripping of fetal bovine serum fbsfbs was purchased from gibco waltham ma usato deplete hormonesin fbsdextrancoated charcoal powder was added to theserum and the mixture was incubated with rotation at degree overnight thereafter the mixture was centrifugedto pellet charcoal and the supernatant was filtered througha 022μm polyvinylidene difluoride membrane thecharcoalstripped serum was used for all experimentstotalthe concentrations oftestosterone and totalvitamin d in the serum were determined using a totaltestosterone test kit abbott japan chiba japan and atotal vitamin d test kit roche basel switzerland according to manufacturers instructions the concentrations of total testosterone in the pre and posttreatmentserum were nm and less than nm limit of detection respectively the concentrations of total vitamin d in the pre and posttreatment serum were and nm respectively thus the basal concentrationsin the culture medium supplemented with fbs wereless than nm total testosterone and approximately nm total vitamin dcell culturethe lncap cell line was obtained from american typeculture collection and cultured in dulbeccos modified 0cigarashi bmc cancer page of eagle medium dmem sigmaaldrich supplementedwith 10charcoalstripping fbs the 293ft celllinewas purchased from invitrogen waltham ma usaand cultured in dmem supplemented with fbs mm of lglutamine sodium pyruvate and nonessentialamino acids the cells were cultured at a temperature of °c in co2humidified condition the mycoplasma contamination was routinely checked and confirmed as negativecell growth assaydmem supplemented with charcoalstripping fbs was used for the cell growth assay the cells were seededat à cells per well in a 6well plate the next daythe medium was replaced with ml of fresh mediumand nm dht andor nm vd3 were added the cellculture was continued throughout the indicated periodthe cultured cells were trypsinized and the number ofcells was assessed using an automated cell countercountess iitm fl invitrogen each assay was repeatedat least three times and similar results were obtainedwestern blottingthe antibodies used for western blotting were mouseantiigfbp3 santa cruz biotechnology mouseantiβactin santa cruz biotechnology mouseantibcl2 santacruz biotechnology and horseradish peroxidaseconjugated secondary antibodies jackson immunoresearch laboratories westgrove pa usa the collected cells were resuspendedin ripa buffer supplemented with protease and phosphatase inhibitors roche basel switzerland and lysedusing bioruptortm ii sonicator cosmo bio tokyojapan cell lysates were resolved by nupagegels invitrogen and transferred onto polyvinylidenefluoride membrane millipore burlington ma usathe signals were developed using enhanced chemiluminescence reagent perkinelmer waltham ma usaand luminograph i atto tokyo japan was used forimage capture quantification of band signal wasanalyzed using cs analyzer software atto at leasttwo biological replicates of each experiment were performed with similar resultsrealtime reverse transcriptionpolymerase chain reactionthe total rna from the cultured cells was extractedusing trizol reagent invitrogen according to the manufacturers instruction the rna was reverse transcribedby the primescript rt reagent kit takara bio shigajapan using oligodt quantitative reverse transcriptionpolymerase chain reaction rtpcr reaction wasperformed using tb green premix ex taq ii takaraand genespecific primers supplementary table bycfx96 touch realtimepcr system bioradlaboratories hercules ca usa gene expression datawere normalized against glyceraldehyde 3phosphate dehydrogenase or hprt1 as internal control at leastthree biological replicates of each experiment were performed and similar results were obtainedflow cytometric analysis of cell cycle and apoptosisfor cell cycle analysis and apoptotic cell detection flowcytometric analysis was performed using the guavaeasycyte plus flow cytometry system millipore andguava cell cycle reagent and annexin v fitc apoptosiskit millipore according to manufacturers instructionas previously described at least three biologicalreplicates of each experiment were performed and similar results were obtainedlentiviral construction and transductionbackbone vectors plko1 puro plasmid andplenti cmv puro dest w118 plasmid were provided by drs bob weinberg eric campeau andpaul kaufman respectively via addgene ref pentr1a plasmid and lentiviral packaging mix plp1plp2 and plpvsvg were purchased from invitrogenfulllength igfbp3 was cloned from lncap cell complementary dna cdna using kod fx neo toyoboosaka japan with a specific primer set supplementarytable and inserted into the pentr1a vector thenthe sequence was confirmed the igfbp3 cdna was introduced into the plenti cmv puro dest vector by recombinaseenzymeinvitrogen to generate the lentiviral expression vectorspecific short hairpin rnas shrnas were designedusing invitrogen or biosettia websites the selectedtarget sequence oligos supplementary table wereannealed and inserted into the plko1 puro vector according to addgenes instruction the lentiviral expressionvector or shrna vector was cotransfected with the lentiviral packaging mix into the 293ft cells using pei maxinstead of lipofectamine according to invitrogensinstruction twentyfour hours posttransfectionthemedium was replaced with the culture medium forlncap cells one day later the lentiviruscontaining supernatants were collected and filtrated through a 045mmpolyvinylidene fluoride filter milliporereaction using lr clonaseiilentivirus infectionone day before infection à lncap cells wereplated into a 10cm dish then the culture medium wasreplaced with the lentiviruscontaining supernatant andculture was continued twentyfour hours post infectionreplaced with fresh culturemedium two days later the medium was replaced withfresh culture medium that contained μgml of puromycin and culture was continued until the noninfectedthe medium was 0cigarashi bmc cancer page of control cells were completely killed the puromycinselected cells were then subjected to each assaystatistical analysisresults are presented as mean ± standard deviation unless otherwise specified pairs of groups were comparedusing a twotailed unpaired students ttest onewayanalysis of variance was used for multiplegroup comparisons rather than specifying three a pvalue was considered statistically significant all statistical analyses were performed using excel software microsoftredmond wa usa and statcel3 addin for exceloms publishing tokyo japanresultsvd3 reduces cell growth rateconsistent with a previous report that treatmentwith vd3 inhibits growth of prostate cancer cells ourresults showed that vd3 treatment reduced the cellgrowth rate in a dosedependent manner and nm fig 1a left as shown below igfbp3 induction activity of vd3 was reached to plateau at nm concentration fig 4a on the other hand testosterone hasbeen reported to stimulate the growth of prostate cancer and the results of this study confirmed that dhttreatment from very low concentration nm stimulated cell growth rate and its activity was reached toplateau at less than nm concentration fig 1a centerthe purpose of our study was to investigate the role ofvd3igfbp3 induction system in cell growth inhibitionand to propose the potency oflowdose vd3 usagewhich could evade sideeffect of vd3 treatment such ashypercalcemia in therapy for patients with prostate cancer thus nm of dht and nm of vd3 concentrations were chosen as minimum but stably workingconcentration for following experiment previouslyithas been demonstrated that simultaneous treatmentwith vd3 and dht enhanced the reduction of cellgrowth rate compared to treatment with vd3 alone anda similar result was reproduced with lowdose dht nm and vd3 nm in this study fig 1a right tofurther characterize growth inhibitory effect with lowdose of dht nm and vd3 nm cell cycle andapoptotic analyses were performed with flow cytometrythe cell cycle analysis revealed that there was no significant change in the cell cycle phase distribution amongfig cellular response of lncap cells treated with vd3 and dht a effect of combined treatment of vd3 and dht on cell growth in lncapcells left vd3 treatment reduced cell growth in a dosedependent manner center dht treatment increased cell growth at lowerconcentrations right simultaneous treatment of vd3 and dht enhanced the reduction of cell growth compared to treatment with vd3 aloneb change in cell cycle phase by vd3 or dht treatment neither vd3 nor dht treatment significantly changed the cell cycle phase c induction ofapoptosis by vd3 or dht treatment neither lowdose vd3 nor dht treatment influenced apoptosis at shortterm d induction of igfbp3expression by vd3 or dht treatment vd3 treatmentinduced igfbp3 expression and cotreatment with dht enhanced the expression level ofigfbp3the ratio indicates the density of igfbp3 band normalized by corresponding βactin band the all experiments were performed in serumcontaining medium condition the uncropped fulllength blot images are presented in supplementary fig 5a 0cigarashi bmc cancer page of control no treatment dht nm vd3 nm anddhtvd3 treatment conditions fig 1b suggestingthat lowdose of vd3 or dhtvd3 treatment did notarrest the cell cycle at a specific phase previous reportshave shown that longterm vd3 treatment has apoptosisinducible activity and dht has apoptosis inhibitingactivity in a dosedependent manner in lncapcells however it was unclear or controversial whetherlowerconcentration of vd3 and dht at shorttermcould influence apoptosis respectively in lncap cellsthus the apoptosis assay was performed with nm ofdht and nm of vd3 for shortterm and found thatneither lowerdose dht vd3 nor dhtvd3 treatmentfor shortterm influenced apoptosis fig 1c these results suggested that the decrease in the cell number induced by lowdose vd3 or dhtvd3 treatment wasmainly due to a decrease in the cell growth rate to further address what was occurring at the molecular levelduring lowdose dht andor vd3 treatment the genesknown to regulate the cell cycle and inducible by dhtandor vd3 treatment were chosen and messengerrna mrna induction was quantitatively measuredfig 1d upper supplementary fig 1a the quantitativertpcr results showed that igfbp3 mrna inductionwas positively correlated to cell growth suppression inresponse to lowdose vd3 or dhtvd3 treatment andthe expression strength was dramatically sensitive tovd3 or dhtvd3 treatment consistent with thatigfbp3 protein was markedly induced by vd3 treatment and it was enhanced by simultaneous treatment ofvd3 with dht a similar response was observed in theexpression of ar the receptor of dht which wasknown to a one oftarget of vdr supplementaryfig 1bmultiple recent studies have revealed that igfbp3functions in cellular response including cell growth andapoptosisin an insulinlike growth factor igfindependent manner considering these findings we believethat igfbp3 can be a key molecule for vd3 treatmentin prostate cancer cellsigfbp3 was a dominant factor in cell growth suppressionto confirm ifigfbp3 dominantly suppresses cellgrowth in lncap cells we applied the gainoffunctionand lossoffunction approach using a lentivirus systemfirst we generated igfbp3overexpressing lncapcells and found that the expression of igfbp3 mrnawas about higher compared to that by lowdosedhtvd3 treatment fig 2a as an infection controlegfpoverexpressing lncap cells were also generatedand it was confirmed that lentivirus infection per se didnot induce igfbp3 expression using these cell linesthe effect of igfbp3 on cell growth was observed fig2b results showed that the cell number of egfpoverexpressed cells treated with nm of dht and nm of vd3 for days was decreased to comparedwith that of untreated cells and the igfbp3overexpressing cells showed comparable cell growth decreasewithout dhtvd3 treatment next we generatedshrna for igfbp3 shigfbp3expressing lncap cellsthe knockdown of igfbp3 mrna and protein inducedby lowdose dhtvd3 treatment was confirmed inshigfbp3expressing lncap cells fig 2c using thiscell line the effect of lowdose dht and vd3 treatmenton cell growth was observed as we expected the suppressive efficacy of lowdose vd3 on cell growth wasweakening and simultaneous treatment with nm ofdht and nm of vd3 increased cell growth fig 2dtaken together these data indicated igfbp3dominantfactor of cell growth suppression induced by lowdosevd3 treatment in lncap cellsacceleration of anticancer drug effect by vd3as previously reported and we demonstrated abovevd3 alone is not cytotoxic at physiological and pharmacological concentrations meanwhile simultaneous treatment with vd3 has been reported to improve theefficacy of anticancer drugs including docetaxel howeverits molecular mechanisms were remained not fully uncovered here we supposed that igfbp3 might be amediator of vd3induced sensitization to anticancerdrugs in prostate cancer cells to confirm this hypothesis lncap cells were treated with lowdose of dhtvd3 in combination with several concentrations of docetaxel to determine the appropriate concentration ofdocetaxel for evaluating the vd3 effect we first screenedthe concentration of docetaxel based on cytotoxic activity and found that a docetaxel concentration nmkilled bulk of the cells treated fig 3a here ic50 ofdocetaxel was nm in our assay and it was consistent with previous reports nm thus a docetaxel concentration nm was chosen to observe theeffect of combinatoriallowdose dhtvd3 treatmentfor the following assay to evaluate the synergistic effectof dhtvd3 on cytotoxicity by docetaxel lncap cellswere treated with or nm of docetaxel withor without dhtvd3 and results showed that lowdosedhtvd3 with docetaxel reduced the living cell numberat the concentration range of nm but the effectwas masked when nm docetaxel was applied fig 3bsimilarlylowdose dhtvd3 with cisplatin reducedthe living cell number at the concentration range of nm supplementary fig to see if these enhancedcytotoxicity effects were dependent on igfbp3 igfbpoverexpressed or shigfbp3expressed lncap cellswere analyzed in the same manner indeed in igfbp3overexpressed cells the living cell number was reducedby docetaxel without dhtvd3 addition fig 3c 0cigarashi bmc cancer page of fig igfbp3 mediates the effect of vd3 on cell growth in lncap cells a overexpression of igfbp3 in lncap cells lncap cells were infectedwith lentivirus containing the igfbp3 gene and its overexpression was confirmed by quantitative reverse transcriptionpolymerase chainreaction b suppression of cell growth by igfbp3 igfbp3overexpressed cells were cultured as they were and control cells were cultured withdhtvd3 for days and then the cell number was measured c knockdown of igfbp3 in lncap cells lncap cells were infected with lentiviruscontaining shrna for igfbp3 and igfbp3 knockdown was confirmed by quantitative reverse transcriptionpolymerase chain reaction andwestern blotting the ratio indicates the density of igfbp3 band normalized by corresponding βactin band d the igfbp3 knockdown cells weretreated with dht andor vd3 for days and then the cell number was measured the all experiments were performed in serumcontainingmedium condition the uncropped fulllength blot images are presented in supplementary fig 5bcorrespondingly in the shigfbp3expressed cells reduction of living cell number by dhtvd3 addition wascanceled rather the cell living number was increasedliving cell number despitefig 3d the increase ofdhtvd3 addition in docetaxeltreated shigfbp3expressed cells was assumed by cancelation of vd3 effect and emerging of dht effect on cell growth basedthese findings lowdose dhtvd3induced enhancedcytotoxicity by docetaxel on lncap cells was dependenton igfbp3 expressioncharacterization of the igfbp3 induction mechanismas demonstrated above igfbp3 had a pivotal role inlowdose dhtvd3induced enhanced cytotoxicity byantitumor drugs to further dissect the igfbp3 induction mechanism and to provide the molecular evidenceof vd3 treatment for clinical research mechanisms ofigfbp3 induction by dht and vd3 were analyzed aspreviously reported in prostate cancer cells vd3 treatment induces cyp24a1 as well an enzyme that catalyzes vd3 to its inactive form as a negative feedbackfactor the induced cyp24a1 limits the efficacy of vd3meanwhile activated ar induced by dht treatmentsuppresses cyp24a1 transcription thus cancelling thenegativefeedback loop to inactivate vd3 consistentwith that cyp24a1 induction by vd3 treatment and itssuppression by simultaneous treatment with dht wereconfirmed even when we applied lowdose dht and 0cigarashi bmc cancer page of fig vd3 enhanced cytotoxicity of docetaxel a dosedependent cytotoxicity of docetaxel lncap cells were treated with docetaxel for daysand then the living cell number was measured ic50 was nm b simultaneous treatment of vd3 with dosedependent docetaxelsimultaneous treatment of vd3 with docetaxel reduced the living cell number at nm range c igfbp3 overexpression was conducive todhtvd3 treatment on docetaxel treatment igfbp3overexpressed or control cells were treated with dhtvd3 and nm of docetaxel andcultured for days and then the cell number was measured d igfbp3 knockdown canceled the effect of vd3 on docetaxel treatment in igfbp knockdown cells dhtvd3 treatment increased the cell number compared to that of the notreatment control and the cell number wasalmost equal to that of the dht treated sample the concentration of docetaxel used was nm the all experiments were performed in serumcontaining medium conditionvd3 which were enough to induce and suppresscyp24a1 expression supplementary fig meaningthat h lowdose dhtvd3 treatment could cancel thecyp24a1driven negativefeedback loop to further dissect the mechanism of igfbp3 expression in lncapcells the cells were treated with vd3 alone or dhtfixed in nm and vd3 in a dosedependent manner and nm when treated with vd3alone the induced igfbp3 reached a plateau at to nm in contrast when treated with vd3 together withdht the amount of induced igfbp3 was increased according to the increment of vd3 concentration fig 4athese results indicated that lowdose dht could improve igfbp3 induction activity of vd3 throughcyp24a1 suppressionclinically highdose vd3 or its derivatives for treatment can cause hypercalcemia thus its continual usageshould be carefully monitored to avoid sideeffect ofvd3 here we wondered if continual vd3 treatmentfig 4btop herewould be required for maintaining igfbp3 induction inprostate cancer cells to address this lncap cells weretreated with vd3 alone or lowdose dhtvd3 for or days followed by washout which was done by replacing the culture medium and continuing the culturefor days in totalintracellularigfbp3 protein was observed by western blottinginterestingly 1day treatment of vd3 or dhtvd3 induced stable igfbp3 expression fig 4b bottom although treatment of vd3 alone showed mild igfbp3induction compared to that by dhtvd3 note thatigfbp3 showed similar strength of expression between and days of vd3 or dhtvd3 treatment this resultclearly indicated that temporal vd3 treatment couldinduce prolonged stable igfbp3 expressionthis nonlinear response suggested the presence of aunique molecular property underlying the igfbp3 expression mechanism generally nonlinear cellular response such as sustained protein expression by temporal 0cigarashi bmc cancer page of fig characterization of igfbp3 expression induced by vd3 and dht treatments a effect of dosedependent vd3 treatment with or withoutdht on igfbp3 induction lncap cells were treated with vd3 alone at the indicated concentration or with vd3 and nm dht top westernblotting image of igfbp3 and bottom quantified graph of igfbp3 induction open circles vd3 alone filled circles dht vd3 b effect oftemporal treatment of dhtvd3 on igfbp3 induction top schematic time course of temporal treatment of vd3 and bottom western blottingimage of igfbp3 the ratio indicates the density of igfbp3 band normalized by corresponding βactin band c effect of mrna transcription andprotein synthesis on the stability of igfbp3 expression induced by dhtvd3 lncap cells were treated with vd3dht for day followed byactinomycin d actd μm or cycloheximide chx μm for another day after vd3dht was washed out or left as is top quantification graphof igfbp3 mrna induction middle western blotting image of igfbp3 and the combination of treatments bottom the ratio indicates thedensity of igfbp3 band normalized by corresponding βactin band the all experiments were performed in serumcontaining medium conditionthe uncropped fulllength blot images are presented in supplementary fig 5ctriggered by positivefeedback loopstimulation wasmechanismin which protein synthesis and transcriptional regulation was included as hysterical responsedriver thus to further dissect if protein synthesis ortranscriptional regulation or both are involved in nonlinear vd3igfbp3 induction actinomycin d and cycloheximide which are transcriptionalinhibitor andprotein synthesis inhibitor respectively were added withvd3 or dhtvd3 and behavior of igfbp3 proteinand its mrna was observed experimentally actinomycin d or cycloheximide was added day after treatmentwith vd3 alone or dhtvd3 and the culture was continued for another day when interfered with μm ofactinomycin d there was no change in igfbp3 mrnaor protein expression fig 4c by contrast when interfered with μm of cycloheximide the igfbp3 proteinwas immediately reduced howeverthe mrna wasunexpectedly increased several times higher than thosewithout cycloheximide interference fig 4c the unexpected mrna increase became stronger when dhtvd3 washout was performed ahead of the cycloheximideinterference these cellular responses on igfbp3 induction interfered by actinomycin d and cycloheximidesuggested that the cells had a protein abundancebasedpositivefeedback loop to maintain the total amount ofigfbp3 via transcriptional controligfbp3independent bcl2 suppression by vd3as shown above although lowdose dht andor vd3treatment did not induce apoptosis fig 1d vd3 treatment rendered lncap cells sensitive to the antitumordrugs fig 3b and supplementary fig suggesting thatany apoptosisrelated factor might be influenced to addressinvestigated the behavior ofidea wethis 0cigarashi bmc cancer page of apoptosisrelated molecules in response to lowdosevd3dht treatment consistent with previous report although the concentration of vd3 was higher thanthat we used here bcl2 protein an antiapoptotic molecule was downregulated by lowdose vd3 treatmentfig 5a compared to that by vd3 alone it seemed thatbcl2 downregulation by dhtvd3 was not seemed tobe enhanced unlike to igfbp3 expression suggestingthat bcl2 downregulation by vd3 was igfbp3 induction independent manner to see whether bcl2 downregulation was igfbp3dependent or not the behaviorof the expression of bcl2 protein and mrna was observed in shigfbp3expressedcells interestingly despitethe igfbp3 disappearance bcl2 downregulation wasobserved according to vd3 or dhtvd3 treatmentfig 5b bottom and it was not significantly different atinigfbp3expressionenhancedprotein and mrna expression level compared to that inshctrlexpressing cells moreover in order to confirmthatconditionshcyp24a1 expression cells were established in whichvd3 effect on igfbp3 induction was expected tostrengthen the knockdown of cyp24a1 under the vd3treatment condition was confirmed by qrtpcr supplementary fig indeed the amount of igfbp3 protein wasincreased in shcyp24a1expressing cellscompared to that in shctrl cells fig 5c bottom despiteigfbp3 expression downregulation bcl2 protein by lowdose vd3 or dhtvd3treatment was not observed compared to that in shctrlcells fig 5c bottom also the expression of bcl2mrna was not significantly changed fig 5ctopthese results suggested that the downregulation of bclenhancement offig igfbp3independent reduction of bcl2 protein expression induced by vd3 a western blotting image of bcl2 reduction by vd3treatment the ratio indicates the density of bcl2 band normalized by corresponding βactin band b effect of igfbp3 suppression on bcl2reduction by vd3 treatment the cells were infected with lentivirus encoding of shrna for igfbp3 and then treated with vd3 and dht topquantification graph of bcl2 mrna expression and bottom western blotting image of bcl2 and igfbp3 induced by vd3 and dht treatmentthe ratio indicates the density of bcl2 band normalized by corresponding βactin band c effect of igfbp3 overexpression on bcl2 reduction byvd3 treatment the cells were infected with lentivirus encoding of the cyp24a1 gene then the cells were treated with vd3 and dht topquantification graph of bcl2 mrna expression and bottom western blotting image of bcl2 and igfbp3 induced by vd3 and dht treatmentthe ratio indicates the density of bcl2 band normalized by corresponding βactin band the all experiments were performed in serumcontainingmedium condition the uncropped fulllength blot images are presented in supplementary fig 5d e 0cigarashi bmc cancer page of protein by lowdose vd3 treatment was independentof igfbp3 induction besides the mrna of bcl | Colon_Cancer |
open accesscitation iburg km mikkelsen l adair t lopez ad are cause of death data fit for purposeevidence from countries at different levels ofsocioeconomic development one e0237539 101371 pone0237539editor brecht devleesschauwer sciensanobelgiumreceived april accepted july published august peer review history recognizes thebenefits of transparency in the peer reviewprocess therefore we enable the publication ofall of the content of peer review and authorresponses alongside final published s theeditorial history of this is available here101371 pone0237539copyright iburg this is an openaccess distributed under the terms of thecreative commons attribution license whichpermits unrestricted use distribution andreproduction in any medium provided the originalauthor and source are crediteddata availability statement the data underlyingthe results presented in the study are available inthe who mortality database appswhointhealthinfostatisticsmortalitywhodpms with the and objectivemany countries have used the new anaconda analysis of causes of national death foraction tool to assess the quality of their cause of death data cod but no crosscountryanalysis has been done to verify how different or similar patterns of diagnostic errors anddata quality are in countries or how they are related to the local cultural or epidemiologicalenvironment or to levels of development our objective is to measure whether the usabilityof cod data and the patterns of unusable codes are related to a countrys level of socioeconomic developmentmethodswe have assessed the quality of national cod datasets from the who mortality database by assessing their completeness of cod reporting and the extent pattern and severityof garbage codes ie codes that provide little or no information about the true underlyingcod garbage codes were classified into four groups based on the severity of the error inthe code the vital statistics performance index for quality vspiq was used to measurethe overall quality of each countrys mortality surveillance systemfindingsthe proportion of garbage codes varied from to across the countries countrieswith a high sdi generally had a lower proportion of high impact ie more severe garbagecodes than countries with low sdi while the magnitude and pattern of garbage codes differed among countries the specific codes commonly used did notsthere is an inverse relationship between a countrys sociodemographic development andthe overall quality of its cause of death data but with important exceptions in particularsome low sdi countries have vital statistics systems that are as reliable as more developedcountries however in lowincome countries where most people die at home the proportion one 101371 pone0237539 august one 0cfollowing selected variables country referenceyear icd10 code 5years age group sex andnumber of deaths data were also obtained fromthe world population prospect unpopulation division populationunwppdownloadstandardpopulation with the followingselected variables country reference year years age group sex and population numberfunding this study was funded under an awardfrom bloomberg philanthropies to the university ofmelbourne to support the data for health initiativewwwbloombergprogrampublichealthdatahealth grant number not applicablethe funders had no role in study design datacollection and analysis decision to publish orpreparation of the manuscriptcompeting interests the authors have declaredthat no competing interests existare cause of death data fit for purposeof unusable codes often exceeds implying that half of all causespecific mortality datacollected is of little or no use in guiding public policy moreover the cause of death patternidentified from the data is likely to seriously underrepresent the true extent of the leadingcauses of death in the population with very significant consequences for health priority setting garbage codes are prevalent at all ages contrary to expectations further researchinto effective strategies deployed in these countries to improve data quality can informefforts elsewhere to improve cod reporting systemsintroductiona key source of evidence for targeting health interventions to improve population health ishigh quality cause of death cod data that reliably document trends in mortality for differentdiseases and injuries however several studies have demonstrated that policy and practicein many countries are based on data that are far from accurate [] in order to target effortsto improve the utility of cod data for policy it is important to first understand what the keydiagnostic errors area major problem with cod data is poor cause of death certification practices that result ingarbage codes ie codes that provide little or no information about the true underlying causeof death garbage codes include what are often called illdefined causes but encompass alarger universe of uninformative diagnoses the major consequence of garbage codes is thatthey obscure the true mortality pattern in a population for example if a death certificate onlystates septicemia as the cause of death there is no way of knowing whether this resulted forexample from an infected wound following an accident from a postoperative amputationdue to diabetes or from meningitis or pneumonia each of which would require different preventive strategies if the underlying cause that led to septicemia is not indicated on the deathcertificate public policy to prevent these deaths would be misinformed potentially leading toinefficient resource allocation to prevent themcod data provide the essential health intelligence for health policies across countries atvarious levels of socioeconomic development our premise is that a better understanding ofgarbage codes ie their levels and patterns in countries at different stages of socioeconomicdevelopment will help to target improvements in cod reporting systems in this study weinvestigate whether the usability of cod data and the patterns of garbage codes are related toa countrys socioeconomic development using the anaconda software tool [ s1 file]to assess the quality of national cod datasets several countries have used this tool to assesshow fit for purpose their data are [] but there has not been any crosscountry analysis ofdata quality across a range of socioeconomic development levels and cod reporting systemsusing the common anaconda frameworkthe implication of our findings for public policy to improve population health is that if therelationship is found to be very weak or nonexistent then efforts to improve national civilregistration and vital statistics systems can expect to make significant progress towardsimproving the evidence base for policy without depending on further socioeconomicdevelopmentdata and methodswe carried out a crosssectional study using publicly available data from the who mortalitydatabase which contains cod data reported by its member states the countries one 101371 pone0237539 august one 0care cause of death data fit for purposewere selected on the basis that they used icd10 had provided data to who for a relatively recent year were located in all major regions of the world and differed in levels of socioeconomic development population data were taken from the un worldpopulation prospects with the youngest age group of years divided into and14years age groups using spragues interpolation we classified a countrys level of development using the socio demographic index sdiscore from the global burden of disease study gbd into three levels high middle and lowthe sdi is a summary measure of development expressed on a scale from to taking intoaccount the total fertility rate years of schooling and gross national income for the lowestsdi level the who mortality database only contained the few countries we selected for themiddle and high sdi levels we selected countries with recent data from different regions of theworld our study included low sdi countries middle sdi and high sdi countrieson the basis of the country specific icd10 codes used in gbd the most severecertification and coding errors that can mislead policy and public health planning were identified and categorized into four groups the fourtier garbage code typology used in anaconda is based on the premise that some garbage codes are worse than others depending onhow serious their impact is for guiding or misguiding policy debates and will thus likely impactdisease and injury control strategies differently ¢ level very highcodes with serious policy implications these are causes for which thetrue underlying cod could in fact belong to any of three broad cause group ie it is impossible to establish whether the true cause was a communicable disease a noncommunicabledisease or because of an injury a good example being septicaemia reported as the underlying cause of death these are the most serious misdiagnoses among the universe of unusable codes since they could potentially grossly misinform understanding of the extent ofepidemiological transition in the population¢ level highcodes with substantial implications for policy these are causes for whichthe true underlying cod is likely to belong to one or two of the three broad cause groupsfor example essential primary hypertension¢ level mediumcodes with important implications for policy these are causes forwhich the true underlying cod is likely to be within the same icd chapter for exampleunspecified cancer and thus are of some policy value¢ level lowcodes with limited implications for policy these are diagnoses for whichthe true underlying cod is likely to be confined to a single disease or injury category egunspecified stroke would still be assigned as a stroke death and not to some other diseasecategory the implications of unusable causes classified at this level will therefore be muchless important for public policy but a more specific code would have increased their utilityfor specific public health analysesa full list of the composition of specific icd10 garbage codes for each of the four severitylevels is given in s2 filegiven the considerable differences in population age structure between countries at highand low levels of socioeconomic development we age standardised the pattern of garbagecodes the point of agestandardising was to investigate whether countries with a comparatively old age structure and hence relatively high average age at death might expect to have agreater fraction of garbage codes simply because of the higher likelihood of multiple comorbidity in the elderly we used the global age distribution of deaths from the latest gbd studyas the standard one 101371 pone0237539 august one 0care cause of death data fit for purposein addition to diagnostic accuracy the ability of any dataset to describe the true mortalitypattern in a population also depends on how complete it is both in terms of capturing alldeaths that occur and in assigning each a cod completeness of the cod reporting ie thepercentage of actual deaths in a population that are assigned a cod for each of the countries was calculated using the adairlopez empirical method incorporated into anaconda the empirical method models the relationship between the crude death rate cdr andits principal determinants namely the age structure of the population and the overall level ofmortality as reflected by the level of child mortality the predicted cdr based on these inputvariables for a population is then compared with the observed cdr to estimate death registration completeness given that the model was built largely from historical data where the levelsof adult mortality and child mortality are closely correlated the predictions of completenessfor populations where this assumption is not valid such as those severely affected by hivshould be interpreted cautiouslydatasets that are both incomplete and have a high proportion of garbage codes provide limited insight into the true health status of a population we combined the proportion of unrecorded deaths with the amount of garbage codes to provide a summary measure of the utilityof the data for policy this indicator is particularly important when investigating data qualityin countries with low completeness where the data available may only come from hospitalsand other health facilities where diagnostic facilities and physician availability is greaterpotentially overstating the policy utility of the dataa key output of any mortality surveillance system is a table showing the leading causes ofdeath for the population in countries where garbage codes are commonly assigned they frequently appear among the or leading causes and can seriously impact the overall utilityof the cod data this is particularly the case when they permeate the top leading causesand are high impact providing little or no useful information for policythe anaconda software tool specifically developed for assessing quality of mortalityand cod data was used to investigate each dataset s1 file to identify the pattern and extentof garbage codes in the data their frequency among the leading causes the completeness ofthe dataset and to provide an overall summary index of the quality of the output of the mortality data system namely the vital statistics performance index for quality vspiq resultsthe proportion of garbage codes in the country datasets varied substantially by countryranging from to see fig while the relationship between sdi and amount of garbage codes in the data is broadly apparent the relatively low r2 arises from the presenceof outliers particularly uzbekistan kyrgyzstan nicaragua and colombia it is quite possiblethat specific certification and coding procedures have been introduced in these countries toavoid the use of garbage codes if these countries are omitted the strength of the inverse relationship is much more apparent further insights into the general characteristics of populationand mortality of the selected countries can be found in s2 filethe mean values for each sdi group indicate that on average countries of high sdi statushad a lower proportion of garbage codes in their data than middle and low sdi countries table interestingly there was large intercountry variation in theuse of garbage codes within each sdi level for example of the high sdi countries finlandhad the lowest amount of garbage codes in their data whereas in japan and france the level was five times higher affecting about one in three deaths for the middle sdigroup argentina thailand and tunisia had more than half of all cods coded to a garbagecode in thailand close to of these were high impact errors while for other countries in one 101371 pone0237539 august one 0care cause of death data fit for purposefig percentage of total garbage codes versus socio demographic index selected countries high sdi middle sdi low sdi101371 pone0237539g001this group notably uzbekistan turkey and colombia the use of garbage codes was much lessprominent surprisingly among the low sdi countries kyrgyzstan and nicaragua had comparatively low levels of garbage and respectively whereas in egypt at a similarsdi level twothirds of all deaths are coded to garbage codes and of these over were ofhigh impact table importantly the fraction of high impact garbage codes ranged from a low of finlandto egypt and for low impact codes from finland to brazil countries withhigh sociodemographic development had a lower proportion of high impact garbage codesmean compared with middle and low sdi countries table rankingcountries according to their percent of high impact garbage codes reveals a very substantialgap between the best and worst performing cod information systems and a surprising mixture of sdi levels fig kyrgyzstan has the secondbest performing system after finlandwith cod data in colombia of almost equal quality to the uk and that in nicaragua fallingbetween australia and canada uzbekistan turkey brazil and jordan all assign less causes tohigh impact garbage codes than both japan and francethe impact of population age structure on the overall level of garbage codes varied acrosscountries but was generally small contrary to what might have been expected table injapan and argentina the ageadjusted fraction of garbage codes was points lower than one 101371 pone0237539 august one 0ctable levels of garbage codes standardised by age and registration completeness for select countries ranked by their sociodemographic index sdiare cause of death data fit for purposecountryfinlandcanadaaustraliajapanfranceyearsdivalueunited kingdom high sdimeanturkeyargentinairanjordanthailandsouth africatunisiabrazilcolombiauzbekistanmiddle sdimeankyrgyzstanegyptnicaraguatajikistanlow sdi meantotal meansdi levelshigh impact gclow impact gca total gc ageadjb completeness of codc deaths of no policygc registration value sdi three levels collapsed from gbd five sdi levelshigh highmiddle high middle middlelow low low middlegarbage code gc levelshigh impact gc levels low impact gc level total gc high lowdeath of no value for policyc 1b a�bthis equation calculates unavailable deaths for policy ie unregistered deaths plus deaths with a garbage code101371 pone0237539t001the unadjusted fraction while in france and south africa it increased by a similar amountcolumn a agestandardisation therefore had no impact on the mean level of garbage codesin each development categoryagestandardisation however masks the age pattern of garbage coding particularly its relative importance at younger adult ages where accurate and specific diagnoses are critical fuiding policies designed to prevent premature deaths the perception that garbage codes arelargely confined to deaths among the elderly due to the presence of comorbidities at oraround the time of death is not confirmed by the agespecific fractions of garbage codes shown one 101371 pone0237539 august one 0care cause of death data fit for purposefig percentage of high impact garbage codes in total causes of death selected countries c high sdi middlesdi low sdi101371 pone0237539g002in fig with exact fractions reported in s3 file garbage codes are prevalent at all ages andoften in similar proportions to what is observed for the age group indeed in some highlydeveloped countries eg finland canada uk the proportion of garbage codes is significantly higher at ages for both sexes than at older ages indeed in tunisia for males andfig garbage codes as a percentage of all deaths by age selected countries101371 pone0237539g003 one 101371 pone0237539 august one 0care cause of death data fit for purposein jordan and kyrgyzstan for both sexes this pattern is already evident from age in uzbekistan garbage coding is more common for deaths of children and adolescents than at ages and abovegiven that the utility of a countrys mortality information system is reduced not only by theamount of garbage codes but also by how complete it is in terms of capturing all deaths wemeasured their combined impact for two countries namely jordan and tajikistan the consolidated indicator of registration completeness and fraction of garbage codes was close to suggesting that useful information on only about one quarter of all deaths that occur injordan and tajikistan is available for policy purposes col c table overall the combinedindicator showed the proportion of deaths for which information was either missing or of littleor no value for guiding health policy was much higher in low and middle sdicountries compared to high sdi countriestable shows the strong influence of garbage codes on the leading cause distribution whenthese are ranked with high impact garbage codes marked in red and low impact in orangethe presence of high impact garbage codes among the leading causes of death will substantially distort the true picture of what are the common cod that most people die from amonghigh sdi countries only japan and france have high impact garbage codes for males amongthe ten leading causes of death canada australia and uk had only low impact causes in thiscategory and finland had neither for the middle and low sdi countries there were manymore high impact garbage codes listed among the leading cod with egypt having seventajikistan five and thailand and tunisia each with four colombia and nicaragua had nonefor females egypt had seven iran tunisia and tajikistan five with the remaining countrieshaving between one and threenotwithstanding some variation in patterns and volume of garbage coding across countriesthe most common garbage codes were remarkably similar in particular other illdefined andunspecified causes of death senility heart failure unspecified neoplasm septicemia respiratory failure unknown cause of death hypertension and unspecified diabetes were observedacross all sdi levels in addition low sdi countries tended to report atherosclerosis hepaticfailure intracerebral hemorrhage and unattended deaths all garbage codes as leading causesfig ranks countries according to a single consolidated summary measure of system performance namely the vital statistics performance index for quality vspiq eleven countries scored and above a level where they could be considered as having wellfunctioningsystems six of the remaining countries achieved scores that would classify them as havingmedium performing systems with lower scores mostly arising from the high proportion ofgarbage codes that bias their cod distributions of the countries only jordan tajikistanand tunisia were classified as having poorly functioning systemsdiscussionin general one would expect that in high sdi countries where all deaths are medically certified with good quality clinical and diagnostic services comparatively few deaths would beassigned unusable garbage codes and certainly much less than in countries where such services are less common our findings based on an analysis of cod datasets from across theworld produced evidence both for and against this hypothesis france and japan do not seemto have more reliable cod data to guide policy than turkey colombia kyrgyzstan and nicaragua in france in deaths is assigned a garbage code with of all deaths being certifiedas due to other illdefined and unspecified causes of death r99 heart failure i509 orrespiratory arrest r092 similarly in japan old age senility r54 is a commonly assignedcause of death accounting for one fifth of all garbage codes other research has found that one 101371 pone0237539 august one 0cdeificepsnuesaesidtraehnoitcrafnilarberecdeificepsnusaercnapainomuenpdeificepsnuevitcurtsboyranomuplhtiwesaesidrewoletucayrotaripsernoitcefnideificepsnulnoocyrtnuocknarselamwolleynisenotcapmiwoldernidekramsedocegabragtcapmihgihxesdnayrtnuocybhtaedfosesuacgnidaelpotelbatcimeahcsicinorhcfoealeuqescinorhcsisohrriccilohoclarevilfoebolreppurosuhcnorbsremiehzlaesaesidgnuldeificepsnulaidracoymnoitcrafnideificepsnuetatsorptesnofomsalpoenetalhtiwesaesidesaesidtraehetucatnangilamsremiehzlacitorelcsorehtadnalnifgnignahybmrahdnanoitalugnartsfleslanoitnetniecalpdeificepsnunoitacoffusdeificepsnusaercnaptonekortssadeificepsegahrromeahnoitcrafnirocitorelcsorehtaesaesidtraehnoitcrafnilarberecfoealeuqesllecrevileudsitinomuenpdeificepsnunoitcrafnilarberecamonicractimovdnadoofotevitcurtsboyranomuplesaesiddeificepsnulaidracoymnoitcrafnideificepsnuetucaaitnemedfomsalpoentraehcimeahcsietatsorpdeificepsnuesaesidytilineseruliaftraehdeificepsnudeificepsnuhcamotslaidracoymnoitcrafnideificepsnurosuhcnorbdeificepsnugnuldeificepsnugnulainomuenpdeificepsnunapajcinorhcdeificepsnutnangilamcinorhcetucarosuhcnorbneilartsuaainomuenpohcnorbdeificepsnutonekortssadeificepsainomuenpdeificepsnuroegahrromeahnoitcrafniesaesidyranomuplrewoletucahtiwyrotaripsernoitcefnietatsorpdeificepsnuesaesidevitcurtsboesaesidtraehfomsalpoenaitnemedtraehcimeahcsilaidracoymnoitcrafnideificepsnudeificepsnugnulmodgnikcinorhccitorelcsorehtatnangilamdeificepsnucinorhcetucarosuhcnorbdetinudeificepsnusaercnapdeificepsnudeificepsnuesaesidesaesiddeificepsnudeificepsnutraehcimeahcsitserralaidracoymnoitcrafnideificepsnufomsalpoendeificepsnudnaetatsorpfosesuacytilatromdeificepsnugnulsremiehzlalnooceruliaftraehcinorhcyrotaripseretucatnangilamdenifedllirehtorosuhcnorbecnarfcinorhcralucsavorbereccitorelcsorehtacinorhceruliaftraehrosuhcnorbetucayekrutesaesidyranomuplroegahrromeahdeificepsnunoitcrafnievitcurtsbocinorhctonekortssadeificepsfomsalpoentnangilametatsorpdeificepsnucitorelcsorehtaaitnemedesaesidtraehlaidracoymnoitcrafnideificepsnudeificepsnugnuletucarosuhcnorbadanacdeificepsnulnoocdeificepsnuesaesidyranomupletatsorpevitcurtsbofomsalpoencinorhctnangilamrewoletucahtiwyrotaripsernoitcefniaimeacitpesdeificepsnuroegahrromeahnoitcrafnitonekortssadeificepsesaesidsremiehzlatesnoetalhtiwdeificepsnuhcamotsainomuenpdeificepsnuesaesidyranomupldeificepsnuevitcurtsboesaesidesaesidtraehevitcurtsboyranomuplesaesiddeificepsnudeificepsnudeificepsnugnullaidracoymnoitcrafnideificepsnudnadenifeddeificepsnufosesuacytilatromdeificepsnugnuldeificepsnullirehtorosuhcnorberuliaftraehetucaainomuenpanitnegraare cause of death data fit for purposeesaesidtraehdnasuhcnorbfodenifedllifomsalpoenesaesidtraehcimeahcsignulfosnoitpircsedhcamotsesaesidsetebaidsutillemdeunitnoceruliaftraehdeificepsnuainomuenpcitorelcsorehtayramirplaitnesserosuhcnorblarberecdeificepsnudeificepsnuesaesidtraehnoisnetrepyhdeificepsnugnulnoitcrafnideificepsnusnoitacilpmocsetebaidsutillemtuohtiwdeificepsnudnaevisnetrepyhesaesidtraehevitsegnoceruliaftraehhtiwfosesuacytilatrommsalpoentnangilamdnasnoitacilpmoctnangilamtraehevisnetrepyhcinorhcdeificepsnutserracaidracdenifedllirehtolaidracoymnoitcrafnideificepsnunoitcrafnilarberecdeificepsnutropsnarttnediccadeificepsnulaidracoymnoitcrafnideificepsnulaidracoymnoitcrafnietucanarietucanadroj one 101371 pone0237539 august one 0cniderunjinosrepnoitcrafnilarberecytilinestserrayrotaripserdeificepsnudeificepsnutnetniidenmretednuesaesidtraehtnevedeificepsnucimeahcsicinorhclaidracoymnoitcrafnietucalarberecartniegahrromeaheruliaftraehdnaaeohrraidlarivrehtonamuhfositiretneortsagtonsesaesidycneicifedonummifomsalpoentnangilamdnasuhcnorbgnultonekortssadeificepsnigirosuoitcefnideifissalcsesaesidcitisarapdnasuoitcefninignitlusernoitcrafnidemuserperehwesleesaesid]ivh[surivroegahrromeahaimeacitpesrehtofomsalpoentnangilamcitapehartnistcudelibdnarevilytilinesainomuenpdeificepsnumsinagrooterusopxedeificepsnurotcafdeificepsnuainomuenpyrotaripsersetebaidsutillemdeificepsnudemrifnocmsinagrotonlsisoucrebutlyllacigooiretcablyllacigootsihrodeunitnocelbatllirehtodnaliahtdnadenifeddeificepsnufosesuacytilatromdnadenifeddeificepsnullirehtofosesuacytilatromhtuosacirfarotomdeificepsnudeificepsnuoteudtnediccaelcihevronoisulccociffartlarberecfosisonetsseiretrahtaeddednettanuyramirplaitnessedeificepsnunoisnetrepyhsnoitacilpmocsetebaidsutillemtuohtiwetatsorpdeificepsnudeificepsnuegahrromeahmraerifnoitcrafnirofosesuacytilatromfomsalpoentnangilamrosuhcnorbgnulybtluassadnarehtoecalptonekortsdenifedllirehtosadeificepsdeificepsnudnaainomuenpdeificepsnulaidracoymnoitcrafnideificepsnuetucalizarbsutillemsetebaidtnevesnoitacilpmocdeificepsnurehtohtiwidenmretednuegahrromeahdeificepstnetninoitcrafnirotonekortssadeificepslaidracoymnoitcrafnideificepsnudeificepsnugnuldnadenifeddeificepsnufosesuacytilatrometucarosuhcnorbllirehtoaisinutdeificepsnueruliafdeificepsnudnagnullanercinorhcrehtoybtluassarosuhcnorbevitcurtsbocinorhcegrahcsidmraerifdeificepsnuesaesidyranomuplfomsalpoentnangilametatsorpsisorelcsorehtaeruliaftraehecalpdeificepsnutonekortssadeificepsegahrromeahnoitcrafnirorewoletucahtiwyrotaripsernoitcefnitraehcimeahcsietucarehtosesaesidlarberecartniegahrromeahgnignahybmrahdnanoitalugnartsemohnoitacoffusfleslanoitnetnideificepsnutraehcimeahcsiralucsavoidracdeificepsnuesaesidosesaesiddebircsedevitcurtsboyranomuplcinorhcesaesidhcamotscinorhccitorelcsorehtadeificepsrehtoegrahcsiddnateertsyawhgihdeificepsnuhcamotsdnasisorbiffosisohrricrevildnadenifeddeificepsnullirehtofosesuacytilatromainomuenpdeificepsnuevitcurtsbodeificepsnudnayranomuplmraerifesaesidteertsegrahcsiddeificepsnuyawhgihdnalaidracoymnoitcrafnideificepsnucinorhcrehtoybtluassaetucaaibmoloclaidracoymnoitcrafniesaesidtraehtraehcimeahcsiesaesidetucasirotcepanignaevisnetrepyhcinorhcnatsikebzunoitcrafnirevilfosisohrricroegahrromeahdeificepsnunoitcrafnilaidracoymetucadeificepsnudnarehtosadeificepsesaesidtraehtonekortscitorelcsorehtanatszygrykare cause of death data fit for purposemsalpoentnangilamnilusninonetatsorpfosetebaidtnednepedlaidracoymnoitcrafnifosisohrriccilohoclalanerhtiwsutillemsnoitacilpmocrevilrevilfosisohrricerehwesletonegahrromeaherehwesletonfosisohrricdeificepsnudeifissalcdnarehtoderunjinosrepdeificepsnunielcihevrotomciffarttnediccaevitcurtsboyranomuplcinorhcesaesiddeificepsnudeifissalctonekortssadeificepsegahrromeahnoitcrafnirorevilainomuenpdeificepsnusisorelcsorehtaytilinesetucaeruliafyrotaripserlarberecartnieruliafcitapehdnasisorbiftserracaidracnoisnetrepyhlanercinorhcdeificepsnueruliafyramirplaitnesseeruliaftraehtpygelaidracoymnoitcrafnideificepsnuetucaaugaracintraehfosnoitpircsedesaesidesaesidfosesuacytilatromnoitcrafnisesaesidroegahrromeahnoisnetrepyhnoitcrafnidenifedllisutillemsetebaidtraehcimeahcsideificepsnudnalaidracoymtraehcimeahcsidnasnoitacilpmocdeificepsnucinorhcsisorelcsorehtadenifedllirehtoetucaetucarehtoytilinestonekortssadeificepsyramirplaitnessenatsikijatdeunitnocknarselamefyrtnuoc one 101371 pone0237539 august one 0clarberecfoealeuqessremiehzlarehtolarbereccimeahcsicinorhcnoitcrafniesaesidnoitcrafnideificepsnuesaesidtraehdeificepsnudeificepsnuaitnemedlaidracoymnoitcrafnideificepsnuevitsegnoceruliaftraehhtiwdeificepsnuesaesidtraehesaesidesaesidtraehetucaevisnetrepyhsremiehzlacitorelcsorehtasremiehzlahtiwesaesidtesnoetaldnalnifdeunitnocelbatdeificepsnulnoocainomuenpdeificepsnudeificepsnutsaerbdeificepsnuhcamotsevitcurtsbocinorhcaitnemedralucsavesaesidyranomuplrewoletucahtiwnoitcefniyrotaripserdeificepsnudeificepsnurettulflairtadnasaercnapnoitallirbiflairtadeificepsnuevitcurtsboyranomuplcinorhcesaesiddeificepsnuevitcurtsboyranomuplcinorhcesaesiddeificepsnulaidracoymnoitcrafnideificepsnuetucaainomuenpdeificepsnusremiehzlaesaesidtonekortssadeificepsdeificepsnuroegahrromeahnoitcrafnideificepsnuesaesidtraehlaidracoymnoitcrafnideificepsnudeificepsnugnulaitnemedtsaerbcitorelcsorehtaetucarosuhcnorbdeificepsnuadanacsremiehzladeificepsnuesaesidnoitcrafnideificepsnularberecsremiehzladeificepsnuesaesiddeificepsnusadeificepstraehcimeahcsignulegahrromeahnoitcrafnirodeificepsnuesaesiddeificepsnulaidracoymnoitcrafnideificepsnuaitnemedtsaerbtonekortscinorhcrosuhcnorbetucadeificepsnuneilartsuanoitcrafnitimovdeificepsnufoealeuqessitinomuenprosuhcnorberuliaftraehlarberecdnadoofoteudgnuldeificepsnuainomuenpdeificepsnuytilinesnapajlaidracoymnoitcrafnideificepsnuetucatonekortssadeificepsegahrromeahnoitcrafnirodeificepsnuesaesiddeificepsnudeificepsnutraehcimeahcsignulaitnemedmodgniktsaerbcinorhcrosuhcnorbdeificepsnudetinudeificepsnulnooclaidracoymetucatonekortstserrayrotaripsereruliaftraehrosuhcnorbdeificepsnudenifedllirehtotsaerbsremiehzlaecnarfare cause of death data fit for purposetraehfosnoitpircseddenifedlliesaesiddnadenifeddeificepsnufosesuacytilatromesaesidnoisnetrepyhtraehcimeahcsiesaesidtraehsutillemsetebaiddnasnoitacilpmocytilinesllirehtoyramirplaitnessecinorhctserracaidracevisnetrepyhdeificepsnunoitcrafnilarbereclaidracoymnoitcrafnietucanarideificepsnutraehfonoitamroflamesaesidtraehdeificepsnulatinegnoccitorelcsorehtaeruliaftraehainomuenpdeificepsnudeificepsnutsaerbnoisnetrepyhyramirplaitnesselaidracoymsutillemsetebaidnoitcrafnideificepsnusnoitacilpmoctuohtiwetucadeificepsnuevisnetrepyhesaesidtraehevitsegnoceruliaftraehhtiwnoitcrafnideificepsnularberecnadrojdeunitnoclarberecartniegahrromeahgnuldnasuhcnorbcitapehartnistcudelibdnarevilmetsyssuovrenerehwesletonfosesaesiddeifissalcfomsalpoenfomsalpoeneruliafevitarenegedsetebaidsutillemtnangilamtnangilamlanercinorhcrehtodeificepsnuaimeacitpesrehtoainomuenpdeificepsnumsinagroytilinesllirehtodnaliahtdnadenifeddeificepsnufosesuacytilatromdeificepsnulnooceruliafyrotaripserrosuhcnorbdeificepsnugnultonekortssadeificepsaimeacitpesdeificepsnudeificepsnuroegahrromeahnoitcrafnidnadenifeddeificepsn | Colon_Cancer |
"cellular recognition of microbial dna is an evolutionarily conserved mechanism by which the innate immunesystem detects pathogens cyclic gmpamp synthase cgas and its downstream effector stimulator of interferongenes sting are involved in mediating fundamental innate antimicrobial immunity by promoting the release oftype i interferons ifns and other inflammatory cytokines accumulating evidence suggests that the activation ofthe cgassting axis is critical for antitumor immunity the downstream cytokines regulated by cgasstingespecially type i ifns serve as bridges connecting innate immunity with adaptive immunity accordingly a growingnumber of studies have focused on the synthesis and screening of sting pathway agonists however chronicsting activation may lead to a protumor phenotype in certain malignancies hence the cgassting signalingpathway must be orchestrated properly when sting agonists are used alone or in combination in this review wediscuss the dichotomous roles of the cgassting pathway in tumor development and the latest advances in theuse of sting agonistskeywords cgassting innate immunity type i interferon sting agonists antitumor response cancerdevelopmentintroductionthe discovery of phagocytosis in advanced the understanding of innate immunity the first line of host defenses protection againston patternrecognition receptors prrs which recognize microbialproducts coordinate antimicrobial defenses and activateinfection dependsagainstinfection byvarious pathogens correspondence zqliucsueducn juyan zheng and junluan mo contributed equally to this work1department of clinical pharmacology hunan key laboratory ofpharmacogenetics and national clinical research center for geriatricdisorders xiangya hospital central south university changsha peoples republic of china2institute of clinical pharmacology engineering research center for appliedtechnology of pharmacogenomics of ministry of education central southuniversity changsha peoples republic of chinafull list of author information is available at the end of the adaptive immunity abnormal rna or dna rnadna hybridization and cyclic dinucleotides derived frommicrobes are usually considered pathogenassociated molecular patterns pamps [ ] cells associated with innate immunity recognize different microbial pampsthrough specific prrs thereby playing key roles in hostresistance to microbial infection the pathways governing rna recognition such as retinoid acid induciblegene i rigilike receptors have been reviewed elsewhere and will not be covered herein in the case of dnarecognition one of the best known prrs is tolllike receptor tlr9 which senses extracellular cpg hypomethylated dna that has entered the cytosol through thephagosomelysosome system in addition the aim2like receptor aim2 inflammasome can be triggered afterthe entry of doublestranded dna dsdna into the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0czheng molecular cancer page of cytosolic compartment which induces the proteolyticmaturation of proinflammatory cytokines such as il1and il18 and the activation of gasdermin d leading topyroptosis [] nevertheless the most notable prr iscgas a direct cytosolic dsdna sensor which was identified by dr chens group in once cgas bindsto dsdna the cgassting pathway is activated to further induce the expression of type i ifns and other inflammatory cytokinesthus triggering innate immuneresponses mounting evidence suggests that cgassting signaling not only plays pivotal roles in the hostdefense against microbialinfection but also modulatestumorigenesis hence in this review we summarize themechanism of cgassting activation and elaboratefindings regarding its dual effects on tumor developmentcurrent advances in the use of sting agonists as a novelstrategy for antitumor therapy are also reviewedinsights into the cgassting signal transductioncascadecgas is an innate immune sensor that identifies variouscytosolic dsdnaincluding dna with viral bacterialmitochondrial micronuclei and retroelement originswhich can be mainly divided into pathogenderiveddna and selfdna table in the cytoplasm cgas isactivated by interacting with dsdna in a sequence[]independent butstructural and biochemical analyses have revealed thatthe cterminal lobe of cgas contains a conserved zinclengthdependent mannerionbinding module that mediates dna binding andcgas dimerization [ ] dna ligands promotecgas activation primarily by inducing conformationalchanges around the catalytic site and in the dnabinding structures of cgasthe gscontaining loopundergoes conformational change to maintain stabilitywhich is a major mechanism of cgas activation bydna in addition to the primary dnabinding sitementioned above the secondary site located beside theprimary site is a helix formed between strands 78and several surfaceexposed loops the proximity ofthe two dnabinding sites in cgas leads to a cgasdna complex assembly in which two cgas moleculesembrace two molecules of dsdna [ ] the cgasdimers are anized in headtohead alignment nextto the dna and thus form stable ladderlike networks between one long curved dsdna helix or two independent dsdna strands [ ] in this way eachindividual cgasdsdna complex can be cooperativelystabilized and can lead to stronger enzymatic activitywhich may provide a possible explanation for longerdsdna as more likely to activate cgas in additionlong dna is more efficient than short dna in drivingthe liquidliquid phase separation of cgas and the formation ofcriticallydependent on the concentration of cgas and dna inthe cytoplasm cgas and dsdna are spatially concentratedcgasdimerization and activation [] once cgas andcgas liquidlike dropletsin liquiddropletsistofacilitatetable classification of the cytosolic dsdna that activates the cgassting signaling axisclassificationselfdnasource of dsdnamicronucleipossible mechanismsrupture of the micronuclei membrane leads to exposureof chromatin dna that is recognized by cgas whichactivates the cgassting pathwayreferences mitochondrionnuclear rnapathogenderived dnadna virushsv1 hsv2 kshv adenovirus vacciniavirus cytomegalovirus papillomavirusmurine gammaherpesvirus retrovirushiv siv murine leukemia virusrna viruswest nile virus dengue virus vsvsarscov2bacterialisteria monocytogenes mycobacteriumtuberculosis listeria shigella francisellachlamydia and neisseriamitochondrial stress induces mtdna leakage into thecytosol thus activating the sting pathway and inducingproduction of cytokinesfacilitated by endogenous retroelements nuclear rnacan be reversely transcribed into dna that activatescgassting signaling dna viruses invade host cells and release pathogenderiveddna to induce sting activation[]dna intermediates generated from reverse transcription maybe recognized by cgas to stimulate downstream stingsignaling infection with rna viruses might cause cellular damage andcell death which results in the release of cellular dna andfurther activation of the cgassting axis sarscov2 bindingto ace2 can lead to excessive angiotensin ii signaling thatactivates the sting pathway in mice[]bacteria produce cdns such as cyclic digmp and cyclicdiamp which can directly bind to and activate sting[ ]hsv1 herpes simplex virus hsv2 herpes simplex virus kshv kaposi sarcomaassociated herpesvirus hiv human immunodeficiency virus siv simianimmunodeficiency virus vsv vesicular stomatitis virus cdns cyclic dinucleotides and sarscov2 severe acute respiratory syndrome coronavirus 0czheng molecular cancer page of dsdna interacts structural switches rearrange the catalytic pocket to enable cgas to catalyze the synthesis of²²cyclic gmpamp ²²cgamp with atp andgtp as substrates the first step in this process is theformation of a linear dinucleotide ²pppg ²²pawith atp serving as the donor and ²oh on gtp serving as the acceptor then the intermediate product flipsover in the catalytic pocket placing gtp at the donorposition and amp at the acceptor position to form asecond ²² phosphodiester bond [ ] notablyalthough dsrna or singlestrand dna ssdna is ableto bind to cgas neither can rearrange the catalyticpocket which may explain the exclusive activation ofcgas by dsdna ultimately cgamp acts as a secondmessenger to bind to and activate sting a small endoplasmic reticulum erlocated protein kd withfour putative transmembrane domains [ ] normally in a resting state sting is retained in the er byinteracting with the ca2 sensor stromalinteractionmolecule stim1 the cytosolic ligandbindingdomain lbd of sting exists as the most functionalunit capable of integrating with ²² cgamp or cdnscyclic dinucleotides such as cdiamp cdigmp or ²²cgamp from bacteria upon interaction the obviousclosure of the ligand binding pocket in the lbd is observed which is related to the activation of sting next sting transforms into a tetramer through a highorder oligomerization reaction and is translocated fromthe er to the perinuclear area facilitated by cytoplasmiccoat protein complex ii copii and adpribosylationfactor arf gtpases [ ] in the golgi sting ispalmitoylated atcys88 andcys91 a posttranslational modification necessary forsting activation modified sting recruits thekinase tankbinding kinase tbk1 in turn the cterminal domains of sting are phosphorylated bytbk1 and then phosphorylated sting recruits interferon regulatory factor irf3 which is also phosphorylated by tbk1 and dimerizes ultimately dimerizedirf3 enters the nucleus and exerts its function in thetranscription of type i ifns and interferonstimulatedgenes isgs in parallel sting can also bind toand stimulate iκb kinase ikk to mediate the production of nuclear factorκb nfκbdriven inflammatorygenes upon signal transduction termination sting istransferred to endolysosomes for degradation considering that cgamp can be transferred through gapjunctions or delivered in viralexosome packages cgassting signaling may be activated in the cytoplasmwithout dsdna [ ] moreover newly produced typei ifns activate heterodimer interferon receptors ifnar1 and ifnar2 through paracrine signaling and thusinduce the transcription of isgs [ ] in summaryonce virusderived dna and selfdna are located intwo cysteine residuesthe cytoplasm they can be sensed by cgas and a cgasdsdna complex is formed to catalyze the synthesis of ²²cgamp with atp and gtp then ²²cgamp and bacteriaderived cdns induce sting activation and mediate the release of downstream type iifns tnfα and il6 which are prerequisites for antimicrobial defense and antitumor effects the wholeprocess shows that the dsdnacgassting axis canlead to the activation of both innate and adaptive immunity fig the antitumor functions of the cgasstingsignaling pathwayrecent evidence has revealed the close association of thecgassting pathway with cancer development thissignaling pathway is generally regarded as a potent regulator of cancer immunity a stingmediated immunesupportive microenvironment can hamper malignancyoccurrence stressbytumor cell cytosolic dsdna induces sting activationunder normal circumstances dna is strictly unaffiliatedwith the cytoplasm in eukaryotic cells to avoid autoimmunity however dna leaks aberrantly in tumorcells [ ] cancer cells share common features including genome instability tumor suppressor gene mutation or deletion oxidativeand vigorousmetabolism under these intense states nuclear andmitochondrial dna is fragile and easily damaged whichleads to eventual dna leakage in the forms of micronuclei chromatin fragments andor free telomeric dna[ ] chromosomal instability cin is the primary source of cytoplasmic dna in malignant cells andis generally associated with tumor progression distantmetastasis and therapeutic tolerance excessive proliferation of cancer cells results in unstable genomes usuallychromosomal missegregation during mitosis due to defects in segregation lagging chromosomes generate micronuclei in a cellcycledependent manner the vulnerable membraneof micronuclei easily exposes the inner dna to the cytoplasm and activates the cgassting signaling axis exogenous stimuli such as chemotherapy and irradiation can also cause dna damage in addition to leakednuclear dna oxidative stressinduced mitochondrialdna leakage is another crucial initiator of sting pathway activation several anticancer treatments that precisely attack mitochondrial membranes result in effluxand cell death therefore the permeabilization of mitochondria membranes provides a reasonable explanationfor mitochondrial dna escape [ ] other sourcessuch as apoptotic cellderived dna exosomal dnaexodna and transposable elements have also beencharacterized 0czheng molecular cancer page of fig the cgassting dna sensing signaling pathway various dna derived from virus dying tumor cells or nucleus and mitochondria binds toand activates the cytosolic dna sensor cgas cgas catalyzes the synthesis of ²²cgamp in the presence of atp and gtp then ²²cgamp bindsto the er adaptor sting which also can be activated by cdns derived from bacteria upon activation sting translocates from er to golgicompartments where it activates tbk1 and ikk which phosphorylate irf3 and iκbα respectively then irf3 and iκbα dimerize and enter nucleusinitiating the transcription of type i ifn tnf and il6 the primary roles of these cytokines are reflected in host defense inflammation andantitumor immunitydemonstrated to evoke cgassting activation intumor cells [ ]type i ifns mediators of sting and adaptive antitumoreffectscgassting signaling exerts antitumor functions incancer cells both in an autonomous and nonautonomousmanner on the one hand dna damage can provokeacute sting signal transduction and induce cellularsenescence an irreversible cell cycle arrest state whichthwarts the aberrant proliferation of tumor cells throughacquisition ofsecretoryphenotype sasp which is associated with the releaseof abundantinflammatory mediators proteases andgrowth factors [ ] in contrast to undergoingsenescence tumor cells also directly propel apoptosisprocesses by upregulating proapoptosis protein bcl2associated x bax and downregulating the bcl2 apoptosis on the other hand stingsenescenceassociatedtheregulatoractivation in tumor cells not only facilitates the transcription of downstream type i ifns to induce dendriticcell maturation but also recruits supportive immunecells for direct nonspontaneous tumor elimination sting activation in nonmalignant cells causes tumorsuppressive effects as well sting signaling protectsagainst colitisassociated carcinomas cacs induced byazoxymethane aom and dextran sulfate sodiumdss which induce dna damage in intestinal epithelialcells and further trigger sting activation downstreamcytokines of sting signaling such as il1 and il18prevent neoplastic transformation by facilitating woundrepair more importantly sting signaling can also provoke cytotoxic t cell responses to control tumorigenesis necrotic cancer cells are commonly engulfed byantigenpresenting cells especially the basic leucine zippertranscription factor atflike batf3drivenlineage of dendritic cells dcs batf3 dcs take intumorassociated antigens and migrate towardsthe 0czheng molecular cancer page of tumordraining lymph node via the lymphatic systemwhere they crossprime tumorspecific cd8 t cellsthen cd8 t cells undergo activation and clonal expansion in the lymph nodes and are trafficked throughblood vessels to kill tumor cells in turn damaged cancercells release more antigens that are further captured bydcs the whole process forms a positive feedback loopcalled the cancerimmunity cycle tumor eradication can be achieved by multiple processes in thecancerimmunity cycle including tumor antigen captureand presentation and t cell priming and activation withtumor antigenspecific t cell priming and activationrelying on dcs and type i ifn release the involvement of type i ifns in innate immune sensing and adaptive immunity provides a reasonable hypothesis forexploring candidate prr pathways as potential immunomodulators mice lacking tlr9 myeloid differentiationprimary response gene myd88 cytosolic rna sensor mavs or the purinergic receptor p2x7r maintainintact antitumor immunity responses whereas mice deficient in sting or irf3 present with impaired cd8 tcell priming and activation [ ] in fact dying tumorcells can release multiple damageassociated molecularpatterns damps to trigger innate immune responsesin dcs among these released stimuli tumor cellderiveddna is a pivotal inducer in general the phagocytosis ofapoptotic cells causesimmune silence because ofdnasebased degradation nevertheless tumor cellreleased dna can be preserved in the dc endolysosomal compartment through an unknown mechanism cgas recognizes dna invading the cytoplasm andinduces the activation of sting cascades excretion oftype i ifns and expression of isgs additionally undersome physiological conditions such as hypoxia andacidic environments nuclear or mitochondrial dnamight be packaged in exosomes exosomal dnaexodna animates sting signaling once it is absorbedby tumorinfiltrating dcs finallytumor cellderived cgamp can also be transferred to host dcs bythe folate transporter slc19a1 and then directly bindsto sting activating it in dcs a recent study moredirectly demonstrated that cellautonomous sting promoted the maintenance of stem celllike cd8 t cellsand augmented antitumor t cell responses and mechanistically cgasstingmediated type i interferon signaling reinforced the stem celllike cd8 t celldifferentiation program mainly by restraining akt activity immune cellderived type i ifns have crucial functions in antitumor immunity control on the one handtype i ifns boost cross presentation by various mechanisms first they stimulate the maturation of dcs secondthey slow the endosomelysosome acidificationprocess to prevent engulfed tumor antigen clearance andelevate the expression of mhc i molecules on the cellsurface [ ] finally they accelerate dc migrationtowardslymph nodes where they can crossprimetumorspecific cd8 t cells on the other handtype i ifns drive the expression of multiple chemokinessuch as cxcl9 and cxcl10 both of which are necessary for cytotoxic t lymphocyte ctl transfer and infiltration similarly type i ifns restrain the defaultimmune suppressive action of regulatory t treg cellsby downregulating phosphodiesterase pde4 and upregulating cyclic amp camp consequently typei ifns serve as bridges linking the cgassting pathway with cd8 t cellmediated antitumor immunitythe antitumor mechanisms of the cgassting signaling axis are illustrated in fig indeed previous studies revealed that sting activation can stimulate antitumor immune responses inleukemia melanoma glioma and hepatocellular carcinoma [] additionally sting expression is downregulated in a wide variety of tumor tissues and celllines according to a pancancer analysis with a smallproportion of tumors approximately bearing silent sting expression lower sting expressionwas found in hepatic carcinoma and gastric cancer compared with its level in corresponding normal tissues andthis lower expression level was correlated with highertumor stage and poorer prognosis [ ] consistentlycompared with that in the mcfg10a mammary epithelial cell line lower sting expression was detected inmalignant breast cancer cellincluding mcf7hbl100 and t47d cells as well as human melanomacell lines and colorectal adenocarcinoma lines [ ] collectivelythat cgassting signaling might act as a tumor suppressor in certain types of cancersthese findings suggestlinessting pathway agonists as cancer therapeuticsthe immunostimulatory potential of the cgasstingpathway makes it an attractive pharmacological targetsince its activation in the tumor microenvironmenttme can induce efficient crosspriming oftumorspecific antigens and facilitate the infiltration of effectort cells recent drug research has focused on the development of sting agonists because of their potential inanticancer therapy [ ] to date various kinds ofsting agonists have been discovered and they aremainly divided into the following categories cyclic dinucleotides and their derivates dmxaa and its analogsand small molecular agonists in addition some conventional antitumor therapeutics can also indirectly activatesting such as chemotherapy radiotherapy rt andtargeted therapy in addition sting agonists areable to enhance the efficacy of other anticancer therapeutic agents when used in combination sting 0czheng molecular cancer page of fig the antitumor immunity effect of the cgassting pathway dna damage leads to the formation of dsdna in tumor cells upon itsstimulation sting signaling is activated and promotes the release of type i ifn which is crucial for dc maturation sting signaling activation indcs is the core step of the whole cancerimmunity cycle which can be initiated through engulfment of dyingdamaged tumor cells exosometransfer and cgamp gap junctions then dcs migrate towards the tumordraining lymph node and crossprime tumor specific cd8 t cells withthe help of type i ifns finally t cells undergo clonal expansion and traffic through the blood vessel to conduct tumor killingagonists and their synergistic use with other remedies isfurther explored in detail belowcyclic dinucleotides cdnscdns constitute a main type of sting agonist whichmainly originate from bacteria the known naturalcdns consist of exogenous cyclic digmp cdigmpcdiamp ²²cgamp and endogenous ²²cgampamong these cdigmp cdiamp and ²²cgampare synthesized by bacteria and identified as secondarymessengers that mediate sting signal transduction inprokaryotic cells while ²²cgamp functions as theinitiator of sting in mammalian cells the antitumor potential of these natural dinucleotides was firstproven by the finding that cdigmp could inhibit theproliferation of human colon cancer cells in vitro andbasal cell proliferation of human cecal adenocarcinomah508 cells was inhibited with μm cdigmp intraperitoneal ip injection of highdose cdigmpdirectly activated caspase3 and triggered t1 tumoripcell apoptosis in vitro nmol of cdigmp reduced thegrowth of t1 tumor cells in vitro by and nmreduced it by while lowdose cdigmp nmol accelerated the adaptive t cell response by converting a subgroup of myeloidderived suppressor cellsmdscs into immune stimulatory cells producing il12injection of ²²cgamp consistentlymgkg expedited dramatic leukemic elimination in eltcl1 transgenic mice bearing chronic lymphocyticleukemia cll and promoted tumor shrinkage of multiple myeloma in vivo from the perspective of endogenous cdns ²²cgamp mgkg was alsoshown to restrain tumorigenesis and improve the survival rate of mice bearing ct26 colon adenocarcinomain a dosagedependent manner relying on dc activationand t cell crosspriming more recently ohkurit further demonstrated that intratumoral it injection of ²²cgamp μg25 μldose on and days after the injection of tumor cells significantly mitigated tumor growth and prolonged the survival of breast 0czheng molecular cancer page of cancer t1luc squamous cell carcinoma mscc1colon cancer ct26 and melanoma b16f10 mousemodels notably the it injection of ²²cgampinhibited not only tumor growth but also lung metastases in mice bearing b16f10 cellderived tumors suggesting that cgampinduced cd8 tcell priming can drivesystemic antitumor immunity to control local and distant tumor growth termedvaccinestingvaxconsidering the superior properties of sting signaling in activating adaptive immunityit is rational toutilize sting agonists such as cdns as cancer vaccineadjuvants to increase tumor immunogenicity fu investigated the in vivo therapeutic efficacy of acancercomprisinggranulocytemacrophage colonystimulating factor gmcsf and bacteriaderived or synthetic cdns theyobserved that after it injection of stingvax with μg of cdns per vaccine dose the volume of b16melanoma tumors was dramatically reduced in a dosedependent manner compared to mice receiving gmcsf cancer vaccine alone stingvaxtreated mice hadmore infiltrating cd8 ifnγ t cells in the tumormicroenvironment the in vivo antitumor effect of stingvax was also verified in models of colon carcinomact26 pancreatic carcinoma panc02 and upper aerodigestive squamous cell carcinoma sccfvii although natural cdns are able to produce robust antitumor immunity their chemical features might hindertheir future application in the clinical setting first native cdns are easily degraded by enzymes inside the cellor in the bloodstream second their negatively chargedproperty hydrophilicity and phosphate moieties severelyimpede cdns from penetrating cell membranes to activate cytosolic sting leading to low bioavailability andpoor retention of the cdns in specific cells and tissuesthird unintentional toxicities and narrow therapeuticwindows are also unavoidable thus new strategies toimprove therapeutic efficacy and reduce adverse effectsare urgently needed including drug delivery carrier engineering original structural modification and nonnucleotide agonist screening regarding agonistdelivery smith reported that biopolymer implantscodelivering cdigmp μg and chimeric antigen receptor t cart cells resulted in significant tumor regression in mice bearing pancreatic tumors moreoveriv administration of cdigmpysk05lip equivalent to μg of cdigmp aysk05liposome delivery system encapsulating cdigmp led to a tremendous decrease in metastatic lesionsin a b16f10 mouse melanoma model with nearly ofthe injected mice showing resistance against tumor relapsethe adaptive immune responsememory was successfully induced chen alsofound thatliposomalindicating thatinjection ofintravenousintravenousivnanopdelivered cgamp cgampnp could activate the sting axis more effectively than solublecgamp and converted the immunosuppressive tme toa tumoricidal state in a transplanted b16f10 cell melanoma model and in a genetically engineered triplenegative breast cancer model moreover a recentstudy creatively suggested that modified bacteria mightbe exploited as a selective carrier of sting agonistsintroduction of a dinucleotide cyclasecoding gene intothe escherichia coli nissle strain was an attempt at realizing this effect however advancements to the systemare needed tobysnakeapartdigestionresistancecompoundatoms the modifiedfrom improving delivery methods cdnswith superior properties are currently being synthesized and tested for instance to prevent enzymatichydrolysis of cgamp the nonbridging oxygen atomsin cgamp phosphodiester linkages were replaced by²²sulfurcgsasmp showed resistance against degradation byenpp1 a major ²²cgamp hydrolasetherebyleading to a longer halflife and sustained high affinity for human sting hsting syntheticdithio mixedlinkage cdns with both rp rp r rand rp sp r s dithio diastereomers possessed notonlyvenomphosphodiesterase but also enhanced affinityforsting a novel superior modified product ml rrs2 cda also termed adus100 had the potencyto activate all hsting variants and mouse stingmsting adus100 had higher efficiency in activating sting signaling than endogenous or exogenous cdns mainly because of its enhanced stabilityand lipophilicity its powerful tumor elimination effect was extensively demonstrated in multiple murinemodelsincluding b16 melanoma t1 breast cancer and ct26 colon cancer with all treated animalsshowing significant and durable tumorregressionafter itinjection of adus100 three mg doseswhen tumor volumes reached mm3 theremarkableforhsting laid the foundation for its clinical use related clinicaltrials of adus100 are outlined intable in addition to adus100 some other novelsting agonists have been well designed iacs8779and iacs8803 are two highly potent ²²thiophosphate cdn analogs that induced striking systemicantitumorin a b16 melanoma murineinjection μg on and daysmodel after itposttumor implantation compared with adus100or cgamp the characteristics and preclinicalapplications of all these mentioned cnds are summarized in table because of the structural modification and optimization of delivery strategiestheapplication range and efficacy of cdns have beenand high affinityresponsescurativeeffect 0czheng molecular cancer page of table characteristics and preclinical applications of different sting agonistsclassificationcharacteristicsapplicationmodelsnatural cdnagonistscdigmppoor membrane permeabilitysuitable for various codeliverytechnologiescolon cancer h508cells t1 metastaticbreast cancertreatmentinformation μm nmol ip nmol ip nmol ip²²cgamp²²cgamphigher binding affinity formsting than for hstinghigher affinity for hsting thanits lineage isomers binds tovarious sting nucleotidepolymorphisms observed inhumans easily degraded byphosphodiesteraseimpermeable to the cellmembranechronic lymphocyticleukemia mgkg ipmultiple myeloma mgkg ipct26 colonadenocarcinoma mgkgbreast cancer t1lucsquamous cellcarcinomasmscc1 μg25 μldose it μg25 μldose itcolon cancer ct26 μg25 μldose itmelanoma b16f10 μg25 μldose ittherapeutic effects references[ ] [ ]inhibitsproliferation tumorregression tumorregressionaccelerates tcellresponseleukemiaeliminationsuppressesgrowthrestrainstumorigenesisimproves survivalratedelays tumrowthdelays tumrowthdelays tumrowthdelays tumrowthstingvaxsyntheticcdnagonistspotent in vivo antitumor efficacyin multiple therapeutic modelsof established cancercgampnpsbiopolymer scaffolds cdigmp and car t cellscdigmpysk05lip²²cgsasmpadus100iacs8779iacs8803noncdnagonistsfaaliposomal nanops npsdeliver cgamp intracellularlymore effectively than realizedwith soluble cgamperadicates tumors moreeffectively than systemicdeliveryysk05 is a lipid that can efficientlydeliver cdigmp to the cytosolpossesses high fusogenic activitywhich enhances endosomalescapemore resistant to degradation byenpp1 tenfold more potent atinducing ifn secretion potentialuse as a cancer vaccine adjuvantimproves stability and lipophilicityhigher affinity for hsting thannatural cdn agonists capable toactivate all hsting variants andmstingstimulates a superior systemicantitumor response thanadus100 and cgampcauses hemorrhagic necrosisfailed in a phase i clinical trialdue to species specificity μg cdns itreduces tumorvolume b16 melanomacolon carcinomact26pancreaticcarcinoma panc02b16f10 melanomaivtnbccreates atumoricidal state pancreatic cancer μg cdigmptumor regression b16f10 mousemelanoma μg cdigmp ivdecreasesmetastasisthp1 monocytesb16 melanomathree mg doses it t1 breast cancerthree mg doses itmc26 colon cancerthree mg doses itdurable tumorregressiondurable tumorregressiondurable tumorregression b16 melanoma μg on day and posttumor implantationantitumorresponse murine colontumorsextensive tumorrejection[ ]dmxaafirst discovered as a vascularrat mammary mgkg iphigh anticancer[ 0czheng molecular cancer page of table characteristics and preclinical applications of different sting agonists continuedclassificationcharacteristicsapplicationmodelstreatmentinformationinduces proinflammatory cytokinesin a stingdependent mannerhuman fibroblastsantiviral activity selectively induces stingdependentsynthesis and secretion of bioactiveifns no evidence of binding directlyto stingactivates sting in openconformation submicromolarlevels induce sting activationand ifn productionhuman fibroblastsantiviral activity colon tumors mgkg iv of a treatedgroup remainedtumor free faa flavone acetic acid dmxaa 56dimethylxanthenone4acetic acid cma 10carboxymethyl9acrid | Colon_Cancer |
emerging evidence reveals that micrornas mirnas play a crucial role in tumor progression but the underlying mechanism of micrornas in lung squamous cell cancer lscc remains unclearmethod westernblotting and quantitative realtime pcr qpcr were carried out to detect mrna and protein expression cell proliferation was evaluated by cell counting kit8 cck8 colonyforming assay or sphereforming assay respectivelyresults mir2143p was markedly deregulated in lscc tissues and was inversely related to the level of yesassociated protein1 yap1 which is the core transcription regulator of the hippo signaling pathway kaplanmeier survival curves illustrated that patients with high mir2143p expression demonstrated more favorable clinical outcomes mir2143p overexpression oe repressed proliferation and cancer stemlike cells cscs properties in vitro and in vivo xenograft mouse model mechanistically luciferase activity assay revealed that mir2143p directly targets yap1 by specifically binding on the ² utr of yap1 mir2143p plays a pivotal role in cscs properties by targeting yap1 which provides a potential treatment strategy for lscc patientskeywords mir2143p yap1 csc properties globally lung cancer lc has high mortality rates targeted drugs have been developed in lung adenocarcinoma ac to inhibit tumor progression which remarkably changed treatment strategies and brought positive news to patients with egfr mutation and alk or ros1 rearrangements compared with ac the mainstream treatment regimens for lung squamous cell cancer lscc are still platinumcontaining chemotherapeutics and immune therapy and the targeted therapy for correspondence shunlusjtueducn tingting lu and ying yang contributed equally to this workdepartment of shanghai lung cancer center shanghai chest hospital shanghai jiao tong university huaihai west road shanghai peoples republic of chinaadvanced lscc is rare therefore it is urgent to clarify the underlying mechanism of lscc to develop new and innovative treatment strategies for lscc patientsmicrornas are a cluster of noncoding rnas that modulate gene expression recently micrornas became known to regulate tumorigenesis increasing evidence has shown that aberrant mirna expression participates in diverse pathogenic processes such as tumor apoptosis and csc properties [] moreover micrornacontrolled cancer stem cells cscs have caused widespread concern as various studies link mirnas to the characteristics of cscs and differentiation of embryonic stem cells [] for instance mir205 oe remarkably decreases the proliferation of cscs in human pancreatic cancer while low expression of mir613 contributed to the the authors this is licensed under a creative commons attribution international license which permits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the creative commons licence and indicate if changes were made the images or other third party material in this are included in the s creative commons licence unless indicated otherwise in a credit line to the material if material is not included in the s creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtain permission directly from the copyright holder to view a copy of this licence visit httpcreat iveco mmons licen sesby40 the creative commons public domain dedication waiver httpcreat iveco mmons publi cdoma inzero10 applies to the data made available in this unless otherwise stated in a credit line to the data 0clu a0et a0al cancer cell int page of expansion of liver cscs by enhancing the dedifferentiation of hepatoma cells hippo is a traditional signaling pathway that regulates various biological processes in both drosophila and human species [ ] the hippo pathway components involve a major kinase cascade and scaffold proteins and yap1 is the core transcription regulator of the hippo signaling pathway briefly in hippo pathway mammalian sterile20like kinase12 mst12 forms a complex with the adaptor protein salvador1 sav1 and ultimately results in yap1 phosphorylation furthermore phosphorylated yap1 is sequestered in the cytoplasm and then degraded through the ubiquitinproteasome increasing evidence suggests that high levels of the yap1 expression are present in distinct human tumors such as in colon liver ovarian cholangiocyte and prostate cancers [] our group previously demonstrated the contribution of yap1 to the maintenance of csc activities in fgfr1amplified lung squamous cell cancer in this study to elucidate the function of mirna in lscc initiation and progression we detected the selected mirna expression profile in lscc human samples using the qpcr additional file a0 figure a0 s1 our results highlighted the low expression of mir2143p in human lscc samples compared to adjacent normal samples for the first time we reported that mir2143p inhibited csc capacity by targeting yap1 in lscc furthermore we verified that mir2143p took part in activating the hippo signaling activity altogether we confirmed that mir2143p exhibited as a tumor suppressor in lscc initiation and it also serves as a possible target for lsccmaterials and a0methodscell linesthe human h520 and hcc95 cell lines were purchased from the american type culture collection mir2143p and negative control nc inhibitors and mimics were from ribobio technologyhuman tissue specimensfor research use the lscc samples were collected from the department of oncology shanghai chest hospital written consent forms were obtained from every lscc patient the institute research ethics committee of shanghai chest hospital approved all protocolswestern blottingyap14912 lats19153 pyap14911 vimentin12020 snail3879and tubulin2148 antibodies were all from cell signaling technology cst danvers ma oct4 ab19875 cd133 ab19898 and sox2 ab171380 were from abcam protein was obtained from cells by using radioimmunoprecipitation assay ripa sigmaaldrich equal quantities of protein lysates were loaded on the sodium dodecyl sulfatepolyacrylamide gel electrophoresis gels sdspage gels and gel electrophoresis was applied to transfer the protein onto a nitrocellulose membrane the protein strips were ultimately detected by enhanced chemiluminescence thermo fisher scientific and captured using the odyssey infrared imaging analysis softwareqrtpcrthrough the trizol reagent life technologies ca usa the total rna was collected according to the manufacturers protocol the qpcr was conducted using the sybr synergy brands synergy brands premix ex taq kit takara briefly the cdna complementary dna was used as the template for qpcr detection using taqman technology on an applied biosystems sequence detection system applied biosystems foster city ca then we detected the expression of yap1 sox2 sryrelated hmgbox a0 cd133 oct4 octamerbinding transcription factor4 and nanog genes by using commercially available primer and probe sequences applied biosystemsmirna transfectionthe mirna mimics were synthesized through the ribobio technology both the h520 and hcc95 cells were grown in 6well plates and transfected with lipofectamine invitrogen thermo fisher scientific in optimem invitrogen thermo fisher scientific after a0h cells were harvested to the following experimentsluciferase activity assayluciferase® reporter assay kit promega madison wi the luciferase assay was performed using the dualbriefly the 293t cells were grown in 24well plates and cotransfected with mirnc or mir2143p mimics pmirglohyap13² untranslated region utr wide type wt or pmirglohyap13²utrmutation type mut or pgl3yap1 promoter and renilla luciferase vector with lipofectamine invitrogen after a0h the firefly luciferase activity normalized to the renilla luciferasecck8 assayusing the cell counting kit8 cck8 dojindo kumamoto japan cell proliferation and viability were measured after transfection cells were grown into 96well plates cellswell n the optical density value od value at a0nm of the cells at the specified time was recorded 0clu a0et a0al cancer cell int page of fig in lung squamous cell cancer lscc the mir2143p is significantly downregulated which is related to better clinical outcomes a correlation analysis of mir2143p with yap1 levels as detected by qpcr n b relative mir2143p levels in lscc cell lines and the normal bronchial epithelial cell line beas2b was used as the control c kaplanmeier analysis of overall survival relying on mir214 levels in lscc patients showed patients with higher mir214 levels possessed a more favorable clinical outcome than patients with lower mir214 levels p colonyforming assayall cells with different treatments were grown into 6well plates the cells were cultured for at least a0days until the colonies became visible and noticeable the cells were washed with phosphate buffer saline pbs fixed with polyformaldehyde sigmaaldrich st louis mo and then stained with giemsa solarbio beijing china image j recorded photos of the coloniessphere formation assaybriefly adherent h520 and hcc95 cells with different treatments were seeded into the 6well ultralow attachment plates at a density of 3000well after a0 days spheres a0 μm diameter were tallied and captured every a0 days the culture medium was refreshed and oncosphere numbers were counted using the leica digital camera the serumfree dmemf12 medium life technologies grand island ny involves the b27 0clu a0et a0al cancer cell int page of table correlations with a0clinicopathological features of a0lscc patientsbetween a0mir2143p variableage years ¥ gender female maletumor size cm ¤ smoke yes notnm stage iii iiiivdifferential degree poorly well moderatenmir3plow n high n plowhigh by the sample median pearson x2 test p was considered statistically significantsupplement life technologies grand island ny a0 ngml basic fibroblast growth factor bfgf gibco and a0ngml epidermal growth factor egf gibcoma after washing with the pbs for at least three times the sections were incubated with the alexa488conjugated or alexa594conjugated secondary antibody dilution life technologies for a0h at room temperature furthermore the nuclei were stained with ²6diamidino2phenylindole dapi dilution beyotime institute of biotechnology china all images were taken using a confocal laserscanning microscope leica tcs sp5 ii germanytumor xenograft modellung squamous cells transfected with mirnc mimics or mir2143p mimics were triplewashed in the pbs and then suspended in matrigel cells were subcutaneously injected into the armpit of each mouse every a0 days each tumor diameter was measured tumor volume was calculated by the equation tumor volume mm3 à length mm à width mm2 after nearly a0weeks tumors were all collected for subsequent analysis all male balbc nude mice slac shanghai were fed at the specific pathogen free spf animal center of shanghai chest hospital all experiments were performed according to the guidelinesorthotropic lung tumor modelcells were suspended in matrigel and orthotropically injected into the left lateral lungs of the mice as described with minor modificationsimmunofluorescence methodall paraffinsections of mouse lung tumors were acquired from a leica cryostat the tumor sections were incubated with one of the following polyclonal antibodies rabbit yap1 antibody rabbit sox2 antibody and rabbit cd133 antibody all at dilution cst danvers statistical analysisgraphpad prism software was employed for statistical analysis all results were presented as mean ± standard error of the mean sem where indicated formats least three independent replicate experiments statistical differences were investigated using the students ttest Ï2 test and repeated measures analysis of variance the table univariate and a0 multivariate analysis of a0 different prognostic variables and a0 overall survival os in a0 lscc patientsvariablesage vs ¥ gender female vs maletumor size ¤ vs smoke yes vs notnm stage iii vs iiiivdifferentiation degree wellmoderate vs poorlymir2143p expression low vs highunivariate analysishr ci p multivariate analysis modelhr cip 0clu a0et a0al cancer cell int page of fig mir2143p overexpression impairs lscc proliferation and csc properties in vitro a cck8 assay indicated that mir2143p reduced cell proliferation b colonyforming assays showed the effect of mir2143p c protein levels of vimentin and snail after overexpression or knockdown of mir2143p d sphere forming assays were used to examine the effects of mir2143p oe on lscc e the qpcr showed the expression of stemrelated genes in both cells data are represented as mean and sem from three independent experiments p p p kaplanmeier method was performed to assess overall survival os and the logrank test was performed to analyze the curves finally the cox model was utilized to identify independent prognostic factors the statistical significance was set at p 0clu a0et a0al cancer cell int page of resultsmir3p is a0downregulated in a0lscc and a0is a0significantly associated with a0better clinical prognosiswhile yap1 is elevated the mir2143p is downregulated in tumor species compared to the matched normal bronchial epithelium using qpcr analysis [ ] in this context our further analysis found a robust negative correlation between the mir2143p expression and yap1 mrna expression in lscc species n fig a01a next we detected the mir2143p rna levels in some lscc cell lines and we observed the reduction of mir2143p in lscc cell lines normalized to the normal bronchial epithelial cell line beas2b accordingly fig a0 1b then we explored the relationship between mir2143p and corresponding clinical features patients were divided into two groups according to the median level of the mir2143p expression we found the association of high mir2143p expression to smaller tumor size and lower tumor node metastasis tnm stage table a0 there was no significant relationship between mir2143p and other clinical features such as age and gender furthermore kaplanmeier km survival curves indicated that patients with high mir214 expression possessed better clinical outcomes p fig a01c n the cox regression analysis model revealed that the mir214 expression and tumor size were independent prognostic indicators for lscc table a0 in summary these results suggested that mir2143p could have a critical role in lscc initiation and progression increased mir3p inhibits lscc cell proliferation and a0csc properties in a0vitroto test the effects of mir2143p in lung cancer we designed subsequent experiments first cck8 6assay demonstrated that the mir2143p overexpression mir2143p oe particularly inhibited proliferation in h520 and hcc95 cells compared to the nc groups fig a02a vimentin and snail are mesenchymal markers involved in many aspects of cellular anization signaling and proliferation westernbolt analysis proved that the mir2143p oe reduced the protein levels of vimentin and snail while the mir2143p knockdown increased vimentin and snail protein levels accordingly fig a0 2c additionally the mir2143p oe remarkably inhibited colony and sphere formations compared with the control group fig a02b and d moreover qpcr proved that the mir2143p oe substantially decreased the rna expression of cscspecific markers involving cd133 sox2 oct4 and nanog fig a02e in addition we detected the proteins level of cscspecific markers and the same was exhibited in additional file a0 figure a0s2mir3p inhibits csc properties of a0lscc in a0vivo and a0decreases yap1 protein levelbased on the above findings regarding in a0vitro we illuminated the role of mir2143p in a0vivo then h520 and hcc95 cells transfected with mir2143p or nc mimics were administered into flanks of nude mice figures a03af exhibited how tumor volume and weight in the mir2143p oe group were smaller than the nc group in both h520 and hcc95 mice models fluorescence confocal microscope analysis implied that mir2143p oe significantly decreased yap1 levels in mouse models fig a0 3g and h the hippo pathway regulates proliferation and apoptosis to control tissue size also we observed that the mir2143p increased yap1 phosphorylation and decreased yap1 levels fig a0 3i altogether the results explained above demonstrated how mir2143p oe suppressed lscc tumor progression by targeting yap1to further thoroughly verify the physiological function of mir2143p we developed orthotropic mice models which closely recapitulated human lung cancer cells were orthotropically administered into the left lateral lungs of nude mice compared to the nc groups the mir2143p oe remarkably decreased tumor occurrence and tumor size fig a04a and b figure a04c illustrated that mouse body weight in the mir2143p oe group is heavier than the nc group after injection mainly caused by cachexia furthermore the mir2143p oe significantly prolonged os than the control group fig a0 4d in the future larger animal models are needed for further research immunofluorescence if staining assays showed a decrease of yap1 and stemrelated biomarkers including sox2 and cd133 this suggests the observable loss of proliferation and csc properties in tumors fig a04egthe mir3p directly interacts with a0yap1 in a0lscc cellsgiven that the mir2143p is downregulated while yap1 is upregulated in lscc patients we speculated that yap1 could be a target of mir2143p bioinformatic see figure on next pagefig mir2143p impairs tumorigenicity of lscc in vivo and activates the hippo pathway decreases yap1 protein level a and b h520 and hcc95 cells transfected with mir2143p were injected into nude mice c and e tumor weight and tumor volume were analyzed in h520 cells d and f tumor weight and tumor volume were analyzed in hcc95 cells g and h fluorescence confocal microscope analysis demonstrated mir2143p overexpression decreased yap1 levels of mice tumors i protein levels of hippo components after transfected with mir2143p in h520 cells at first mice models were established in each group but mice died in hcc95 groupsp p p 0clu a0et a0al cancer cell int page of 0clu a0et a0al cancer cell int page of fig mir214 overexpression impairs lscc tumor initiation and progression of orthotropic models a images of primary tumors in the left lungs at day from each group after injection of h520 cells transfected with mir2143p mimics and nc mimics b and c mean primary tumor volume and body weight from two groups error bars represent sem p d survival for the mice in each group evaluated p eg upper panel representative if images of yap1 and stemrelated biomarkers including sox2 and cd133 lower panel quantification of biomarker expression error bars represent sem n p p 0clu a0et a0al cancer cell int page of fig mir2143p directly targets yap1 in lscc cells a binding sites of mir214 in the yap1 ²utr b luciferase activity of each combination was assessed c the yap1 mrna levels in h520 cell lines d the yap1 protein levels in lscc cell lines transfected with mir214 mimics p p 0clu a0et a0al cancer cell int page of see figure on next pagefig rescue experiments confirm that yap1 liberated the growth and csc properties repressed by mir214 mir2143p represses growth and csc properties by inhibiting yap1 a cck experiments in lscc cells transfected with different plasmids b colony formation in cells transduced with different plasmids c sphereforming experiments in lscc cells transfected with different plasmids d reintroduction of yap1 rescued the reductions of cscspecific markers in both cell lines d e vimentin and snail protein levels were detected in lscc cells transfected with different plasmids e f in subcutaneous mice models the reexpression of yap1 remarkably decreased the antitumor effects of mir2143palgorithms such as miranda and targetscan recommended that yap1 possesses potential complementary sites for the mir2143p seed region in the ² untranslated region ²utr fig a05a to verify this hypothesis we transfected both the h520 and hcc95 cells with mir or mir214 inhibitors and yap1 mrna and protein levels were measured results showed that the mir214 oe significantly decreased yap1 mrna and protein levels in both cell lines while mir214 knockdown kd led to increased yap1 expression fig a0 5c and d furthermore to determine the direct binding site between the mir2143p and yap1 we performed a luciferase assay then we constructed the wild type of and mutated seed sequences in the ² utr of yap1 mrna the yap1wt3²utr and its mutant type yap1mt3²utr were amplified and cloned downstream of a luciferase reporter gene in the pgl3basic vector the luciferase reporter experiment revealed that the cotransfection of pcdnamir214 and yap1wt3²utr in h520 and hcc95 cells remarkably inhibited luciferase activity compared to the nc cells fig a05b however there was no difference in the luciferase activity in cells that cotransfected with yap1mt3²utr and pcdnamir214 fig a0 5b therefore we confirmed that mir2143p directly targets yap1 by specifically binding on the ² utr of yap1 in lscc cell linesrescue experiments illustrated that a0yap1 liberated the a0growth and a0csc properties inhibited by a0mirthese findings encouraged us to explore whether the mir2143p repressed lscc growth and csc properties via yap1 our previous work revealed that yap1 kd remarkably decreased sphere and colony formations and aldehyde dehydrogenase gene aldh proportion preveiously our group have proved that yap1 kd substantially reduced stemrelated genes levels of lung cancer cells these results suggested that yap1 kd could mimic the role of mir2143p oe our findings above confirmed that silenced yap1 led to the same phenotypes as mir2143p oe did implying that mir2143p inhibited proliferation and csc phenotype via yap1 to validate this hypothesis we performed a subsequent rescue experiment in h520 and hcc95 cells cells transfected with mir214 were transduced with a pcdna31yap1 plasmid interestingly yap1 oe partially rescued the proliferation capacity inhibited by the mir214 fig a06a the yap1 plasmid transduction liberated the inhibited growths of mir2143p oe colonies and spheres fig a06b and c we also found that reintroduction of yap1 rescued the reductions of cscspecific markers in both h520 and hcc95 cell lines fig a06d in addition the reintroduction of yap1 rescued the reductions of vimentin and snail protein expressions caused by the mir2143p oe fig a0 6e furthermore in mice models the restoration of yap1 expression significantly liberated the decreased proliferation and selfrenewal abilities caused by the mir2143p oe in lscc fig a06fdiscussionthis study confirmed that yap1 was overexpressed while mir2143p was downregulated in lscc tissues which was inversely correlated with each other the km survival curves indicated that patients with high mir214 expression possessed better clinical outcomes than those with low expression in this case we initially reported that the mir2143p strongly modulated csc phenotypes of lscc in a0vitro and in a0vivo we provided robust evidence on how mir2143p served as a tumor suppressor and a promising target in therapies against lscc because it imitated and displayed excellent antitumor efficacylike many other mirnas mir214 has reportedly been abnormally expressed in various tumors it plays a dual role by modulating tumor initiation based on the tumor types either as oncogenes or tumor suppressors as a tumor suppressor mir214 represses tumor progression in hepatocellular gastric ovarian and cervical cancers however it is also reported to promote tumor migration in melanoma [] furthermore mir214 acts as either an oncogene or a suppressor depending on the genetic or the cancer type interestingly in lung cancer wu et a0 al reported that mir214 oe significantly diminished the migration and invasion of nonsmall cell lung cancer nsclc cells while long et a0al found that mir214 expression contributed to epithelialmesenchymal transition emt and metastasis of lung adenocarcinoma [ ] however the impact and regulatory mechanisms of mir214 in lscc remain to be extensively exploredwe demonstrated the role of mir214 as a tumor suppressor and how mir214 impaired the csc properties of lscc by gain and lossoffunction assays to our knowledge the crucial role of mirna is to regulate the 0clu a0et a0al cancer cell int page of 0clu a0et a0al cancer cell int page of expression of its downstream genes through the mrna cleavage andor by inhibition of translation depending on the extent of complementarity with the ²utr of target genes our group previously confirmed that the mir2143p was downregulated whereas yap1 was upregulated in lscc tissues in this context we reported that mir214 was inversely correlated with yap1 which stimulated our attention and interest further investigations validated our hypothesis that yap1 was a direct downstream gene of mir214 in lscc first mir214 oe evidently reduced yap1 mrna and protein expression in lscc second ectopic mir214 levels significantly impaired a luciferase reporters activity which contains the ²utr sequence of yap1 third rescue experiments showed that yap1 reexpression abrogated the antitumor effect of mir214 on lscc to a certain extentto conclude mir214 acts as a critical regulator of csc properties in lscc we also reported that mir inhibited lscc proliferation and csc capacities via targeting yap1 to activate the hippo signaling pathway the identification of tumorspecific mirnas and target genes is necessary to understand the underlying mechanisms of lscc initiation this is of great value to design new strategies in the future mir2143p might become a promising prognostic marker finally targeting the mir214yap1 axis could be identified as a promising strategy in lsccour data revealed that mir214 could inhibit lscc cell proliferation through the hippo signaling pathway collectively these findings provide new insight into the role of mir214 which might serve as a novel therapeutic target for lscc treatmentsupplementary informationsupplementary information accompanies this paper at https doi101186s1293 additional file a0 microrna expression profile in lscc human samples additional file a0 mir2143p oe substantially decreased the proteins level of cscspecific markersabbreviationsmirnas micrornas lscc lung squamous cell cancer yap1 yesassociated protein1 cscs cancer stemlike cells lc lung cancer ac lung adenocarcinoma mst12 mammalian sterile20like kinase12 sav1 salvador fgfr1amplified fibroblast growth factor receptor1amplified nc negative control lats1 large tumor suppressor pyap1 phosphoyap1 ripa radioimmunoprecipitation assay sdspage gels sodium dodecyl sulfatepolyacrylamide gel electrophoresis gels sybr synergy brands synergy brands cdna complementary dna sox2 srysex determination region of ychromosomerelated hmgbox oct4 octamerbinding transcription factor4 optimem reducedserum medium wt wide type mut mutation type cck8 cell counting kit8 od value optical density value dmem dulbecco modified eagle medium pbs phosphate buffer saline bfgf basic fibroblast growth factor egf epidermal growth factor dapi 46diamino2phenyl indole spf specific pathogen free sem standard error of mean os overall survival tnm tumor node metastasis km kaplanmeier oe overexpression if immunofluorescence ²utr ²untranslated region kd knowdown aldh aldehyde dehydrogenase gene nsclc nonsmall cell lung cancer emt epithelialmesenchymal transitionacknowledgementsnot applicableauthors contributionstl and sl designed the study tl and yy performed the experiments and prepared the figures tl yy zl contributed to drafting the manuscript all authors read and approved the final manuscriptfundingthis study was supported by the national key rd program of china 2016yfc1303300 to s lu shanghai chest hospital project of collaborative innovationyjxt20190105yjxt20190209 to sl and zl the national natural science foundation of china to s lu and to w xia the national key grant of china 2016yfc0906400 to w xia wu jie ping medical foundation the key project of shanghai health family planning to s lu shanghai municipal science technology commission research project to s lu shanghai scientific research projects to s lu and astrazeneca research funding clinical research plan of shdc 16cr3005a to s l shanghai science and technology commission guidance projects18411968200 to zl medicalengineering joint funds of shanghai jiao tong university yg2017ms81 to zl and shanghai youth top talent project to zlavailability of data and materialsthe datasets used andor analyzed during the current study are available from the corresponding author on reasonable requestethics approval and consent to participatethe study was approved by the ethics review committee of shanghai chest hospital shanghai china the animal study followed the guidelines for the animal care and use approved by shanghai chest hospitalconsent for publicationnot applicablecompeting intereststhe authors declare that they have no competing interestsreceived may accepted august references torre la et al global cancer statistics ca a cancer j clin bartel dp micrornas genomics biogenesis mechanism and function cell nicoloso ms et al micrornasthe micro steering wheel of tumour metastases nat rev cancer calin ga et al microrna signatures in human cancers nat rev cancer catalanotto c et al microrna in control of gene expression an overview of nuclear functions int j mol sci kent oa et al a small piece in the cancer puzzle micrornas as tumor suppressors and oncogenes oncogene chakraborty c et al mirnaregulated cancer stem cells understanding the property and the role of mirna in carcinogenesis tumor biol melton c et al microrna regulation of embryonic stem cell selfrenewal and differentiation cell biol stem cells 0clu a0et a0al cancer cell int page of liu c et al microrna regulation of cancer stem cells cancer res chaudhary ak et al chemosensitization and inhibition of pancreatic cancer stem cell proliferation by overexpression of microrna205 cancer lett li b et al mir613 inhibits liver cancer stem cell expansion by regulating sox9 pathway gene harvey kf et al the drosophila mst ortholog hippo restricts growth and cell proliferation and promotes apoptosis cell udan rs et al hippo promotes proliferation arrest and apoptosis in the salvadorwarts pathway nat cell biol zhao b et al tead mediates yapdependent gene induction and growth control genes dev snijders am et al rare amplicons implicate frequent deregulation of cell fate specification pathways in oral squamous cell carcinoma oncogene zhao b et al inactivation of yap oncoprotein by the hippo pathway is involved in cell contact inhibition and tissue growth control genes dev zhang x et al the hippo pathway transcriptional coactivator yap is an ovarian cancer oncogene oncogene rizvi s et al a hippo and fibroblast growth factor receptor autocrine pathway in cholangiocarcinoma j biol chem lu tt et al the hippoyap1 pathway interacts with fgfr1 signaling to maintain stemness | Colon_Cancer |
covid19 has had an impact on the provision of colorectal cancer care the aim of the crccovid study is to describe the changes in colorectal cancer services in the uk and usa in response to thepandemic and to understand the longterm impactmethods and analysis this study comprises phases phase is a survey of colorectal units that aims toevaluate adherences and deviations from the best practice guidelines during the covid19 pandemicphase is a monthly prospective data collection of service provision that aims to determine the impactof the service modiï¬cations on the longterm cancer speciï¬c outcomes compared to the national standards phase aims to predict costs attributable to the modiï¬cations of the crc services and additionalresources required to treat patients whose treatment has been affected by the pandemic phase aims tocompare the impact of covid19 on the nhs and usa model of healthcare in terms of service provisionand cost and to propose a standardised model of delivering colorectal cancer services for future outbreaksethics and dissemination this study is a service evaluation and does not require hra approval or ethicalapproval in the uk local service evaluation registration is required for each participating centre in theusa ethical approval was granted by the research and development committee the results of thisstudy will be disseminated to stakeholders submitted for peer review publications conference presentations and circulated via social mediaregistration details nil the authors published by elsevier ltd on behalf of surgical associates ltd this is an open access under the cc byncnd license httpcreativecommonslicensesbyncnd40 introductionthe covid19 pandemic has had a significant impact on theprovision of healthcare worldwide as of the 29th june covid19 has resulted in conï¬rmed cases and corresponding author at department of surgery and cancer imperial collegelondon chelsea and westminster and the royal marsden campus unitedkingdomemail address ckontovounisiosimperialacuk c kontovounisiosdeaths in the uk at a local level hospitals have been forcedto make a number of workforce modiï¬cations and changes toserviceprovision to combat the crisis and maintain standards ofcare for our patients facetoface consultations have beendissolved or minimized in favour of telephone or virtual clinicsprovision of investigations including ct scans and endoscopieshave been significantly reduced and all benign surgical procedurespostponed furthermore the treatment algorithm for conï¬rmed colorectal cancer cases has proved challenging101016jisjp20200700524683574 the authors published by elsevier ltd on behalf of surgical associates ltdthis is an open access under the cc byncnd license httpcreativecommonslicensesbyncnd40 0ca courtney international of surgery protocols in the uk over forty thousand patients are diagnosed with colorectal cancer each year deviation from nice colorectal cancercrc guidelines may lead to significantly poorer outcomes however the current model of cancer services delivery cannot be maintained because of both resource limitation and the potential risksto patients and staff during the pandemic there is a lack of highdependency beds which are being utilized for covid19 patientsthere is the risk of exposing colorectal cancer patients the majority of whom are elderly and have significant comorbidities to thevirus during their treatment within the hospital patients requiringneoadjuvant or adjuvant therapy are at particular risk finallystaff safety must also be considered particularly around aerosolgenerating procedures such as endoscopy and laparoscopic surgery intercollegiate general surgery guidance on covid19 outlinedgeneral principles on the provision of a safe surgical service duringthe pandemic however there has been no speciï¬c guidance todate on how to best modify colorectal cancer crc service provision during the pandemic in the absence of a national consensusthe onus is on individual hospital trusts and multidisciplinaryteams to make very challenging decisions about individual patientcare lack of a uniï¬ed approach may have important consequencesat patient and healthcare institution levelsdelay in cancer diagnosis or treatment due to service modiï¬cation is likely to create an increased demand in resources once thecrisis has passed predicting the economic impact and planningfor this is essentialhow hospitals approach the new constraints on crc care andallocate resources may vary between the uk and usa it is hopedthat gaining insights from both perspectives will improve the problem solving methods and analysis aims and objectivesthe aim of the crc covid study is to describe the changes incolorectal cancer services in the uk and usa in response to thecovid19 pandemic and to understand the longterm impactour primary and secondary objectives relevant to each phase ofthe study are listed in table study designthis is a multicentre service evaluation conducted through aresearch collaborative with the support of the crc covid steeringcommittee all colorectal units continuing to provide cancer services in the uk ireland and the usa have been invited to participate all study and recruitment information is available on thetable primary and secondary study objectiveswebsite crccovid this service evaluation has been endorsedby the royal college of surgeons of england rcsthis service evaluation will be carried out in phases fig phase uses a questionnaire to assess the modiï¬cationsadopted by each colorectal unit in order to continue provision ofthe colorectal cancer services during the covid19 pandemic ithas been developed using an iterative process after research ofall relevant guidelines to construct the standard against which services would be evaluatedthe following guidelines relevant to the management of colorectal cancer have been used as standards for this serviceevaluation nice guidelines colorectal cancer [ng151] nice guideline suspected cancer recognition and referral[ng12] association of coloproctology of great britain irelandacpgbi guidelines for the management of cancer of thecolon rectum and anus [] british society of gastroenterologyassociation of coloproctology of great britain and irelandpublic health england postpolypectomy and postcolorectal cancer resection surveillanceguidelines informal consultations with consultants nurse specialists andpatients have been used to develop the tool and then it has beenmodiï¬ed after clinician review for face validity ï¬ow and relevance the ï¬nal instrument comprises questionsphase investigates the provision of colorectal cancer servicesduring the covid19 pandemic by evaluating the performance ofeach unit against the national bowel cancer audit outcomes all centres participating in phase will be required to registerthis service evaluation as per local protocol prior to commencement of data collection on redcap this will be the responsibilityof the local leadphase of the study will develop a prediction model of the economic burden of the modiï¬cations in cancer service delivery thismodel will be designed jointly by two international businessschools based on previous publications and national statisticsphase will evaluate and compare the impact of the covid19pandemic on the nhs and the usa healthcare using data collectedduring phase and the predictive mode utilized in phase speciï¬cdifferences in modiï¬cations of crc services will be examined recruitmentphase survey has been distributed to all colorectal consultantsin the uk and ireland through personalised emails social mediaand the rcs the recruitment of colorectal cancer units in thephasephase phase phase phase primary objectiveevaluate adherences and deviations from best practiceguidelines on colorectal cancer during covid19pandemicative crc service deliverysecondary objectives 0f describe modiï¬cations to screening process for crc 0f describe modiï¬cations to preoperative intraoperative and postoper 0f demonstrate global effect of covid19 pandemic on crc service provi 0f outline consensus recommendations for sustainable modiï¬cations to 0f predict the impact of modiï¬cations on the incidence and prevalence of 0f plan adjustments to crc service provision after the end of pandemicsion irrespective of the type of healthcare systemdetermine the impact of crc service provision followingmodiï¬cations on longterm cancer speciï¬c outcomescompared to national standards 0f predict the costs attributable to modiï¬cations of crc services during covid19 pandemic 0f predict additional resources required to treat patients whose treatment has been affected by covid19 0f compare the impact of covid19 on the nhs and usa model of healthcare in terms of service provision and cost 0f propose a standardised model of delivering colorectal cancer services for future outbreaksdifferent crc stages in monthscrc services 0ca courtney international of surgery protocols fig phases of crc covidusa will use similar approach all units recruited into phase arerecruited into phase participation in phase is not mandatory inorder to participate in phase data collectionall surveys and data collection follow the gdpr requirementsand comply with caldicott principles individual patient identiï¬able data is not collected in this study study data is collectedand managed using redcap electronic data capture tool hostedat the kennedy institute of rheumatology at the university ofoxford redcap research electronic data capture is asecure webbased software platform designed to support datacapture for research studies providing an intuitive interfacefor validated data capture audit trails for tracking data manipulation and export procedures automated export procedures forseamless data downloads to common statistical packages and procedures for data integration and interoperability with externalsources after the close of the phase data collection period all data setswill be checked for missing data where possible centres will begiven an opportunity to rectify missing data centres where of data is missing will be excluded from data analysis and localleads will be notiï¬ed a nominated data validator will need toensure data accuracy prior to submission if during this processmajor discrepancy is identiï¬ed within the data set the centresdata will be excluded completely from the analysisfor details of the data collected in phase and phase pleaserefer to supplement data analysisphase responses to the survey pertaining to deviations fromdiagnostic and treatment protocols during the covid19 pandemicsee supplement will be converted to a numerical scale where will denote no deviation and will denote complete cessation ofservice provision the scores will be summarized using appropriatesummary statistics and analyzed using unsupervised learningkmeans and hierarchical clustering to identify clusters of homogeneous response to the pandemicphase every month participating centres will report theirdiagnostic and treatment activity see supplement to determine the impact of covid19 on colorectal cancer activity we willuse timeseries methods and data on historical activity and patientoutcomes to estimate a baseline of expected monthly activity that would have taken place in the absence of the pandemicthe baseline and a conï¬dence interval will be estimated atthe national regional and individual nhs trust level the baselineestimate will then be compared to the actual activity as reportedby publicly available data and data collected by this studythe difference between expected and actual activity will providean estimate of the reduction in activityto quantify the impact on patient outcomes associated with theestimated reduction in activity and deviation from standardizedcare protocols we will use estimates of disease progression available in peerreviewed literature [] a similar methodologywill be used to predict the impact of the pandemic on the incidenceand prevalence of different colorectal cancer stages in the following months under different scenarios predictions will be madeat the regional and national level and depending on data granularity at the trust levelphase will estimate the ï¬nancial costs of modiï¬cations to thecrc service provision due to the covid19 pandemic this willallow prediction of the expenditure and the additional resourcesrequired to resume routine services we will base this on the literature regarding the price of treatment at different disease stages and the information about the cost of resource utilizationconsultations diagnostic tests operating theatre time and hospital stay phase will compare the results from phases in theuk with those in the us discussioncancer care and maintaining high standards of diagnosis andtreatment has long been a priority of the nhs and internationalhealth care systems the pandemic has shifted this focus awayfrom the cancer services colorectal cancer patients are particularly 0ca courtney international of surgery protocols vulnerable to the disruption of their care as diagnosis throughendoscopy was stopped due to concerns about virus aerosolysation this study is important because it is the ï¬rst study to ask howindividual units had to modify their services and adapt to the newconstraintsin addition to describing the changes and understandingwhether different units had different approaches we wish to gofurther by understanding the effects of diagnostic and treatmentdelay by prospective data collection of cancer cases referrals diagnosis staging and treatment and comparing them to nationally collected audit data these data will allow us to model the economic impact of thedelay and what resources are required to restore cancer servicesto precovid19 standardsthe strengths of this study are in the multimodal approach tothe issues international collaboration and support from the royalcollege of surgeons our diverse team of management and business academics colorectal surgeons nurse specialists and patientadvisors enable us to have a range of approaches to collect andanalyse the datathe main limitation to the study is nonresponder or samplingbias as we require voluntary participation from colorectal teamswe will ensure that we adjust statistical analysis for any underrepresentation we expect that even with minimal participationuseful models can be generated to understand future resourcerequirements at an individual hospital level the methodologyemployed by other units will demonstrate the utility of the modelin summary this is a novel and important multiphase studythat is vital to understand how to best care for cancer patientsand ensure that the effects of the pandemic are mitigated ethics and disseminationthis study is a service evaluation and does not require hraapproval or ethical approval in the uk departmental approvalhas been granted by the university each participating centre mustseek local permission from their local audit department prior tocommencement of data collection in the usa ethical approvalwas granted by the research and development committeedata for phase will be submitted for publication as soon as theresults become available interim data analysis will be presented tothe royal college of surgeons covid19 research collaborativedata for other phases will be submitted for publication once thedata collection has been completed which is anticipated to be afterthe routine service provision resumes all data will be presented atnational and international conferences circumstances permitting guarantornone research registration numbernoneethical approvalnoneauthor contributionsac designed the study wrote the initial proposal drafted themanuscript based on the study proposal and is part of the auditadvisory groupamh ns nd ow sm sr gm nt mg td bs jee ad ptadvised on the study design and the protocol and is part of thesteering committeeck is a project piall authors read commented on and approved the study designthe protocol and the ï¬nal manuscriptfundingthis research received no speciï¬c grant from any fundingagency in the public commercial or notforproï¬t sectorsdeclaration of competing interestthe authors declare that they have no known competing ï¬nancial interests or personal relationships that could have appearedto inï¬uence the work reported in this paperreferences world health anization the united kingdom who coronovirus diseasecovid19 dashboard covid19whointregioneurocountrygbaccessed june covidsurg collaborative global guidance for surgical care during the covid pandemic br j surg a spinelli g pellino covid19 pandemic perspectives on an unfolding crisisbr j surg british society of gastroenterology endoscopy activity and covid19 bsgand jag guidance apr wwwbsgukcovid19adviceendoscopyactivityandcovid19bsgandjagguidance accessed may nhs england nhs improvement letter to chief executives of all nhs trustsand foundation trusts ccg accountable ofï¬cers gp practices and primary carenetworks and providers of community health services mar wwwenglandnhsukcoronaviruswpcontentuploadssites52202003urgentnextstepsonnhsresponsetocovid19lettersimonstevenspdfaccessed may research uk cancerincidencecancerbowelstatisticswwwcancerresearchukhealthprofessionalcancerstatisticsstatisticsbycancertypebowelcancerincidenceheadingzeroaccessed may royal college of surgeons of england updated intercollegiate general surgeryguidance on covid19 apr wwwrcsengacukcoronavirusjointguidanceforsurgeonsv2 accessed may national institute for health and care excellence nice colorectal cancernice guideline ng151 wwwniceukguidanceng151accessed accessed march national institute for health and care excellence nice suspected cancerrecognition and referral nice guideline ng12 wwwniceukguidanceng12 accessed accessed march c cunningham k leong s clark a plumb s taylor i geh s karandikar bmoran association of coloproctology of great britain ireland acpgbiguidelines for the management of cancer of the colon rectum and anus diagnosis investigations and screening colorectal dis suppl s gollins b moran r adams c cunningham s bach as myint a renehans karandikar v goh d prezzi g langman s ahmedzai i geh association ofcoloproctology of great britain ireland acpgbi guidelines for themanagement ofmultidisciplinary management colorectal dis suppl rectum and anusthe coloncancer of glangman m loughrey n shepherd p quirke association ofcoloproctology of great britain ireland acpgbi guidelines for themanagement of cancer of the colon rectum and anus pathologystandards and datasets colorectal dis suppl k leong j hartley s karandikar association of coloproctology of greatbritain ireland acpgbi guidelines for the management of cancer of thecolon rectum and anus follow uplifestyle and survivorshipcolorectal dis suppl b moran c cunningham t singh p sagar j bradbury i geh s karandikarassociation of coloproctology of great britain ireland acpgbi guidelinesfor the management of cancer of the colon rectum and anus surgicalmanagement colorectal dis suppl md rutter j east cj rees n cripps j docherty s dolwani pv kaye kjmonahan mr novelli a plumb bp saunders s thomasgibson djmtolan s whyte s bonnington a scope r wong b hibbert j marsh bmoores a cross l sharp british society of gastroenterologyassociation ofcoloproctology of great britain and irelandpublic health england postpolypectomy and postcolorectal cancer resection surveillance guidelines gut 0ca courtney international of surgery protocols healthcare quality improvement partnership hqip national bowel canceraudit annual report an audit of the care received by people with bowelcancer in england and wales v20 wwwnbocaukcontentuploads202001nboca2019v20pdf accessed june pa harris r taylor r thielke j payne n gonzalez jg conde researchelectronic data capture redcapa metadatadriven methodology andworkï¬ow process for providing translational research informatics support jbiomed inform pa harris r taylor bl minor v elliott m fernandez l oneal l mcleodg delacqua f delacqua j kirby sn duda re consortium the redcapconsortium building an international community of software platformpartners j biomed inform nhs england and nhs improvement cancer waiting times wwwenglandnhsukstatisticsstatisticalworkareascancerwaitingtimesaccessed june national bowel cancer audit nboca datagov weblink accessed junewwwnbocaukresourcesnbocadatagovweblink cancer research uk incisive health saving lives averting costs an analysis ofthe ï¬nancial implications of achieving earlier diagnosis of colorectal lung andovarian cancer wwwcancerresearchuksitesdefaultï¬lessaving_lives_averting_costspdf accessed may s sun f klebaner t tian a new model of time scheme for progression ofcolorectal cancer bmc syst biol suppl s2 j emery p vedsted new nice guidance on diagnosing cancer in generalpractice br j gen pract hb keshava je rosen mr deluzio aw kim fc detterbeck dj boffawhat if i do nothing the natural history of operable cancer of the alimentarytract eur j surg oncol yh lee pt kung yh wang wy kuo sl kao wc tsai effect of length oftime from diagnosis to treatment on colorectal cancer survival a populationbased study plos one e0210465 d roder cs karapetis i olver d keefe r padbury j moore r joshi dwattchow dl worthley cl miller c holden e buckley k powell dburanyitrevarton k fusco t price time from diagnosis to treatment ofcolorectal cancer in a south australian clinical registry cohort how it variesand relates to survival bmj open e031421 cancer research uk bowel cancersurvivalstatistics wwwcancerresearchukhealthprofessionalcancerstatisticsstatisticsbycancertypebowelcancersurvival accessed june kk turaga s girotra are we harming cancer patients by delaying theircancer surgery during the covid19 pandemic ann surg 0c' | Colon_Cancer |
" chemotherapy is one of the most commonly used treatments for patients with advanced colon cancer yet the toxicity of chemotherapy agents such as fluorouracil 5fu limits the effectiveness of chemotherapy ginsenoside rg3 rg3 is an active ingredient isolated from ginseng rg3 has been shown to display anticancer effects on a variety of malignancies yet whether rg3 synergizes the effect of 5fu to inhibit the growth of human colon cancer remains unknown the present study was designed to ascertain whether rg3 is able to enhance the anticolon cancer effect of 5fu the results revealed that combined treatment of rg3 and fu significantly enhanced the inhibition of the proliferation colony formation invasion and migration of human colon cancer cells sw620 and lovo in vitro we also found that combined treatment of rg3 and fu significantly enhanced the apoptosis of colon cancer cells by activating the apaf1caspase 9caspase pathway and arrested the cell cycle of the colon cancer cells in g0g1 by promoting the expression of cyclin d1 cdk2 and cdk4 in addition the pi3kakt signaling pathway in colon cancer cells was suppressed by rg3 and 5fu in vivo rg3 synergized the effect of 5fu to inhibit the growth of human colon cancer xenografts in nude mice similarly combined treatment of rg3 and 5fu altered the expression of colon cancer protein in vivo and in vitro collectively the present study demonstrated that ginsenoside rg3 enhances the anticancer effect of 5fu in colon cancer cells via the pi3kakt pathwaycorrespondence to dr xiangbo chen endoscopy center the quanzhou first hospital affiliated to fujian medical university east street licheng quanzhou fujian pr chinaemail coloboyeahnetkey words ginsenoside rg3 colon cancer 5fluorouracil pi3kaktintroductioncolon cancer is a common malignant tumor of the digestive tract located in the colon which mainly occurs at the junction of the rectum and the sigmoid colon statistics show that the highest incidence of colon cancer is in the age group of years the ratio of male to female is and the incidence of colon cancer ranks third among all cases of gastrointestinal tumors the 5year survival rate of patients with colon cancer is approximately yet the 5year survival rate of patients with advanced stage disease is as low as since the early symptoms of patients with colon cancer are not obvious only about of patients can be diagnosed at the early stage of the disease chemotherapy is one of the most important treatments for patients with advanced colon cancer of which fluorouracil fu is the most widely used 5fu inhibits the proliferation invasion and migration of tumor cells by interfering with the nucleic acid metabolism of tumor cells but it is also toxic to normal cells causing serious adverse reactions even endangering the life safety of patients severely limiting its clinical application previous research has shown that 5fu combined with other agents may reduce the required dosage of 5fu consequently reducing the adverse reactions caused by 5fu without affecting or even improving the efficacy of chemotherapy compared with chemical drugs and biopharmaceuticals multicomponent multitarget and less adverse reactions are unique advantages of traditional chinese medicine for the treatment of diseases in patients with colon cancer chinese medicine can improve patient immunity reduce the side effects of radiotherapy and chemotherapy or enhance drug sensitivity inhibiting the expression of oncogenes helps to inhibit the migration of cancer cells and has a good effect on the treatment of colon cancer ginsenoside rg3 rg3 an active ingredient isolated from ginseng is a tetracyclic triterpenoid saponin that inhibits neovascularization induces tumor cell apoptosis and selectively inhibits tumor cell metastasis and enhances immune function previous studies have shown that rg3 exhibits an inhibitory effect on proliferation invasion and migration of human tumor cells such as lung cancer gastric carcinoma and prostate cancer in colon cancer rg3 was found to activate the ampk signaling pathway to accelerate apoptosis in colon 0chong effects of ginsenoside rg3 on colon cancercancer cell line ht29 in vitro and also to block colon cancer progression by targeting inhibition of cancer stem cells and tumor angiogenesis in vivo although numerous studies have shown that rg3 increases the efficacy and decreases the toxicity of chemotherapeutic drugs and suppresses the chemotherapeutic resistance in cancer its combination with chemotherapeutic agent fu to achieve extra benefits in anticolon cancer treatment warrants detailed investigationin the present study the effects of a combined treatment of rg3 and 5fu on the biological properties of sw620 and lovo cells were investigated in vivo and in vitro we found that a combined treatment of rg3 and 5fu not only enhanced the inhibition of colon cancer cell proliferation migration and invasion but also promoted apoptosis of colon cancer cells and arrested the cells in the g0g1 phase in addition it was also found that rg3 could synergize the capacity of 5fu to inhibit the growth of human colon cancer xenografts in nude mouse and the combined treatment of rg3 and 5fu enhanced the inhibition of the pi3kakt pathway in colon cancer cellsmaterials and methodscell lines and agents sw620 ccl atcc american type culture collection manassas va usa and lovo ccl229 atcc cell lines were cultured with dmem medium cat no thermo fisher scientific inc supplemented with fetal bovine serum fbs cat no thermo fisher scientific inc and penicillinstreptomycin cat no thermo fisher scientific inc the cell lines used in the present study were cultured at Ëc with co2rg3 cat no sigmaaldrich merck kgaa and 5fu cat no sigmaaldrich merck kgaa were dissolved in dmso for the cell experiments the diluted culture solution of rg3 or 5fu was dissolved in dmso to achieve the experimental concentration and was administered to the cells for h for animal experiments pbs diluted rg3 or 5fu was dissolved in dmso to the experimental concentration the experiments were approved by the ethics committee of the quanzhou first hospital affiliated to fujian medical university quanzhou fujian chinamtt assay a total of 2x103 cellswell were inoculated in a 96well culture plate containing the indicated medium dmem plus fbs we evaluated the viability of the sw620 and lovo cells by mtt assay in short after h of culture mtt µl mgml which was dissolved in dmso was added to the cells and incubated the cell supernatant was removed and then µl dmso was added after min the optical density od570 was determined using a plate reader elx808 biotek instrumentscell colony formation assay a total of 2x103 cellsml were seeded in 6well plates with ml mediumwell and medium was exchanged once every days cells were routinely cultured for about weeks when visible clones appeared in the well the culturing was stopped the supernatant culture medium was drawn washed with pbs times and fixed with formaldehyde for min the supernatant was drawn stained with crystal violet for min and slowly rinsed with sterile water plates were placed in a sterile purification table and images were captured after drying the relative proliferation was measured by measuring the absorbance at nm using a plate reader elx808 biotek instrumentswestern blot analysis ripa lysate buffer cat no p0013c beyotime institute of biotechnology shanghai china was used to extract total cellular protein and the bca kit cat no p0009 beyotime institute of biotechnology was used to determine the protein concentration then cell lysates of sw20 and lovo cells were separated by sdspage with µg total protein and transferred to a pvdf membrane the following primary antibodies were selected as follows antincadherin antibody ab18203 dilution antiecadherin antibody ab1416 dilution antimmp ab38898 dilution antiactivecaspase antibody ab2324 dilution antiactivecaspase3 antibody ab2302 dilution antiapaf1 antibody ab2324 dilution antipi3kp85 antibody ab191606 dilution antipi3k110 antibody ab32569 dilution antipanakt phospho t308 antibody ab38449 dilution antipanakt antibody ab8805 dilution antipdk1 antibody ab52893 dilution and antigapdh ab9484 dilution the secondary antibody was selected as follows goat antirabbit ab150077 dilution or goat antirat ab150117 dilution the blocking protocol was with skim milk for h at room temperature the primary antibody was incubated overnight at Ëc and the secondary antibody was incubated for h at room temperature the beyoecl plus kit cat no p0018s beyotime was used for the chromogenic protein bands with beckman coulter immunoassay system unicel dxi beckman coulter and imagej v2147 national institutes of health was used for the densitometric analysis of protein bands all antibodies were purchased from abcam unless otherwise statedtranswell invasion experiment the cell density was adjusted to 05x106 cellsml and then the cells were added to a 24well transwell upper chamber corning corning ny usa medium containing fbs gibco thermo fisher scientific inc was added into the lower transwell chamber and the transwell was incubated at Ëc for h the transwell was taken out and the medium was removed it was washed twice with pbs methanol was added and dried after being fixed for min after the membrane was dried it was stained with crystal violet for min and the relative migration was determined by measuring the absorbance at nm using a plate reader elx808 biotek instruments inccell scratch test a total of 5x105 cells were placed in a 6well plate mlwell a scratch was made as far as possible perpendicular to the back of a horizontal line by using tips against a ruler tips should be vertical and cannot be tilted the cells were washed with pbs for three times and the scratched cells were removed and serumfree dmem was added cells were cultured at Ëc in a co2 incubator for h and images were captured in and h using an ckx41 olympus inverted microscope magnification x100 olympus corp 0concology reports figure effect of the combined treatment of rg3 and 5fu on proliferation of colon cancer cells in vitro sw620 and lovo cells were treated with different doses of a rg3 mmoll or b 5fu µmoll and then the mtt assay was used to detect cell viability c and d after treatment with rg3 mmoll or 5fu µmoll or their combination the colony formation of the colon cancer cells was photographed wt group was used as a baseline for cell viability and cell colony formation three independent repetitions were performed for each experiment p005 p001 and p0001 compared with the wt group fu fluorouracil rg3 ginsenoside rg3 flow cytometric analysis cells that had been treated in different manners were collected and precooled ethanol prechilled pbs and waterfree configuration was added at Ëc overnight then the cells were washed with pbs and stained with propidium iodine pi for cell cycle macsquant® analyzer flow cytometer miltenyi biotec was used to detect the cell cycle and the annexin v fitcpi kit invitrogen thermo fisher scientific inc was used for flow cytometry to detect apoptosisanimal experiment human colon cancer cells 5x10602 ml in the logarithmic phase were selected a total of female nude mice weeks of age g shanghai lingchang biological technology co ltd that were adaptive for feeding [room temperature of Ëc half day light and night dark cycle air humidity of ] for one week were selected mice were anesthetized [ sodium pentobarbital mgkg intraperitoneal ip] and then the lateral skin of the nude mice was selected as a cell inoculation site to inoculate 5x10602 ml human colon cancer cells at the logarithmic phase of growth when the tumor tissue grew to a volume of approximately mm3 then the mouse were randomly assigned to the solvent group equal amount of pbs dmso rg3 group mgkg gavage administration once every two days 5fu group mgkg ip injection once every two days and rg35fu group combined rg3 and 5fu group administration after weeks of treatment the mice were sacrificed using cervical dislocation and breathing and heartbeat for min were observed to determine death and tumor tissues were extracted and weighed with an analytical balance bsa124s beijing sartorius instruments ltd beijing china all animal experiments were approved by the ethics committee of quanzhou first hospital affiliated to fujian medical universitystatistical analysis all data are expressed as mean ± standard deviation and spss ibm corp was used to analyze the data student's ttest was used to compare differences between two groups and multiple groups were compared with oneway anova followed by duncan test as a post hoc test p005 was assigned to indicate that a difference was statistically significantresultscombined treatment of rg3 and fu enhances inhibition of cell proliferation after treatment with different doses of rg3 or 5fu mtt assay was used to measure the cell viability the results revealed that the cell viability of sw620 and lovo cells was significantly and gradually decreased with an increasing dose of rg3 thus we chose mmoll rg3 for subsequent experiments fig 1a as shown in fig 1b the proliferative activity of the colon cancer cells in the combined treatment group of rg3 and fu was significantly lower than 0chong effects of ginsenoside rg3 on colon cancerfigure effect of the combined treatment of rg3 and 5fu on migration and invasion of colon cancer cells in vitro treatment of colon cancer cells with combined treatment of rg3 mmoll and 5fu µmoll inhibited the migration a and invasion b abilities of the sw620 and lovo cells scale bar µm c western blot analysis was used to detect the expression of emtrelated protein ncadherin ecadherin and mmp9 the solvent group was used as a baseline for the migration and invasion of cells three independent repetitions for each experiment were performed p005 p001 and p0001 compared with the rg35fu group fu fluorouracil rg3 ginsenoside rg3 emt epithelialmesenchymal transition mmp matrix metalloproteinase that of the 5fu treatment alone group in addition the cell viability of sw620 and lovo cells gradually decreased with the increasing dose of 5fu however after treatment with the combination of mmoll rg3 and µmoll fu for h the cell viability of sw620 cells was only which was not conducive to subsequent protein detection experiments thus mmoll rg3 and µmoll 5fu were chosen for subsequent experimentationcell clone formation assays were also used to detect in vitro proliferation of colon cancer cells as shown in fig 1c and d the number of colonies formed by the colon cancer cells treated with rg3 and fu was significantly lower than that of rg3 or fu alone these findings indicated that combined treatment of rg3 and 5fu enhanced the inhibition of colon cancer cell proliferation in vitrocombined treatment of rg3 and fu enhances the inhibition of cell migration and invasion the ability of tumor cells to invade and migrate is the key to tumor progression in the present study we compared the effects of different treatment conditions on the invasion and migration of colon cancer cells it was demonstrated that the invasion and migration ability of the colon cancer cells treated with rg3 combined with fu was significantly lower than that of rg3 or 5fu alone fig 2a and b epithelialmesenchymal transition emt is the source of tumor cell ability to acquire higher invasion and migration capacity thus we determined the levels of three emtrelated proteins ncadherin ecadherin and mmp9 and found that the expression of ncadherin and mmp9 protein in the rg35fu group was significantly lower than that of rg3 or fu alone group but 0concology reports figure effect of the combined treatment of rg3 and 5fu on the apoptosis of colon cancer cells in vitro a the percentage of apoptotic sw620 and lovo cells in the different groups b apoptosisrelated proteins [cleaved clcaspase clcaspase and apaf1] were assessed by western blot analysis in sw620 and lovo cells three independent repetitions for each experiment were carried out p0001 compared with the rg35fu group fu fluorouracil rg3 ginsenoside rg3 apaf1 apoptotic protease activating factor the expression of ecadherin protein was significantly higher fig 2ccombined treatment of rg3 and fu promotes apoptosis of colon cancer cells fist we found that the apoptosis of the colon cancer cells treated with rg3 combined with 5fu was significantly higher than that of rg3 or fu alone fig 3a the levels of apoptosisrelated proteins in the sw620 and lovo cells were assessed by western blot analysis as shown in fig 3b expression levels of apaf1 cleaved clcaspase and clcaspase protein in colon cancer cells sw620 and lovo treated with rg3 were significantly increased and the expression of these apoptosisrelated protein in colon cancer cells following fu treatment was significantly higher than that treated with rg3 more importantly expression levels of these apoptosisrelated proteins in colon cancer cells treated with the combination of rg2 and 5fu were significantly higher than levels treated with rg3 or 5fu alonewe analyzed the cell cycle distribution of the colon cancer cells after treatment with the different agents as shown in fig 4a the percentages of colon cancer cells in the g0g1 phase treated with the rg3 and 5fu combination were significantly higher than the percentages following rg3 or 5fu alone similarly we also detected cell cycleassociated protein by western blot analysis as shown in fig 4b the expression levels of cyclin d1 cdk2 and cdk4 protein in colon cancer cells which were treated with the rg3 and 5fu combination were significantly lower than levels following treatment with rg3 or 5fu alonecombined treatment of rg3 and fu suppresses pi3kakt signaling in colon cancer cells the pi3kakt signaling 0chong effects of ginsenoside rg3 on colon cancerfigure effect of the combined treatment of rg3 and 5fu on cell cycle progression of colon cancer cells in vitro a flow cytometry was used to analysis the cell cycle in colon cancer cells after treatment with rg3 mmoll or 5fu µmoll or the combination b cell cycleassociated protein cyclin d1 cdk2 and cdk4 were assessed by western blot analysis three independent repetitions were performed for each experiment p005 p001 and p0001 compared with the rg35fu group fu fluorouracil rg3 ginsenoside rg3 cdk cyclindependent kinase pathway is a signaling pathway involved in cancer cell proliferation invasion and migration and its abnormal activation can confer high proliferation invasion and migration ability of cancer cells in the present study we found that the expression levels of pp85 p110 ppdk1 and pakt protein in the colon cancer cells which was treated with rg3 and 5fu combination were significantly lower than levels in the cells treated with rg3 or 5fu alone fig these results indicated that the combined treatment of rg3 and 5fu enhanced the inhibition of the pi3kakt signaling pathway in colon cancer cells in vitrocombined treatment of rg3 and fu suppresses tumor growth in nude mice based on the results of in vitro studies we further investigated the effects of the rg3 and 5fu combination on colon cancer cell proliferation and protein expression in nude mice sw620 cells were injected into the armpits of nude mice after weeks of treatment the mice were sacrificed and the weight and volume of tumor tissues were measured it was found that the weight and volume of tumor tissues in the rg35fu group were significantly lower than these parameters in the groups treated with rg3 or 5fu alone fig 6a and bmoreover western blot analysis was used to detect the expression of emtrelated proteins cell cyclerelated proteins and key proteins in the pi3kakt signaling pathway it was found that although the effects of the rg3 and 5fu combination were not as obvious as the in vitro results compared with rg3 of 5fu alone the overall trend in protein expression was consistent fig 6ce these results demonstrated that rg3 0concology reports figure effect of the combined treatment of rg3 and 5fu on pi3kakt signaling in colon cancer cells in vitro a and b western blot analysis was used to detect the expression of key proteins in the pi3kakt signaling pathway after treatment with rg3 mmoll or 5fu µmoll or the combination three independent repetitions were performed for each experiment p005 p001 and p0001 compared with the rg35fu group fu fluorouracil rg3 ginsenoside rg3 synergizes the effect of 5fu to inhibit the growth of human colon cancer xenografts in nude micediscussionthe anticancer effect of 5fluorouracil 5fu is exerted mainly by interfering with tumor cell dna replication and it is a commonly used antitumor agent for the treatment of advanced colon cancer however since 5fu displays nonspecific cytotoxicity it also causes damage to normal cells causing irreversible renal dysfunction and severe gastrointestinal reactions these adverse effects limit its clinical application and further improvements in the efficacy of chemotherapy are needed therefore it is urgent to discover a drug that can enhance the chemotherapeutic effects of 5fu and reduce the 5fu toxicity when used in combination with 5fuginsenoside rg3 rg3 is one of the main active ingredients extracted from ginseng research has shown that ginsenoside rg3 has certain inhibitory effects on lung cancer breast and prostate cancer the antitumor mechanism of rg3 was that rg3 reduced the neovascularization probability of tumor recurrence proliferation and metastasis in tumors by inhibiting kdrvegf protein expression and blocking hif1αcox2vegf pathway in the present study we found that the combined treatment of rg3 and 5fu promoted the inhibition of colon cancer cell proliferation in vivo and in vitro tumor growth development and metastasis are closely related to cell proliferation the previous study found that rg3 inhibits the proliferation of tumor cells such as rg3induced egfrmapk pathway deactivation was found to inhibit melanoma cell proliferation by decreasing fut4ley expression rg3 was found to inhibit the proliferation of multiple myeloma cells by inducing the secretion of igf1 promoting tumor cell apoptosis is also a method of inhibiting tumor cell proliferation in the present study we found that the combined treatment of rg3 and fu significantly enhanced the apoptosis of colon cancer cells by activating the apaf1caspase 9caspase pathway in the mitochondrial pathway of apoptosis apoptosisrelated signals release cytochrome c by stimulating the mitochondrial outer membrane cytochrome c enters the cytoplasm which activates caspase9 by binding with apaf1 activation of caspase9 further activates caspase3 while the activated caspase3 can activate caspase leading to apoptosis in addition we also found that the rg3 and 5fu combination enhanced the number of g0g1 phase colon cancer cells and decreased expression of cyclin d1 cdk2 and cdk4 the cell cycle refers to the whole process that the cell undergoes from the completion of one division to the end of the next division and 0chong effects of ginsenoside rg3 on colon cancerfigure effects of the combined treatment of rg3 and 5fu on tumor growth and protein expression of colon cancer cells in vivo after weeks of treatment the mice were sacrificed tumor tissues were excised and the weight a and volume b of tumor tissues were measured ce total protein was extracted from the colon cancer tumor tissues and the expression of proteins was detected by western blot analysis five nude mice in each group and at least tumor tissues were used to evaluate protein expression p005 p001 and p0001 compared with the rg35fu group fu fluorouracil rg3 ginsenoside rg3 the regulation of the cell cycle is mainly achieved by the retention of the g1 phase when a cell is in the g1 phase there is an important node regulating the cell cycle the r point when the cell cycle is before the r point the cell needs the external growth factor to achieve the normal operation of the cell cycle after the cell cycle crosses the r point the cell cycle becomes a process that is controlled autonomously by the cell and no longer depends on the presence of external cytokines cyclin d1 is a g1sspecific cyclin and its main function is to promote the cell cycle from g1 to s by binding and activating the cyclindependent kinase cdk24 a unique cyclindependent kinase of g1 so as to promote cell proliferation invasion and migration of tumor cells are the most important features of malignant tumors and the important causes of death in patients with malignant tumors ncadherin ecadherin and mmp9 are three proteins that play important roles in cell epithelialmesenchymal transition emt whereas emt provides cells the ability to transfer and invade promoting tumor cell emt can inhibit the expression of intercellular junction protein resulting in decreased intercellular connectivity which is beneficial to the invasion and migration of tumor cells to surrounding healthy tissues previous studies have found that rg3 not only inhibits metastasis and invasion of lung cancer cells by inhibiting emt induced by transforming factor 1 but also inhibited the metastasis of prostate pc3m cells by downregulating the expression of aqp1 by downregulating mmp rg3 affected the metastasis and invasion ability of melanoma cells the present study demonstrated that the combined treatment of rg3 and fu significantly suppressed the invasion and migration ability of human colon cancer cell in vitro by altering emtrelated proteinfurthermore we also found that rg3 and 5fu combination inhibited the conduction of the pi3kakt signaling pathway in vivo and in vitro many studies have shown that the occurrence and development of tumors are the result of multifactor multigene and multipathway processes and the cell signal transduction pathway is crucial in the process of tumor development invasion and metastasis the phosphatidylinositol 3kinaseserinethreonine kinase b pi3kakt signaling pathway plays an important role in the regulation of solid tumors [eg liver cancer breast cancer colon cancer gastric cancer neuroblastoma ] and blood tumors [eg leukemia ] pi3k acts as a bridge molecule for the relationship between extracellular signals and cellular responses under the influence of a series of upstream or bypass signaling molecules it acts on the downstream of the effects of a variety of molecules thus promotes cell migration 0concology reports inhibits cell apoptosis accelerates the process of the cell cycle and promotes cell proliferation many previous studies have shown that traditional chinese medicine or traditional chinese medicine monomers can play an antitumor role by inhibiting the pi3kakt signaling pathway in conclusion rg3 enhances 5fu inhibiting proliferation invasion and migration of colorectal cancer cells and helps 5fu block g1 phase induced apoptosis in more colorectal cells all in all our study found that rg3 enhanced the anticancer effect of 5fu on colon cancer cell via pi3kakt pathwayacknowledgementsnot applicablefundingno funding was receivedavailability of data and materialsthe datasets used during the present study are available from the corresponding author upon reasonable requestauthors' contributionsxc made substantial contributions to the conception and design of the study and critically revised it for important intellectual content sh contributed to the acquisition of the data wc zh yw xm yh and zl analyzed and interpreted the data all authors read and approved the final manuscriptethics approval and consent to participateall animal and cell experiments were approved by the ethics committee of the quanzhou first hospital affiliated to fujian medical university quanzhou fujian chinapatient consent for publicationnot applicablecompeting intereststhe authors state that they have no competing interestsreferences siegel rl ward em and jemal a trends in colorectal cancer incidence rates in the united states by tumor location and stage cancer epidemiol biomarkers prev siegel rl miller kd and jemal a cancer statistics ca cancer j clin chen w zheng r baade pd zhang s zeng h bray f jemal a yu xq and he j cancer statistics in china ca cancer j clin chen w zheng r zhang s zeng h zuo t xia c yang z and he j cancer incidence and mortality in china in an analysis based on urbanization level chin j cancer res li j hou n faried a tsutsumi s and kuwano h inhibition of autophagy augments fluorouracil chemotherapy in human colon cancer in vitro and in vivo model eur j cancer sanoff hk carpenter wr freburger j li l chen k zullig ll goldberg rm schymura mj and schrag d comparison of adverse events during fluorouracil versus fluorouraciloxaliplatin adjuvant chemotherapy for stage iii colon cancer a populationbased analysis cancer cheah ky howarth gs bindon ka kennedy ja and bastian sep low molecular weight procyanidins from grape seeds enhance the impact of fluorouracil chemotherapy on caco human colon cancer cells plos one e98921 gao y xiao x zhang c yu w guo w zhang z li z feng x hao j zhang k melatonin synergizes the chemotherapeutic effect of fluorouracil in colon cancer by suppressing pi3kakt and nfbinos signaling pathways j pineal res doi 101111jpi12380 wang sf wu my cai cz li m and lu jh autophagy modulators from traditional chinese medicine mechanisms and therapeutic potentials for cancer and neurodegenerative diseases j ethnopharmacol ernst e traditional chinese medicine for cancer br j cancer sun hy lee jh han ys yoon ym yun cw kim jh song ys and lee sh pivotal roles of ginsenoside rg3 in tumor apoptosis through regulation of reactive oxygen species anticancer res tang yc zhang y zhou j zhi q wu my gong fr shen m liu l tao m shen b ginsenoside rg3 targets cancer stem cells and tumor angiogenesis to inhibit colorectal cancer progression in vivo int j oncol wang j tian l khan mn zhang l chen q zhao y yan q fu l and liu j ginsenoside rg3 sensitizes hypoxic lung cancer cells to cisplatin via blocking of nfκb mediated epithelialmesenchymal transition and sternness cancer lett joo e ha yw and kim ys abstract lb23 molecular mechanisms of ginsenoside rg3 related to apoptosis in human lung and pancreatic adenocarcinomas cancer res lb23 kim bj nah sy jeon jh so i and kim sj transient receptor potential melastatin channels are involved in ginsenoside rg3induced apoptosis in gastric cancer cells basic clin pharmacol kim sm lee sy cho js son sm choi ss yun yp yoo hs yoon dy oh kw han sb and hong jt combination of ginsenoside rg3 with docetaxel enhances the susceptibility of prostate cancer cells via inhibition of nfkappa b eur j pharmacol yuan hd quan hy zhang y kim sh and chung sh 20sginsenoside rg3induced apoptosis in ht29 colon cancer cells is associated with ampk signaling pathway mol med rep liu tg huang y cui dd huang xb mao sh ji ll song hb and yi c inhibitory effect of ginsenoside rg3 combined with gemcitabine on angiogenesis and growth of lung cancer in mice bmc cancer sun my ye y xiao l duan xy zhang ym and zhang h anticancer effects of ginsenoside rg3 review int j mol med longley db harkin dp and johnston pg fluorouracil mechanisms of action and clinical strategies nat rev cancer hokmabady l raissi h and khanmohammadi a interactions of the fluorouracil anticancer drug with dna pyrimidine bases a detailed computational approach struct chem rateesh s luis sa luis cr hughes b and nicolae m myocardial infarction secondary to fluorouracil not an absolute contraindication to rechallenge int j cardiol e331e333 shan x aziz f tian ll wang xq yan q and liu jw ginsenoside rg3induced egfrmapk pathway deactivation inhibits melanoma cell proliferation by decreasing fut4ley expression int j oncol luo y zhang p zeng hq lou sf and wang dx ginsenoside rg3 induces apoptosis in human multiple myeloma cells via the activation of bcl2associated x protein mol med rep cardone mh roy n stennicke hr salvesen gs franke tf stanbridge e frisch s and reed jc regulation of cell death protease caspase by phosphorylation science 0chong effects of ginsenoside rg3 on colon can | Colon_Cancer |
gon§ales junior w effect of treatment with injectable progesterone at the onset of super precocious tairessincronization protocol on tai pregnancy rates of nellore heifers in animal reproduction for the annual meeting of the brazilian embryo technology society sbte august florianopolis brazil animal reproduction suppl pugliesi g a novel strategy for resynchronization of ovulation in nelore cows using injectable progesterone p4 and p4 releasing devices to perform two timed inseminations within days reprod domest anim 101111rda13475 pugliesi g use of doppler ultrasonography in embryo transfer programs feasibility and field results anim reprod 1021451 19843143ar201 siqueira l g b color doppler flow imaging for the early detection of nonpregnant cattle at days after timed artificial insemination j dairy sci 103168jds20136814 pugliesi g efficacy of doppler ultrasonography to detect nonpregnant nelore cows and heifers submitted to three timedai in days in reproduction ed international ruminant reproduction symposiumirrs vol foz do igua§u brazil melo g d applied use of interferontau stimulated genes expression to detect pregnancy status in nelore cattle submitted to timedai anim reprod in the annual meeting of the brazilian embryo technology society sbte vol ilha de comandatuba zaied a a concentrations of progesterone in milk as a monitor of early pregnancy diagnosis in dairy cows theriogenology 1010160093691x7990052 mansouriattia n pivotal role for monocytesmacrophages and dendritic cells in maternal immune response to the developing embryo in cattle1 biol reprod 101095biolr eprod shirasuna k vascular and immune regulation of corpus luteum development maintenance and regression in the cow domest anim endocrinol 101016jdoman iend201203007 toji n evaluation of interferonstimulated genes in peripheral blood granulocytes as sensitive responders to bovine early conceptus signals vet j 101016jtvjl201710007 binelli m bovine interferontau stimulates the janus kinasesignal transducer and activator of transcription pathway in bovine endometrial epithelial cells biol reprod 101095biolr eprod spencer t e forde n lonergan p insights into conceptus elongation and establishment of pregnancy in ruminants reprod fertil dev 101071rd163 schmaltzpanneau b early bovine embryos regulate oviduct epithelial cell gene expression during in a0vitro coculture anim reprod sci 101016janire prosc i201406022 xu b pan q h liang c role of mxb in alpha interferonmediated inhibition of hiv1 infection j virol 101128jvi00422 forde n conceptusinduced changes in the endometrial transcriptome how soon does the cow know she is pregnant biol reprod 101095biolr eprod bazer f w spencer t e johnson g a interferons and uterine receptivity semin reprod med 101055s002811080 forde n lonergan p transcriptomic analysis of the bovine endometrium what is required to establish uterine receptivity to implantation in cattle j reprod develop 101262jrd2011021 sanchez j m mathew d j passaro c fair t lonergan p embryonic maternal interaction in cattle and its relationship with fertility reprod domest anim 53suppl 101111rda13297 moir s decreased survival of b cells of hivviremic patients mediated by altered expression of receptors of the tnf superfamily j exp med 101084jem20032 sedy j bekiaris v ware c f tumor necrosis factor superfamily in innate immunity and inflammation cold spring harb persp biol 101101cshpe rspec ta0162 groebner a e enhanced proapoptotic gene expression of xaf1 casp8 and tnfsf10 in the bovine endometrium during early pregnancy is not correlated with augmented apoptosis placenta 101016jplace nta200912017 bauersachs s mitko k ulbrich s e blum h wolf e transcriptome studies of bovine endometrium reveal molecular profiles characteristic for specific stages of estrous cycle and early pregnancy exp clin endocrinol diabetes 101055s200810767 bauersachs s embryoinduced transcriptome changes in bovine endometrium reveal speciesspecific and common molecular markers of uterine receptivity reproduction 101530rep100996 okumu l a endometrial expression of progesteroneinduced blocking factor and galectins1 and binding protein in the luteal phase and early pregnancy in cattle physiol gen 101152physi olgen omics scientific reports 101038s41598020706168vol0123456789wwwnaturecomscientificreports 0c van baarsen l g m regulation of ifn response gene activity during infliximab treatment in rheumatoid arthritis is associated with clinical response to treatment arthritis res therapy 101186ar291 ullrich a the secreted tumorassociated antigen 90k is a potent immune stimulator j biol chem hasegawa m altered expression of dermokine in skin disorders j eur acad dermatol venereol 101111j14683083201204598 x toulza e the human dermokine gene description of novel isoforms with different tissuespecific expression and subcellular location j invest dermatol 101038sjjid57000 leclerc e a huchenq a kezic s serre g jonca n mice deficient for the epidermal dermokine beta and gamma isoforms display transient cornification defects j cell sci 101242jcs14480 miyamoto a shirasuna k sasahara k local regulation of corpus luteum development and regression in the cow impact of angiogenic and vasoactive factors domest anim endocrinol 101016jdoman iend200904005 tsai m h plscr2 a novel stat3interacting protein negatively regulates type i ifnmediated antiviral response allergy revelo x s waldron m r in a0vitro effects of escherichia coli lipopolysaccharide on the function and gene expression of neutrophils isolated from the blood of dairy cows j dairy sci 103168jds20114616 holm d e thompson p n irons p c the value of reproductive tract scoring as a predictor of fertility and production outcomes in beef heifers j anim sci 102527jas20081579 jiemtaweeboon s evidence that polymorphonuclear neutrophils infiltrate into the developing corpus luteum and promote angiogenesis with interleukin8 in the cow reprod biol endocrinol 10118614777827979 rocha c c ultrasonographyaccessed luteal size endpoint that most closely associates with circulating progesterone during the estrous cycle and early pregnancy in beef cows anim reprod sci 101016janire prosc i201812003 ginther o j colordoppler ultrasonography schurch n j how many biological replicates are needed in an rnaseq experiment and which differential expression tool should you use rna 101261rna05395 dobin a star ultrafast universal rnaseq aligner bioinform oxf engl 101093bioin forma ticsbts63 robinson m d mccarthy d j smyth g k edger a bioconductor package for differential expression analysis of digital gene expression data bioinformatics 101093bioin forma ticsbtp61 gentleman r c bioconductor open software development for computational biology and bioinformatics genom biol robinson m d oshlack a a scaling normalization method for differential expression analysis of rnaseq data genom biol 101186gb2004510r80 r25 101186gb2010113r25 robinson m d smyth g k smallsample estimation of negative binomial dispersion with applications to sage data biostatistics 101093biost atist icskxm03 benjamini y hochberg y controlling the false discovery rate a practical and powerful approach to multiple testing j r stat soc ser b methodol 101111j251761611995tb020 31x huang d w sherman b t lempicki r a systematic and integrative analysis of large gene lists using david bioinformatics resources nat prot 101038nprot andersen c l jensen j l orntoft t f normalization of realtime quantitative reverse transcriptionpcr data a modelbased variance estimation approach to identify genes suited for normalization applied to bladder and colon cancer data sets cancer res 10115800085472can040496 pfaffl m w a new mathematical model for relative quantification in realtime rtpcr nucl acids res 101093nar299e45 acknowledgementsthis study was funded by the s£o paulo research foundation fapesp the authors would like to thank all students from the molecular endocrinology physiology laboratory lfemfmvzusp project 03gdcr17 we thank the administration of pirassununga campus of the university of s£o paulo the laboratory of animal biotechnology of the esalqusp and ourofino animal health and alta genetics brazil for donating the hormones and semen respectivelyauthors contributionsccr conceived the study performed pcr analyses and wrote the manuscript scdsa performed the bioinformatics analysis gddm assisted with sample processing igm performed the ultrasonography evaluations llc contributed with the rnaseq analysis amgd assisted with data analyses and provide expertise in pcr data analysis mb contributed with critical review of the manuscript gp is the pi of the project provided the financial support expertise in experimental design statistical analysis and correct the manuscript all authors reviewed and approved the manuscriptcompeting interests the authors declare no competing interestsadditional informationsupplementary information is available for this paper at 101038s4159 correspondence and requests for materials should be addressed to gpreprints and permissions information is available at wwwnaturecomreprintspublishers note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliationsscientific reports 101038s41598020706168vol1234567890wwwnaturecomscientificreports 0copen access this article is licensed under a creative commons attribution international license which permits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the creative commons license and indicate if changes were made the images or other third party material in this article are included in the articles creative commons license unless indicated otherwise in a credit line to the material if material is not included in the articles creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtain permission directly from the copyright holder to view a copy of this license visit httpcreat iveco mmons licen sesby40 the authors scientific reports 101038s41598020706168vol0123456789wwwnaturecomscientificreports 0c' | Colon_Cancer |
s case reports duplicate studies and those with insufficient available data were excludeddata extraction and managementdata were independently extracted by two investigators yao xl and wu ww according to the same inclusionand exclusion criteria disagreements were adjudicated by a third reviewer qu kthe following data will be extracted from eligible literatures the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commons attributionlicense cc by 0cbioscience reports bsr20202509101042bsr20202509¢¢¢study characteristics name of the first author year of publication and sample size of included studiesparticipant characteristics tumor stage and age of patientsinterventions intervening methods and dosage administration route cycles and duration of treatment of huaiergranule¢ outcome and other data overall response rate orr disease control rate dcr overall survival osdiseasefree survival dfs quality of life qol immune indexes [cd3 cd4 cd8 natural killer cellsnk percentage and cd4cd8 cell ratios] and adverse effects we will attempt to contact the authors to request the missing or incomplete data if those relevant data are notacquired they will be excluded from the analysisquality assessmentto ensure the quality of the metaanalysis the quality of the included randomized and nonrandomized controlledtrials was evaluated according to the cochrane handbook tool and methodological index for nonrandomizedstudies minrrs supplementary table s2 respectively types of outcome measuresmain outcomesthe primary outcomes in present analysis included shortterm and longterm clinical efficacy and adverse effectsaccording to response evaluation criteria in solid tumors recist criteria i shortterm clinical efficacy the shortterm tumor response included orr and dcr orr was defined as thesum of complete and partial response rates and dcr was defined as the sum of complete response partialresponse and stable disease ratesii longterm clinical efficacy year os the time from the date of randomization to death from any cause year dfs the time from date of random assignment to date of recurrence or deathiii adverse events gastrointestinal adverse effects myelosuppression and hepatotoxicity secondary outcomesi qol qol was evaluated using the qualityoflife improved rate qir and karnofsky score kpsii immune function indicators the immune function of breast cancer patients was assessed in terms of cd3cd4 cd8 nk cells percentage and cd4cd8 cell ratiosstatistical analysisstata stata corp college station tx usa and review manager nordic cochran centre copenhagendenmark statistical software were used for statistical analyses cochranes q test and i2 statistics were used to assessheterogeneity among the studies if p01 or i2 a fixed effects model was used for the metaanalysisotherwise a random effects model was used the mantelhaenszel method will be applied for pooling of dichotomous data and results will be presented as risk ratio rr with their confidence intervals cis inverse variancemethod will be used for pooling of continuous data and results will be presented as standardized mean differencesmd with their cis a twotailed p005 was considered statistically significantthe presence of publication bias was investigated using the funnel plots beggs and eggers test if or more studiesare included in the metaanalysis [] if publication bias existed a trimandfill method should be applied tocoordinate the estimates from unpublished studies and the adjusted results were compared with the original pooledrr sensitivity analysis was performed to explore an individual studys influence on the pooled results by deleting onesingle study each time from pooled analysisresultssearch resultsthe initial search retrieved a total of s of which were excluded due to duplication after title and review s were further excluded because they were noncomparative clinical trials n19 were the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commons attributionlicense cc by 0cbioscience reports bsr20202509101042bsr20202509figure study selection process for the metaanalysisnot related to huaier granule n9 were nonpeer reviewed s n8 were literature review or metaanalysisn3 and were case report and series n5 leaving studies as potentially eligible after detailed assessment offull texts studies with breast cancer patients n5 trials with insufficient data n8 and inappropriate criteriafor the experimental or control groups n11 were excluded ultimately trials [] involving patients with breast cancer were included in the final analysis figure patient characteristicsall included studies were performed in different medical centers in china in total patients with breast cancerwere treated using conventional methods in combination with huaier granule while patients were treated usingconventional methods alone huaier granule was manufactured by qidong gaitianli pharmaceutical co ltd andgranted a manufacturing approval number issued by the chinese sfda z20000109 study and patient characteristics are summarized in table quality assessmentquality assessment of the risk of bias is shown in figure and table the results revealed that the literature retrievedfor the present study was of medium and high quality the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commons attributionlicense cc by 0cbioscience reports bsr20202509101042bsr20202509table clinical information from the eligible trials in the metaanalysisincluded studieschen qj chen y dai yg guo fd han sj lei ss liang yq li zh lu mq2017lu y qun sx ren xb shan cy tan zd tang y wang mh wang w wu yb xiong y xu f yang z2017yin x iiiiiiiviiiiinotprovidediiiiiiiiiviiiiiiviivnotprovidediiiiiiiiiiiiiiiiiiiiiiiiiviiiiiiiiiiiinotprovidedtumorstagepatientsconexpage year control vsexperimentalintervening methods range median ct vs cthuaier granules vs ct vs cthuaier granulesoa meanoadosage ofhuaiergranulesduration oftreatments20gtime times monthcourse daycourses20gtime times weeks for a course daycourses months vs range vs vs meanmeanct vs cthuaier granules20gtime timesoadayct vs cthuaier granules20gtime times monthoadayct vs cthuaier granules20gtime times weeks for a course oadaycourses yearsnot providedct vs cthuaier granules20gtime timesoaday vs range vs vs meanmeanct vs cthuaier granules20gtime times monthsoadayct vs cthuaier granules20gtime times months for a course oadaycoursesct vs cthuaier granules20gtime times weeks for a course oadaycourses vs medianct vs cthuaier granules20gtime times weeks for a course not providedct vs cthuaier granules20gtime timesoadaycourses years mean vs vs vs meanmeanoadayct vs cthuaier granules20gtime times weeksoadayct vs cthuaier granules20gtime times weeks for a course oadayct vs cthuaier granules20gtime timesoadaycoursesnot providednot providedct vs cthuaier granules20gtime times monthoadaynot providedct vs cthuaier granules20gtime times months vs meanoadayct vs cthuaier granules20gtime times monthsoaday range mean ct vs cthuaier granules20gtime times monthsoaday vs rangect vs cthuaier granules20gtime times weeks for a course vs range vs vs meanmeanoadayct vs cthuaier granules20gtime timesoadaycoursesnot providedct vs cthuaier granules20gtime times weeks for a course oadayct vs cthuaier granules20gtime timesoadaycourses monthparametertypes1cid2 3cid21cid2 3cid25cid22cid22cid2 3cid25cid24cid2 5cid22cid2 3cid25cid22cid24cid2 5cid23cid2 4cid25cid24cid2 5cid21cid2 2cid24cid22cid2 3cid22cid2 3cid24cid25cid22cid23cid22cid22cid2 5cid25cid24cid2 5cid22cid22cid22cid21cid2 5cid22cid22cid2 3cid2zhang jg iiii vs rangect vs cthuaier granules20gtime times monthsoadayzhang y notprovidedzhao zw iiiiv vs range vs meanct vs cthuaier granules20gtime times monthsoadayct vs cthuaier granules20gtime times weeksoadayzhong sw zhou p iviiii74range median ct vs cthuaier granules20gtime times monthsoaday vs rangect vs cthuaier granules20gtime times monthsoadaynotes control group conventional treatments alone group experimental group conventional treatments and huaier granule combined group1cid2 overall response rate and disease control rate 2cid2 overall survival or diseasefree survival 3cid2 adverse events 4cid2 quality of life 5cid2 immunefunction indexabbreviations ct conventional treatments oa oral administration the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commons attributionlicense cc by 0cbioscience reports bsr20202509101042bsr20202509figure risk of bias summaryreview of authors judgments about each risk of bias item for included studies note each color represents a different level ofbias red for highrisk green for lowrisk and yellow for unclearrisk of biastable quality assessment of nonrandomized comparative studiesnonrandomized studiesadditional criteria in comparativestudytotalabcdefghijklstudyguo fdhan sjlei ssren xbwu ybyin xzhang yzhongsw zhou pa a clearly stated aim b inclusion of consecutive patients c prospective collection of data d endpoints appropriate to the aim of the study eunbiased assessment of the study endpoint f followup period appropriate to the aim of the study g loss to follow up less than h prospectivecalculation of the study size i an adequate control group j contemporary groups k baseline equivalence of groups l adequate statistical analysesnotes the items are scored not reported reported but inadequate and reported and adequatetherapeutic efï¬cacy assessmentsorr and dcrfour clinical trials involving patients compared orr and dcr between the two groups as shown in figure the pooled results revealed that patients who underwent combination therapy experienced improved orr rr ci p002 and dcr rr ci p019 compared with those whoreceived conventional treatments alone although the dcr did not reach significant difference dcr p095 i2 was not heterogeneous among the studies therefore a fixedeffect model was used to analyze rr otherwise arandomeffect model was used the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commons attributionlicense cc by 0cbioscience reports bsr20202509101042bsr20202509figure comparisons of orr and dcr between experimental and control groupforest plot of the comparison of orr a and dcr b between the experimental and control group control group conventionaltreatment alone group experimental group conventional treatment and huaier granule combined grouplongterm survival1year 2year 3year and 5year oseleven clinical trials with breast cancer patients reported os figure metaanalysis revealed that the 2yearrr ci p002 3year rr ci p00001 and 5year os rr ci p0004 of patients in the combined treatment group were significantly prolongedcompared with the control group there was statistical heterogeneity in 1year os p009 i2 and 2year osp00001 i2 according to the heterogeneity test therefore a randomeffect model was used to pool thismetaanalysis otherwise the fixedeffect model was used1year 2year 3year and 5year dfsten clinical trials with breast cancer patients reported dfs figure metaanalysis revealed that the 1yearrr ci p0003 2year rr ci p000001 3year rr ci p000001 and 5year dfs rr ci p003 of patients in thecombined treatment group were all significantly prolonged compared with the control group there was statisticalheterogeneity in 5year dfs p005 i2 according to the heterogeneity test therefore a random effectsmodel was used to pool this metaanalysis otherwise the fixedeffect model was usedqol assessmentfour trials with participants evaluated qir and three trials including patients reported kps data figure results demonstrated that the qol of breast cancer patients in the combined group was significantly better than thatof the control group indicated by significantly increased qir rr ci p000001 and kpsrr ci p000001 qir p084 i2 was not heterogeneous among the studiestherefore a fixedeffect model was used to analyze rr otherwise a randomeffect model was usedimmune function evaluationimmune status of the patients was examined between the two groups in eleven controlled studies including patients figure the percentages of cd3 cd3 rr ci p005 cd4 rr ci p000001 and nk cells rr ci p00001 and cd4cd8 ratio rr ci p000001 in the combined treatment group were significantly increased compared with the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commons attributionlicense cc by 0cbioscience reports bsr20202509101042bsr20202509figure comparisons of os between experimental and control groupforest plot of the comparison of 1year a 2year b 3year c and 5year os d between the experimental and control groupcontrol group conventional treatment alone group experimental group conventional treatment and huaier granule combinedgroup the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commons attributionlicense cc by 0cbioscience reports bsr20202509101042bsr20202509figure comparisons of dfs between experimental and control groupforest plot of the comparison of 1year a 2year b 3year c and 5year dfs d between the experimental and control groupcontrol group conventional treatment alone group experimental group conventional treatment and huaier granule combinedgroup the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commons attributionlicense cc by 0cbioscience reports bsr20202509101042bsr20202509figure comparisons of qol between experimental and control groupforest plot of the comparison of qir a and kps b between the experimental and control group control group conventionaltreatment alone group experimental group conventional treatment and huaier granule combined groupthose in the conventional treatment alone group whereas the proportions of cd8 rr ci to p033 did not differ significantly between the two groups a randomeffect model was used to pool thismetaanalysis due to significant heterogeneityassessment of adverse eventsas shown in figure patients treated with huaier granule and conventional methods exhibited lower incidences ofmyelosuppression rr ci p0001 and hepatotoxicity rr ci p005 whereas analysis of gastrointestinal adverse effects rr ci p014 leukopeniarr ci p006 nausea and vomiting rr ci p052 andalopecia rr ci p020 did not differ significantly between the two groups there wasstatistical heterogeneity in gastrointestinal adverse effects p006 i2 according to the heterogeneity testand a random effects model was used to pool this metaanalysis otherwise the fixedeffect model was usedpublication biasas shown in figure the funnel plots beggs and eggers regression tests results showed that there was publicationbias in cd4cd8 ratio begg egger to determine whether bias affected the pooled risk ofcd4cd8 ratio a trimandfill analysis was performed the adjusted rr indicated a trend similar to the resultsof the primary analysis before p00001 after p00001 reflecting the reliability of the primary conclusionsparameters discussed less than papers were not conducted publication bias analysessensitivity analysisas figure signified the results revealed that no individual studies significantly affected the primary indicatorscd4 and cd4cd8 ratio which indicated statistically robust results parameters discussed less than paperswere not conducted sensitivity analysesdiscussionhuaier granule the active ingredient of huaier extract appears as a lightyellow powder through hotwater extractionethanol precipitation deproteinization and lyophilization procedures as a type of tcbp huaier granule hasbeen clinically applied as an effective adjuvant drug in cancer treatment for decades although several studies havereported that addition of huaier granule could be beneficial to patients with advanced breast cancer but the the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commons attributionlicense cc by 0cbioscience reports bsr20202509101042bsr20202509figure comparisons of immune function between experimental and control groupforest plot of the comparison of immune function indicators including cd3 a cd4 b cd8 c and nk d cells percentage andcd4cd8 ratio e between the experimental and control group control group conventional treatment alone group experimentalgroup conventional treatment and huaier granule combined group the random effects metaanalysis model inverse variancemethod was used the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commons attributionlicense cc by 0cbioscience reports bsr20202509101042bsr20202509figure comparisons of adverse effects between experimental and control groupforest plot of the comparison of adverse effects including gastrointestinal adverse effects a myelosuppression b hepatotoxicity c leukopenia d nausea and vomiting e and alopecia f between the experimental and control group control groupconventional treatment alone group experimental group conventional treatment and huaier granule combined group the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commons attributionlicense cc by 0cbioscience reports bsr20202509101042bsr20202509figure funnel plot of cd4 a and cd4cd8 bexact therapeutic effects have yet to be systematically evaluated thus indepth knowledge of the efficacy and safetyof huaier granule is needed this systematic review will provide a helpful evidence for clinicians to formulate thebest postoperative adjuvant treatment strategy for patients with breast cancer and also provide scientific clues forresearchers in this fielddata from trials [] including patients with breast cancer were included in our metaanalysishuaier granule in all of the included studies was manufactured by qidong gaitianli pharmaceutical co ltd thedosages of huaier granule were g per day via oral administration the pooled results revealed that the combinationof huaier granule and conventional treatment for breast cancer achieved more beneficial effects compared withthose treated solely with conventional therapy compared with conventional treatment alone huaier granule couldsignificantly improve orr and qol in patients with breast cancer p005 the study also assessed whether huaiergranule could prolong the longterm survival rates of breast cancer patients and the results showed that the and 5year os and and 5year dfs of patients were all significantly prolonged compared with the controlgroup these results indicated that using huaier granule could improve the short and longterm curative effects ofconventional treatment for breast cancert lymphocyte subsets cd3 cd4 cd8 cell subsets and cd4cd8 ratio and nk cells play an importantrole in antitumor immunity studies have shown that patients with advanced cancer showed decreased immunefunction and nk activity and exhibiting imbalance of t lymphocytes percentage many studies have reportedthat huaier granule can enhance the ability of the bodys immunity and resistance to tumors our analysisdemonstrated that the percentages of cd3 cd4 and nk cells and cd4cd8 ratio were all significantly increasedin breast cancer patients treated with huaier granule indicating that immune function of breast cancer patients wasimproved after huaier granule adjuvant therapysafety is the top priority of clinical treatment seven clinical trials with breast cancer patients reported adverse events according to world health anization standards metaanalysis revealed that patients who underwent huaier granule plus conventional treatment demonstrated a lower risk for myelosuppression and hepatotoxicitycompared with conventional treatment alone whereas analysis of other toxic side effects did not differ significantlytherefore huaier granule appears to be a safe auxiliary antitumor medicine for individuals with breast cancerthere were some limitations to our analysis currently five clinical trials table in which breast cancer are being treated by huaier granule in conjunction with conventional regimens have been registered onclinicaltrialsgov nct02615457 and nct02627248 and chinese clinical trial register chictr1800015390chictroic16007737 and chictrtrc11001250 however except for two studies most of the includedtrials were not registered before the first participant enrolled second as an important chinese patent medicinehuaier granule was mainly applied in china which may bring an unavoidable regional bias and subsequently influence the clinical application of huaier granule worldwide third different trials evaluated the treatment efficacy withdifferent outcomes resulting in a reduction in the size of the statistical sample making it difficult to summarize theresults at the same scale fourth several results demonstrated significant heterogeneity among the included trialswhich may be due to the different tumor stage tumor subtypes ages of the breast cancer patients and duration oftreatment however based on the currently available literature there are insufficient data to perform more statistical analysis to evaluate correlations in addition the efficacy of monotherapy of huaier granule in the treatment of the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commons attributionlicense cc by 0cbioscience reports bsr20202509101042bsr20202509figure sensitivity analysis for cd4 a and cd4cd8 b the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commons attributionlicense cc by 0cbioscience reports bsr20202509101042bsr20202509table search results of clinical trial registrationregistrationnumbernct02615457nct02627248chictr1800015390chictroic16007737chictrtrc11001250titlephaseconditionsinterventionslocationshuaier granule intreating women withtriple negative breastcancerneoadjuvantchemotherapy with orwithout huaiergranule in treatingwomen with locallyadvanced breastcancer that can beremoved by surgeryhuaier granule forstage ii and iii triplenegative breastcancer with lymphnode metastasis amulticenterrandomizeddoubleblindplacebocontrolledclinical triala multicenter doubleblind randomizedplacebocontrolledstudy on stage iiiiitriplenegative breastcancer with lymphnode metastasistreated by huaiergranulesextract of fungi ofhuaier used for triplenegtive breastcancera prospectiverandomized controlledtrialivivtriple negative breasthuaier granuleqilu hospital ofcancershandong universityjinan shandong chinabreast cancerhuaier granule otherqilu hospital ofchemotherapyshandong universityjinan shandong chinaivtriple negative breasthuaier granulecancerthe first aï¬iliatedhospital of amusouthwest hospitalchongqing chinaibreast cancerhuaier granuleivtriple negative breasthuaier granulecancersouthwest hospital thethird military medicaluniversity chongqingchinasouthwest hospital thethird military medicaluniversity chongqingchinabreast cancer also needs highquality evidence to verify however up to now huaier granule is mainly combinedwith radiotherapy chemotherapy or surgery and other conventional treatment methods for breast cancer we willkeep paying close attention to upcoming highquality clinical trials in our later studies and carry out further analyseson studies conducted huaier granule monotherapy against breast cancer finally publication bias was exists in someindicators which might because some authors tended to deliver positive results of s to editors therefore anyconclusions need to be made with cautionconclusionin summary findings of this metaanalysis indicate that the combination of huaier granule and conventional treatment is effective in treating patients with breast cancer the clinical application of huaier granule not only clearlyenhanced the therapeutic effects of conventional treatment but also effectively improved qol and immune functionin patients with breast cancer thus we anticipate that our study will provide valuable evidence for further evaluationof huaier granule on the other hand the low quality of some of the included publications increased the risk of biaswhich to some extent affects the reliability of this research therefore additional studies with highquality evidenceto verify the effectiveness of huaier granulemediated therapy for breast cancer are warrantedcompeting intereststhe authors declare that there are no competing interests associated with the manuscriptfundingthis study was supported by grants from national science foundation of china [grant numbers and ] the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commonsattribution license cc by 0cbioscience reports bsr20202509101042bsr20202509author contributionxi w and yao xl conceived and designed the methods yao xl and wu ww extracted the original data and drafted themanuscript yao xl wu ww and qu k performed statistical analysis xi w and yao xl interpreted results xi w and quk revised the manuscript all authors had full access to all data in the study and take responsibility for the integrity of the data andthe accuracy of data analysisabbreviationscbm chinese biological medicine database ci confidence interval cnki china national knowledge infrastructure csjdchinese scientific journal database dcr disease control rate dfs diseasefree survival kps karnofsky performance scoreminrrs methodological index for nonrandomized studies nk natural killer cells orr overall response rate os overallsurvival prisma preferred reporting items for systematic reviews and metaanalyses qir qualityoflife improved rate qolquality of life rct randomized controlled trial recist response evaluation criteria in solid tumors rr risk ratio sfdastate food and drug administration smd standardized mean difference tcbp traditional chinese biomedical preparationreferences ferlay j colombet m soerjomataram i mathers c parkin dm pi Ëneros m estimating the global cancer incidence and mortality in globocan sources and methods int j cancer 101002ijc31937 bray f ferlay j soerjomataram i siegel rl torre la and jemal a global cancer statistics globocan estimates of incidenceand mortality worldwide for cancers in countries ca cancer j clin 103322caac21492 watkins ej overview of breast cancer jaapa 10109701jaa0000580524957333d akram m iqbal m daniyal m and khan au awareness and current knowledge of breast cancer biol res 101186s4065901701409 reeder jg and vogel vg breast cancer prevention cancer treat res 1010079780387731612Ë waks ag and winer ep breast cancer treatment jama 101001jama201820751 peart o breast intervention and breast cancer treatment options radiol technol 535m558m quiz merino bonilla ja torres tabanera m and ros mendoza lh breast cancer in the 21st century from early detection to new therapiesradiologia 101016jrx201706003 greenlee h dupontreyes mj balneaves lg carlson le cohen mr deng g clinical practice guidelines on the evidencebaseduse of integrative therapies during and after breast cancer treatment ca cancer j clin 103322caac21397 wong ky tan ey chen jj teo c and chan pm the use of traditional chinese medicine among breast cancer patients implications forthe clinician ann acad med singapore zhu l li l li y wang j and wang q chinese herbal medicine as an adjunctive therapy for breast cancer a systematic review andmetaanalysis evid based complement alternat med wang w xu l and shen c effects of traditional chinese medicine in treatment of breast cancer patients after mastectomy ametaanalysis cell biochem biophys 101007s120130140348z mcpherson l cochrane s and zhu x current usage of traditional chinese medicine in the management of breast cancer a practitionersperspective integr cancer ther 1011771534735415607656 chen y wu h wang x wang c gan l zhu j huaier granule extract inhibit the proliferation and metastasis of lung cancer cellsthrough downregulation of mtdh jak2stat3 and mapk signaling pathways biomed pharmacother 101016jbiopha201802028 hu z yang a fan h wang y zhao y zha x huaier aqueous extract sensitizes cells to rapamycin and cisplatin through activatingmtor signaling j ethnopharmacol 101016jjep201603069 su d zhang x zhang l zhou j and zhang f a randomized doubleblind controlled clinical study on the curative effect of huaier onmildtomoderate psoriasis and an experimental study on the proliferation of hacat cells biomed res int 10115520182372895 wang m hu y hou l pan q tang p and jiang j a clinical study on the use of huaier granules in postsurgical treatment oftriplenegative breast cancer gland surg 1021037gs20191208 chen q shu c laurence ad chen y peng bg zhen zj effect of huaier granule on recurrence after curative resection of hcc amulticentre randomised clinical trial gut 101136gutjnl2018315983 zhang y wang x and chen t efï¬cacy of huaier granule in patients with breast cancer clin transl oncol 101007s1209401819594 zhao gs liu y zhang q li c zhang yw ren zz transarterial chemoembolization combined with huaier granule for thetreatment of primary hepatic carcinoma safety and efï¬cacy medicine baltimore e7589 101097md0000000000007589 zhou l pan lc zheng yg du gs fu xq zhu zd novel strategy of sirolimus plus thymalfasin and huaier granule on tumorrecurrence of hepatocellular carcinoma beyond the ucsf criteria following liver transplantation a single center experience oncol lett sun y sun t wang f zhang j li c chen x a polysaccharide from the fungi of huaier exhibits antitumor potential andimmunomodulatory effects carbohydr polym 101016jcarbpol201209006 the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commonsattribution license cc by 0cbioscience reports bsr20202509101042bsr20202509 xie hx xu zy tang jn du ya huang l yu pf effect of huaier on the proliferation and apoptosis of human gastric cancer cellsthrough modulation of the pi3kakt signaling pathway exp ther med 103892etm20152600 moher d liberati a tetzlaff j and altman dg preferred reporting items for systematic reviews and metaanalyses the prisma statementj clin epidemiol 101016jjclinepi200906005 zeng x zhang y kwong js zhang c li s sun f the methodological quality assessment tools for preclinical and clinical studiessystematic review and metaanalysis and clinical practice guideline a systematic review j evid based med 101111jebm12141 slim k nini e forestier d kwiatkowski f panis y and chipponi j methodological index for nonrandomized studies minorsdevelopment and validation of a new instrument anz j surg 101046j14452197200302748x schwartz lh litiere s de vries e ford r gwyther s mandrekar s recist 11update and clariï¬cation from the recistcommittee eur j cancer 101016jejca201603081 jackson d white ir and riley rd quantifying the impact of betweenstudy heterogeneity in multivariate met | Colon_Cancer |
"melatonin is a sleepregulating hormone created by the pineal glandand is released at night it has been found to have biological activityin almost all living anisms including plants animals and microbes itcan quickly enter cells through the bilipid bilayer and exhibit scavenging activity towards oxygen free radicals as well as antioxidant properties due to its low molecular weight and amphiphilic nature peripheral tissues have been found to show a high afï¬nity towardsthis hormone melatonin and hence act on receptors and bindingsites studies reported by tan show that melatonin can alsobe produced in the mitochondria and hence tissue melatonin levelsare more than that of serum levels hence can be used as a moleculethat targets mitochondria the main physicochemical and biologicalproperties of melatonin are sleepinducing effects antioxidant behavior [] antiammatory activity [] antiapoptotic effects and neuroprotective effects it also regulates various corresponding authoremail address renjithsbcollegeacin r thomas101016jmolliq2020114082 elsevier bv all rights reservedphysiological functions of the brain as melatonin can diffuse quicklythrough the bloodbrain barrier it is effectively used in the treatmentof brain injuries rapid eyemovement sleepbehavior disorder patients are managed by a combination treatment of melatoninand clonazepam [] the sensing of bacteria through tolllikereceptor4 and regulation of bacteria through altered goblet cells andantimicrobial peptides are all involved in the anticolitic effects of melatonin in ammatory bowel disease melatonin is involved in theaging process growth towards puberty and modulation of blood pressure this versatile compound blocks proangiogenic andantiangiogenic effects caused by docetaxel and vinorelbine which areantitumor drugs and it enhances their tumorï¬ghting behavior this molecule can modify the redox state of the rat pancreaticstellate cellmelatonin is an endogenous hormone that is involved in circadianrhythm control it is inexpensive and safe as it has a significant effectas an antioxidant and antiammatory melatonin chemically nacetyl5methoxytryptamine is a tryptophan derivative has multiplephysiological effects and can be used to treat many diseases related tovirus infections especially respiratory diseases in covid19 patientswith digestive complications melatonin has positive effects 0cn alzaqri of molecular liquids melatonin can thus be used as an adjunctive or even as a regular therapyas no antiviral treatment is currently available electrochemical measurements of melatonin overï¬ow demonstrate that melatonin secretiondecreases with age melatonin treatments result in the enhancement of essential oil production in salvia species in the context of the recent covid pandemic melatonin can beresearched as a potential molecule to control the dangerous effect ofthis disease rising patients' tolerance and decreasing the mortality infatal virus infections would control the innate immune response and reduce ammation during this period melatonin is a molecule with respective properties as it decreases the overreaction of the innateimmune response and overshoots ammation but also facilitatesadaptive immune function even though melatonin is a critical biomolecule few works havebeen reported on the electronic structure and reactivity of this moleculeexcept for a preliminary work reported by turjanski and coworkers in using semiempirical methods in this manuscript we describe a detailed investigation into the quantum mechanical propertiesof melatonin its spectral features reactivity preferences and the resultsof docking studies with three known structural protein receptors of thenovel coronavirus2 we found that melatonin docks strongly with thethree proteins we hypothesise that this compound can be used as anadjuvant medicine for the treatment of covid19 also significant restby a person peacefully sleeping in dark surroundings will enhance theproduction of this hormone which could help in the management ofcurrent patients or as a preventive measure in the vulnerablepopulation material and methodsthe melatonin molecule was optimised using the gaussian09 software package with the dftb3lyp functional and the 6311g 2dp basis set b3lyp is a commonly used functional and 63112dpbasis set is mediumsized basis set with diffused functions over heavyatoms and polarization functions to bring accuracy we performed frequency calculations to ensure that no imaginary frequency exists suchthat the geometry determined would correspond to a global minimumfor reaching the optimised geometry we used the same geometry for calculating frontier molecular analysis natural bonding orbitals and nonlinear optical studies for uvvisible spectrum simulation we usedtimedependent density functional theory tddft with longrangecorrected camb3lyp functionals with 6311g 2dp as the basis setbecause electronic transitions are timedependent phenomena tddftcalculations are done using the optimised geometry obtained fromb3lyp6311g2dp simulations the frontier molecular orbitals wereviewed from the checkpoint ï¬le generated during the optimisation calculations a wavefunction ï¬le was generated during a single point groundstate calculation job using which the subsequent analysis performedthe melatonin molecule has more than two reaction sites for examplemethoxy carbonylamide and purine ring reaction sites of melatonincalculated using the multiwavefunction software for calculatingtotal electrostatic potential average localised ionization energies and noncovalent interactions as it is reported that melatonincan be used as an adjuvant therapeutic material to ï¬ght covid19 we decided to dock the molecule with three ncov2019 protein's rcsb site melatonin is effective in critical care patients by decreasing vessel permeability anxiety use of sedation and increasing the quality ofsleep which may also be beneï¬cial to covid19 patients for improvedclinical outcome melatonin especially has a high health proï¬le significant data indicate that melatonin reduces virusrelated diseases and willpossibly also be effective in patients with covid19 the target proteinswere downloaded cleaned removed alien atoms and molecules andthen used for docking the energy received from the swissdock software and the score values received from patchdock as well as thedocked results collected from biodiscovery studio software arepresented results and discussion geometry of melatoninthe geometry of the molecule explains its rigid structure thestructure of melatonin can be explained based on its physical parametersof bond lengths and bond angles between important atoms or groups asshown in fig the bond lengths of and arebonds of 1c14n 7c14n and 12h14n respectively and the corresponding bond angles are1090343° ° and ° for 1c14n7c 1c14n12h and 7c14n12h respectively the bond angle of° for 4c25o26c having bond lengths of and for 4c\\\\25o and 25o\\\\26c respectively the bond lengths of 18c21n21n22h 21n23c 23c\\\\24o and 23c\\\\30c are and respectively with the corresponding bondangles of ° ° ° ° ° and° for 18c21c22h 18c21c23c 22h22n23c 21n23c24o21n23c30c and 24o23c30c respectively frontier molecular orbital fmo properties of melatoninthe frontier molecular orbitals are highly reactive orbitals of othermolecules and some chemical descriptors are shown in table the higher occupied molecular orbital homo lower unoccupied molecular orbital lumo and energy gap of melatonin are and kcalmol respectively the energygap is significant indicating that the molecule is inherently stable theionization energy electron afï¬nity hardness softness chemical potential electronegativity electrophilicity index and nucleophilicity indexof melatonin are and kcalmol respectively interactionof the melatonin with the biological target can be explained by the softness value the softness value is high kcalmol indicatingthat the molecule can positively interact with biological systems andshow the desired effect electron transition study and excitedstate properties of melatonin insolutionthe electron transition study explains electrontransfer excitedstates we used the tddft formalism using camb3lyp functionalsand 6311g 2dp basis sets in an implicit solvation atmosphere ofmethanol using the iefpcm model as transmission occurs some energyis also emitted melatonin electron transitions to homo having a pyrrole ring and oxygen in methoxy homo1 over the pyrrole ring andethyl carbons and homo2 over acetamide oxygen nitrogen andethyl carbons with energies of and ev respectively melatonin electron transitions to lumo which is over the pyrrolering lumo which is over the pyrrole ring oxygen in methoxyethyl carbons and acetamide nitrogen and carbon and lumo2 having acetamide carbons acetamide nitrogen and carbons with energiesof and ev respectively the electronic spectral datausing tddft simulations indicate a significant λmax of nm in amethanol solvent the transitions are due to the movement of electronsfrom homo1 to lumo and homo to lumo with an oscillator strength of the electronic transitions are due to chargetransfer transitions from one region of the molecule to another whichindicates its inherent stability due to electronic excitations nonlinear optical behavior of melatoninscientists and technologists working in the molecular electronics ï¬eldare continuously searching for compounds with substantial nonlinearoptical nlo activity such compounds ï¬nd immense application in electronic displays surveillance equipment and consumer electronic gadgetscomputationally the ability of a molecule to act as an nlo material can be 0cn alzaqri of molecular liquids fig geometry of melatonindetermined from the polarizability and hyperpolarizability data []the nlo properties of melatonin are shown in table this is an essentialbehavior of melatonin that has a light absorption nature movement ofelectrons or protons as compared with a standard nlo material such asurea the dipole moment of melatonin is d which is times greater than urea hyperpolarizability mean polarizabilityand anisotropy of the polarizability of melatonin are and esu and which are and times greater than urea respectively the compound is not centrosymmetric hence generates secondorder spherical harmonics and betahyperpolarizability functions this compound can hence be used as an anic nonlinear optically active substance in anic electronicappliances nature of nbo study of melatonina molecule especially one with profound biological activity may havemany intramolecular electron delocalisation and hyperconjugativestabilisation regions natural bond orbital analysis which is a quantummechanical method is useful for this type of study the molecular orbitaltable frontier molecular orbital properties for melatoninchemical descriptorshomolumoionization energy i Éhomo homoelectron afï¬nity a Élumo lumoenergy gap homo lumoglobal hardness η i a global softness s ηchemical potential μ i a electronegativity Ï Î¼electrophilicity index Ï Î¼2 2ηnucleophilicity index n Ïenergy in kcalmolproperties of melatonin for the occupancy of the natural orbitals wereperformed by the nbo suite embedded in the gaussian softwarefrom donorbonding orbital Ï c1c2 with occupancy is toacceptor antibonding orbitals Ï c3c4 Ï c5c6 and Ï c7c8exhibiting the transition the energies are and kcalmol respectively from Ï c3c4 with occupancy is to Ï c5c6 and the rydberg orbital r c30 with the energies are and kcalmol respectively from Ï c5c6 having an occupancy is to Ï c3c4 r and r h33 with the energies are and kcalmol respectively from Ï n21c23 has occupancy to rydberg orbital r c30 and r h33 with the energies are and kcalmol respectively from Ï c23o24 having the occupancy is to Ï c1c2 Ï c2c3 r c23 and r h33 withthe energies are and kcalmol respectivelyfrom Ï c23c30 has occupancy is to r h17 r c18 rh19 r c23 r o24 r c30 r h33 Ï c1c2 Ï c1c6Ï c2c3 Ï c26h27 Ï c30h31 Ï c30h32 and Ï c30h33 having the energies are and kcalmol respectively from Ï o25c26 having occupancy is to r c30 andÏ c1c2 with the energies are and kcalmol respectivelyfrom Ï c26h27 to Ï c1c2 having the energy kcalmol withthe occupancy is from Ï c30h31 with the occupancy is to r h17 r c18 r h19 r c23 r o24 r c30 rh33 Ï c1c2 Ï c2c3 Ï c23o24 Ï c30h31 and Ïc30h32 having the energies are and kcalmol respectively from Ï c30h32 with occupancy is to r h17 rc18 r h19 r c23 r o24 r c30 r h33 Ï c1c2 Ïc2c3 Ï c30h32 and Ï c30h33 having the energies are and kcalmol respectively and from Ï c30h33 with the occupancy is to r h17 r c18 r c19 r c23 r c24 ro25 r c26 r h28 r c30 r h33 Ï c1c2 Ï c1c6Ï c2c3 Ï c2c4 Ï c18h19 Ï c23o24 Ï c26h27 Ï 0cn alzaqri of molecular liquids table nonlinear optics property for melatoninnonlinear propertydipole moment μhyperpolarizability βmean polarizability α0anisotropy of the polarizability δαmelatonin d esu esu esuurea d esu esu esucomparison of melatonin with urea times greater than urea times greater than urea times greater than urea times greater than ureac30c31 Ï c30h32 and Ï c30h33 having the energies are and kcalmol respectivelyfrom core bonding orbital c c23 which has the occupancy electrons move to antibonding r c23 r c30 and r h33 withthe transition energies are and kcalmol respectively from c c23 with the occupancy is to r h33 Ï c2c8 and Ï c26h28 having the energies are and kcalmol respectively from c o24 to Ï c26h29 has the energy is kcalmol with occupancy is and from c c30 having the occupancy is to r h17 r c23 r c30 r h33 Ïc2c8 Ï c26h28 Ï c30h31 and Ï c30h32 with the energies are and kcalmol respectively from lone pair orbital n n14 with the occupancy is to Ï c7c8 with the energy kcalmol from nn21 has the occupancy is to Ï c23o24 having the energy kcalmol and from n o24 having the occupancy is to rc23 r c30 r h33 Ï c1c2 and Ï c2c3 with the energiesare and kcalmol respectively fromantibonding orbital Ï c1c2 having the occupancy to rc30 and Ï c2c3 with the energies are and kcalmolrespectively from Ï c5c6 has occupancy is to r c30 withthe energy is kcalmol and from Ï c23o24 has occupancy is to r c30 with the energy is kcalmol the inherentstabilisation offrom the series ofhyperconjugative interactions presented above these interactions canalso be between the melatonin and the surrounding solvent moleculeswhich reveals its stabilisation in biological medium and also betweenthe molecule and the target proteins used in the dockingthe molecule is evident total electrostatic potentials esp and average localised ionization energy alie of melatoninthe electrostatic potential explains how reactive sites canundergo nucleophilic or electrophilic addition or substitution reactionsof melatonin shown in fig within bohr and a color changefrom blue to red indicates charges on elements from to the blue color appears in both methoxyoxygen and acetamideoxygen these are electronrich sites and electrophiles can quickly attack them the red color appears on all of the hydrogens these areelectronpoor sites and nucleophiles can quickly attack themthe alie clariï¬es the stability of any molecule based on saturatedand unsaturated bond electron movements which are localised ordelocalised the number of the resonance structure is proportionalto the stability of the molecule the alie of melatonin shown in fig iswithin the range of ± bohr color is from indigo to red and the numerical value is from to the blue color of protons in themethoxy group three protons in the sixmembered ring in the indolegroup methyl protons and both adjacent carbons in the acetamidegroup are all sites that act as electrophiles the red color ofacetamidecarbon conjugated carbon with oxygen atoms and bothacetamideamide and methoxy groups are all sites that act as nucleophiles these blue and red regions represent saturated bonds thebluishgreen regions are on indole rings to methoxy carbons via oxygenand acetamide chains this indicates that delocalised electrons and unsaturated bonds lead to several resonance structures and explains thestability of melatonin the electrophilic and nucleophilic reactive centres identiï¬ed above interact with the covid virus proteins and providevarious electrostatic and noncovalent interactions and increases drugafï¬nity noncovalent interaction nci properties of melatoninnoncovalent interactions are a valuable biological property of molecules and are nonbonded directly but are bound by some forces suchas hydrogen bonding van der waals bonding andor steric constraintsnoncovalent interactions of melatonin are shown in fig plotted as agraph with energy plotted versus a reduced density gradient hydrogen bonds appear from to au between oxygenand protons from the acetamide group the van der waals force rangesfig electrostatic potentials of melatoninfig average localised ionization energy for melatonin 0cn alzaqri of molecular liquids fig noncovalent interactions of melatoninfrom to au between acetamideoxygen and its adjacentprotons in both methyl and methoxy groups the steric force rangesfrom to au between the indole ring the methyl groupand the carbonylamide group noncovalent interactions are a groupof interactions like hydrogen bond pistacking hydrophobic interactions van der waal's forces iondipole interactions and dipoledipoleinteractions responsible for the stabilisation of the molecule and thedocking between melatonin and the covid proteins molecular dockingmolecular docking is one of the essential functions of biologically active molecules this is the theoretical evidence to design the structureand reactivity relationship of a molecule at present the covid19 pandemic caused by a new strain of the coronavirus is creating havocthroughout the world we made efforts to dock the melatonin withthe three proteins isolated from the virus represented through thepdb id 6lu7 6m03 and 6w63 were deposited in the database as mentioned in the methodology sectionwith the rapid spread of the novel coronavirus globally the designof vaccines is of great importance sarscov2 is an enveloped nonsegmented and single stranded positive sense rna virus the bestdrug target among coronaviruses is the main protease mpro also called3cl protease this is a key coronavirus enzyme and plays a vitalrole in mediating viral replication and transcription it is identiï¬ed ashaving a mechanismbased inhibitor the main targeting protease protein pdb 6lu7 is widely studied a series of frontier molecular orbital based interaction analyseswere performed on the complex between the main protease ofcovid19 and the peptidelike inhibitor whose fundamental structure was obtained from the protein pdb 6lu7 anothertargeted protease protein in an apo form pdb 6m03 shows themost stable form after binding with the selected drug threonine residue with the help of several covalent bond interactionwith a kcalmol docking afï¬nity lasinavir brecanavirtelinavir rotigaptide 13bis2ethoxycarbonylchromon5yloxy2lysyloxypropane and pimelautide can be consideredas the main protease inhibitors of covid19 by docking them tothe binding cavities of apo pdb 6m03 and holo pdb 6lu7 another protease protein pdb 6w63 is a reversible inhibitorthe ï¬avonoid narcissoside is reported to have a high afï¬nity towards the protease protein pdb 6w63 according to moleculardocking studies thus these three protease proteins pdb 6lu7pdb 6m03 and pdb 6w63 can be included in the category ofnonstructural proteins in the structure of sarscov2 from table the result from swissdock explains the biological activity of melatonin with coronavirus proteins pdb id 6lu7 6m03and 6w63 in general the total δg is more than kcalmol isright active luckily melatonin has a total δg of and kcalmol with coronavirus2 proteins pdb id 6lu7 6m03 and6w63 respectively and the total δg is directly proportional to the fullï¬tness energy values which are and kcalmol respectively it can also be seen from this table theinterfull ï¬tness intrafull ï¬tness full solvent ï¬tness full surface ï¬tnessδg complex polar solvent δg complex nonpolar solvent δg proteinpolar solvent δg protein nonpolar solvent δg ligand polar solventδg ligand nonpolar solvent δg van der waals force and δg electricforce relationships between melatonin and coronavirus2 proteins asreferredthe result from patchdock are as follows the score values are and total surface interacting area and and the minimum atomic contact energies are and kcalmol for melatonin with coronavirus2 proteins pdb id 6lu7 6m03 and 6w63 respectively figs and s1show the skeletal structure and protein residue interactions betweenmelatonin and coronavirus2 protein pdb id 6lu7 6m03 and 6w63table explains what protein residues are interacting with melatoninand details the residue names labels hydrophobic values pka valuesaverage isotropic displacements secondary structures residue solventaccessibility sidechain solvent accessibility percent solvent accessibility and percent sidechain solvent accessibility values of coronavirus2proteinstable and fig s2 show the residue structure of the favorable nonbond interactions between melatonin and coronavirus2 proteinstable lists favorable nonbond interactions of 6lu7 having conventional hydrogen bonds carbonhydrogen bonds pidonor hydrogenbonds pisulfur and pialkyl with melatonin 6m03 has pisigmapipi tshaped and pialkyl with melatonin while 6w63 has pipi tshaped pialkyl and pialkyl with melatonin along with the bond distance from chemistry fig s2 and table show the residue structure 0cn alzaqri of molecular liquids table swissdock result for melatonin with coronovirus2 proteins pdb id 6lu7 6m03 and 6w63energysimple ï¬tnessfull ï¬tnessinter full ï¬tnessintra full ï¬tnesssolvent full ï¬tnesssurface full ï¬tnessextra full ï¬tnessδg complex solvent polarδg complex solvent nonpolarδg protein solvent polarδg protein solvent nonpolarδg ligand solvent polarδg ligand solvent nonpolarδg van der waals forceδg electric forcetotal δg6lu7 kcalmol kcalmol kcalmol kcalmol kcalmol kcalmol kcalmol kcalmol kcalmol kcalmol kcalmol kcalmol kcalmol kcalmol kcalmol kcalmol kcalmol 6m03 kcalmol kcalmol kcalmol kcalmol kcalmol kcalmol kcalmol kcalmol kcalmol kcalmol kcalmol kcalmol kcalmol kcalmol kcalmol kcalmol kcalmol6w63 kcalmol kcalmol kcalmol kcalmol kcalmol kcalmol kcalmol kcalmol kcalmol kcalmol kcalmol kcalmol kcalmol kcalmol kcalmol kcalmol kcalmolof the unfavorable nonbond interactions between melatonin and coronavirus2 proteins protein 6lu7 does not have any unfavorablestericinteractions protein 6m03 having three unfavorable nonbond interactions and protein 6w63 having unfavorable bumpnonbond interactions fig s2 and table show unsatisï¬ed bonds within melatonininteracting with coronavirus proteins when interacting protein 6lu7has one hydrogen donor and one oxygen acceptor protein 6m03 hastwo hydrogen donors and two oxygen acceptors and protein 6w63has one hydrogen donor and two oxygen acceptors with melatonin table shows noncovalent interactions between melatonin and coronavirus2 proteins hydrophobic groups of protein 6lu7 residues areahis41 aleu141 acys145 ahis164 amet165 and aglu166those of protein 6m03 residues are ahis41 amet49 aphe140 aleu141 acys145 amet165 aglu166 and aleu167 and those ofprotein residues 6w63 are ahis41 acys44 amet49 aleu50 amet165 aglu166 aleu167 and agln189 with melatonin as shownin fig s2 and table the hydrophilic groups of protein 6lu7 residuesare ahis41 aasn142 ahis164 aglu166 ahis172 aasp187 aarg188 and agln189 those of protein 6m03 residues are ahis41 aasn142 ahis163 ahis164 aglu166 ahis172 and agln189 andthose of protein 6w63 residues are ahis41 ahis164 aglu166 aasp187 aarg188 agln189 and agln192 with melatonin as shownin fig s3 and table neutral groups of protein 6lu7 residues are atyr54 agly143 and aser144 those of protein 6m03 residues are agly143 aser144 and apro168 and those of protein 6w63 residuesare apro52 atyr54 aarg188 and athr190 with melatonin asshown in fig s4 and table acidic groups of protein 6lu7 residuesare aglu166 and aasp187 that of protein 6m03 residue is aglu166 and protein 6w63 residues are aglu166 and aasp187 withmelatonin as shown in table and fig s5 basic group interactions ofprotein 6lu7 residues are ahis41 ahis164 ahis172 and aarg188those of protein 6m03 residues are ahis41 ahis163 ahis164 andahis172 and those of protein 6w63 residues are ahis41 ahis164and aarg188 as shown in table and fig s6 tan etal has shownthat melatonin and derivatives has excellent biological responses likeacting against oxidative stress and free radical scavenging []our studies show that melatonin molecule can interact with differentproteins present in the ncov19 virus and inhibit their proliferationthese results need further clinical follow up and could assist in the management of covid pneumonia conclusionswe conducted a detailed quantummechanical investigation of thehormone melatonin and regulation of the sleepwake cycle naturalbonding orbital studies revealed the intensity of several intramolecularinteractions the various frontier molecular orbital data explain the nature and physical parameters of melatonin and the nonlinear opticalproperties are compared with urea which is a standard materialfig skeletal structure of interactions between melatonin and 6lu7 a 6m03 b and 6w63 c coronavirus2 protein residues 0cn alzaqri of molecular liquids table interactions between melatonin and coronavirus2 protein residuesnamelabelhydrophobicitypkapdbidsavg isotropicdisplacementsecondarystructureresidue solventaccessibilitysidechain solventaccessibilitypercent solventaccessibilitypercent sidechainsolvent accessibility6m03 histidine6lu7 histidineahis41amet49methionineatyr54tyrosinealeu141leucineaasn142asparagineagly143glycineaser144serineacys145cysteineahis164histidinemethionineamet165glutamic acid aglu166ahis172histidineaasp187aspartic acidaarg188arginineglutamineagln189ahis41methionineamet49phenylalanine aphe140aleu141leucineaasn142asparagineglycineagly143aser144serineacys145cysteineahis163histidineahis164histidinemethionineamet165glutamic acid aglu166aleu167leucineapro168prolineahis172histidineagln189glutamine6w63 histidineahis41cysteineacys44methionineamet49leucinealeu50prolineapro52tyrosineatyr54histidineahis164methionineamet165glutamic acid aglu166aleu167leucineapro168prolineaasp187aspartic acidarginineaarg188agln189glutamineathr190threonineglutamineagln192helixturnhelixcoilturnturncoilturnsheetsheetsheetsheetcoilcoilcoilhelixhelixcoilcoilturnturncoilturnsheetsheetsheetsheetcoilturnsheetcoilhelixcoilcoilcoilcoilhelixsheetsheetsheetcoilturncoilcoilcoilcoilcoilwavefunction studies gave information about electrostatic potentialsaverage localised ionization and noncovalent interactions these datahelped to predict the reactivity and identify the active site of the reactivity of the molecule melatonin docks with novel coronavirus proteinsand shows a variety of interactions with an excellent docking scorewhich leads to inhibition of the virus proteins leading to its destructionhence clinicians can consider incorporating melatonin also in thecovid19 treatment regime after further studiestable favorable nonbond interactions between melatonin and coronavirus2 proteinspdb idsdistance categorytypefromfrom chemistrytoto chemistry6lu76m036w63hydrogen bondhydrogen bondhydrogen bondhydrogen bond otherhydrophobichydrophobichydrophobichydrophobichydrogen bondhydrophobichydrophobichydrophobicconventional hydrogen bondconventional hydrogen bondcarbon hydrogen bondpidonor hydrogen bond pisulfurpialkylpisigmapipi tshapedpialkylconventional hydrogen bondpipi tshapedpialkylpialkylaglu166nunk0hunk0hacys145sgunk0amet165caahis41unk0unk0hahis41unk0unk0hdonorhdonorhdonorhdonor sulfurpiorbitalschpiorbitalspiorbitalshdonorpiorbitalspiorbitalspiorbitalsunk0oahis164oaglu166ounk0acys145unk0unk0acys145aglu166ounk0amet165amet165hacceptorhacceptorhacceptorpiorbitals piorbitalsalkylpiorbitalspiorbitalsalkylhacceptorpiorbitalsalkylalkyl 0cn alzaqri of molecular liquids table unfavorable nonbond between melatonin and coronavirus2 proteinspdb idsdistance categorytypefromfrom chemistrytoto chemistry6lu76m036w63nilunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorable bumpunfavorable bumpunfavorable bump carbon hydrogen bondunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpacys145sgaglu166ounk0hagln189caagln189caagln189cbagln189cgagln189cgagln189cgagln189cgagln189cdagln189cdagln189cdagln189cdagln189oe1agln189oe1agln189ne2agln189ne2agln189ne2agln189ne2agln189ne2agln189ne2agln189ne2agln189haagln189haagln189haagln189hg1agln189hg2agln189hg2agln189he21agln189he21agln189he21agln189he21agln189he22agln189he22agln189he22stericstericsteric hdonorstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericunk0cunk0caglu166ounk0cunk0hunk0cunk0cunk0hunk0nunk0hunk0cunk0cunk0hunk0hunk0cunk0hunk0nunk0cunk0ounk0cunk0hunk0hunk0hunk0cunk0hunk0hunk0hunk0cunk0hunk0nunk0cunk0cunk0hunk0cunk0cunk0hstericstericsteric hacceptorstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericsterictable unsatisï¬ed bonds in melatonin with coronavirus2 proteinspdb ids6lu76m036w63nameunk0hunk0ounk0hunk0hunk0ounk0ounk0hunk0ounk0oatomunsatisï¬ed typehohhoohoodonoracceptordonordonoracceptoracceptordonoracceptoracceptorrenjith thomas conceptualization formal analysis investigation methodology project administration resources softwaresupervision validation visualization writing review editingdeclaration of competing interestthe authors declare that they have no known competing ï¬nancialinterests or personal relationships that could have appeared to uence the work reported in this paperacknowledgementsresearchers supporting project number rsp202078 king saudcredit authorship contribution statementuniversity riyadh saudi arabianabil alzaqri cenceptualization funding acquisition tpooventhiran investegation methodology original draft alialsalme investegation original draft ismail warad resourcesreview methods athira m john methodology writing draftappendix a supplementary datasupplementary data to this article can be found online at 101016jmolliq2020114082table noncovalent interactions between melatonin and coronavirus2 proteinshydrophobicityhydrophilicityneutral groupacidic groupbasic grouppdbids6lu7ahis41 aleu141 acys145 ahis164 amet165 and aglu1666m03 ahis41 amet49 aphe140 aleu141 acys145 amet165 aglu166 and aleu1676w63 ahis41 acys44 amet49 aleu50 amet165aglu166 aleu167 and agln189ahis41 aasn142 ahis164 aglu166 ahis172 aasp187 aarg188 and agln189ahis41 aasn142 ahis163 ahis164 aglu166 ahis172 and agln189ahis41 ahis164 aglu166 aasp187 aarg188 agln189 and agln192atyr54 agly143 andaser144agly143 aser144 andapro168apro52 atyr54 aarg188 and athr190aglu166 andaasp187aglu166aglu166and aasp187ahis41 ahis164 ahis172 and aarg188ahis41 ahis163 ahis164 and ahis172ahis41 ahis164 andaarg188 | Colon_Cancer |
objective this study aimed to test the hypothesis that levobupivacaine has antitumour effects on breast cancer cellsresults colony formation and transwell assay were used to determine breast cancer cells proliferation flow cytometry annexin v and pi staining was used to investigate breast cancer cells apoptosis the effects of levobupivacaine on cellular signalling and molecular response were studied with quantitative polymerase chain reaction and western blot induction of apoptosis was confirmed by cell viability morphological changes showed cell shrinkage rounding and detachments from plates the results of the western blot and quantitative polymerase chain reaction indicated activation of active caspase and inhibition of foxo1 the results of the flow cytometry confirmed that levobupivacaine inhibited breast cancer cell proliferation and enhanced apoptosis of breast cancer cells quantitative polymerase chain reaction and western blot analysis showed increased p21 and decreased cyclin d quantitative polymerase chain reaction and western blot analysis showed that levobupivacaine significantly increased bax expression accompanied by a significant decreased bcl expression and inhibition of pi3kaktmtor signalling pathway these findings suggested that levobupivacaine inhibits proliferation and promotes breast cancer cells apoptosis in vitrokeywords levobupivacaine proliferation invasion apoptosis breast cancerintroductionbreast cancer is one of the most recorded cancer illness among women in the united states it is estimated that more than women die every year from breast cancerrelated illness despite the advance in chemotherapy and targeted treatments correspondence yanqiu63126com wqp89163com department of anaesthesiology dalian medical university dalian china department of biochemistry and molecular biology dalian medical university dalian chinafull list of author information is available at the end of the molecular signalling pathways that are involved in breast cancer transformation have become targets for treatment the mechanisms of the pi3kaktmtor signalling pathway have present some promising targets for cancer treatments this signalling pathway hinders the functions of several tumour suppressor genes such as bad gsk3 foxo transcription factors and tuberinhamartin complex which control cell survival proliferation and growth [] suppressing this signalling pathway may inhibit cancer cells proliferation and also stimulate them toward cell deaththe growing evidence of local anaesthetics inhibiting cancer cell growth seems promising though limited the authors this is licensed under a creative commons attribution international license which permits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the creative commons licence and indicate if changes were made the images or other third party material in this are included in the s creative commons licence unless indicated otherwise in a credit line to the material if material is not included in the s creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtain permission directly from the copyright holder to view a copy of this licence visit httpcreat iveco mmons licen sesby40 the creative commons public domain dedication waiver httpcreat iveco mmons publi cdoma inzero10 applies to the data made available in this unless otherwise stated in a credit line to the data 0ckwakye a0et a0al bmc res notes page of at the tissue level administration of a certain amount of local anaesthetics topical or local has shown to have a direct inhibitory effect on the action of epidermal growth factor receptor egfr which is a potential target for antiproliferation in cancer cells evidence also shows that ropivacaine and lidocaine hinder cancer cells growth invasion migration and enhance apoptosis of lung cancer cells [] to the best of our knowledge the effect of levobupivacaine on breast cancer cells is yet to be determined the present study therefore aimed to investigate the antitumour effects of levobupivacaine on breast cancer cellsmain textmaterials and a0methodsethics statementthe ethical committee of the dalian medical university first affiliated hospital approved for this study to be carried outcell culturewe purchased mcf7 and mdamb231 breast cancer cells from the atcc beijing zhongyuan limited china we maintained the mcf7 and mdamb231 cells with highglucose dmem or dmemf12 gibco usa medium the medium was supplemented with fetal bovine serum fbs gibco usa penicillin a0unitsml and streptomycin a0µgml transgen biotech china to maintain the cells the mcf7 and mdamb231 cells were then maintained in an incubator at a0ºc humidified air with co2 atmospheric condition the cells were routinely subcultured subsequentlyantibodies and a0reagentsepr17671 akt monoclonal antibody abcam china y391 mtor polyclonal antibody abcam china a2845 bcl2 polyclonal antibody abclonal technology a11550 bax polyclonal antibody abclonal technology a0265 pik3ca polyclonal antibody abclonal technology a2934 foxo1 polyclonal antibody abclonal technology epr21032 active caspase monoclonal antibody abcam china afo931 cyclin d1 polyclonal antibody affbiotech china af6290 p21 polyclonal antibody affbiotech china antimtor phospho s2448 antibody abcam china pa517387 phosphopi3k p85p55 tyr458 tyr199 polyclonal antibody themofisher scientific posphopanakt123 ser473 antibody affbiotech china peroxidaseconjugated goat antirabbit igg proteintech china prap antibodies proteintech china and gapdh antibodies proteintech chinacell viability assay and a0ic50we determined the mcf7 and mdamb cells viability using cck8 assay levobupivacaine at a concentration of or a0mm was used to treat mcf7 and mdamb cells plated in 96well plates a0cellswell and then incubated for or a0h respectively in an incubator at the atmospheric condition of a0 °c with co2 the rest of the procedures used for the cck8 assay were the same as described elsewhere flow cytometryannexin v and propidium iodide pi staining assay were used to investigate the apoptosis of mcf7 and mdamb cells following levobupivacaine treatment after treating the cells for a0h trypsin was used to harvest the treated cells and centrifugation at rcf for a0min the mcf7 and mdamb treated cells were again suspended with binding buffer and then a0 µl of fluorochromeconjugated annexin v sigmaaldrich saint louis usa was added into a0µl of the cell suspension to stain intracellular phosphatidylserine ps the cells were then incubation in a dark under room temperature the cells were again suspended and a0 µl propidium iodide staining solution sigmaaldrich saint louis usa added into a0µl of the cell suspension we detected the percentage of the apoptotic cells via flowjo software treestar ashland usa and flow cytometry facs calibur becton dickinson and sunnyvale ca usaquantitative polymerase chain reaction qpcrthe procedures used for the qpcr were the same as previously described the primers sequences were bax 5tgg cag ctg aca tgt ttt ctg3 f 5tcc cgg agg aag tcc aat g3 bcl2 5acg gtg gtg gag gag ctc tt3 f 5gcc ggt tca ggt act cag tcat3 r p21 5gcg act gtg atg cgc taa tg3 f 5gaa ggt aga gct tgg gca gg3 r gapdh ²cat gtt cgt cat ggg tgt gaa² f ²ggc atg gac tgt ggt cat gag3² rr western blotat the log phase of treated mcf7 and mdamb cells growth we harvested the cells and then washed twice with icecold pbs the rest of the procedures used for the western blot were the same as described elsewhere colony formation assaythe procedures used for the colony formation assay were the same as previously described 0ckwakye a0et a0al bmc res notes page of transwell assaythe mcf7 and mdamba231 cells that were pretreated with different dose of levobupivacaine a0mm for a0h and resuspended in culture medium with the same concentrations of levobupivacaine were seeded onto the coated membrane in the upper chamber of the transwell 24well millicell cell culture insert a0mm diameter a0μm pores merck kgaa p18p01250 china the procedures used for the transwell assay were the same as previously describe data analysisvalues were expressed as the mean ± sd statistical analysis was performed with graphpad prism version 501graphpad software la jolla ca us oneway anova was used to measure significance p dunnetts post hoc tests were used to test the difference between groupsresultslevobupivacaine decreases breast cancer cell invasiontranswell assay analysis showed significantly decreased in the invasion ability of mcf7 and mdamb231 cells in a dosedependent manner compared with the untreated cells additional file a0 fig s1a b levobupivacaine inhibits proliferation in a0breast cancer cellsthe mcf7 and mdamba231 cell viability decreased as the concentrations of levobupivacaine or a0mm increased the mcf7 cells showed a cytotoxic effect while the mdamb231 cells showed a similar cytotoxic effect of fig a01a under a fluorescence microscope cells treated with levobupivacaine showed morphological changes including cell rounding cell shrinkage and cells detachment from the plates additional file a0 fig s2a b the viability of breast cancer cells decreased in a dosedependent manner the results showed significantly decreased in the number of clones of the treated cells compared with the untreated cells fig a01b c the data showed that the mrna level of p21 significantly increased following levobupivacaine treatment fig a0 1d e western blot analysis showed a similar increased in p21 and decreased in foxo1 and cyclin d1 expressions in a dosedependent manner compared with the untreated cells fig a01f g additional file a03f glevobupivacaine promote apoptosis in a0breast cancer cellslevobupivacaine significantly reduced the number of cells showing nuclear staining when compared with the untreated cells fig a0 2a b the qpcr data showed a decreased in bcl2 and increased in bax expressions in mcf7 and mdamb231 cells compared with the untreated cells fig a0 2c d western blot analysis also showed a similar decreased in bcl2 and increased expressions of active caspase and bax compared with the untreated cells fig a02e f additional file a03e flevobupivacaine inhibits proliferation and a0promotes apoptosis in a0breast cancer through a0pi3kaktmtor signalling pathwaywestern blot analysis showed a significant decreased in the expression of the nuclear localization of ppi3k pakt and pmtor compared with the untreated cells fig a03a b additional file a03a bdiscussionbreast cancer remains a common cause of mortality among women worldwide though current orthodox drugs have demonstrated promise in breast cancer therapy its treatment options remain limited these therefore supports the concept that effective therapeutic approaches for breast cancer are critically needed several retrospective studies have demonstrated that regional anaesthesia is associated with a decreased risk of recurrence or metastasis of multiple carcinomas including breast prostate and cervical cancers [] recent growing evidence demonstrates that local anaesthetics have an antitumour effect and may suppress the motility of cellular function and invasiveness more likely via voltagegated sodium channel inhibition a study report indicates that lidocaine inhibits the growth of human hepatocellular carcinoma cells hcc by increasing the caspase activity whereas ropivacaine inhibits the growth of hcc cells by stopping the cell cycle in g2 phase lee et a0al demonstrated that local anaesthetics potentiate tnfα mediated apoptosis in hk2 cells the cellular modification of treated cells is likely dependent on the duration of exposure and the dose of the local see figure on next pagefig levobupivacaine inhibits proliferation in breast cancer cells mcf and mdamb cells were treated with different concentrations of levobupivacaine a cell viability was measured by cck assay ic50 results of levobupivacaine on mcf and mdamb cells b c colony formation of mcf and mdamb cells treated with various concentrations of levobupivacaine and stained with crystal violet d e the mrna expression levels of p21 and gapdh were analysed by qpcr f g protein expression assessment of mcf and mdamb cells by western blot against antibodies foxo1 p21 cyclin d1 and gapdh used as control the data was statistically significant at indicates p indicates p indicates p compared with untreated cells this data corresponds to the mean ± sem of three independent experiments 0ckwakye a0et a0al bmc res notes page of 0ckwakye a0et a0al bmc res notes page of fig effects of levobupivacaine on apoptosis of breast cancer cells a b mcf and mdamb cells were treated with different concentrations of levobupivacaine for h the cells were then stained with fluoresceinconjugated annexin v and pi and analysed by flow cytometry error bars represent standard error of the mean p versus the control c d relative gene expression of bax and bcl following the treatment of breast cancer cells with different concentrations of levobupivacaine for h and analysed by qpcr e f mcf and mdamb cells were treated with different concentrations of levobupivacaine for h and the activities of bax bcl and active caspase were examined by western blot analysis using specific antibodies gapdh was used as internal controls the data was statistically significant at indicates p indicates p compared with control the data correspond to the mean ± sem of three independent experiments 0ckwakye a0et a0al bmc res notes page of fig mcf and mdamb cells were treated with different concentrations of levobupivacaine for h a b the cells were lysed and subjected to sdspage and analysed by western blotting and probed with specific antibodies ppi3k pakt and pmtor the results showed a decrease in the expressions of ppi3k pakt and pmtor proteins gapdh was used as internal controls the data represent the mean ± sd of three independent experimentsanaesthetic [] in this study we employed mcf and mdamb231 cells as models and found that different concentrations of levobupivacaine could effectively inhibit breast cancer cell proliferation and promote apoptosis in a0vitro the antiproliferation and apoptosis effects observed in this study suggest that levobupivacaine may have therapeutic effects on breast cancerpi3kaktmtor signalling pathway plays a vital role in cell proliferation survival development metabolism motility and regulation of the immune response breast cancer cell resistance to therapies can result from the activation of pi3kaktmtor signalling pathway [] this has made the pi3kaktmtor signalling pathway an important object of study for understanding the development and progression of breast cancer in patients with breast cancer pi3kaktmtor signalling pathway can be a target for diagnostic prognostic and treatment purposes [] akt plays a role in the activation and inactivation of many transcription factors activation of akt correlated with the activation of mtor phosphorylation of the foxo proteins by akt may results in cytoplasmic retention by interacting with other proteins thereby isolating them from their targeted genes cyclin d1 classified as a pro oncogene is often overexpressed in several human malignancies including breast colon lung and prostate cancers reports show that overexpression of cyclin d1 and underexpression of tumour suppressor p21 is required for cancer initiation as it is confirmed that downregulation of cyclin d1 and overexpression of p21 in xenograft model discontinues the formation of cancer in the early stages datta et a0al reported that akt can phosphorylate the proapoptotic bcl2 family member bad causing its isolation from the mitochondrial membrane by other proteins local anaesthetics modify the protein levels of key members of the bcl2 family in a manner that presents an increase in the ratio of baxbcl2 which may contribute to the response of cancer cells to apoptosis in the present study the role of levobupivacaine on the expression of pi3k akt and mtor was investigated to illustrate the potential molecular mechanism we observed a significantly decreased expression of pakt ppi3k pmtor and subsequent decreased expression of foxo cyclin d1 and bcl2 following levobupivacaine treatment which correlated with decreased breast cancer cells proliferation and increased apoptosis these emerging pieces of evidence suggest that levobupivacaine may inhibit proliferation and promote apoptosis by suppressing pi3kaktmtor signalling pathway which demonstrated an antitumour effect on breast cancer cells in this studyconclusionlevobupivacaine has the potency of reducing breast cancer cell viability proliferation and also causes cell death by suppressing the pi3kaktmtor signalling pathway these findings could lead to clinical studies which will seek to examine the anticancer effects of levobupivacaine and may also increase the benefits in cancer patient as well as improve patient care 0ckwakye a0et a0al bmc res notes page of limitationsnumerous studies have reported on the antitumour effects of local anaesthetics on various cancer cells [] however our work is not without limitations in a0vivo and clinical studies on the antitumour effects of levobupivacaine are neededbiology dalian medical university dalian china department of anaesthesia and critical care school of medicine university of health and allied sciences ho ghana department of biochemistry and molecular medicine school of medicine and health sciences university for development studies tamale ghana departments of anaesthesia and critical care ridge hospital accra ghana department of medicine princefied university ho ghana received june accepted july supplementary informationsupplementary information accompanies this paper at https doi101186s1310 additional file a0 figure s1 levobupivacaine decreases breast cancer cell invasionadditional file a0 figure s2 effect of levobupivacaine on the morphology of mcf and mdamb cellsadditional file a0 original gelsblots scan used in fig 1f g fig 2e f and fig 3a b for mcf and mdamb cellsabbreviationsegfr epidermal growth factor receptor hcc hepatocellular carcinoma cells nc nitrocellulose pi propidium iodide ps phosphatidylserine qpcr quantitativepolymerase chain reactionacknowledgementswe thank the first affiliated hospital and the department of biochemistry of dalian medical university for making available all the necessary materials needed for this work we also thank the key laboratory of liaoning provincial education department grant no lz2016002 and liaoning natural science foundation grant no of china for supporting this work our thanks also go to the china scholarship council and the government of the republic of ghana for giving financial aid to some of the authors to study at dalian medical universityauthors contributionsakk sk qy and qpw conceived and designed this study qpw and qy were responsible for the supervision and coordination of this study akk sk jl mnr qy and qpw conducted the data collections sk led the data analysis with inputs from akk qy and qpw akk and sk wrote the first draft of the manuscript and jl mnr sar aaf ja and ean contributed to revising and reviewing the manuscript all authors read and approved the final manuscriptfundingthis study was supported by the key laboratory of liaoning provincial education department grant no lz2016002 and liaoning natural science foundation grant no availability of data and materialsthe data used andor analysed in this study are available from the corresponding author upon reasonable requestethics approval and consent to participatethe ethical committee of the first affiliated hospital of dalian medical university approved the study protocol and because this study used breast cancer cells consent to participate was not applicable for the studyconsent for publicationsnot applicablecompeting interestsauthors declare that they have no competing interestsauthor details department of anaesthesiology dalian medical university dalian china department of anaesthesiology first affiliated hospital of dalian medical university dalian china department of biochemistry and molecular references american cancer society breast cancer facts and figures atlanta american cancer society american cancer society cancer facts and figures atlanta ameri can cancer society siegel r naishadham d jemal a cancer statistics ca cancer j clin chang yc hsu yc liu cl huang sy hu mc cheng sp local anaesthetics induce apoptosis in human thyroid cancer cells through the mitogenactivated protein kinase pathway plos one 20149e89563 gomezgutierrez jg souza v hao hy de montes ocaluna r dong yb zhou hs mcmasters km adenovirusmediated gene transfer of fkhrl1 triple mutant efficiently induces apoptosis in melanoma cells cancer biol ther sunters a de fern¡ndez mattos s stahl m brosens jj zoumpoulidou g saunders ca coffer pj medema rh coombes rc lam ew foxo3a transcriptional regulation of bim controls apoptosis in paclitaxeltreated breast cancer cell lines j biol chem fu z tindall dj foxos cancer and regulation of apoptosis oncogene barnes dm gillett ce cyclin d1 in breast cancer breast cancer res treat sherr cj roberts jm cdk inhibitors positive and negative regulators of g1phase progression genes dev pelengaris s khan m evan g cmyc more than just a matter of life and death nat rev cancer di padova m barbieri r fanciulli m arcuri e florida a effect of local anaesthetic ropivacaine on the energy metabolism of ehrlich ascites tumour cells oncol res 199810491e8 xing w chen dt pan jh chen yh yan y li q xue rf yuan yf zeng wa lidocaine induces apoptosis and suppresses tumour growth in human hepatocellular carcinoma cells in vitro and in a xenograft model in vivo anesthesiology drasner k lidocaine spinal anaesthesia a vanishing therapeutic index anesthesiology wang hw wang ly jiang l tian sm zhong td fang xm amidelinked local anaesthetics induce apoptosis in human nonsmallcell lung cancer j thorac dis https doi1021037 jtd20160966 piegeler t vottavelis eg liu g place at schwartz de beckschimmer b minshall rd beat a antimetastatic potential of amidelinked local anaesthetics inhibition of lung adenocarcinoma cell migration and inflammatory src signalling independent of sodium channel blockade anesthesiology lirk p berger r hollmann mw feigl h lidocaine time and dosedependently demethylates deoxyribonucleic acid in breast cancer cell lines in vitro br j anaesth villargarea a fraga mf espada j esteller m procaine is a dnademethylating agent with growthinhibitory effects in human cancer cells cancer res 4984e634984e9 kampo s ahmmed b zhou t owusu l anabah tw doudou nr kuugbee ed cui y lu z yan q wen qp scorpion venom analgesic peptide bmk agap inhibits stemness and epithelialmesenchymal transition by downregulating ptx3 in breast cancer front oncol hirata m sakaguchi m mochida c sotozono c kageyama k yoshihiro k munetaka h lidocaine inhibits the tyrosine kinase activity of the epidermal growth factor receptor and suppresses proliferation of corneal epithelial cells anesthesiology 0ckwakye a0et a0al bmc res notes page of grouselle m tueux o dabadie p geescaud d mazat jp effect of local anaesthetics on mitochondrial membrane potential in living cells biochem j fraser sp diss jkj chioni am mycielska me pan h yamaci rf pani f siwy z krasowska m grzywna z brackenbury wj theodorou d koyuturk m kaya h battaloglu e de tamburo bella m slade mj tolhurst r palmieri c jiang j latchman ds coombes rc djamgoz mba voltagegated sodium channel expression and potentiation of human breast cancer metastasis clin cancer res le gac g angenard g clement b laviolle b coulouarn c beloeil h local anaesthetics inhibit the growth of human hepatocellular carcinoma cells anesth analg lee ht xu h siegel cd krichevsky ie local anaesthetics induce human renal cell apoptosis am j nephrol unami a shinohara y ichikawa t baba y biochemical and microarray analyses of bupivacaineinduced apoptosis j toxicol sci villargarea a fraga mf espada j esteller m procaine is a dnademethylating agent with growthinhibitory effects in human cancer cells cancer res sakaguchi m kuroda y hirose m the antiproliferative effect of lidocaine on human tongue cancer cells with inhibition of the activity of epidermal growth factor receptor anesth analg chang yc liu cl chen mj hsu yw chen sn lin ch chen cm yang fm hu mc local anaesthetics induced apoptosis in human breast tumour cells anesth analg kawasaki c kawasaki t ogata m sata t chaudry ih lidocaine enhances apoptosis and suppresses mitochondrial functions of human neutrophil in vitro j trauma hodgkin al huxley afa quantitative description of membrane current and its application to conduction and excitation in nerve j physiol besson p driffort v bon © gradek f chevalier s roger s how do voltagegated sodium channels enhance migration and invasiveness in cancer cells biochim biophys acta roger s gillet l le guennec jy besson p voltagegated sodium channels and cancer is excitability their primary role front pharmacol roger s potier m vandier c besson p le guennec jy voltagegated sodium channels new targets in cancer therapy curr pharm des driffort v gillet l bon e marionneaulambot s oullier t joulin v collin c pag¨s jc jourdan ml chevalier s bougnoux p le guennec jy besson p roger s ranolazine inhibits nav15mediated breast cancer cell invasiveness and lung colonization mol cancer nelson m yang m dowle aa thomas jr brackenbury wj the sodium channelblocking antiepileptic drug phenytoin inhibits breast tumour growth and metastasis mol cancer yuan t li z li x yu g wang n yang x lidocaine attenuates lipopolysaccharideinduced inflammatory responses in microglia j surg res brocco mc paulo dns almeida ced carraretto ar cabral sa silveira ac gomez rs baptista jf a study of interleukin il and tumour necrosis factoralpha tnfα serum levels in rats subjected to faecal peritonitis and treated with intraperitoneal ropivacaine acta cirurgica brasileira piegeler t schl¤pfer m dull ro schwartz de beat a minshall rd beckschimmer b clinically relevant concentrations of lidocaine and ropivacaine inhibit tnfαinduced invasion of lung adenocarcinoma cells in vitro by blocking the activation of akt and focal adhesion kinase br j anaesth shankar s chen q srivastava rk inhibition of pi3kakt and mekerk pathways act synergistically to enhance antiangiogenic effects of egcg through activation of foxo transcription factor j mol signal qian j zou y rahman jsm lu b massion pp synergy between phosphatidylinositol kinaseakt pathway and bclxl in the control of apoptosis in adenocarcinoma cells of the lung mol ortega ma frailemartınez o asunsolo a bujan j garcıahonduvilla n coca s signal transduction pathways in breast cancer the important role of pi3kaktmtor j oncol royds j khan ah buggy dj update on existing ongoing prospective trials evaluating the effect of anaesthetic and analgesic techniques during primary cancer surgery on cancer recurrence or metastasis int anesthesiol clin 2016544e76 burgering bmt medema rh decision on life and death foxo forkhead transcription factors are in command when pkbakt is off duty j leukoc biol chen q ganapathy s singh kp shankar s srivastava rk resveratrol induces growth arrest and apoptosis through activation of foxo transcription factors in prostate cancer cells plos one 20105e15288 arnold a papanikolaou a cyclin d1 in breast cancer pathogenesis j clin oncol santarius t shipley j brewer d stratton mr cooper cs a census of amplified and overexpressed human cancer genes nat rev cancer datta sr brunet a greenberg me cellular survival a play in three akts genes daev lirk p hollmann mw fleischer m weber nc feigl h lidocaine and ropivacaine but not bupivacaine demethylate deoxyribonucleic acid in breast cancer cells in vitro br j anaesth 2014113suppl 1i32i3838 li k yang j han x lidocaine sensitizes the cytotoxicity of cisplatin in breast cancer cells via the upregulation of rarβ2 and rassf1a demethylation int j mol sci publishers notespringer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations¢ fast convenient online submission ¢ thorough peer review by experienced researchers in your ï¬eld¢ rapid publication on acceptance¢ support for research data including large and complex data types¢ gold open access which fosters wider collaboration and increased citations maximum visibility for your research over 100m website views per year ¢ at bmc research is always in progresslearn more biomedcentralcomsubmissionsready to submit your research choose bmc and benefit from 0c' | Colon_Cancer |
" exercise has been extensively utilised as an effective therapy for overweight and obesityassociatedchanges that are linked to health complications several preclinical rodent studies have shown that treadmillexercise alongside an unhealthy diet improves metabolic health and microbiome composition furthermorechronic exercise has been shown to alter hypothalamic and adipose tissue gene expression in dietinduced obesityhowever limited work has investigated whether treadmill exercise commenced following exposure to anobesogenic diet is sufficient to alter microbiome composition and metabolic healthmethods to address this gap in the literature we fed rats a highfathighsugar westernstyle cafeteria diet andassessed the effects of weeks oftreadmill exercise on adiposity dietinduced gut dysbiosis as well ashypothalamic and retroperitoneal white adipose tissue gene expression fortyeight male spraguedawley rats wereallocated to either regular chow or cafeteria diet and after weeks half the rats on each diet were exposed tomoderate treadmill exercise for weeks while the remainder were exposed to a stationary treadmillresults microbial species diversity was uniquely reduced in exercising chowfed rats while microbiome compositionwas only changed by cafeteria diet despite limited effects of exercise on overall microbiome composition exerciseincreased inferred microbialreduced fat mass and altered adipose andhypothalamic gene expression after controlling for diet and exercise adipose il6 expression and liver triglycerideconcentrations were significantly associated with global microbiome compositions moderate treadmill exercise induced subtle microbiome composition changes in chowfed rats but didnot overcome the microbiome changes induced by prolonged exposure to cafeteria diet predicted metabolic functionof the gut microbiome was increased by exercise the effects of exercise on the microbiome may be modulated byobesity severity future work should investigate whether exercise in combination with microbiomemodifyinginterventions can synergistically reduce diet and obesityassociated comorbiditieskeywords obesity microbiome exercise hypothalamus white adipose tissue cafeteria dietfunctions involved in metabolism correspondence mmorrisunsweduau1department of pharmacology school of medical sciences unsw sydneynsw australiafull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cleigh nutrition metabolism page of introductionoverweight and obesity leads to reductions in physicaland mental health and quality of life resulting in increased direct and indirect costs to the global economy along with gross metabolic changes obesity is associated with altered fecal microbial species diversity and composition separate studies involving transferof obese human fecal microbiome samples induced fatgain in na¯ve mice and supplementation withakkermansia muciniphila improved insulin sensitivityand reduced body weightin overweight and obesepeople providing some evidence for a potential roleof diet and obesityassociated gut microbiota changes inadiposity and metabolic dysfunctionweight loss through lifestyle intervention is an effective strategy for reducing obesityrelated comorbidities one such intervention is moderate exercise a practical and sustainable approach for people with overweight and obesity while exercising at this intensityis unlikely to cause weight loss independent of caloricrestriction it confers cardiovascular and metabolic benefits and assists with weight maintenance [ ] furthermore regular exercise is known to improve glucoseregulation and insulin sensitivity as well as reducingcardiovascular disease and cancer risk andthere is increasing interest in the effects of exercise onthe gut microbiotathe first study to indicate an effect of exercise on fecalmicrobiome showed that elite professional athletes exhibited a distinct microbiome composition with increased microbial species diversity however sinceathletes consume a distinct diet from healthy people inthe community further work has been undertaken in rodents to identify the specific effects of exercise on fecalmicrobiome a recent systematic review of primarily rodent studies concluded that while there was no consistent effect of exercise on microbial species richnessexercise increases the relative abundance of firmicutes the different types of exercise used forced versusvoluntary have been shown to exert different effects onfecal microbiome composition in mice [ ] whichmay in part explain the inconsistent findingsfurthermore there were considerable differences inexercise duration used weeks which may contribute to the range of responses observed microbiomecompositional changes were observed in mice maintained on a healthy diet following weeks of moderatetreadmill exercise and after weeks in mice fed ahighfat diet in contrast eight weeks of lowtomoderateconfer microbiomecompositional changes in mice fed a highfat diet furthermore most studies examining the effects oftreadmill exercise on fecal microbiome in dietinducedobesity used a design wherecoexercise wasexercisedidnotadministered with highfat diet making it difficult totranslate the findings to people in terms of implementing exercise after a history of unhealthy eating andobesityhere we sought to examine whether moderate treadmill exercise in rats could exert benefits to gut microbiome composition following exposure to either ahealthy or a highfat highsugar westernstyle cafeteriadiet we aimed to investigate whether any changes ingut microbiome were associated with altered gene expression in white adipose tissue wat and the hypothalamus which are known to be affected by bothobesogenic diets and exercisematerials and methodssubjects and diet manipulationthis protocol was approved by the animal care andethics committee of unsw sydney in accordance withthe australian guidelines for the use and care of animalsfor scientific purposes national health and medicalresearch councilfortyeight male spraguedawley rats weeks g animal resource centre australia werehoused 3box °c h lightdark and handleddaily for one week while maintained on standard chow kjg premium rat and mouse maintenance dietgordons specialty stock feeds australia and water adlibitumfollowingacclimatization weightmatched groupswere randomly allocated to chow plus water or cafeteria diet n rats n cages per group ad libitumwhich comprised sucrosesolution alongsidecommerciallyproduced cakes cookies and savoury foods in addition to chow and water body weight and h food intake were measured twice weekly and food intake was calculated assuming equalintake per rat ineach cage body composition was measured during week by echomri900 echomri llc usacafsedcex cafeteria sedentarytreadmill exercisefollowing weeks half the rats in each dietary conditionwere allocated to treadmill exercise generating weightmatched groups chow sedentary csed chowexerciseandcafeteria exercise cafex for weeks until the day before sacrifice moderate exercise consisted of mmin for min five days a week at zero incline the firstweek comprised two training sessions mminafter which time spent at mmin was gradually increased sedentary rats were placed in stationary treadmills during the exercise protocol exercising rats wereclosely monitored three cafex rats showed signs of fatigue distress or vocalisation and were removed fromthe treadmill and thereafter exercised at a lower 0cleigh nutrition metabolism page of intensity mmin for min these rats were analysed together with moderately exercised cafex ratssample collectionat weeks of diet rats were deeply anaesthetized ketaminexylazine mgkg intraperitoneally bodyweight nasoanal length girth and blood glucose weremeasured following induction of anesthesia rats weredecapitated for trunk bloodthe hypothalamus within coronal block defined byrostrocaudal limits of circle of willis was rapidly dissected and collected retroperitoneal and gonadal watand liver were dissected and weighed one fecal pelletwas removed from the distal colon tissue and feceswere snap frozen in liquid nitrogen and stored at °cprotein and triglyceride measurementsplasma leptin and insulin concentrations were measuredusing commercial kits cat90040 and cat90060crystalchem inc usa plasma and liver triglyceridecontent were measured spectrophotometrically usingtriglyceride reagentroche diagnostics australia ptyltd australia at °c alongside a standard curvegenerated from glycerol standard g77935ml sigmaaldrich pty ltd australia livers were extracted byhomogenization in chloroformmethanol and incubated overnight nacl was added and samples werevortexed and centrifuged g for min the lowerphase was then evaporated at °c under nitrogen gasdried extract was redissolved in absolute ethanol andmeasured spectrophotometrically retroperitoneal watil6 content was determined using a duoset il6 ratelisa dy506 rd systems inc following manufacturers recommendationsstatistical analysesdata were analyzed using twoway betweensubjectsanova while measures over time were analysedusing 3way mixed anova posthoc pairwise comparisons were performed using a tukey adjustmentwhere appropriate thsd and presented in the associated figures and tables only when p pearsonscorrelations were used to identify associations allanalyses were completed using ibm spss statistics australiafecal dna extraction microbiome community sequencingand statistical analysesdna extraction was performed using the powerfecaldna isolation kit mobio laboratories usa microbial community composition was assessed by illuminaamplicon sequencing bp miseq chemistry v4region 515f806r primer pair using a standard protocol sequence data were analyzed using mothur using modified commands from miseq sop including alignment with the silva database singletonremoval chimera checking with uchime and classification against the latest rdp training set sequence depthwas normalized by subsampling to total clean readsper samplecompletedusing calypso withotuoperationaltaxonomic unitcorrelationswerethebenjaminihochberg false discovery rate fdr procedure used to control for multiple tests fdrcorrected deseq2 was performed using the phyloseq r package for the negative binomial walk test indeseq2 otu abundances were analyzed usingspss with kruskalwallisfollowed by nonparametric bonferronidunn posthoc testing whereappropriate otus ofinterest were identified usingsina aligner testsrna extraction and gene expression assaysrna was extracted from hypothalamus and retroperitoneal wat using tri reagent sigmaaldrich pty ltdaustralia following dnase i treatment catalogue merck australia or μg of rna wat andhypothalamus respectively were reverse transcribed toproduce cdna high capacity reverse transcriptasekit thermofisher scientific usa gene expression wasassessed using taqman inventoried gene expression assays life technologies australia pty ltd australia seesupplementary table genes of interest were normalized against the geometric mean of the two most stablehousekeeping genes gapdh and hprt1 for wat hprt1and b2m for hypothalamus identified by the normfinder package analysis of relative gene expressionwas performed using the δδct method normalized toan independent calibrator made from all samplesdblmalpha diversity metrics distancedbased linear modellingpermutational multivariate anovapermanova nonmetric multidimensional scalingnmds and canonical analysis of principal coordinateswere completed using primer v6 primere ltdplymouth united kingdom all primer analysesutilized a braycurtis similarity matrix constructed atthe otu levelphylogenetic investigation of communities by reconstruction of unobserved states picrust was performed using galaxy web to predict putative functionsthrough metagenomic prediction from the 16s otudata using greengenes for taxonomic classification pathway counts were compared across groupsusing fdrcorrected kruskalwallis tests followed bynonparametric bonferronidunn posthoc testing whereappropriate 0cleigh nutrition metabolism page of resultsenergy intake body weight and composition and watgene expressionover the 7week study cafeteriafed rats ate more thantwice the energy consumed by chowfed groups fig 1acsed kjrat cex kjrat cafsed kjrat cafex kjrat when energy intake wasstratified into pre and duringexercise intervention asignificantinteraction between time and diet wasobserved fig 1b f p which appeared to be due to increased cafeteria diet intake whilerats were exercising although this comparison did notreach statistical significance significant interactions between time and diet were also identified for fat andfig shortterm treadmill exercise reduces body weight gain and fat mass and alters wat expression without affecting energy intake a energyintake over the study and b average weekly energy intake before and during treadmill exercise intervention c body weight of the study and dbody weight gain during treadmill exercise intervention e fat mass as a percentage of body weight and f absolute lean mass data expressedas mean ± sem n for cage data n for individual data data were analyzed by twoway anova followed by tukeyadjusted posthoctesting ap relative to csed bp relative to cex cp relative to cafsed 0cleigh nutrition metabolism page of carbohydrate intakes supplementary figure 1a f p and supplementary figure 1c f p respectively where both macronutrient intakes increased in the cafeteriafed rats during exercise protein intake while unaffected by time orexercise was elevated by cafeteria diet supplementaryfigure 1b f p sucrose intake increased over time supplementary figure 1d f p but was not affected by exerciseall rats gained body weight over time fig 1c andcafeteriafed rats gained significantly more than chowfed controls prior to f p and during fig 1d f p exercise exercisesignificantly reduced weight gain overall f p thsd posthoc comparisons showed thatcompared to csed and cex cafsed p and p and cafex p and p gained significantly more weight over the exercise intervention ofnote cafsed rats gained significantly more weight thancafex rats over the exercise intervention p body composition data following weeks of treadmillexercise showed that relative fat mass was significantlyincreased by cafeteria diet fig 1e f p absolute fat mass presented in table and reduced by exercise f p thsdposthoc comparisons revealed that relative fat mass inboth cafeteriafed groups were significantly greater thanboth chowfed groups lean mass however showed onlyan overall diet effect fig 1f f p with significantly more lean mass in cafeteriafed ratsthan chowfed ratscafeteria di so increased nasoanallength girthplasma insulin and triglycerides with no effect of exercise table plasma leptin levels were significantly increased by diet f p and reducedby exercise f p unfasted bloodglucose did not differ between groups table in line with plasma leptin retroperitoneal and epidydimal fat pad weights were significantly greater in cafeteria than chowfed rats f p andf p for retroperitoneal and epidydimal fat pads respectively and were significantly reduced by exercise overall f p and f p for retroperitoneal and epidydimalfat pads respectively see table microbial species diversity and microbiome compositionmicrobial species diversity was assessed using shannonsdiversity index microbial species richness and microbialspecies evenness shannons diversity and evenness weresignificantly reduced by cafeteria diet overall f p fig 2a and f p fig 2c respectively and thsd posthoc comparisonsshowed that cafex rats exhibited reduced evenness relative to cex p and reduced shannons diversityrelative to csed p no significant differenceswere observed for bacterial species richness fig 2bmicrobiome composition at the otu level was signifipseudof p cantly affected by diet and cage pseudof p butnot by exercise when assessed using 4way permanova permutations and confirmed with nontable anthropometric measures at tissue collection and plasma measuresmeasurecafsedcsedcexterminal body weight gnasoanal length cmgirth cmliver scoreheart weight g ± ± ± 262ab ± ± ± ± ± ± ± ± cafex ± 124ab ± 01b ± 04ab ± 022ab ± 003ab ± 681ab ± 047ab ± 021ab ± ± 043ab ± 121ab ± 025ab ± 241abmain effects pvalueexercisediet interaction ± ± 06ab ± 017ab ± 007ab ± 1627ab ± 043ab ± 054ab ± ± 032ab ± 143ab ± 032ab ± 252ababsolute fat mass g echomri ± ± rp fat pad weight bw ± ± epidydimal fat pad weight bw ± ± blood glucose mmoll ± ± plasma insulin ngmlplasma leptin ngml ± ± ± ± plasma triglycerides mmoll ± ± liver triglycerides mgg tissueblood and plasma measures performed unfasted data expressed as mean ± sem n data were analyzed using twoway anova followed by posthocmultiple comparisons with a tukey hsd correction liver score was analysed using a kruskalwallis test followed by nonparametric bonferronidunnposthoc testingap relative to csed bp relative to cex ± ± 0cleigh nutrition metabolism page of fig see legend on next page 0cleigh nutrition metabolism page of see figure on previous pagefig impact of cafeteria diet and treadmill exercise intervention on fecal microbiota and inferred microbiome function at weeks a shannonsdiversity b microbial species richness and c evenness data expressed as mean ± sem n data were analyzed by twoway anovafollowed by tukeyadjusted posthoc testing d nonmetric multidimensional scaling braycurtis permutations showing similarity betweenfecal microbiota samples at weeks e muribaculum_otu72 identified by deseq2 adjusted p as differentially abundant with exercise inchowfed rats f relative abundance of otu72 at weeks data expressed as boxandwhisker plots min iqr max n data were analyzedusing kruskalwallis test followed by nonparametric dunnbonferroni posthoc testing g amino acid metabolism h overall energy metabolism idglutamine and dglutamate metabolism and j one carbon pool by folate predicted using picrust from fecal microbiome data at weeks dataare expressed as boxandwhisker plots min iqr max n were analyzed by kruskalwallis tests fdradjusted overall pvalue to accountfor multiple relevant pathways included in analysis followed by nonparametric bonferronidunn posthoc comparisons posthoc symbols ap relative to csed bp relative to cex cp relative to cafsedmetric multidimensional scaling fig 2d supplementary figure shows groups differences in microbiomecomposition at the phylum leveldeseq2 analyses were used to identify otus differentially enriched with exercise exposure amongst the top otus while exercise was not associated with differentially expressed otus in cafeteriafed rats muribaculum_otu72 was significantly depleted in cex ratsrelative to csed fig 2e relative abundance in fig 2fthis otu was originally classified as akkermansia whenaligned with the rdp reference library muribaculum_otu72 was putatively identified as an unknown bacterium from the genus muribaculum using sina aligner alignment identitypredicted microbiome functionto determine whether the subtle microbiome composition changes observed with exercise affected microbiome function we inferred microbiome function usingpicrust following an fdr correction amino acidmetabolism fig 2g h p overallenergy metabolism fig 2h h p dglutamine and dglutamate metabolism fig 2i h p and one carbon pool by folate fig 2jh p exhibited overall group differences amino acid metabolism was significantly elevatedin cafex relative to csed p and cafsed p rats while the other processes were significantlyelevated in both exercised groups relative to csedwat and hypothalamic gene expressionexamination of wat inflammatory signaling andbrowning genes fig 3a revealed a significant interaction effect for ucp1 f p whilethsd posthoc comparisons showed that cafex ratsexhibited elevated ucp1 expression relative to cafsedp lep expression was significantly elevatedby cafeteria diet consumption f p while lepr expression was increased with exercise overall f p no significantdifferences wereproinflammatorymarkers or adipoq gene expressionobservedinhypothalamic gene expression was analyzed to determine if weeks of moderate exercise was sufficient toreverse dietinduced changes in expression of genes involved in feeding fig 3b blood brain barrier integrityand proinflammatory signaling fig 3c a significantinteraction effect was observed for npy gene expressionin the hypothalamus f p andthsd showed that cafsed rats exhibited downregulatednpy relative to csed p cex p andcafex rats p no significant differences wereobserved for pomc agrp npy1r lepr or insr crh geneexpression was significantly increased in cafeteriafedrats overall f p but was unaffected by exercise f p posthocthsd analysis revealed that cafex rats exhibited significantly upregulated crh relative to csed p andcex p both cln5 and glut1 were significantly increased incafeteriafed rats overall f p andf p respectively while no groupdifferences were apparent for cln5 using thsd comparisons glut1 expression was significantly elevated incafsed rats relative to csed controls p no significant differences were observed in ocln tjp1 or anyof the proinflammatory genes assessedassociations between variables of interest andmicrobiomewhen variables were assessed for their unique contribution to the variance in overall microbiome compositionseveral adiposity measures as well as retroperitonealwat il6 gene expression and hypothalamic crh andnpy expression were identified as significant predictorsof global microbiomecomposition supplementarytable when the contribution of variables of interestto overall microbiome composition was assessed whilecontrolling for diet and treadmill exercise both livertriglyceride concentration r2 p andretroperitoneal wat il6 gene expression r2 p were significant predictors of microbiomecomposition attable completemodel predicts of the variance in microbiomecompositionthe otu level 0cleigh nutrition metabolism page of fig gene expression in retroperitoneal wat hypothalamus and associations with gut microbiome changes a adipokine metabolic andinflammatory gene expression in retroperitoneal wat b feeding and stressrelated gene expression in the hypothalamus c bloodbrain barrierand proinflammatory gene expression in the hypothalamus adipoq adiponectin agrp agoutirelated protein cln5 claudin5 crh corticotrophinreleasing hormone glut1 glucose transporter il6 interleukin il10 interleukin10 il1b interleukin beta insr insulin receptor lep leptin leprleptin receptor npy neuropeptide y npy1r neuropeptide y receptor ocln occludin pomc proopiomelanocortin tjp1 tight junction protein tnf tumour necrosis factor ucp1 uncoupling protein data expressed as mean ± sem n data were analyzed by twoway anovafollowed by tukeyadjusted posthoc testing ap relative to csed bp relative to cex cp relative to cafsedporphyromonadaceae unclassified_otu106 wasincreased with cafeteria diet fig 4a h p and significantly associated with wat il6 geneexpression fig 4b interestingly this otu was alsocorrelated with hypothalamic npy genenegativelyexpression fig 4c il6 protein in wat exhibited asignificant interaction effect f p fig 4d and was positively associated with porphyromonadaceae unclassified_otu106 fig 4efollowing asimilar although nonsignificant trend to that observedwith il6 gene expression like il6 gene expression watil6 content was a significant independent predictor ofoverall microbiome composition supplementary table but was not a significant predictor in the final model 0cleigh nutrition metabolism page of table the shared contributions of diet exercise and variables of biological relevance on the variance observed in microbiomecomposition using distancebased linear modellingvariabledietpseudofpvaluessr2cumulative r2exerciseliver triglyceride contentwat il6 expressionsequential multiple regression captured by the braycurtis similarity matrix at the otu level max permutations involves interrogating the conditionalcontribution of each variable in order of entry into the model to determine whether variables contribute significantly to the variance explained in the presence ofother variables here diet and exercise conditions were added before any metabolic predictors were considered and the final model containing only statisticallysignificant covariates is shown metabolic predictors included in the sequential regression were selected based on their predictive value while trying to eliminatevariables with high covariance n after controlling for diet and exercise sina alignerputatively identified porphyromonadaceae unclassified_otu106 as a strain of bacteroides eggerthii a gramnegative bacterium known to hydrolyze carbohydratesincluding simple sugars discussionwe found that weeks of moderate treadmill exercisereduced fat mass and plasma leptin concentrations andaltered wat expression ofsome adipokine andmetabolismassociated genes overall microbiome composition and microbial species diversity was changed bycafeteria diet but not by exercise however predictedmicrobial functions associated with metabolism were increased by exercise cafeteria dietinduced changes inhypothalamic npy and glut1 gene expression werereturned to control levels by exerciseexercise induced modest changes in gut microbiomecomposition that were statistically significant in chowfed rats only and the relative abundance of otu72 wassignificantly reduced in chowfed rats that exercisedcex rats relative to sedentary controls csed whilecafex rats exhibited reduced microbial species diversitythis reduction appeared dependent on cafeteria dietexposure rather than exercise to date work examiningthe effects of exercise on microbial species diversity hasproduced inconsistent results some rodent studieshave shown that exercise is associated with reductions infecal microbial species richness while others havereported no such effect after weeks of treadmill exercise we did not observechanges in overall microbiome composition our dataare in line with findings in humans showing thatchanges in microbiome composition are dependent onobesity status such that more severe obesity is associated with smaller effects of exercise since thecafeteria diet used here tends to produce a more severe metabolic phenotype than purified highfat diets[] which to our knowledge have been used in allstudies investigating the interrelationship between exercise and diet on microbiome compositionthe dietinduced effects on microbiome composition here may bemore resistant to the effects of exercise than previouslyreported additionally a number of rodent studies report no differences in overall microbiome compositionwith exercise [ ] and there is evidence that this effect may be moderated by age which may have contributed to the inconsistent findings in the literaturepredicted microbial functions associated with metabolism specifically overall energy metabolism amino acidmetabolism one carbon pool by folate and dglutamineand dglutamate metabolism were increased in exercised rats this is in line with metagenomic resultswhere fecal microbiome from male elite athletes exhibited increased amino acid biosynthesis and overall energy metabolism relative to sedentary normalweightcontrols while picrust analysis produces predicted functional data unlike metagenomic analysis thisis an interesting finding that warrants followup to determine if and how exercise shifts the metabolic profileof the gut microbiome and whether any such shift is affected by exercise intensity and duration furthermoreconfirming these results across a range of diets would beuseful to determine whether the shift in microbial function with exercise is modulated by the macro andmicronutrients availablehere a moderate exercise intervention reduced fatmass and plasma leptin concentrations and increasedwat lepr gene expression in both exercised groupsand ucp1 gene expression in exercised cafeteriafedcafex rats uniquely increased ucp1 in wat depots isa marker of adipocyte beiging known to be promoted by exercise and is most likely related toexerciseinduced fat loss while there were no significanteffects of exercise on wat proinflammatory gene expression after controlling for diet and exercise wat il6expression wassignificantly associated with globalmicrobiome compositionwat is one of the major sources of il6 in obesehumans and mice [ ] which is a key component of the lowgrade systemic inflammation observedin overweight and obesity and is associated with 0cleigh nutrition metabolism page of fig relationship between otu106 and hypothalamic npy and wat il6 expression a relative abundance of otu106 data expressed as boxandwhisker plots min iqr max n data were analyzed using kruskalwallis test followed by nonparametric dunnbonferroni posthoctesting scatterplots for otu106 abundance and b il6 gene expression in wat and c npy gene expression in the hypothalamus showingoverall lines of best fit n d wat il6 protein content data expressed as mean ± sem n data were analyzed by twoway anovafollowed by tukeyadjusted posthoc testing e scatterplot for otu106 abundance and il6 protein content in wat showing overall lines of bestfit n ap relative to csed bp relative to cex cp relative to cafsedinsulin resistance wat il6 expression was stronglyassociated with the relative abundance of a strain ofbacteroides eggerthii otu106 a gramnegative sugarscavenging bacterium which is enriched in obesechildren relative to normalweight controls theassociations between wat il6 expression global microbiome composition and otu106 abundance are therefore likely to be due to the effects of cafeteria diet onboth adipose inflammatory processes and the gut microbiome however probiotic treatment with a strain of 0cleigh nutrition metabolism page of bifidobacterium in mice fed a highfat diet reducedwat macrophage infiltration and plasma il6 concentration indicating that changes to the gut microbiome may contribute to wat inflammatory signalingand il6 production furtherstudies determiningwhether specific bacterial species can modulate watil6 production are warranted as interventions thatcould reduce wat il6 expression in obesity may provide an avenue for preventing insulin resistance and type diabetesin contrast to the overall effect of exercise on watgene expression hypothalamic genes disrupted by cafeteria diet were typically normalized with exercise withno differences observed in chowfed rats npy wasdownregulated in cafsed rats as shown previously but normalized to control levels with exercise which isin line with other rodent work showing increased hypothalamic npy mrna and protein in response to bothacute and chronic exercise [] hypothalamic glut1gene expression was increased in cafsed rats and reduced to control levels with exercise this is in contrastto studies showing that acute exercise increased glut1protein expression across the rat brain and prolonged exercise increases whole brain resting glucoseuptake in people further work investigating acuteand chronic exerciseinduced changes in glut1 expression in the hypothalamus and other brain regions isrequiredactivatescafeteria dietinduced crh upregulation in the hypothalam | Colon_Cancer |
"gut microbiota composition influences the balance between human health and disease increasing evidencesuggests the involvement of microbial factors in regulating cancer development progression and therapeuticresponse distinct microbial species have been implicated in modulating gut environment and architecture thataffects cancer therapy outcomes while some microbial species offer enhanced cancer therapy response othersdiminish cancer treatment efficacy in addition use of antibiotics often to minimize infection risks in cancer causesintestinal dysbiosis and proves detrimental in this review we discuss the role of gut microbiota in cancerdevelopment and therapy we also provide insights into future strategies to manipulate the microbiome and gutepithelial barrier to augment therapeutic responses while minimizing toxicity or infection riskskeywords intestinal dysbiosis cancer development cancer therapy microbial therapy human intestinal microbiota is essentialfor microbialhomeostasis regulation of metabolism and immune tolerance intestinal dysbiosis occurs when there are alteredratios of healthy microbial flora along with changes in theirdiversity and density such changes may lower mucus layerthickness reduce antimicrobial defense and disrupt theepithelial tightjunction barriers to allow increased translocation of intestinal bacteria and bacterial products intothe systemic circulation and trigger inflammation andimmune responses circulating bacterial products such asendotoxin genotoxin and trimethylamine oxide have beenimplicated in many human disorders including metabolicsyndrome cardiovascular complications atherosclerosisand thrombosis and various neoplastic conditions intestinal dysbiosis may also affect adaptive immunity by correspondence seahhlimyahoocom1division of hematology and oncology suny downstate health sciencesuniversity clarkson avenue room b5495 brooklyn new york usa2division of hematology and oncology department of medicine new yorkmedical college valhalla new york usamodulating the functions of t lymphocytes and promotingtumor immune escapewhile increased translocation of intestinal luminal content is associated with carcinogenesis and poor therapeuticresponse the causeeffect relationship is often bidirectionalin this review we will discuss the role of gut microbes inmodulating tumor immunity intestinal permeability andcancer development next we will highlight the effects ofintestinal dysbiosis and increased permeability in cancertherapy finally we will explore the options to improve guthealth to enhance the efficacy of cancer therapyintestinal immunity and permeabilitythe intestinal architecture and microbiota regulateinnate and adaptive immunity disruption of the architecture andor microbiota affects these functions therelationships between the different players in the intestinal microenvironment is summarized in fig the composition of microbes in the gut dictatesmucus layer thickness and production of antimicrobialsignals in germfree mice mucus layer and effector t the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cdutta and lim biomarker research page of fig interplay between different factors involved in gut immunity and permeability a the intestinal epithelial cells containing paneth cellsgoblet cells enterocytes and enteroendocrine cells coordinate with intraepithelial lymphocytes to generate a functional immune responsepaneth cells secrete antimicrobial peptides and goblet cells produce mucus to cover the epithelial layer this mucus layer prevents adhesion ofmicrobes to the epithelial cells lamina propria situated under the mucus layer contains peyers patches and immune cells including antigenpresenting cells apcs like dendritic cells dcs t cells and b cells pattern recognition receptors prrs such as tolllike receptors tlrs onepithelial cells interact with microbederived pathogenassociatedmolecular patterns pamps such as lipopolysaccharide lps to activatemyd88dependent signaling dcs travel to mesenteric lymph nodes mln and promote the differentiation of na¯ve t cells to regulatory t tregcells that migrate to other sites treg cells secrete il10 to elicit an antiinflammatory response b dysbiosis decreases mucus layer thickness andshortchain fatty acid scfas production this affects the secretion of antimicrobial peptides and allows microbes to come in close proximity tothe epithelial cells reduction in scfas influences gut barrier dysfunction as a result the gut luminal content also translocated and spreadedthrough the systemic circulation to trigger local and systemic immune responses in addition to pamps damps released from damaged intestinalepithelium interact with prrs to facilitate expression of macrophages and maturation of dcs mature dcs promote the differentiation of na¯ve tcells to effector t cells such as t helper cells th1 th2 th17 th1 release tnfα and ifnÎ and th17 secrete il17 to recruit polymorphonuclearneutrophils pmns these cytokines create a proinflammatory condition 0cdutta and lim biomarker research page of cells are absent [ ] microbes secrete shortchain fattyacids scfas such as propionate and butyrate thatprevent microbial binding to the epithelial cells and helpmaintain barrier function and immune homeostasisbutyrate promotes tightjunction formation [ ] andactivates peroxisome proliferatoractivated receptor gammapparÎto enhance epithelial oxygen consumptionresulting in reduced emanation of oxygen from the mucosalsurface it helps in maintaining an anaerobic condition inthe gut lumen needed for colonization of obligate anaerobes this intestinal microenvironment determines thecomposition of resident bacterial species for example onlyclostridium lactobacillus and enterococcus are enrichedon the epithelial surface and in the mucus layer whereasbacteroides bifidobacterium streptococcus enterobacteriaceae enterococcus clostridium and lactobacillus are allpredominant in the intestinal lumen dysbiosis increases inflammatory signals that shiftthe metabolism of enterocytes epithelial hypoxia iseliminated and increased oxygenation results in therelease of more oxygen from the mucosal surfacesince only facultative anaerobes can respire oxygendysbiosisinduced shift in epithelial oxygenation altersgut microbial community from obligate to facultativeanaerobes intestinal pathogens such as proteobacteria produce genotoxins like colibactin and cytolethaldistending toxin cdt to induce inflammation andhost deoxyribonucleic acid dna damage that initiates tumor formation dysbiosis also decreasesmucus layer thickness reduces scfa production anddamages mucosal barrier allowing pathogenassociatedmolecular patterns pamps to interact with pattern recognition receptors prrs and activate tolllike receptortlr 24myeloid differentiation primary response protein myd88 signaling pathways in addition changes inmicrobial composition and density triggers epithelial releaseof damageassociated molecular patterns damps such asextracellular adenosine triphosphate atp cytoplasmiccalreticulin high mobility group box hmgb1 proteinsendogenous nucleic acids and intracellular proteins tointeract with prrs prr engagementtriggers a proinflammatory condition that causes tissue damage and localinflammation microbiotadriven tlr immune signalinghas been implicated in cancer formation and modificationof treatment efficacy [] for example cpg oligodeoxynucleotides that mimic bacterial dna acts as a pamp totrigger a tlr9dependent tlr4 activation and tumor necrosis factor tnfα production by tumorinfiltratingmyeloidderived cells mice bearing el4 lymphomamc38 colon carcinoma and b16 melanoma when treatedwith cpg oligodeoxynucleotides show reduced tumrowth and enhanced survival rate the beneficial effects ofcpg oligodeoxynucleotides were positively associated withthe abundance of alistipes shaii in the gut effects of intestinal microbiota on cancerdevelopmentintestinal microbes can influence local and distantcarcinogenesis through infection and microbial productsor by modulating tumor immunosurveillance this isaccomplished via altering the balance between the rateof cell proliferation and apoptosis triggering chronicinflammation andor immunosuppression or changingthe metabolism of the products produced by host andmicrobes in this section we will discuss how intestinaldysbiosisrelated permeability may contribute to tumorigenesis in different anscolorectal cancerfusobacterium nucleatum a gramnegative mucosaadherent anaerobic bacteria has been implicated in theinitiation and progression of colorectal cancer crc[ ] fada an adhesion molecule on f nucleatumbinds to host ecadherin to enter epithelial cells this activates the wntβcatenin pathway leading toan increased secretion of inflammatory cytokines including il6 il8 and tnfα and upregulation of nuclearfactor kappa light chain enhancer of activated b cellsnfκb that facilitates crc development in addition itattracts myeloidderived suppressor cells and the autotransporter protein fap2 interacts with the human inhibitoryreceptor t cellimmunoreceptor with ig and itimdomains tigit to create a tumor immunosuppressivemicroenvironment f nucleatum may also induce chemoresistance by modulating the tlr4myd88 signalingpathway following 5fluoruracil treatment in crc patients an increased abundance of f nucleatum along with clostridium difficile and species ofstreptococcus campylobacter and leptotrichia has beendemonstrated in tumor tissue and fecal materials []f nucleatummediated colorectal carcinogenicity occursdownstream of apc introduction of f nucleatum resultedin rapid onset of colonic tumors in mice deficient in onecopy of adenoma polyposis coli apc apcmin gene both intestinal dysbiosis and loss of apc disruptepithelial tightjunctions and mucus layer [ ] andallow increased infiltration of f nucleatum and other nonresidential microbes to drive crc development the roleof defective gut barrier in crc has been confirmed inmucin 2knockout muc2 mice in which the lack ofgastrointestinal mucin resulted in spontaneous crc development therefore dysbiosisinduced gut permeabilitymay play an important role in tissue enrichment of fnucleatum and increased risks for crchepatobiliary cancerthe liver is chronically exposed to intestinal microbiotaand its products via the portal vein intestinal dysbiosisand increased permeability enhance translocation of gut 0cdutta and lim biomarker research page of microbiota to trigger inflammation and chronic liver disease that predisposes patients to the development of hepatocellular cancer alteration in bile acid metabolism due tochanges in clostridium spp suppress anticancer immunity in mice eradication of grampositive bacteria by oralvancomycin inhibits secondary bile acid conversion resulting in the upregulation of chemokine cxc motif ligandcxcl16 in liver sinusoidal endothelial cells cxcr16recruits natural killer t nkt cells in the tumor microenvironment and kill tumor cells in a cd1ddependentmanner in addition gut microbiotaderived lipopolysaccharides lps promote tumor progression in liver cancerby activating the tlr4 signaling in a study involving cholangiocarcinoma patients bile ducttissues haddistinct dominance of dietziaceae pseudomonadaceae andoxalobacteraceae members pancreatic cancergut microbiota influences the development of pancreaticcancer through activating tlr4 signaling the stromain pancreatic tumor harbors an abundance of microbiotaespecially bifidobacterium pseudolongum compared tonormal pancreas this helps in creating an immunosuppressive environment by differentially activating distincttlrs in monocytes pancreatic adenocarcinoma has anenrichment of proteobacteria synergistetes and euryarchaeota longer survival is observed in patients with amore diverse intratumor microbial composition primarilyof sachharopolyspora pseudoxanthomonas streptomycesand bacillus clausii tumoral colonization with mycoplasma hyorhinis and gammaproteobacteria is associatedwith gemcitabine resistance antibiotics diminishmyeloidderived suppressor cells and increase antitumorm1 macrophages to promote th1 differentiation of cd4t cells and cd8 t cell activation in the tumor cotreatment of gemcitabine with ciprofloxacin abrogatedgammaproteobacteriainduced chemotherapy resistance the efficacy of immune checkpoint inhibitors icistherapy is also enhanced by antibiotics lung cancerwhile local microbiota is important there are reportsthat gut microbiome may also contribute to lung cancerdevelopment lung cancer patients demonstrated an abundance in intestinal enterococcus and depletion in bifidobacterium and actinobacteria they are also enrichedwith veillonella bacteroides and fusobacterium depletedof dialister enterobacter escherichiashigella fecalibacterium and kluyvera in nonsmall celllung cancernsclc patients butyrate producers such as faecalibacterium prausnitzii clostridium leptum clostridial cluster iruminococcus spp clostridial cluster xiva and roseburiaspp were significantly reduced since butyrate isessential for preserving mucosal homeostasis reduction ofintestinal butyrate producers may imply a compromised intestinal barrier in these patientshematologic malignanciesdysbiosisinduced intestinal permeability affects mucosaassociated lymphoid tissue malt and plays a significantrole in hematologic malignancies composition of intestinalmicrobiota is responsible for maintaining the pool of bonemarrow myeloid cells preleukemic myeloproliferationis driven by microbial signals in teneleven translocation2tet2deficient mice [ ] these mice show increasedinfiltration of inflammatory cells disrupted mucosal barrierand increased translocation of bacteria [ ] it wassuggested that dysfunction of small intestinal barrier andleakage of microbes can occur due to tet2 mutation in occurrence of tet2hematopoietic compartmentmutation intestinal dysbiosis and leaky gut is common inleukemia and lymphomaacute myeloid leukemia aml and acute lymphoblasticleukemia all patients have a compromised intestinalbarrier [] fecal microbiota in all patients showedlower microbial diversity they were enriched in enterococcaceae porphyromonadaceae and bacteroidetes mainlyb fragilis and depleted in blautia erysipelotrichiales lachnospiraceae and clostridiales members [ ] abundanceof staphylococcaceae and streptococcaceae have also beenreported in pediatric all and adult aml [ ]helicobacter pylori is associated to malt lymphoma and chamydophila psittacito ocular maltlymphoma while borrelia burgdorferi was linked tocutaneous bcell nonhodgkin lymphoma two studiesdid not find significant risk of borrelia burgdorferi in thedevelopment of nonhodgkin lymphoma [ ] abundance of proteobacteria is a predictor for neutropenicfever and enrichments of enterococcaceae and streptococcaceae are strong predictors of infectious complications inall similarly higher gut microbiota diversity inmultiple myeloma is associated with reduced risk fordisease relapse all patients with infectious complications have an abundance of brevundimonas diminuta andagrobacterium tumefaciens whereas faecalibacteriumprausnitzii producer of scfas is completely absent similar findings have been reported in nonhodgkinlymphoma with infectious complications effects of intestinal microbiota on cancer therapythe efficacy of cancer treatment is in parts dependenton normal immune function since gut microbiota playsa crucial role in modulating immune response it is notsurprising that dysbiosis affects treatment outcomesprophylactic antibiotics are commonly used for cancerpatients undergoingallogeneichematopoietic stem cell transplantation allohsct toreduce the risk of neutropeniaassociated infectionchemotherapyand 0cdutta and lim biomarker research page of however antibiotic use causes intestinal dysbiosis thatresults in negative outcomes including poor treatmentresponse and toxicity and the development of clostridium difficile infection cdi in addition to antibioticsopioid analgesics for cancer pain management may alsotrigger dysbiosis opioid analgesics impair intestinal motility and promote bacterial overgrowth resulting in dysbiosisand gut permeability intestinal dysbiosis induces mucosal injury and triggers the release of damps damps have a dual andbidirectional effect on cancer although damps exertimmunosurveillance and immunemediated cell death toeliminate tumor cells and protect against cancer development chronic inflammation induced by damps maypromote tumor initiation damps released by apoptoticcells from cancer therapy may also induce chemoresistance and promote metastasis for example tlr78expressed on tumor cells may bind damps loxoribinefor tlr7 and poly u for tlr8 and promote chemoresistance through the activation of nfκb and the upregulation of bcl2 damps may also activate tlr9on human breast prostate and lung cancer cells to trigger tumor invasion and metastasis [ ] given theclinical significance of dysbiosismediated mucosal injuryand permeability in cancer we will in this section discusshow the treatment outcome by various cancer therapymay be affected by intestinal microflora and permeabilitychemotherapy and radiation therapyintestinal microbial composition and mucosal barrier function influence chemotherapeutic outcome and the effect isbidirectional while dysbiosis can exacerbate chemotherapy drug toxicity and reduce its efficacy chemotherapy canitself cause dysbiosis although prevalence of certain intestinal microbes in the gastrointestinal tract offer beneficialeffects others contribute to chemoresistance and drug toxicity this multiplepathway effect is best covered by timer mechanisms translocation of microbes immunomodulation metabolism and enzymatic effects on drugsand reduced microbial diversity these mechanistic effectsalter chemotherapy efficacy and toxicity and risks forinfections for example translocation of microbes due tochemotherapy induceddysbiosis and disruption of mucosal barrier can increase the risk of infection howevercertain chemotherapy drugs such as cyclophosphamideand doxorubicin damage intestinal barrier for the translocation of commensal bacteria into secondary lymphnodes to elicit antitumor immune response vancomycin prophylaxis inhibits antitumor effects of cyclophosphamide in fibrosarcoma inoculated mice irinotecanused for crc treatment is transformed into its active formsn38 by tissue carboxylesterase it is detoxified in theliver by host udpglucuronosyltransferases into inactiveglucuronide sn38g and excreted into the gut via bileducts in the gut bacterial βglucuronidases reconvertssn38g into active sn38 which causes severe intestinaltoxicity and diarrhea streptomycin inhibits irinotecanabsorption and reduces epithelial carboxylesterase activityand diarrhea ciprofloxacin inhibit βglucuronidases and low dose amoxapine βglucuronidases inhibitorsuppress irinotecanassociated diarrhea in rats table provides a selection of chemotherapeutic agents affectingand affected by intestinal microbial composition andpermeabilitylocal pelvic irradiation damages intestinal epitheliumand barrier integrity and produce reactive oxygen speciesirradiation increase alistipes and decrease prevotella inmice in gynecologic cancer patients receiving pelvicradiotherapy firmicutes and fusobacterium were significantly decreased in addition to reduced diversitysignificant enrichment of clostridium iv roseburia andphascolarctobacterium was associated with radiationenteropathy in pelvic cancer patients the effects oftotal body irradiation which is a preparative regimen forallohsct that causes dysbiosis and gastrointestinaltoxicity is discussed in more details in the allohsctsection belowimmunotherapycancer cells often create an immunosuppressive microenvironment to mediate tumor immune escape this immune escape mechanism may be reversed by icis directedat cytotoxic t lymphocyteassociated antigen ctla4programmed death receptor pd1 or pd1 ligandspdl1 since intestinal microbes influence local and systemic antitumor immune reaction by modulating prrspamps and dampsintestinal dysbiosis may impacttreatment outcome figure illustrates how the potentialmechanisms ofthe antitumor immune responses aredownregulated by intestinal dysbiosis the effects of intestinal microbiome on responses to icis have been discussed previously [ ] broadspectrum antibioticsbefore during or after icis therapy alter intestinal microbiome and resulted in lower tumor response rate inferiorprogressionfree survival and reduced overall survival responses to inhibition of ctla4 by ipilimumab inmouse models of mca205 sarcoma ret melanomaand mc38 colon carcinoma were inferior in germfreeor in broadspectrum antibiotic treated mice poorresponses were associated with decrease in intestinalbacteroides thetaiotaomicron bacteroides uniformis andburkholderia cepacia and increase in clostridiales suchdysbiosis was also associated with mucosal damage andcolitis oral feeding with either bacteroides thetaiotaomicron or bacteroides fragilis individually or with acombination of bacteroides fragilis and burkholderiacepacia restored the antitumor effects of ctla4 blockade through augmentation of th1 responses in tumor 0cdutta and lim biomarker research page of table selection of chemotherapeutic agents and the bidirectional effects between the chemotherapy and intestinal microbiotachemotherapy drugcisplatintoxicity infectioncdi ototoxicity effects on gut changes in microbiotadamages mucosal barrier by impairing dnareplication of rapidly proliferating epithelialcells facilitates translocation of gut bacteriacommensal gut bacteria influencesgenotoxicity by inducing reactive oxygenspecies ros production and recruitment ofpathogenic th17 cells in the tumormicroenvironment independently ofimmunity elicited by immunogenic celldeath microbial interventionantibiotics against grampositive bacteriaabrogate antitumor chemotoxicityincrease tumor size and decrease survivalratecisplatin alone show better responsecompared to a combined treatment ofcisplatin and antibiotics in mice with lungcancer the combination treatmentincreased tumor size and decreasedsurvival ratelactobacillus acidophilus restores antitumorefficacy following antibiotic treatment[ ]restoration of gut microbiota andepithelial integrity by fmt andtreatment with dmethionine [ ]prevent infections and ototoxicity withoutaffecting tumor chemotoxicityfmt increases a muciniphilaabundance and reduces cipn oral butyrate supplementation improvesgut barrier by reducing inflammation andmucositis antibiotics reduce mucositis and cytokineproduction but also diminish antitumorefficacy and promote chemotherapyresistance antibiotics against grampositive bacteriareduce th17 responses and subsequentdevelopment of cyclophosphamideresistancereestablishment of e hirae alone restoresantitumor activity e hirae decreases tumorinfiltrating tregsbarnesiella intestinihominis accumulates inthe colon and increases the number ofintratumoral ifnÎproducing Îδt cellse hirae and b intestinihominissynergistically stimulate local and systemicimmunity to improve anticancer effects nod1nod2 mice having abundant bintestinihominis demonstrate increased Îδtcells in tumor beds and enhancedcyclophosphamide efficacy paclitaxel5fluoruracilincreases gut permeability as indicated by5fold elevation in circulating lpsbindingprotein and systemic inflammation reduces abundance of roseburiaporphyromonadaceae and akkermanisamuciniphila [ ]reduces clostridium spp and increasesmembers of proteobacteria mainlyenterobacteriaceae damages mucosal barrierchemotherapyinducedperipheral neuropathicpain cipn cdi [ ]mucositis along the entiregastrointestinal tract cdi [ ]cyclophosphamidecdi triggers disruption of gut barrier by alteringbacterial compositiongrampositive bacteria such asenterococcus hirae lactobacillus johnsoniiand l murinus translocate from gut intomesenteric lymph nodes and spleen this enhances immune responses by theproduction of interferon gamma ifnÎ andactivation of th17 cellsdraining lymph nodes and promotion of maturation oftheintratumoral dendritic cells dcscombination treatment of bacteroidesandburkholderia cepacia prevented intestinal damage andrefractory colitisin additionfragilisfecal microbiota analysis of melanoma patients beforeand after ipilimumab treatment showed a change in therelative proportions ofenterotypeclusters cluster a was dominated by prevotella spwhereas clusters b and c by different bacteroides sppfecal microbiota transplantation fmt from patientsinto tumorbearing germfree mice showed that onlyfecal material from cluster c resulted in colonizationthree dominantwith bacteroides thetaiotaomicron or bacteroides fragilisand enhanced ipilimumab response in another study ofipilimumab in mice vancomycin treatment resulted in amore severe manifestation of colitis whereas oraladministration of bifidobacterium ameliorated the sideeffects similarly melanoma patients with increasedabundance of bacteroidaceae rikenellaceae and barnesiellaceae members responded better to ctla4 antibodies however a different study in ipilimumabtreated melanoma patients found that bacteroides spp were associatedwith decreased response whereas faecalibacterium andother firmicutes members improved clinical outcome 0cdutta and lim biomarker research page of fig potential antitumor immune mechanisms induced by intestinal dysbiosis a in the presence of intact mucosal barrier and signals fromcommensal microbiota effector t cell activation is modulated by t cell receptor tcr ligation with major histocompatibility complex mhc classi and costimulation of cd80cd86 and cd28 binding of cytotoxic t lymphocyteassociated antigen ctla4 receptor to antictla4 antibodyon treg impairs its effector tcell inhibitory function it also downregulates ctla4 expression on apc ligation of repressive receptorprogrammed death receptor pd1 and its ligand pdl1 to antipd1 and antipdl1 antibodies respectively activate effector tcell proliferationand function activated effector t cells interact with tumor cells and release cytokines to induce tumor cell death b signals from unfavorablemicrobes due to dysbiosis upregulates ctla4 pd1 and pdl1 expression to inhibit tcell activation ctla4 on treg binds to cd80cd86 onantigen presenting cell apc cd80cd86 on apc also disengages from cd28 and binds to ctla4 on effector t cells pdl1 the ligand of pd1is expressed on antigen presenting cell apc and tumor cells pd1 on effector t cells ligates to pdl1 on apc and tumor cells these activitiesinhibit effector tcell activation reduces immune checkpoint inhibitor ici efficacy and causes tumor escape 0cdutta and lim biomarker research page of patients with higher abundance of faecalibacteriumand improved response to ctla4 antibodies showedhigher incidence of enterocolitis and lower level of treg inperipheral blood thus the beneficial effects of specificand isolated gut microbes may depend on the commensalassociation with other microbial species and may differ between humans and micepd1 blockade may also be modulated by intestinalmicrobiota melanoma patients who responded to pd1blockade had increased abundance of enterococcus faeciumcollinsella aerofaciens bifidobacterium adolescentis klebsiella pneumoniae veillonella parvula parabacteroidesmerdae lactobacillus sp and bifidobacterium longumwhereas in nonresponders the intestinal microbiome wasenriched in ruminococcus obeum and roseburia intestinalis another study found higher abundance of faecalibacterium species in responders and enrichment with bacteroides thetaiotaomicron escherichia coli and anaerotruncuscolihominis in nonresponders clinically nonsmallcell lung cancer nsclc and renal cell carcinoma rccpatients experienced increased resistance to pd1 blockadeafter antibiotic treatment these patients had shorterprogressionfree survival as well as overall survival in thisstudy response to pd1 blockade correlated with higherfecal abundance of akkermansia muciniphila fmt fromresponders to germfree or antibiotictreated mice improved the outcome of pd1 blockade administration ofa muciniphila after fmt from nonresponders restoredresponsesimilarly intestinal microbiota may influence the outcome of chimeric antigen receptor t cell car t therapypatients with complete response to cd19 car ttherapyexhibited enrichment of oscillospiraceae ruminococcacaeae and lachnospiraceae in their intestinal microbiomewhereas patients who did not attain a complete responseshowed increased abundance of peptostreptococcaceae effectiveallogenic hematopoietic stem cell transplantationalthough allohsct issomein treatinghematological malignancies the immunosuppressive agentsbroadspectrum antibiotics and chemoradiation used withthe transplant often induce intestinal dysbiosis gut permeability and impaired systemic immune response highermicrobiota diversity is associated with longterm survivaland lower diversity in gut microflora is associated with reduced overall survival and higher transplantrelated mortalityfollowing allohsct [ ] severe infections that occurdue to intestinal dysbiosis such as cdi and vancomycinresistant enterococci vre infections are also associatedwith higher treatmentrelated mortality [] allohsctdisrupts the equilibrium of bacterial composition in feceswith a dominance of enterococcus streptococcus and proteobacteria members [ ] and reduces beneficial bacteriasuch as faecalibacterium and ruminococcus higherabundance of blautia was found to be associated with improved overall survival moreover allohsct patientswith reduced risk of relapse had an enrichment of eubacterium limosum ofgraftversushost diseaseone of the major complications of allohsct is thedevelopmentgvhdoccurrence of cdi during allohsct increases the riskof gvhd besides the loss of overall microbial diversityreduction in beneficial faecalibacterium blautia lactobacillus and ruminococcus and increased abundance ofenterococcus and clostridiales was observed in gvhd[ ] patients without gvhd had increasedabundance of parabacteroides and bacteroides in theirpretransplant feces in a preclinical study reducedgvhd and improved overall survival was observed afterthe administration of the probiotics lactobacillus rhamnosus gg alone or in combination with ciprofloxacindue to the preservation of gut mucosal integrity in therecipient mice restoration of normalintestinalmicrobiome by fmt has been found to benefit patientswith steroidrefractory gvhd [ ] multipleclinical trials are currently ongoing to investigate howmanipulation of gut microbiota using dietary intervention and fmt might reduce the risk of gvhdmanipulation of intestinal microbiome and barrierto improve outcome of cancer therapeuticsifintestinal dysbiosis and its associated increased gutpermeability are associated with cancer development andtherapyrelated complications and treatment outcomes itfollows that intervention of the intestinal microbiomeandor gut barrier may alter cancer outcome in thissection we will explore three broad approaches fig that might be investigated nonselective modificationof intestinal microbiome using fmt semiselectivemodification of intestinal microbiome using antibioticsand biologic modification of intestinal barrier we willdiscuss the challenges and obstacles each of the approaches may encounternonselective modification of intestinal microbiome usingfmtmodification of the intestinal microbiome is theoreticallybest accomplished by fmt unmanipulated fmt will notonly replete the dysbiotic intesti | Colon_Cancer |
" among eukaryotic anisms alternative splicing is an important process that can generate multipletranscripts from one same precursor messenger rna which greatly increase transcriptome and proteome diversitythis process is carried out by a superprotein complex defined as the spliceosome specifically splicing factor branchpoint binding protein sf1bbp is a single protein that can bind to the intronic branchpoint sequence bpsconnecting the ² and ² splice site binding complexes during early spliceosome assembly the molecular functionof this protein has been extensively investigated in yeast metazoa and mammals however its counterpart inplants has been seldomly reportedresults to this end we conducted a systematic characterization of the sf1 gene family across plant lineages inthis work a total of sequences from plant species were identified phylogenetic relationships of thesesequences were constructed and subsequent bioinformatic analysis suggested that this family likely originatedfrom an ancient gene transposition duplication event most plant species were shown to maintain a single copy ofthis gene furthermore an additional rna binding motif rrm existed in most members of this gene family incomparison to their animal and yeast counterparts indicating that their potential role was preserved in the plantlineage our analysis presents general features of the gene and protein structure of this splicing factor familyand will provide fundamental information for further functional studies in plantskeywords alternative splicing expression profile phylogenetics plants promoter splicing factor correspondence fyzhunjfueducn kailu zhang zhen feng jingfang yang and feng yang contributedequally to this work1coinnovation center for sustainable forestry in southern china college ofbiology and the environment nanjing forestry university nanjing jiangsu province chinafull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0czhang bmc plant biology page of in eukaryotes canonical splicing removes noncoding intronic sequences and assembles the coding elements intomature mrnas while alternative splicing as generatesdifferent multiple transcripts that encode proteins withdistinct structures and functions by differential usage ofexons or splice site [ ] the resulting transcripts ofas greatly contribute to posttranscriptional regulationbiological complexity and proteome diversity in eukaryotes [ ] given that on average there are approximately exons in each transcript in the humantranscriptome and the degenerative nature of corresponding splice sites premrna splicing is sophistically catalysed by the spliceosome spliceosome is amultimegadalton protein complex which consists offive u1 u2 u4 u5 and u6 small nuclear ribonucleoprotein ps snrnps and over spliceosomalproteins furthermore the early assembly of spliceosome complex e or the commitment complex is anatpindependent process and contains u1 snrnps sf1and u2 snrnp auxiliary factors u2af large and u2afsmallthe prespliceosome complex a is formed by replacing sf1 withsf3b155sap155 of u2 snrnps [ ] stepwiseassembly of the following spliceosome during the splicing reaction has been reported as well [ ] however splice site recognition is a critical step during earlyassembly of the spliceosome the current model describes the binding of u1 snrnp and u1 snrna to ashort stretch of nucleotides at the ² splice site of splicing factor sf1mammalian branch point bindingprotein mbbp at the branch point and of u2 snrnpauxiliary factors at the ² splice site these threeciselements are necessary but usually insufficient to define a specific exonintron boundary thus additionalsplicing enhancers or silencers located at exons and introns may allow the recognition of genuine splice sitesduring early spliceosome assembly [ ] subsequentlysubunitsimportantly sf1 preferentially binds to the intronbranch point sequence bps which is adjacent to thebinding site polypyrimidine tract py of u2af largesubunits mammal u2af65 and fission yeast u2af59bridging u1 and u2af to form an intermediate lariatstructure [ ] in particular sf1 is characterized bythe presence of two types of rna binding motifs at thenterminus a k homologyquaking khqua2 domain which originated from the human heterogeneousribonucleoprotein hnrnp k protein [ ] and oneor two zinc knuckle motifs cx2cx4hx4c x represents any amino acid sf1 also contains a prolinerichregion at cterminus [ ] intriguingly the yeast khdomain specifically binds to the bps of premrnas witha glyproarggly motif and the variable loop of thekh domain and is necessary for spliceosomeassembly the first but not the second zinc knuckledomain in yeast has been demonstrated to bind rnawith high affinity moreover the stability of thesf1u2af65rna complex is further affected by thephosphorylation status of several sf1 serine residuesser20 ser80 and ser82 in vitro the prolinerichregion of sf1 interacts with u1 snrnp prp40fbp11 inyeast and human [ ] in regards to its interactionpartner the u2af large subunit the nterminal of sf1interacts with its noncanonical rna recognition motifsrrm or u2af homology motif[ ]whereas the other two rrms of u2af large subunitbind to the py region uhmsaccharomycescerevisiae common fruita previous study in fission yeast schizosaccharomycespombe suggests that the initial corecognition of thebranch site and ² splice site is pivotal for correct splicing of target premrnas because of the importance of splice site recognition for gene expression andprotein diversity sf1 has been demonstrated to play essential roles in a number of eukaryotic species includinghuman homo sapiens mice mus musculus buddingyeastflydrosophila melanogaster and roundworm caenorhabditis elegans [ ] for example in humansmissense mutation of splicing factors which are responsible for splice site recognition such as sf1 has beenlinked to tumourigenesis similarly heterozygoussf1 knockdown mice are susceptible to colontumourigenesis induced by an anotrophic carcinogenazoxymethane and sf1 has been found to associatewith betacatenintcf4 complex suggesting its role incarcinogenesis in contrast knockdown of sf1 suppresses the development of germ cell tumours in mice indicating its tissue dependency in cancer researchfurthermore the molecular function of sf1 has been extensively studied in yeast for instance a sf1 mutantstrain causes frequent exon skipping in fission yeast additionally sf1 has been proposed to recognize suboptimal sequences in specific introns and lead to nuclearaccumulation of premrna with aberrant splicing however increasing evidence indicates that this proteinis a regulator of splice site recognition and does not reduce general splicing specifically during alternative splicing by targeting a subset of genes [ ] thishypothesis is supported by the fact that knockdown ofsf1 in both yeast and human extracts only slightly affects the splicing outcome rnai targeting of thisgene has been demonstrated to not affect the splicingpattern of several splicing marker genes tested in comparison to studies in human and yeast few reports have been published related to plant sf1 genessimilar functions of the arabidopsis sf1 gene were proposed in an early study in this plant sf1homologue is reportedly responsible for the splicing of a 0czhang bmc plant biology page of to maintain itsgroup of transcripts the lossoffunction mutant atsf1 of this gene leads to abnormal development earlyflowering and dwarfism and aba or heat stress sensitivity in arabidopsis [ ] subsequently the domainstructure and its functional relationships have been substantially investigated and the rrm domain is considered crucialfunction in plantsmoreover sf1 may have a different mechanism of ²splice site recognition in plant because the plant sf1 homologs contain a different rrm domain compared withfungal and metazoan counterparts [ ] on the otherhand a study found that atsf1 may be likely to play afunctional role in the cytoplasm because it was found toshuttle between the nucleus and cytoplasm however no related investigations have been conducted onthe phylogenetic analysis of plant sf1 genes and theirregulatory mechanisms although it is a highly conserved family and has conserved functions in eukaryotesplant sf1 genes may have overlapping and distinct rolescompared to the mammalian genes hence studying thephylogenetic relationship and regulatory mechanism ofplant sf1 genes may make us understand the evolutionary history characteristics an expression profile of thisgene family and predict specific functions in plants thiscan lay the foundation for further functional studies inviridiplantae to this end we systematically identified sf1 sequences from plant species ranging fromalgae to higher plants meanwhile the gene and proteinstructure potential regulation at promoter regions andexpression pattern of these genes were further investigated in this study we hypothesize that plant sf1 isstructurally different from its counterparts in animalsand yeast but it is conserved among lower and higherplants indicating its specific role in alternative splicingin branch point recognitionthalianasf1methodssequence acquisition and identification of plant sf1genesthe arabidopsisprotein sequenceat5g51300 was used to search similar sequences inall available plant species from the phytozome v121databasehttpsphytozomejgidoegovpzportalhtml by running the blastp program with an evaluecutoff 1e10 the other parameters were the default settings then the retrieved protein sequences wereexamined and filtered using the hmmer score defaultsettings which contained pf16275 splicing factorkhomology domain kh_1 and pf00076 rna recognition motif rrm_1 finally putative sf1 sequencesfrom plant species were identified detailed information including groups plant species common namesand number of sf1 homologs reported for each planthelixhairpinsf1hhdomainpf00013species for subsequent analysis are listed in table s1subcellular location prediction of identified sf1 proteinswas carried out using wolf psort httpswolfpsorthgcjp construction of molecular phylogenetic tree of plant sf1genesprotein sequences of the aforesaid plant sf1 genes wereextracted from phytozome v121 database for phylogenetic relationship analysis the sequences with the longestcoding sequences were chosen for genes with multipledifferent splicing isoforms then multiple sf1 proteinsequences were aligned with the muscle v38 softwarewith default settings the molecular phylogenetictree of plant sf1 genes was then constructed using themaximum likelihood method ml jtt g i modelvia phyml v30 program with the following parametersinitial tree bionj discrete gamma model yes numberof categories gamma shape parameter proportion of invariant subtree patterns aliasing no figtree v143 was used to visualize and edit the phylogenetic treegene structure protein domain and multiple em for motifelicitation meme analysisrequired genomic cdna and peptide sequences and allsf1 gene structures were downloaded from the phytozome v121 database corresponding intron phases weregenerated using the online program gene structure display server gsds20httpgsdscbipkueducn correlation analysis of sf1 exons were performedby using the piece2 webserver httpwwwbioinfogenomenetpiecesearchphp tdsourcetags_pctim_aiomsg sf1 protein sequences were used to search formatching pfam families using the hmmer websitehttpswwwebiacuktoolshmmer then protein domain patterns were drawn by using tbtools software according to the full pfam resultanttableconserved motifs of plant sf1 cdna sequences andprotein sequences were analysed on the meme onlineprogram httpmemesuitetoolsmeme considering a maximum of the most preserved motifs predicted for each sequence and leaving other settings onthe default parametersmotif prediction in promoter regions of plant sf1 genesthe 15kb ²flanking sequences of plant sf1 geneswere extracted from genomic data available in phytozome database prediction of plant putative ciselementswas performed with the online server plantcarehttpbioinformaticspsbugentbewebtoolsplantcarehtml motifs related to tissuespecific expressioninternal hormones and external environmental stress response were selected for further analysis and discussion 0czhang bmc plant biology page of expression analysis base on microarray datasets and geneexpression experimentsexpression data of arabidopsis s tuberosum g max slycopersicum p trichocarpa and b distachyon includingtissue specificity and stress responses were extracted fromthe efp browser series of the bioanalytic resource forplant biology httpbarutorontoca expressionvalues of selected plant sf1 genes were log transformedlg to generate visualize expression difference heatmapsby using bar heatmapper tool program httpbarutorontocantoolscgibinntools_heatmappercgigene expression experimentstotal rna of samples from different plant tissues wereextracted by rneasy mini kit qiagen usa and subsequently reversed transcribed into cdna by fastkinggdna dispelling rt supermix fastking tiangenchina according to the manufacturers instruction rtpcr amplification were programmed asfollowings °c min °c s °c s °c s cycles °c min sybr premix ex taqtm accuratebiotechnology co ltd hunan china was used forquantitative realtime rtpcr analysis which was conducted on the stepone plus realtime pcr system following optimized program °c s °c s °c s cycles the data were normalized to the expression of internal reference genes table s6 and the transcript abundance was determined by the comparativect value method analysis of proteinprotein interaction network andstructural conservationa proteinprotein interaction network was generated bythe string website httpsstringdb withrepresentative protein sequences from arabidopsis thefollowing basic settings were employed meaning of network edges evidence line colour indicates the type ofinteraction evidence and active interaction sourcesexperimentsthere are three domains in the arabidopsis sf1 protein the phosphorylation and u2af65 binding of thenterminal domain of splicing factor during ² splicesite recognition of homo sapiens pdbid 2m0g identity evalue 7e17 was similar to that of the khomology domain the structure for recognition of theintron branch site rna by splicing factor of homo sapiens pdbid 1k1g identity evalue 9e27 canbe used as the template for the splicing factor helixhairpin domain therefore homology modelling wasperformed with modeller based on two crystalstructures the amino acid conservation scores were calculated using the consurf web server based on the mlmethod input attributes were the 3d model andmultiple sequence alignment figure s4 related figureswere created based on pymol with default settings analysis gene structure evolution with orthologue groupof sf1 genesreconstruction of the evolutionary history of the structure of the plant sf1 family of orthologous genes wascarried out by searching at5g513001 in the piece severhttpwwwbioinfogenomenetpieceindexphpthis provided an exonintron display for orthologousgenes from gene structure data sets linked to the phylogenetic treeresultssequence identification and phylogenetic analysis of theplant sf1 gene familyto identify sf1 gene family members in plants we carried out a blastp search using the arabidopsis atsf1at5g51300 amino acid sequence against the phytozome database v121 after filtering the sequence without sf1 signature or truncated sequences a total of sequences from plant species were retrieved whichwere roughly classified as algae bryophyta basicangiosperm monocots and eudicots table s1specifically the only species with four copies of plantsf1s was eutrema salsugineum salt cress table s1 inparticular three copies of sf1 genes were observed infive species including panicum virgatum switchgrasstriticum aestivum common wheat daucus carotacarrot kalanchoe laxiflora milky widows thrill andsalix purpurea purple osier willow additionally plant species contained two copies and species including the model plant arabidopsis possessed only onecopy of plant sf1s respectively the relatively largernumber of sf1 genes and higher number of plant speciesin this work demonstrated the universality and complexity of the sf1 gene family the retrieved sequences of plant species provided us with more complete information to analyse the phylogenetic relationship of the sf1gene family subsequently a rooted phylogenetic treewas constructed based on the abovementioned protein sequences by using the maximum likelihoodmethod the trees bootstrap threshold was represented by a colour gradient fig in general all sf1protein sequences were clustered into four major cladesincluding alga in yellow other land plants in greenmonocots in pink and eudicots in blue and one species amborella trichopoda belonged to basic angiosperm shown in colourless the phylogenetic tree ofsf1s figs and left panel with clear topology andoverall high bootstrap values was similar to evolutionarytrend from lower plants to higher plants reported inother studies for example the genes of algae in the yellow branch were representative members of the lineage 0czhang bmc plant biology page of fig circular phylogenetic tree of the sf1 gene family available in plants the phylogenetic tree of sf1 genes in plants was constructed basedon maximumlikelihood with jtt g model by using phyml v3037 a total of protein sequences from plant species were chosen tocalculate the phylogenetic relationship for tree construction bootstrap values are labelled at each major branch the corresponding informationof each transcript such as species name common name number of identified transcripts and their transcript id nomenclature are shown intable s1 taxonomies based on apgiv system 0czhang bmc plant biology page of fig see legend on next page 0czhang bmc plant biology page of see figure on previous pagefig gene structure comparisons and conserved motif identification among plant sf1 genes from left panel to right panel verticalphylogenetic tree genomic anization and identified cdna conserved motifs by meme analysis intron phase and are shown on thegene structure the conserved sequence of identified motifs represented by different coloured boxes are listed below some long genes werereduced to onehalf of their original length to fit this picturethat diverged before the evolution of land plants whichwas the basal part of the phylogeny in the blue branchfive sequences from kalanchoe with higher bs valuesformed a subclade showing their closer evolutionary relationships additionally cagra3782 s00261p fromcapsella grandiflora and carubv10025900m from c rubella formed a subclade with the arabidopsis sequencesbecause they all belong to brassicaceae which is consistent with the apg iv system fig and table s1 usually some homologous sf1 sequences from the samespecies were clustered in the same small branch next toeach otherthese species included cashew soybeanapple woodland strawberry quinoa carrot coloradoblue columbine maize common wheat cereal grassmoss and bog moss fig and table s1 in contrastsome other homologous sf1 members from the samespecies were clustered into the different subclades suchas purple osier willow poplar eastern cottonwood saltcress potato diploid kalanchoe milky widows thrillhalls panicgrass switchgrass green algae and volvoxfig and table s1gene structure and conserved motif analysisit is necessary to compare the exonintron anizationand conserved motifs of the plant sf1 gene family toclarify their evolutionary process and potential functionthe gene structure models of sf1 genes were attachedto the phylogenetic tree fig and the correspondingintron phase of each was also displayed fig tables2 figure middle panel shows that the gene lengthand structure of each member of the sf1 family exhibitssignificant differences for example the gene structureof members of sf1 family genes did not containintron sequences this subset accounts for of thetotal number of members fortyeight sequences of sf1genes had exon1 intron anizations accounting for of all genes in particular some genes from algaehad multiple exons including vocar0008 s02941p volvox carteri which contained the most exons exonsmoreover different gene structures were also observedat the same subbranch for instance two sequencesfrom zea mays maize zm00008a037777_p01 exonsand zm00008a007621_p01 exons were observed tohave distinctive gene structures although the dissimilation of gene structure of each member of sf1s was substantial we found that the length of cdss did notsignificantly change fig thus whether it influencesthe differentiation of their gene function needs to befurther investigated further investigation on conservedmotifs by using multiple em for motif elicitationmeme search tool demonstrated that most sf1 genes sequences exhibited similar sequence signatures andthe same order and all contained the analysed motifsexcept one sequence of micromonas pusilla hada different position fig right panel although no obvious differences in identified conserved motifs werefound among basal angiosperm monocots and eudicotssequences from the same species were found to have different motifs fig for example aqcoe5g4069001pand aqcoe7g0393001p from the eudicot aquilegiacoerulea had motifs and motifs respectively thesame situation was found in d carota dcar_006843dcar_008506 and dcar_004968 had motifs motifs and motifs respectively intriguingly the cdslength of dcar_008506 was the longest notably somesequences from algae and moss had fewer conservedmotifs for examplein bryophyta the sequences ofphyscomitrella patens pp3c7_10890v31p and pp3c11_24710v31p sphagnum fallax sphfalx0015s00771pand sphfalx0010s01971p and marchantia polymorphamapoly0009s01891p had nine motifs in algal plantsthe sequences of and from micromonashad only motifs and motifs respectively moreoveralthough the sequences of volvox carteri vocar0007s03451p and vocar0008 s02941p and chlamydomonasandcre09g386731t11 contained multiple exons they had motifs indicating their sequence variation had little influence on function classes further correlation analysisof the sf1 exon regions were carried out to elucidate thegainloss of introns correlations between transcripts ofplant sf1s are shown in fig providing additional information for phylogenetic analysis for example thereis more similarity between pgsc0003dmt400081859and migutd025312 because of multiple exact matchesbetween the exons of the two transcriptscre12g553750t11reinhardtiianalysis of protein domain and conserved motifs inpeptidesthe protein domains were analysed by using the aboveselected peptide sequences from plant species thepeptides annotations were splicing factorrelated andconserved protein motifs were predicted according tothe retrieved peptide sequences by meme analysisfig consequently all sf1s were found having sf1_hh nterminal domain on the nterminal ofthe 0czhang bmc plant biology page of fig analysis of gene structure evolution with orthologue group of sf1 genes exonintron structure and intron phase right panel are linked tothe plant species tree left panel genes with red colour represent the members of the plant sf1 genes different coloured lines mean differentexon comparison results between species 0czhang bmc plant biology page of fig comparisons of protein domains and conserved motif identification among plant sf1 genes protein domain middle panel and identifiedprotein conserved motifs right panel identified by meme analysis are shown against the vertical phylogenetic tree left panel the conservedsequence of identified motifs represented by different coloured boxes are listed below 0czhang bmc plant biology page of peptides followed by a kh domain and a cterminal domain namely an rna recognition motif rrm fig middle panel interestingly in algae peptides from mpusilla v carteri vocar0008 s02941p andc reinhardtii cre09g386731t11 had two rrm domains the amino acid lengths of sf1 proteins rangedfrom aa to aa and most of them possessed to amino acids table s3 consistently most ofthem are approximately to amino acids inlength subcellular location prediction showed that themajority of sf1 proteins were had nuclear localization table s3 moreover proteins of 147m014250 ricinus communis and migutf011911pmimulus guttatus were located in the vacuoles proteins of traes_2dl_6f03f05fa4 t aestivum and m pusilla were predicted to be cytoplasmic proteins of gsmua_achr5p25100_001 musa acuminataand cre09g386731t11 c reinhardtii were located inthe chloroplast and endoplasmic reticulum respectivelymeme analysis for sf1 peptide sequences was used topredict a total of conserved motifs which are presented as coloured boxes and cover most of the proteinfig right panel further analysis showed that peptides had all motifs accounting for approximately of all sf1 protein sequences analysed in the studyinterestingly all sequences from moss have conservedmotifs in the analysis suggesting the conservation ofsf1 proteins in bryophyta furthermore almost all eudicots had conserved motifsexcept anacardium occidentalegrandifloracagra3782 s00261p which lacked motif and malusdomesticavescamrna211921v10hybrid and brassica rapa brarac014811p which lacked motif while most monocots had eight conserved motifs in contrast algal plantsonly possess approximately half of the predicted motifs due to their peptides with integrant protein domainsimplying the least degree of conservation and divergenceof plant sf1 proteins in algae t motifs that all algaeshared were motif motif motif and motif anaoc0018 s04251pmdp0000558834fragariaand canalysis of promoter and tissuespecific expression of sf1genesto further analyse the regulation of plant sf1 genes atthe transcriptional level the 15kb upstream sequencesof plant sf1 genes were obtained from the phytozomedatabase then the ciselements of each promoter wereidentified by using the plantcare program table s4 consequently a total of motifs were predictedgenerally eight ciselements related to tissuespecificexpression among them were selected fig and tables4 including hdzip1 for differentiation of the palisademesophyll cells the ryelement which regulates seedspecific expression the aaca_motif and gcn4_motif waspresentfurther hdzipinvolved in endosperm expression and the catboxccgtccbox doct and oct for meristem expression further analysis showed that there were only promoters of sf1 genes which had tissuespecific regulatory ciselements particularly the catbox and ccgtccbox turned up at the highest frequency and greatestabundance in the promoters of sf1 genes both of themregulate meristemspecific expression and play key rolesduring development and growth of plants consistentlypurple false brome brachypodium distachyon of monocots not only had a catbox and ccgtccbox butwas also highly expressed in young leaves internode adventitious roots and roots fig and figure s2however no motifs were found to link the highexpression of two sf1s of glycine max soybean insam and roottip figure s1 additionally the aaca_motif was only detected in solanum tuberosumpgsc0003dmp400032853 of potato suggesting itsspecific role in regulating endospermspecific negativeexpressioninpodel03g1132001p of populus deltoideseasterncottonwood and spipo17g0046100 of spirodela polyrhiza greater duckweed the ryelement was detectedin the promoter of the dicot model plant arabidopsisand low expression was also reported in dry seed inarabidopsis fig suggesting that the ryelement isinvolved in seedspecific negative expression of arabidopsis moreover expression levels in the same tissuetype showed significant differences during differentgrowth stages for example the expression level in stamen of flower stage of arabidopsis was obviouslyhigher than that of the other flower development stageshowever the expression levels of different growth stagesof solanum lycopersicum were not only similar butlower and no motifs were found in the promoter in tomato figs and s1 furthermore different expressionpatterns were detected in several sf1 genes with multiple copies figs s1 and s6 for instance similar tissue expression profiles were detected in two sf1trichocarpahomologuespotri001g1264001 and potri003g1072001 and themonocotandzm00008a037777_p01 figure s1 and s5 in contrasttwo sf1 genes of s tuberosum showed differential expression patterns similar to in g max figs and s1zm00008a007621_p01dicot populuszea maysfrom theanalysis of promoter and internal and external hormonesexpression of sf1 genesin longterm evolution and development plants havegradually formed mechanisms of adaptation and resistance to adversity to maintain their life and sustaingrowth to understand the regulatory mechanisms of internal and external stimuli on plant sf1s cisacting elements involved in hormone and stress were studied with 0czhang bmc plant biology page of fig see legend on next page 0czhang bmc plant biology page of see figure on previous pagefig analysis of motifs related to tissue specificity in the plant sf1 promoter regions eight cisacting motifs are represented in different colortriangles positions of these identified motifs are labeelled along the kb ²flanking regions of each sf1 gene the line solid and dottedrepresents regions with basic pairs and regions of no sequences or annexed base n respectively symbols on above the line represent the motifsat the plus strand whereas symbols on below the line represent the motifs at the minus strand function of motifs aacamotif involved inendospermspecific negative expression catbox cisacting regulatory element related to meristem expression ccgtccbox cisactingregulatory element related to meristem specific activation doct cisacting regulatory element related to meristem specific activationgcn4_motif cisregulatory element involved in endosperm expression hdzip1 element involved in differentiation of the palisade mesophyllcells ryelement cisacting regulatory element involved in seedspecific regulation the black vertical lines represent break at that particularbranch oct cisacting regulatory element related to meristem specific activationlowtemperatureltr droughtthe plantcare database fig table s4 finally hormone and stressrelated motifs were selected from promoter sequences of plant sf1s there are hormonerelated motifs including abscisic acid abreauxin auxre auxrecore tgabox tgaelementethylene ere gibberellin garemotif pbox tatcbox meja cgtcamotif tgacgmotif and salicylic acid tcaelement and five stressrelated motifsincludingmbswound wunmotif and anoxic are gcmotif motifs almost each sf1 sequence had a great diversity ofciselements in its promoter regions except some sequences such as araha13031 s00021 and traes_2al_3d67296921 which did not contain a single motif dueto the sequences contain n or no promoter suggestingthat multiple hormonesmediated signalling pathwaysare closely related to sf1 plants resistance analysisshowed that more than half of sf1 promoters containedabre cgtcamotif tgacgmotif and are respectively moreover external hormone signals also affect theabundance of sf1 transcripts figure s3 for examplein arabidopsis at5g513001 mj methyl jasmonateinhibited its expression fig and treatment withother hormones like acc a precursor of ethylene iaaauxin | Colon_Cancer |
"although the clinical development of immune checkpoint inhibitors icis therapy has ushered in a new era of antitumor therapy with sustained responses and significant survival advantages observed in multiple tumors mostpatients do not benefit therefore more and more attention has been paid to the identification and developmentof predictive biomarkers for the response of icis and more indepth and comprehensive understanding has beencontinuously explored in recent years predictive markers of icis efficacy have been gradually explored from theexpression of intermolecular interactions within tumor cells to the expression of various molecules and cells intumor microenvironment and been extended to the exploration of circulating and host systemic markers with thedevelopment of highthroughput sequencing and microarray technology a variety of biomarker strategies havebeen deeply explored and gradually achieved the process from the identification of single marker to thedevelopment of multifactorial synergistic predictive markers comprehensive predictivemodels developed byintegrating different types of data based on different components of tumorhost interactions is the direction offuture research and will have a profound impact in the field of precision immunooncology in this review wedeeply analyze the exploration course and research progress of predictive biomarkers as an adjunctive tool totumor immunotherapy in effectively identifying the efficacy of icis and discuss their future directions in achievingprecision immunooncologykeywords neoplasm immune checkpoint inhibitor predictive biomarker tumor mutation burden programmeddeath ligand1 immune checkpoint inhibitors icis therapy has usheredin a new era of antitumor therapy with sustained responses and significant survival advantages observed inmultiple tumors antiprogrammed cell death1programmed cell deathligand pd1pdl1 antibody hasbeen approved for secondline or firstline treatment in avariety of malignant neoplasms including melanoma lungcancer renal cell carcinoma rcc head and neck squamous cell carcinoma hnscc and gastroesophageal correspondence cuijwjlueducncancer center the first hospital of jilin university xinmin streetchangchun jilin chinacancer [ ] however despite the breakthrough in clinical treatment with icis most patients do not benefitpembrolizumab or nivolumab has an objective responserate orr of in firstline melanoma and insecondline nonsmall cell lung cancer nsclc []therefore in recent years more and more attentions havebeen paid to the identification and development of predictive biomarkers for the efficacy of icis and more indepth and comprehensive understanding has also beenobtained in recent yearsincluding new data on biomarkers of tumor genome and neoantigen tumor immune microenvironmentbiopsybiomarkers hostrelated factors and all of which havephenotypeliquid the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cbai biomarker research page of technologyimmunohistochemicalmade many new advances in the corresponding fieldswith the development and continuous improvement ofmultiplexhighthroughput sequencing and microarray technology a variety of biomarker strategies have emerged and graduallyrealized the process from the identification of singlemarker to the development of multifactorial synergisticpredictive markers the development of predictive biomarkers contributes to revealing the therapeutic mechanisms of icis and the interaction mechanisms betweentumor and host immunity achieving decisionmaking ofindividualized antitumorimmunotherapy monitoringefficacy and disease development guiding clinical trial design as well as for further understanding of drug resistance mechanisms and tumor prognosis in this review wedeeply analyze the exploration course and research progress of predictive biomarkers as an adjunctive tool totumor immunotherapy in effectively identifying the efficacy of icis it should be pointed out here that when reading and collating we try to read and include all therelevant s in the process of selecting s we include the authoritative s published in highlevel s or the latest research results and objectively describe and analyze their roles in this field as well as discuss the reasons that different research results may beinvolvedadvances of multiple predictive biomarkers toicis efficacyi tumor genome and neoantigen biomarkerstumor mutation burdensignificant correlations between high tumor mutationburden tmb and response to icis have been reported inseveral cancer types including urothelial carcinoma small cell lung cancer sclc nsclc []melanoma and human papilloma virus hpvnegative hnscc a metaanalysis of cancer typesshowed that the mean response rate was positively correlated with log tmb the national comprehensivecancer network nccn guidelines have adopted tmbas the recommended test for patients with nsclc receiving immunotherapy although the results in some clinicalstudies of rcc hpvpositive hnscc and melanoma receiving antipd1 after recurrence showedthat tmb alone also did not clearly distinguish respondersand predict os it is still exciting that multiple studies inthe american society of clinical oncology ascomeeting have confirmed the predictive value of tmb inimmunization or combination therapy keynote061study [ ] condor study eagle study epoc1704 study etc consolidating its status oftmb as an independent predictor and in april theus food and drug administration fda prioritized theapproval of tmb as a companion diagnostic biomarkerfor pembrolizumabnonetheless the cutoff values of tmb were defineddifferently across studies and assay platforms such asatezolizumab mtmb in urothelial cancer pembrolizumab mtmb in nsclc and atezolizumab¥ ¥ or ¥ mtmb in nsclc [] andnivolumab plus ipilimumab ¥ mtmb in nsclc which needs further study to confirm the optimalcutoff value in different tumors moreover the ngspanels have approved by the fda that can be used to estimate tmb include the mskimpact and foundationone cdx panel the detection results of which arehighly consistent with whole exome sequencing wes[ ] and other solutions are under development astudy detecting tmb cutoff value at mtmb in nsclc patients with the foundationone platformcontaining a gene panel found that compared withtmbl patients overall survival os and dcr was significantly improved in tmbh patients treated withantipd1l1 drug both wes and targeted ngs a422cancergene panel performed in patients withnsclc treated with antipd1l1 demonstrated thattmbh population has a significantly better durableclinical benefitdcb and progressionfree survivalpfs these findings demonstrate the feasibility ofcomprehensive genomic profiling cgp but the designof optimal next generation sequencing ngs panel thatis more accurate comprehensive and costeffective isstill not clear in addition given that btmb was identified as a predictor of pfs but failed to differentiate patients with os benefits researchers consider the need toexplore other more precise factors eg allele frequencyaf a study that developed a new btmb algorithm intwo independent cohorts poplar and oak showedthat modified btmb low af btmb lafbtmb mutation counts with an af was significantly associated with favorable hr 95ci p pfs hr 95ci p andorr p after immunotherapy but required tobe prospectively validated finally static biomarkersare insufficient to accurately predict response due to thecomplexity of tumorimmune interactions a recent analysis of tumor genomewide dynamic detection in pretreatment and ontreatment melanomasfound thatpretreatment tmb was only associated with os in untreated patients while early 4week ontreatment changein tmb δtmb was strongly associated with antipd1response and os in the entire cohort the detectionof δtmb is helpful for early evaluating the response totherapy of patient but its clinical usability limited by thedifficulty in obtaining tissue samples and high price whileliquid biopsy discussed below might better 0cbai biomarker research page of in addition epigenetic changes are associated withtmb the latest study investigated the association between tmb and dna methylation dnam to explorepotential complimentary biomarkers for nsclc immunotherapies the results showed that high tmbnsclcs had more dnam aberrance and copy numbervariations cnvs showing certain value in predictingefficacy such as hox gene methylation status and tmb thus the correlated exploration of epigenetics hasattracted more attention in recent years and liquidbiopsybased epigenetic studies may become a future research direction exploration in chinese nsclc patientsshowed that nsclcs with high tmb had dnam aberrance and cnvs some insertion and deletion indelmutations can lead to frameshifts and more immunogenic neoantigens in the pancancer analysis of cancer types evaluated in the cancer genome atlastcga rcc had the highest indel mutation load andframeshift indel mutations were found to produce threetimes more candidate neoantigens per mutation thannonsynonymousnssnvs somatic copy number alterations scnas are another feature of the genomic landscape of tumors andpancancer tcga analysis revealed an inverse correlation between scnas atthe singlearm or wholechromosomelevel and immune infiltration in tumortypes tested and this result was subsequently replicated in a larger study of tcga single nucleotide variantsdna damage response pathwaysgenetic variation involved in dna mismatch repairmmr pathway can lead to microsatellite instabilitymsi a specific type of high tmb tumors and increased numbers of cd8 tumor infiltrating lymphocytestils pd1tils and indoleamine 23dioxygenaseido tumor cells have been shown in mmr deficiencydmmr colorectal cancer recently five clinical trials keynote016 including multipletumor types have shown that patients with dmmrmsih can achieve durable responses to pembrolizmabbased on this pembrolizumab is approved by the usfda for the treatment of any advanced solid tumor withdmmrmsih and nivolumab in combination with ipilimumab has also shown promising response in dmmrmsih colorectal cancer in addition dmmr canalso cause mutations in the dna polymerase gene epsilondelta polepold1increasing the mutationload and neoantigen load analysis of polepold1mutations in patients with different cancer typesshowed that patients with these mutations had significantly higher tmb and os therefore it may be an infordependentinidentifying patients who benefitaddition pathways of base excision repairberand prognostic markerfrom icis risk factorhomologous recombination repair hrr mmr in thedna damage response ddr signaling network contribute more significantly to tmb or neoantigens whichhave the highest levels when comutated it hadbeen identified that comutations in the ddr pathwaysof hrr and mmr or hrr and ber defined as comutare associated with increased levels of tmb neoantigenload and immune gene expression signatures comutpatients showed a higher orr and longer pfs or os indicating that comut can be used as predictors of response to icis and provide a potentially convenientmethod for future clinical practice specific mutated gene pathways in tumor cellsit is worth noting that alterations of signaling pathwaysin tumor cells affect the responsiveness to immunotherapy patients with mutations in the interferon ifnγpathway genes ifngr12 jak12 and irf1 are poorlyresponsive to icis treatment and confer resistance a study found that in patients receiving immunotherapytumor cells can downregulate or alter ifnγ signalingpathways such as lossoffunction alleles of genes encoding for jak12 and changes in stat1 to escape the influence of ifnγ resulting in poor efficacy andresistance recent studies suggest that inactivating mutations in a mammalian analog of the chromatin remodeling swisnf complex and unique genes of the pbafcomplex pbrm1 arid2 and brd7 lead to sensitivitiesto icis [ ] loss of function of the pbaf complexincreased chromatin accessibility to transcription regulator elements of ifnγinducible genes within tumorcells and subsequently increased production of cxcl9cxcl10 chemokines leading to more efficient recruitment of effector t cells into tumors in human cancers expression of arid2 and pbrm1 are related toexpression of t cell cytotoxicity genes which confirmedin pbrm1deficient murine melanomas with strongly infiltrated by cytotoxic t cells and responsive to immunotherapy [ ]in addition doublestranded rnadsrna editing enzyme adenosine deaminase acting onrna adar1 protein can block the ifnγ signalingpathway and lead to poor icis efficacy and resistanceloss of function of adar1 in tumor cells can reduce atoi editing of interferoninducible rna species and leadto dsrna ligand sensing by pkr and melanomadifferentiationassociated protein mda5 this resultsin growth inhibition and tumor inflammation respectively and profoundly sensitizes tumors to immunotherapy finally demethylation positively regulates thetranscriptional activity of some immunerelated genesincluding pdl1 and ifn signaling pathway genes sensitizingto anticytotoxic tlymphocyteassociatedprotein4 ctla4 therapy it 0cbai biomarker research page of in addition to the ifnγrelated signaling pathway alterations in other tumor genome such as tumor oncogenes and suppressor genes pathways and pathwaysrelated to tumor cell proliferation and infiltration canalso affect immunotherapy efficacy epidermal growthfactor receptor egfr and anaplastic lymphoma kinasealk mutations have been shown to be associated withreduced response rates to icis and low tmb and therefore the fda does not recommend firstline icistreatment in patients with egrf or alk positive tumors[ ] certain types of mutations in mdm2mdm4and arid1a can predict nonresponse to icis in hightmb tumors nsclc with kras and stk11 comutated was associated with reduced response andshorter survival in three independent cohorts of patientstreated with antipd1 therapy and stk11 deficiency was an independent indicator of poor antipd1response in nsclc with kras mutant however at the american association for cancer research aacrmeeting of patients in the keynote042 studynct02220894 update data were tested for stk11 andkeap1 and the results showed that patients could benefit from pembrolizumab regardless of stk11 and keap1status but patients with stk11 mutations did not respond well to chemotherapy but given that only ofall patients had mutation detection the results may beaffected in initial data from studies using targeted ngspanels suggested that duration of icistreatment was associated with certain braf and m terations butnot tmb status notch signaling pathway is associated with the occurrence development and prognosisof tumors especially with the biological function of cancer stem cells recent breakthrough findings have distinguished deleterious notch mutation showing that itcan be used as a potential predictor of favorable ici response in nsclc potentially via greater transcription ofgenes related to dna damage response and immune activation another tumorspecific inheritance thatmay influence icis efficacy is the aberrant expression ofendogenous retroviruses ervs pancancer analysisidentified a positive correlation of transcript expressionof ervs with tcell activity in various tumors andpatient prognosis furthermore with the improvement of precision detection technology the accurateanalysis of negative mutation sites helps to identify thepossibly effective ones for example the analysis of studydata of secondline pd1l1 inhibitor therapy found thatthe mpfs of patients with kras g12c or g12v was significantly better than that of patients with kras mutations at other sites in addition several pancancer biomarkers are recentlyapproved by the fda for example given the effectiveorr of and a disease control rate dcr of in secondline cholangiocarcinoma patients treated withanalysispemigatinib a new targeted therapy the recent fda approval of pemigatinib for the treatment of previouslytreated patients with locally advanced or metastatic cholangiocarcinoma with fibroblast growth factor receptor fgfr2 fusion or rearrangement and the comprehensive genomicassay foundationone cdxdeveloped by foundation medicine as a companiondiagnostic also exciting is the recent fda approval ofthe targeted anticancer drug capmatinib for the treatment of metastatic nsclc with met exon skippingmetex14 mutations including firstline patients andpreviously treated patients also using foundationonecdx as a companion diagnostic to help detect specificmutations present in tumor tissueimmunogenicity ofneoantigen loadneoantigen load the number of mutations actually targeted by t cells may be directly related to the responseto icis [] a retrospective study showed thatclonal neoantigen burden was associated with the longeros in primary lung adenocarcinomas p traditionallycomputational neoantigen predictionshave focused on major histocompatibility complexmhc binding of peptides based on anchor residueidentities however neoantigen loads identified by thismethod are generally not superior to overall tmb inpredicting icis efficacy or survival in recent practice this neoantigen can be assessed by the difference inpredicted mhci binding affinity between the wildtypepeptide and the corresponding mutant peptide knownas the differential agretopicity index dai reflectingclinically relevanttumor peptide a high dai value indicates that the mutant peptidesignificantly increases binding affinity to mhc compared to the wildtype sequence and can generate moreimmune responses studies on previously published cohorts treated with three icis have shown that dai outperforms tmb and the traditionally defined neoantigenload in predicting survival [ ] in additionlowneoantigen intratumour heterogeneity might also be important for icis response analysis of the lung adenocarcinoma tcga database found that combining highmutational load and low intratumoral neoantigen heterogeneity was significantly associated with osand longer lasting clinical benefit than either variablealone anotherreported method for assessingneoantigen foreignness is based on sequence homologyof experimentally validated immunogenic microbial epitopes in the immune epitope database iedb butit does not account for all possible human leukocyteantigen hla contexts in addition the detection forneoantigen can be reflected from different levels such aspeptides or genomes a study developed the neopepseealgorithm using a machine learning approach incorporating 0cbai biomarker research page of integration of nine immunogenicity features and gene mutation expression levels and its application to melanoma and leukemia patients could improve the sensitivityand specificity of neoantigen prediction recently it has alsobeen shown that promoter hypermethylation of neoantigengenes may be an important mechanism for immune editingand tumor immune evasion indicating that combineddetection of tumor genome and epigenetics may providemore information for immunotherapy efficacyii tumor immune microenvironment phenotypebiomarkerscells is also considered separately as one of the biomarkersto distinguish the benefit population called immune positive score ips herbst showed that response toatezolizumab treatment was significantly associated withhigh levels of pdl1 expression on the surface of tils before treatment but not with pdl1 expression on tumorcells p finally other inhibitory immune pathways may affect the response to icis therapy including tcelllymphocyte activationgene3 lag3 and vdomain ig suppressor of tcell activation vista which can be used as potential biomarkers for icis responseimmunoglobulin3 tim3pdl1 expressiongiven that multiple studies in a variety of tumors havedemonstrated a positive correlation between pdl1 expression and response to icis or os even in firstlinecombination therapy [] pembrolizumab is currently approved by the fda for use in patients with pdl1 pdl1 ¥ of tumor cells in firstline treatmentand ¥ in secondline treatment nsclc and pdl1immunohistochemistry ihc as a companion diagnosticfor antipd1 therapy in nsclc patients [ ] however some studies have not detected a significant correlation between pdl1 expression and response to icis[ ] and pdl1 negative patients can still benefitclinically with treatment with ici or combination treatment with icis with orrs ranging from to therefore pdl1 cannot yet be a comprehensive and independent biomarker in clinical practice in assessing efficacy with following challenges still existing firstlypdl1 assay and antibody are not standardized secondly pdl1 expression is temporally and spatiallyheterogeneous a study of metastatic nsclctreated with icis showed that pdl1 varies substantiallyacross different anatomic sites and during clinicalcourse being highest in adrenal liver and lymph nodemetastases and lower in bone and brain metastases andthe predictive value of pdl1 at different biopsy sites forthe benefit of icis in nsclc may vary higher pdl1 inlung or distant metastasis specimens was significantly associated with higher response rate pfs and os whilepdl1 in lymph node metastasis biopsy was not associated with either response or survival thirdly positive score and cutoff value of pdl1 expression is notstandardized at present pdl1 positive scoremainly focuses on the pdl1 expression level of tumorcells that is tumor proportion score tps but pdl1is also expressed on immune cells such as lymphocytesand macrophages and stromal cells thus the investigators introduce the concept of combined positive scorecps which is the proportion score of the sum of pdl1 expressed by tumor cells and tumorassociated immune cells in addition pdl1 expression on immuneresponseto icisimmunetreatmentbiomarkers of tumorinfiltrating immune cellsoverall immune status of tumor microenvironmentthe pattern of tumor immune infiltration can be broadlyclassified into immuneinflamed immuneexcluded andimmunedesert immuneinflamed is characterizedby the presence of cd8 and cd4 t cells in the tumorparenchyma accompanied by the expression of immunecheckpoint molecules indicating a potential antitumor immuneexcluded is characterized by the presence ofdifferent immune cell types in the aggressive margin orstroma of tumor but cannot infiltration into tumor parenchyma [ ] analysis of pretreatment samples forantipd1pdl1 revealed a relatively high abundance ofcd8t cells at the invasive margin in responders andserial sampling during treatment showed an increasedinfiltration of cd8t cells into tumor parenchyma while immunedesert phenotype is characterized by theabsence of abundant t cells in the parenchyma orstroma of tumors and poor response to icitreatment recentlyimmunoscore has been proposed as avalid marker for characterizing the immune status oftumor microenvironment tme classifying tumors aswell as predicting treatment response and prognosis which involves the density of two lymphocyte populations cd8 and memory [cd45ro] t cells in thecenter and invading margin of tumor mlecnik evaluated immunoscore in specimens of stageiiv colorectal tumor and confirmed that it was significantly associated with pfs dfs and os and multivariate analysis also showed the superiority of immunoscorein predicting disease recurrence and survival the valueof immunoscore to predicting icis efficacy is being validated internationally in clinical trials of melanoma andnsclc a wider assessment of active immune responses withintme by immune gene expression profiling might effectively predict clinical benefit to icis strategies analysisof total rna and genes that were substantially differentbetween the patient groups in pretreatment tumor biopsies revealed atleast a 25fold increase in the 0cbai biomarker research page of expression of immunerelated genes in clinically active patientsincluding cytotoxic t cell markers egcd8a perforin granzyme b th1 cytokines or chemokines mhcii and other immunerelated genes egnkg7 ido1 ascierto screened morethan immunerelated genes in patients with recurrent breast cancer years after treatment and thosewithout recurrence more than years later and foundthat five genes igk gbp1 stat1 igll5 and oclnwere highly overexpressed in patients with recurrencefree survival in addition ifnγinduced immune genesignatures may be effective biomarkers for predicting theclinical benefit of treatment with icis the study developed ifnγ scores combining multiple immune variablesbased on gene signatures which were then extendedto gene signatures in a validation set of melanomapatients including genes encoding ifnγ granzymes ab perforin ido1 and other immunerelated genesboth gene scores showed significant associations withbest overall response rate and pfs optimized cutoffvalues for ifnγ scores based on receiver operatingcharacteristic curve roc curve can achieve a positivepredictive value of for responders and a negativepredictive value of for nonresponders immune cells with specific phenotypes in tmethe phenotype of tils also influences the efficacy oficis the study used singlecell mrna sequencingscrnaseq data analysis to identify two major cd8tcell phenotypes within melanoma memorylike andexhausted the proportion of which is strongly correlated with response to icis the research furtherfound that the transcription factor tcf7 is selectivelyexpressed in memorylike t cells so the ratio ofcd8tcf7 to cd8tcf7tils is strongly correlatedwith improved response and survival in melanoma patients treated with antipd1 balatoni found that of immune cells in tme were positivelyassociated with os after treatment including cd4 andcd8 t cells foxp3 t cells cd20 b cells cd134and cd137 cells and nkp46 cells and different immune cells at different sites were differently associatedwith clinical outcomes researchers found that only asmall proportion of cd8 tilsin tumors couldrecognize tumor mutationassociated antigens while another population bystander cells was insensitive anddifferential cd39 expression was the key molecule thatdistinguished the two populations analysis of peripheral blood from a patient with colorectal cancer whoresponded rapidly to pembrolizumab treatment showedhigh expression of cd39 on cd8 tils indicating thatcd39cd8til may be a promising predictive biomarker the fact of very low level of cd39 expression on cd8tils in of egfrmutant nsclc isconsistent with their low response rate to antipd1immunotherapyin addition a study showed that fc domain glycan ofthe drug and fcγ receptor fcγr expressed by the hostbone marrow cells could determine the ability of pd1tumorassociated macrophages tams to capture antipd1 drugs from the surface of t cells which leads topd1 inhibitor resistance and the association oftams and poor antipd1 response was reported inmelanoma cohorts antipd1 response was associated with an increase in cd8t cells and natural killercells nk cells and a decrease in macrophages andhigh intratumoral myeloid markers were associated witha nearly 6fold decrease in mpfs after antipdl1 therapy in rcc emphasizing the inhibitory role of myeloidcells in response to icis in conclusion immunecells in tme show a great promise in the developmentof predictive biomarkers for icisimmunerepertoirediversity of immune repertoires in tmeeffective t cell responses involve the activation and expansion of specific antigenreactive t cell clones so diversity ofin intratumoral orperipheral may correlate with icis responses and can bequantified as richness and clonality however theresults seem to be complex with some studies finding apositive correlation between til clonality and the response to icis before or after treatment whileothers showing that only an increase in til clonalityduring treatment is associated with the response to antipd1 [ ] others show that intratumoral t cellclonality is not associated with survival while peripheralt cell clonality is inversely associated with pfs and os tumeh further investigated whetherbaseline tils have a narrow t cell receptor tcr repertoire focusing on tumorspecific immune responsesand whether this narrow tcr repertoire correlates withpembrolizumab responses they found that respondingpatient had more restricted usage of the tcr beta chainie a more clonal less diverse population than patientswith progressive disease and showed a 10times increasein these clones after treatmentimplying a tumorspecific response to treatment in these patients notablybaseline tcr clonality was not highly correlated withtil density suggesting that some patients with restricted tcr clonality specific for tumor antigens maystill benefit from antipd1 therapy even though tildensity is low recently researchers have proposed theimmune repertoire irindex the average frequency ofshared tcr clones in t clones in tils and peripheralpd1cd8 t cells they found that neoantigenstimulated tcr agreed with irindex and patients withhigh irindex had better immune activation and highergene expression profiles geps score subsequently they 0cbai biomarker research page of confirmed the predictive value of irindex to icis efficacy dcrpfs but considering that it is difficult tosort out pd1cd8 t cells in tumor tissue based ontwo separate patient cohorts a research confirmed thattcr repertoire diversity and clonality of peripheral pd1cd8t cells may serve as noninvasive predictors ofclinical outcomes after icis in patients with nsclc the viewpoints of t cell diversity and tcr clonality as markers of icis efficacy need to be further validated in a large patient populationiiiliquid biopsy biomarkersperipheral blood cell biomarkersperipheral blood is a noninvasive source to explore potential biomarkers for icis and although associationswith clinical benefit and survival have been observed itseffectiveness has not been validated in prospective studies analysis of melanoma treated with ipilimumabshowed that improved os and pfs were associated withbaseline values of peripheral blood components including low absolute neutrophil countlow neutrophiltolymphocyte ratio nlr low absolute monocyte countlow frequency of myelogenous suppressor cells high frequency of foxp3 treg cells high lymphocyte frequencyhigh eosinophil count and clinical benefit also associated with the dynamic changes of blood markers duringincluding decreased foxp3treg concentratreatmenttions and increased lymphocyte and eosinophil counts reports in patients with melanoma treated withpembrolizumab and in patients with nsclc treatedwith nivolumab have shown that nlr is associated withworse tumor response [ ] multivariate analysis inmelanoma patients treated with antipd1 antibodiesshowed that nlr was the only factor associated withworse orr and shorter pfs indicating that nlr is astrong predictor of worse outcome in patients treatedwith ici low baseline lactate dehydrogenase ldhlevels high relativeabsolute eosinophil counts and relative lymphocyte counts were associated with prolongedos in antipd1 and ctla4 treated melanoma given that previous studies have proposed the importance of baseline derived nlr dnlr and ldhlevels as prognostic markers a recent study proposed acomposite prognostic index that comprehensively takesthe two factors into account lung immune prognosticindex lipi which characterized risk groups goodintermediate and poor the analysis of patients with advanced nsclc in randomized trialss | Colon_Cancer |
ammation is an established risk factor for colorectal cancer we and others have shown that colorectal cancerpatients with elevated cysteinyl leukotriene receptor cyslt2r and 15hydroxyprostaglandin dehydrogenase15pgdh levels exhibit good prognoses however both cyslt2r and 15pgdh which act as tumour suppressorsare often suppressed in colorectal cancer we previously reported that leukotriene c4 ltc4induced differentiationin colon cancer via cyslt2r signalling here we investigated the involvement of hedgehog hhgli1 signallingwhich is often hyperactivated in colorectal cancer we found that the majority of colorectal cancer patients hadhighgli1 expression which was negatively correlated with cyslt2r 15pgdh and mucin2 and overall survivalcompared with the lowgli1 group ltc4induced 15pgdh downregulated both the mrna and protein expressionof gli1 in a protein kinase a pkadependent manner interestingly the ltc4induced increase in differentiationmarkers and reduction in wnt targets remained unaltered in gli1knockdown cells the restoration of gli1 in15pgdhknockdown cells did not ameliorate the ltc4induced effects indicating the importance of both 15pgdhand gli1 ltc4mediated reduction in the dclk1 and lgr5 stemness markers in colonospheres was abolished incells lacking 15pgdh or gli1 both dclk1 and lgr5 were highly increased in tumour tissue compared with thematched controls reduced mucin2 levels were observed both in zebraï¬sh xenografts with gli1knockdown cellsand in the cysltr2 colitisassociated colon cancer cac mouse model furthermore gli1 expression waspositively correlated with stemness and negatively correlated with differentiation in crc patients when comparingtumour and mucosal tissues in restoring 15pgdh expression via cyslt2r activation might beneï¬tcolorectal cancer patientsintroductioncolorectal cancer crc one of the most prevalentcancers in the world has a high metastatic efï¬cacy and alow 5year survival rate1 a nontargeted therapeuticapproach combined with late diagnosis leads to poorprognosis and treatment failure more than of crccorrespondence anita sj¶lander anitasjolandermedluse1cell and experimental pathology department of translational medicine lunduniversity sk¥ne university hospital malm¶ sweden2chemical biology therapeutics group department of experimental medicalscience lund university lund swedenfull list of author information is available at the end of the intracellular mechanismscases exhibit anomalous apcwntcatenin signallingwhich regulates the progression of crc by adopting differentthus affecting cancerstem cells and interactions with the tumour microenvironment hedgehog hh signalling which regulatesdifferentiation under physiological conditions has attracted attention because of its emerging role in the promotion and maintenance of crc2 in the untransformedcolon hh ligands are secreted by epithelial cells targetingmesenchymal cells as a classic paracrine hh signallingpathway to ensuring the proper size and location of thecryptvillus axis5 as also observed in other tissues6 the authors open access this is licensed under a creative commons attribution international license which permits use sharing adaptation distribution and reproductionin any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the creative commons license and indicate ifchanges were made the images or other third party material in this are included in the s creative commons license unless indicated otherwise in a credit line to the material ifmaterial is not included in the s creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this license visit httpcreativecommonslicensesby40oncogenesis 0csatapathy oncogenesis page of in crc abnormal hh signalling functions in a liganddependent manner and is activated in human cc celllines7 and xenograft models4 however the role of hhsignalling and its importance in cell survival in crc arenot well deï¬ned although some previous studies havefailed to derive a positive correlation between hh signalling and crc initiation and maintenance89 major bodiesof evidence point to a positive correlation347 moreoverprevious reports have suggested high activity ofthehhsmogli axis in crc cell survival and metastasiswhich is coordinated by either canonical signalling viasmo or a noncanonical mode of activation via therasmap kinase pathway47 within these pathways themost prominent factors are gliomaassociated oncogenehomologue gli and and the transcriptional regulators downstream of smo which keep the oncogenicpathway activecrc which is considered to be an ammationassociated cancer is greatly uenced by ammatorymediators such as leukotrienes and prostaglandins whichbelong to the gproteincoupled receptor family thecysteinyl leukotriene receptors cysltrs cyslt1r andcyslt2r are activated by binding with their highafï¬nityligands leukotriene d4 and c4 ltc4 respectively1011these proammatory lipid mediators are derived fromthe arachidonic acid pathway via 5lipoxygenase and theyplay crucial roles in pathological ammation such asthat observed in ammatory bowel disease we previously showed that elevated cyslt1r levels were associated with poor prognoses in crc patients whilepatients with high cyslt2r expression had better prognoses12 another important group of eicosanoids areprostaglandins pgs which are produced via the cox2pathway the upregulation of cox2 in crc increasesthe pge2 level which promotes cancer cell proliferationangiogenesis survival migration and invasion these areimportant hallmarks of cancer1314thetumoursuppressor15hydroxyprostaglandindehydrogenase 15pgdh is an enzyme responsible forthe degradation of pge2 into an inactive metabolite15 pgdh is abundantly expressed in normal colon mucosabut its expression is lost in crc cells1617leading todisease progression recently researchers have exploredthe efï¬cacy of 15pgdh as a potential antitumour agentagainst colon cancer18 in a recent study we established that 15pgdh is induced by ltc4 via cyslt2rsignalling by phosphorylating cjun nterminal kinaseand ap1 to induce 15pgdh promoter activity andfurther guide colon cancer cells toward redifferentiation20however the detailed mechanism underlying this phenomenon remains unclearin this study we elucidated the mechanism by whichltc4induced 15pgdh promotes differentiation incolon cancer cells through cyslt2r activation with theoncogenesisinvolvement of hhgli signalling we observed thatgli1 was involved in the regulation of the redifferentiation and reduction in stemness induced by ltc4 via pgdh in colon cancer cellsresultsgli1 expression is negatively correlated with cyslt2r pgdh and mucin2 expression in crc patientsto elucidate the regulatory activity of gli1 on theantitumorigenic proteins cyslt2r and 15pgdh and thedifferentiation marker mucin2 in colon cancer we used atissue microarray tma of primary crcs from patients22 after ihc analysis we found only ï¬ve patientswith negative gli1 staining patients with weak stainingintensity patients with moderate staining intensityand patients with strong staining intensity the mean ±standard deviation sd of the immunoreactive score irsfor gli1 expression was ± then we grouped thepatients with negative and weak staining intensity anddeï¬ned them as the lowgli1 expression group n and those with moderate and strong staining intensitywere deï¬ned as the highgli1 expression group n fig 1a seventy patients had missing or incomplete coresand were excluded from the ï¬nal analysisafter ihc evaluation of cyslt2r and 15pgdh weobserved that patients with highgli1 expression hadsignificantly lower levels of cyslt2r irs ± and15pgdh irs ± expression than those with lowgli1 expression fig 1bd furthermore there was asignificant negative correlation between gli1 and pgdh r p suggesting hyperactivatedhhgli signalling with suppressed 15pgdh fig 1ehowever no significant correlation was found betweengli1 and cyslt2r expression fig 1eexhibitedhighgli1on the other hand patients with lowgli1 expressionhad a significantly higher irs for cyslt2r ± and15pgdh ± expression fig 1c d which indicates the adverse effects of cyslt2r15pgdh axis activation on gli1 we also noticed that the majority ofpatientspatients and when stratiï¬ed according to the tumournodemetastasis tnm staging a significantly strongerassociation for the patients with tnm stages iii and ivwas found fig 1e in addition to the above observationstranscriptome data from a public database23 also suggested a significant negative correlation between gli1 andthe tumour suppressors cysltr2 fig 1f and hpgd15pgdh fig 1gexpressionimportantly we observed that patients with lowgli1expression had a lower risk of overall mortalityhazard ratio ci than patientswith highgli1 expression after adjusting for age andtnm stage fig 1h the unadjusted survival curve isprovided in supplementary fig s1a the median follow 0csatapathy oncogenesis page of fig see legend on next pageoncogenesis 0csatapathy oncogenesis page of see ï¬gure on previous pagefig gli1 expression exhibited a negative correlation with cyslt2r and 15pgdh expression in colorectal cancer patient tissuesa matched pair immunohistochemistry ihc images of gli1 cyslt2r and 15pgdh expression in patients with low and highgli1 expressionshown at magniï¬cation the staining immunoreactivity was quantiï¬ed by the mean immunoreactive score irs calculated according to thefollowing formula irs staining intensity of stained cells the mean irs for the groups of patients with low and highgli1 expression b theyaxis represents irs for gli1 in these patients c cyslt2r and d 15pgdh expression according to patients with low and highgli1 expression in crctissue e distribution of tumournodemetastasis tnm stages of crc according to low and highgli1 expression pairwise pearson correlationcoefï¬cient r between the expression of gli1 and that of cyslt2r and 15pgdh p value according to chisquare test xyscatter plots showing mrnalevels of f gli1 and cyslt2r and g gli1 and 15pgdh hpgd gene expression from a public database containing crc patients kaplanmeiercurves for overall survival adjusted for age and tnm stage for patients with h low and highgli1 expression and subgroups of patients with bothgli1 and i cyslt2r or j 15pgdh expression compared by the logrank test the last patient group served as the reference category k western blotanalysis showing the protein expression of cyslt2r 15pgdh and gli1 in matched pairs of six patients with normal n and tumour t areas kdaindicated on the left side of the immunoblots kda represents protein size markers graphical representation of the densitometricanalysis showing the relative protein expression for cyslt2r 15pgdh and gli1 in matched pairs of patient samples n from normal mucosa mwhite and tumour t black areas l qrtpcr analysis of gli1 in matched pairs of normal mucosa m white and tumour t black tissues from crcpatients n scale bar as indicated in the images data represent the mean ± sd p p p mannwhitney testup time was months years with total eventsthisrole of gli1 in coloncarcinogenesissuggests a prominentwe have previously shown that crc patients with lowcyslt2r andor low15pgdh expression have a poorprognosis1220 in this study we noted that patients withhighgli1 expression coupled with either lowcyslt2rexpression orlow15pgdh expression had poorerprognoses than patients with either lowgli1 and low15pgdh expression orlowgli1 and lowcyslt2rexpression fig 1i j western blot analysis of six matchedpairs of crc patients showed significantly higher proteinexpression of cyslt2r and 15pgdh in normal tissuethan in matched tumour tissue fig 1k howevergli1 showed elevated expression in tumour tissue compared with matched normal tissue fig 1kfurthermorethe mrna analysis ofthese pairedtumour tissues with matched mucosa tissues from crcpatients n showed significantly higher mrnaexpression of gli1 in the tumour tissue compared with itsmatched normal mucosa the normal mucosa as referenceset to and tumour tissue ± mean ±sem mannwhitney test p fig 1l furthermore the muc2 mucin2 mrna analysis of thesepaired tumour tissues t and matched normal mucosam showed significantly lower mrna expression ofmuc2 in the tumour tissue compared with its matchednormal mucosa the normal mucosa as reference set to and tumourtissue ± mean ± semmannwhitney test p fig 2a a representativematched pair of high and lowgli1 and the corresponding mucin2 is shown fig 2b we observed thatpatients with highgli1 expression had lower levels ofmucin2 expression than those with lowgli1 expressionfig 2c and by combining these data a significantnegative correlation between elevated expression of gli1and decreased expression of mucin2 in tumour tissueswas found fig 2d moreover grouping the patients intooncogenesismucinous n and nonmucinous n typesrevealed that of patients with nonmucinous status had highgli1 expression suggesting anegative correlation with mucin2expressing cells while of patients in the mucinous category showedhighgli1 expression fig 2d furthermore we found asignificant negative correlation between gli1 and thedifferentiation marker mucin2 expression in these crcpatients n fig 2e we found a better overallsurvival for patients with lowgli1 than those with highgli1 expression regardless of mucin2 expressionfig 2e taken together these results suggest that gli1expression is negatively correlated with cyslt2r pgdh and mucin2 expression but positively with themucinous status of the patientscyslt2r is essential for differentiation in a colitisassociated colon cancercacmouse modelto further validate the role of cyslt2r in promotingdifferentiation in crc we adopted an ammatorymouse model that was induced by azoxymethane aomand dextran sodium sulfate dss brieï¬y c57bl6nwildtype and cysltr2 mice were subjected to aomand two dss cycles24 fig 2f as described in thematerials and methods section we found that all miceformed polyps in the colon regardless the phenotype butcysltr2 mice n developed significantlargerpolyps ¥ mm p in the colon compared withtheir wildtype wt n littermates supplementaryfig s1b c this result indicates that cysltr2 micedevelop a more progressive disease supplementary figs1 shows a representative image of colon polypsfig s1d and a likewise representative haemotoxylinand eosinstained imageshowing premalignant areas aberrant cryptfoci and metaplasiaareas from both wt and cysltr2 mice colonfig s1ecolon tissue sections from wt mice showed abundantmucin2expressing cells compared with sections from 0csatapathy oncogenesis page of fig see legend on next pageoncogenesis 0csatapathy oncogenesis page of see ï¬gure on previous pagefig gli1 expression was negatively correlated with differentiation a qrtpcr analysis of mucin2 in matched pairs of normal mucosa mwhite and tumour t black tissues from crc patients n b immunohistochemistry ihc images for gli1 and mucin2 expression in matchedpaired tissue samples from colorectal cancer crc patients with low and highgli1 expression represented at magniï¬cation c quantiï¬cation ofthe staining immunoreactivity by the mean irs for mucin2 expression according to patients with low and highgli1 expression in crc tissued distribution of tumour type mucinous adenocarcinomas versus nonmucinous adenocarcinomas according to low and highgli1 expressionpairwise pearson correlation coefï¬cient r between the expression of gli1 and mucin2 p value according to chisquare test e kaplanmeier curvesfor overall survival adjusted for age and tnm stage subgroups of patients with both gli1 and mucin2 expression compared by the logrank test thehighgli1 and lowmucin2 patient group was set as the reference category f experimental schematic of the aomdss mouse model gli1expression exhibited a negative correlation with differentiation in the cysltr2 aomdss mouse model immunohistochemical evaluation showingthe protein expression of g mucin2 and h gli1 in wt and cysltr2 aomdsschallenged mice graph bars showing the irs scores for mucin2 andgli1 respectively compared between wt and cysltr2 n micegroup scale bar as indicated in the images data represent the mean ± sdp mannwhitney testcysltr2 mice ± n fig 2g gli1 exhibited aslightly higher but nonsignificant overall expression incysltr2 mouse tissue sections compared with wt tissuesections ± n fig 2h taken together theabove observations encouraged us to further investigatehhgli signalling using both in vitro and in vivo coloncancer model systems and to delineate the involvement ofcyslt2r and 15pgdh in promoting differentiationltc4induced 15pgdh downregulates gli1 in coloncancer cellswe determined whether hhglisignalling wasinvolved in the ltc4induced 15pgdhpromoted differentiation of cc cells20 interestingly ltc4 stimulationsignificantly downregulated gli1 expression at both themrna and protein levels compared with unstimulatedht29 and caco2 cells fig 3a b in addition immunoï¬uorescence analysis of gli1 expression revealed adecrease in nuclear gli1 upon ltc4 stimulation fig 3cto determine the mechanism of 15pgdhmediateddepletion of gli1 we examined the expression of proteinkinase a pka which is a known gli1 antagonist2526the activation of the pka αδ catalytic subunit incaco2 cells after stimulation by ltc4 was significantlyincreased as indicated by the levels of phosphorylatedpka threonine but remained unchanged in stimulated ht29 cells fig 3b moreover phosphorylation atserine on the catalytic subunit of pka wasincreased after ltc4 stimulation in ht29 cells but wasnot present in caco2 wholecell lysates fig 3b15pgdhspeciï¬c shrna shhpgd was employed toinvestigate the involvement of 15pgdh in the ltc4mediated downregulation of gli1 expression comparedwith cells transfected with control shrna shctrl cccells transfected with shhpgd showed no response toltc4 stimulation we observed that shrnamediatedknockdown of hpgd did not affect the expression of theintestinal differentiation markers cdx2 or cdhr2 or thewnt target axin2 supplementary fig s2ad for ht29cells and unaltered gli1 expression at both the mrnaoncogenesisand protein levels fig 3df see supplementary figs3af for caco2 cells similarly 15pgdh knockdownreduced the effect of ltc4 stimulation on pka activationin both ht29 and caco2 cells fig 3e supplementaryfig s3fto further conï¬rm the role of pka as an intermediatemolecule in ltc4induced 15pgdhmediated downregulation of gli1 we used h89 a pkaspeciï¬c inhibitor nm prior to stimulating the cells with ltc4 wefound that neither ht29 nor caco2 cells treated withthe pka inhibitor affected ltc4induced 15pgdhexpression at either the mrna or protein levels however no significant alteration was observed in either themrna or protein level of gli1 poststimulation withltc4 fig 3g h supplementary fig s4a b which wasalso supported by immunoï¬uorescence analysis fig 3isupplementary fig s4c these data indicate that pkaplays a role in the 15pgdhmediated downregulation ofgli1 in colon cancer cellsgli1 regulates 15pgdhpromoted differentiation in coloncancer cellsnext we determined the mechanism by which ltc4induced 15pgdh promoted differentiation in coloncancer cells20 based on the above evidence we investigated the effects of gli1 knockdown fig 4aj for ht29cells and supplementary fig s5ak for caco2 cells onhpgd 15pgdh expression we used shgli1 todetermine possible changes in the expression of pgdh however gli1 knockdown in these cells did notaffect ltc4induced 15pgdh expression at either themrna or protein level compared with the correspondingshctrltransfected cells these data indicate that theeffect of ltc4 signalling on 15pgdh expression isindependent of gli1 suggesting that gli1 is downstreamof 15pgdh we next validated the involvement of gli1in differentiation by testing the mrna expression of sisucraseisomaltase and muc2 mucin2 which arerepresentative intestinal differentiation markers followingltc4 stimulation the observed increases in the mrna 0csatapathy oncogenesis page of fig 15pgdh regulates the ltc4mediated downregulation of hhgli signalling in colon cancer cells a qrtpcr analysis of gli1 mrnaexpression in ht29 and caco2 cells with or without ltc4 stimulation for h b western blot analysis of 15pgdh gli1 and phosphopka αγ subunitand subunit levels in ht29 and caco2 cells with or without ltc4 stimulation αtubulin served as the loading control c immunoï¬uorescence analysisof gli1 in ht29 cells with or without ltc4 stimulation for h d qrtpcr analysis of ht29 cells transfected with control shrna shctrl or pgdhspeciï¬cshrna shhpgd with or without ltc4 stimulation for h e western blot analysis of ht29 cells transfected with control shrna shctrl or pgdhspeciï¬cshrna shhpgd blotted with antibodies against 15pgdh gli1 or phosphopka αγ subunit and subunit with or without ltc4 stimulation for hαtubulin served as the loading control f immunoï¬uorescence analysis of gli1 in ht29 cells transfected with control shrna shctrl or pgdhspeciï¬cshrna shhpgd with or without ltc4 stimulation for h g qrtpcr analysis of 15pgdh and gli1 in ht29 cells treated with the pka inhibitor h89 pkainh for h followed by ltc4 for h h western blot analysis showing the expression of 15pgdh gli1 and phosphopka αγ subunit and subunit inht29 cells treated with the pka inhibitor h89 pkainh for h followed by ltc4 for h i immunoï¬uorescence analysis of gli1 in ht29 cells treatedwith the pka inhibitor h89 pkainh for h followed by ltc4 for h hprt1 was used as the housekeeping gene for normalisation of the qrtpcr geneexpression data graphs represent the mean ± sem of data from to independent experiments p p oncogenesis 0csatapathy oncogenesis page of fig 15pgdh promotes differentiation in colon cancer via gli1 qrtpcr validation of gene expression in shctrl control shrna or shgli1gli1speciï¬c shrnatransfected ht29 cells with or without ltc4 stimulation for h marker of tumour suppression in a 15pgdh markers ofdifferentiation in b si in c mucin2 and in d cdhr2 marker of differentiation regulation as in e cdx2 markers of wnt activation in f axin2 andg cmyc marker of proliferation in h ccnd1 hprt1 was used as the housekeeping gene for normalization of the gene expression data i western blotanalysis of wholecell lysates for 15pgdh gli1 si cdx2 and cdhr2 expression in shctrl control shrna or shgli1 gli1speciï¬c shrnatransfected ht29 cells with or without ltc4 stimulation for h αtubulin served as the loading control j immunoï¬uorescence analysis of gli1 andmucin2 in cells with or without ltc4 stimulation for h and transfected with shctrl or shgli1 graphs represent the mean ± sem of data from threeindependent experiments p p p oncogenesis 0csatapathy oncogenesis page of and protein expression of si after ltc4 stimulation inboth ht29 fig 4b and caco2 cells supplementaryfig s5c were abolished in gli1knockdown cellsfig 4b supplementary fig s5c j furthermore similarresults were observed for the mrna expression of otherdifferentiation markers such as muc2 fig 4c supplementary fig s5d and for both the mrna and proteinexpression of cdhr2 and cdx2 fig 4d e supplementary fig s5e f j ltc4 induced a reduction inaxin2 a potential wnt signalling target fig 4f supplementary fig s5g furthermore myc and ccnd1mrna expression was also abolished in gli1knockdowncells exposed to ltc4 fig 4g h supplementary fig s5hi to further validate the above observations we performed western blot fig 4i and immunoï¬uorescencemicroscopy using double staining for gli1 and mucin2in shctrl and shgli1transfected cells and found thatmucin2 expression was downregulated in cells lackinggli1 fig 4j supplementary fig s5k these ï¬ndingssupport our hypothesis of a possible regulatory effect ofgli1 in ltc4induced 15pgdhpromoted differentiation in colon cancer cellsgli1 suppresses differentiation in the absence of 15pgdhwe next investigated the ability of gli1 to regulate pgdh by overexpressing gli1 using the pegfphgli1construct in combination with the simultaneous knockdown of hpgd with shhpgd supplementary figs s6ab s7a b we found significant downregulation in theltc4induced increase in si muc2 cdhr2 and cdx2mrna expression levels in ht29 cells fig 5ad andalso in caco2 cells supplementary fig s7cf thatoverexpressed gli1 and lacked 15pgdh compared withcells expressing the control vector shctrl we alsofound that the ltc4induced reduction in axin2 mycand ccnd1 mrna was abolished although we observedincreased basal levels of these mrnas fig 5eg supplementary fig s7gi we next investigated the proteinexpression of si by western blot fig 5h supplementaryfig s7j and mucin2 by immunoï¬uorescence microscopy fig 5i supplementary fig s7k the ltc4induced increase in si was abolished in hpgdknockdown and gli1overexpressing cells fig 5h supplementary fig s7j a similar pattern was found regardingmucin2 protein expression in these cells which wasobserved using immunoï¬uorescence microscopy fig 5isupplementary fig s7k finallythe ltc4inducedreduction in gli1 was abolished fig 5iltc4induced cyslt2r signalling downregulates gli1 in a15pgdhdependent mannerto determine whether ltc4 acts via cyslt2r as ltc4is the highafï¬nity ligand of cyslt2r we investigated thespeciï¬c involvement of cyslt2r signalling in the ltc4oncogenesisstableofknockdownmediated downregulation of gli1 in colon cancer cellswe ï¬rst constructed hct116 cells with doxycyclinedoxinduciblecyslt2rshcysltr227 we examined the mrna and proteinexpression levels of cyslt2r 15pgdh and gli1 withand without ltc4 stimulation in this cell line with doxinduction and compared them with the levels in cellsgrown in the absence of dox the mrna expression ofcysltr2 and hpgd wassignificantly upregulatedfig 6a b while gli1 mrna expression was downregulated in cells cultured without dox fig 6c howin the doxinduced cells gli1 gene expressioneverremained unaltered most likely due to the low mrnaexpression levels of cysltr2 and hpgd these observations were also reï¬ected in the levels of proteinexpression fig 6d supplementary fig s8acto further study the involvement of cyslt2r we treatedht29 and caco2 cells with the cyslt2rspeciï¬cantagonist ap100984 µm followed by ltc4 stimulation for h20 ap100984 treatment efï¬ciently blocked boththe mrna supplementary fig s8d e and proteinexpression supplementary fig s8f of cyslt2r as well asof its downstream signal 15pgdh in ht29 cells as well asin caco2 cells supplementary fig s8gj ap100984 pretreatment abolished the effect of ltc4 stimulation on gli1at both the mrna and protein levels fig 6e f supplementary fig s8i j taken together the above resultsshowed that cyslt2r signalling plays a role in the ltc4induced 15pgdhmediated downregulation of gli1ltc4induced 15pgdh expression reduces stemness incolonosphereswe extended our study to determine whether ltc4induced 15pgdh affected stemness in colon cancer cellsas well as the possible role of gli1 we created a 3dmodel of multicellular colonospheres derived from coloncancer cells fig 6g we observed that shctrltransfected ht29 or caco2 cellderived colonospheresshowed reduced numbers and sizes with ltc4 stimulation fig unlike the shctrl group shhpgdtransfected cellderived colonospheres showed increases innumber and size howeverthe absence of gli1 inshgli1transfected cellderived colonospheres resulted inno significant alteration in size or number even afterltc4 stimulation the mrna expression levels of thecolon cancerspeciï¬c stemness markers dclk1 lgr5and aldh1a1 were elevated in ht29 as well as in caco cellderived colonospheres and were downregulatedafter ltc4 stimulation fig 6i supplementary figs9ah the mrna and protein levels of the cancer stemcell markers dclk1 and aldh1a1 remained unchangedin both shhpgd and shgli1knockdown ht29 andcaco2 cellderived colonospheres even after ltc4 stimulation compared with their unstimulated counterparts 0csatapathy oncogenesis page of fig gli1 negatively regulates the differentiation and promotes the proliferation of colon cancer cells in the absence of 15pgdh ht29cells were either transfected with shctrl alone or cotransfected with shhpgd and pegfphgli1 followed by ltc4 stimulation for h qrtpcranalysis of the differentiation markers a si b mucin2 and c cdhr2 d the differentiation regulation marker cdx2 e the wnt activation marker axin2f the prooncogene cmyc and g the proliferation marker ccnd1 h western blot analysis of wholecell lysates for 15pgdh gli1 and si expressionαtubulin was used as the loading control i immunoï¬uorescence analysis of gli1 and mucin2 in unstimulated or ltc4stimulated cells transfectedwith shhpgd or shgli1 hprt1 was used as the housekeeping gene for normalization of the qrtpcr gene expression data graphs represent datafrom to independent experiments and represent the mean ± sem p p p fig 6ik supplementary fig s9ah gli1 gene andprotein expression in ht29 and caco2derived colonospheres was also downregulated after ltc4 stimulation we next analysed the mrna expression levels intumour and adjacent mucosa samples from pairedcrc patients which also suggested a significantly positivecorrelation between gli1 and the stemness markersdclk1 and lgr5 with elevated expression of all threegenes in tumour tissues figs 1l 6ln the normalmucosa as reference set to and the tumour tissue fordclk1 ± and lgr5 ± mean± sem respectively mannwhitney test p asexpected the expression level of dclk1 showed a negative correlation with cysltr2 and hpgd expressiononcogenesis 0csatapathy oncogenesis page of fig see legend on next pagefrom our previously published results see ref whilemuc2 had a positive correlation fig 2c20 this suggeststhat a poorly differentiated tumour could occur due toenriched stemnessltc4induced 15pgdh promotes differentiation inzebraï¬sh xenograftsnext we used the zebraï¬sh xenograft model28 to further evaluate and visualise the differentiationpromotingoncogenesis 0csatapathy oncogenesis page of see ï¬gure on previous pagefig ltc4induced 15pgdh expression negatively regulates gli1 via cyslt2r graphs showing qrtpcr analysis of a cysltr2 b 15pgdhand c gli1 mrna expression in hct116 cells with stable transfection of doxycyclineregulated shcysltr2 cultured with or without doxycycline µm treatment followed by ltc4 nm stimulation for h d western blot analysis of cyslt2r 15pgdh gli1 and si expression in wholecelllysates αtubulin served as the loading control e qrtpcr analysis showing mrna expression of gli1 and f western blot analysis of gli1 proteinexpression in ht29 cells stimulated with or without ltc4 and with or without ap100984 a cyslt2r antagonist hprt1 was used as the housekeepinggene and αtubulin was used as the loading control in the western blot assay g schematic illustration of colonosphere formation gli1 regulates theeffect of ltc4 on stemness in multicellular colonospheres the cells were cultured in ultralowattachment conditions on matrigel containing serumfree medium for days h representative images of colonospheres from ht29 cells transfected with shctrl shhpgd or shgli1 and stimulated ornot stimulated with ltc4 bar graphs showing the number of colonospheres formed per well and the size of colonospheres with or without ltc4stimulation and comparing the shctrl shhpgd and shgli1transfected groups qrtpcr analysis of the stemness markers i dclk1 j gli1 andm lgr5 in colonospheres derived from shctrl shhpgd or shgli1transfected ht29 cells with or without ltc4 stimulation for h k western blotanalysis showing the expression of dclk1 15pgdh and gli1 in transfected ht29 cells as indicated αtubulin served as the loading control qrtpcr analysis of l dclk1 and n lgr5 in matched pairs of mucosa m and tumour t tissues from crc patients n hprt1 was used as thehousekeeping gene for normalization of the qrtpcr gene expression data data represent the mean ± sem from to independent experimentsp p p role of 15pgdh in colon cancer transgenic zebraï¬sh tgï¬i1egfp embryos were injected with u | Colon_Cancer |
" preproof 0c highlights f0b7 propolis made by bees from bioactive plant resins has antiviral activity f0b7 propolis potentially can interfere with host cell invasion by sarscov2 f0b7 propolis blocks proinflammatory pak1 a kinase highly expressed in covid19 patients propolis a resinous material produced by honey bees from plant exudates has long been used in traditional herbal medicine and is widely consumed as a health aid and immune system booster the covid19 pandemic has renewed interest in propolis products worldwide fortunately various aspects of the sarscov2 infection mechanism are potential targets for propolis compounds sarscov2 entry into host cells is characterized by viral spike protein f0b7 propolis is a safe widely consumed functional food with medicinal properties f0b7 standardized propolis has consistent properties for lab and clinical research preprooffactor in advanced covid19 disease propolis has also shown promise as an aid in the treatment of various of the comorbidities that are particularly dangerous in covid19 patients including induced lung inflammation fibrosis and immune system suppression propolis components have inhibitory effects on the ace2 tmprss2 and pak1 signaling pathways in addition antiviral activity has been proven in vitro and in vivo in preclinical studies propolis promoted α this immunoregulation involves monocytes and macrophages as well as jak2stat3 nfkb and inflammasome pathways reducing the risk of cytokine storm syndrome a major mortality interaction with cellular angiotensinconverting enzyme ace2 and serine protease tmprss2 this mechanism involves pak1 overexpression which is a kinase that mediates coronavirusimmunoregulation of proinflammatory cytokines including reduction in il6 il1 beta and tnfrespiratory diseases hypertension diabetes and cancer standardized propolis products with consistent bioactive properties are now available given the current emergency caused by the covid19 pandemic and limited therapeutic options propolis is presented as a promising and 0crelevant therapeutic option that is safe easy to administrate orally and is readily available as a natural supplement and functional food keywords propolis sarscov2 covid19 antiviral antiinflammatory pak1 blocker introduction the covid19 pandemic is of grave concern due its impact on human health and on the deadly disease most require more robust data in clinical trials before they can be widely and safely used isolation and stayathome measures do not effectively protect essential workers economy it is much more deadly than influenza and other types of diseases that recently have had worldwide impact forcing countries to take unusual measures such as limiting travel closing schools businesses and other locations where many people can come into contact with each other various public healthcare strategies have been adopted in an attempt to reduce the impact of the disease but with limited effectiveness as the virus continues to spread often through asymptomatic patients unfortunately tests to determine if people are infectious or were previously infected are not widely available often are costly and frequently do not provide timely and accurate results various therapeutic alternatives have been proposed and tested however preproofnonessential professions return to the workplace they become more at risk for infection in this scenario any options that could help ameliorate disease progression and its consequences even marginally would be useful the world needs safe alternatives to help reduce the impact of this especially health care personnel who have become infected and are dying at alarming rates economic and other necessities limit how well and how long these isolation measures can be maintained especially in poor countries and in poor communities such as slums and favelas [ ] as populations gradually try to get back to normalcy reducing social distancing and people in natural products which have historically been widely used to help avoid and alleviate diseases are among the options being considered as an adjuvant treatment for sarscov2 infection because they are generally inexpensive widely available and rarely have undesirable side effects some have proven antiviral activity an important advantage of using natural remedies is that people who have other health problems or who have mild flurelated 0c infection infection by sarscov2 the virus that causes covid19 is characterized by binding symptoms but do not have the means or courage to visit an already overcrowded medical facility could take simple and inexpensive measures to help reduce the impact of infection with sarscov2 considering the large number of deaths and other types of damage that the covid19 pandemic is causing there is an urgent need to find treatments that have been approved as safe and potentially able to inhibit the new coronavirus reduce its infectivity andor alleviate the symptoms of infection [ ] along this line propolis and its components emerge as potential candidate materials that could help to reduce the pathophysiological consequences of sarscovfactors increased pak1 levels also suppress the adaptive immune response facilitating viral replication [ ] sarscov2 infection is associated with increased levels of chemokines and activated proinflammatory cytokines that lead to the development of atypical pneumonia with rapid respiratory impairment and pulmonary failure immunologicalinflammatory between viral spike proteins and angiotensinconverting enzyme ace2 activation of the spike protein is mediated through proteases such as tmprss2 which play important roles in the viral infection after entry followed by endocytosis coronavirus infection causes pak1 upregulation a kinase that mediates lung inflammation lung fibrosis and other critical mortality preproofphenomena such as cytokine release syndrome have been shown to be important in the spectrum of sarscov2 infection these mechanisms are associated with an dysfunction more than the viral load per se along this line a retrospective observational study found higher serum levels of proinflammatory cytokines such as il6 il1 and tnfα in patients with severe inflammatory diseases by affecting various metabolic cycles recently several studies have shown that propolis extract and some of its components act against several important targets in the pathophysiological context of the disease caused by sarscov2 such as reducing tmprss2 expression and reducing ace2 anchorage which would otherwise facilitate entry of covid19 compared to individuals with mild disease there is considerable evidence that propolis can reduce and alleviate the symptoms of the virus into the cell this is in addition to immunomodulation of monocytes macrophages reducing production of and eliminating il1 beta and il6 reduction of the transcription factors 0cviral replication and antiinflammatory action [ ] propolis and its properties are particularly relevant to sarscov2 infection such as immune system fortification reduced nfkb and jak2 stat3 and blocking pak1 which determine inflammatory activities and fibrosis caused by covid19 various comorbidities have been associated with severe covid19 symptoms and a greater chance of patients requiring intensive care these include hypertension and diabetes also mortality rates of covid19 patients are much higher in those with cardiovascular disease chronic respiratory disease and diabetes [ ] there is considerable evidence that these conditions could be alleviated by treatment with propolis this includes research in animal models of diabetes [ ] hypertension [ ] and cardiovascular disease [ ] propolis has properties that preproofthese plant products to make propolis which they use to protect the colony the production and use of propolis by honey bees evolved to the point that these social bees have considerably fewer immune genes than solitary insect species bees in colonies that produce more propolis are healthier and live longer and propolis consumption by the bees augments their immune [ ] as this variability can affect their medicinal properties standardized propolis products have been developed to help meet the need for a product that does not vary in the main bioactive components and is safe with minimal interaction with pharmaceutical drugs and proven efficacy in clinical trials in recent decades it has been shown to have antimicrobial including the composition of propolis varies according to the plant species available in each region propolis is a product derived from resins and plant exudates plants defend themselves from pathogens mainly by producing phytochemicals many of which have been extracted and used in medicine plant defense substances collected by bees include phenols and terpenoids phytochemical compounds that show promise for the inhibition of coronavirus in humans include quercetin myricetin and caffeic acid all components of propolis honey bees and many other species of social bees recognize these antimicrobial properties and selectively collect and process response to bacterial challenge antiviral antiinflammatory immunomodulatory antioxidant and anticancer properties propolis has historically been widely used to alleviate various diseases [ ] it also has been considered among other natural alternatives as an adjuvant treatment for sarscov2 infection 0ccompared to the propolis that the bees make from these plant materials because it is generally inexpensive widely available and rarely causes undesirable side effects some types of propolis that are highly valued for their medicinal properties such as brazilian green propolis are mainly produced by bees from materials they collect from specific plants in this case baccharis dracunculifolia after the botanical origin of the propolis has been identified extracts of the plant can be made to develop useful products such as a medicinal mouthwash however the medicinal properties of these plant extracts are often inferior can be safely consumed these propolis products and the raw material for their manufacture are extensively exported by brazilian companies especially to asian countries including japan south among natural medicine alternatives propolis has been widely studied and is already extensively consumed in many countries [ ] for example propolis products such as throat sprays and extracts are produced by hundreds of companies in brazil and are sold as a health aid in nearly every pharmacy throughout the country demonstrating on a practical basis that they preproofin fact propolis has a wide spectrum of pharmacological properties and is a dietary supplement that is commonly consumed by both healthy and sick people as a preventative precaution and for treatment it is also used in veterinary medicine due its antibacterial antifungal antiviral korea and china [ ] the importance of propolis in chinese japanese russian and korean medicine is reflected in the number of patents for propolis containing products registered by including about by china and each for japan russia and korea since about new propolisrelated patents were applied for in the us patent office it is a key ingredient in traditional chinese medicine japanese scientists have isolated and patented various brazilian propolis components for cancer treatment demonstrating their usefulness why propolis may be a good fit for dealing with covid19 antiparasitic hepatoprotective and immunomodulatory activities in the wake of the coronavirus outbreak south korea has seen a boon in the use of functional foods according to their ministry of food and drug safety health functional foods are nutrients that have been proven to be beneficial to health in march of this year in response to the coronavirus pandemic the ministry eased regulations for propolis which is considered a 0ccould help fight against the covid19 pandemic active site of this enzyme is a relevant target for drug discovery functional food and allowed new oral formulations however despite considerable evidence that propolis can reduce and alleviate disease symptoms its acceptance as a healthpromoting supplement in human medicine has been limited in many countries such as the usa because of a relevant criticism that propolis products are not standardized and vary in their components and biological activity in part this is because propolis varies with the species of plants available in each region from which the bees collect resins to produce it [ ] however standardized propolis products have recently become available to help fill the need for a product that does not vary in the main bioactive components and effectiveness [ ] one such option a standardized brazilian propolis extract blend has been tested for safety and effectiveness in clinical trials for treating kidney disease and diabetes denture stomatitis and burn patients therefore propolis as a nutraceutical or functional food should be considered as a resource that preproofcaffeic acid phenethyl ester cape galangin chrysin and caffeic acid substances found in several different types of propolis around the world appeared as potential drugs against this viral target table specifically cape was predicted to interact with sarscov2 mpro in a potential targets in order to inactive the virus and reduce the damage that it causes the main protease of coronavirus sarscov2 mpro 3chymotrypsinlike cysteine enzyme is essential similar study therefore although it will be necessary to run in vitro assays to evaluate the potential antisarscov2 effects of propolis andor its constituents these in silico results are along this line hashem evaluated various natural compounds with an in silico approach molecular docking to try to find useful options for treating sarscov2 infection curiously for coronavirus processing of polyproteins and for its life cycle and therefore inhibition of the some propolis compounds can potentially interact with sarscov2 mpro the research community has examined the genetic code of coronavirus and the mechanisms underlying the damages caused by sarscov2 to help search for drugs andor well boding propolis can interact with ace and tmprss2 potentially blocking or reducing sarscov2 invasion of the host cell 0c sarscov2 strongly binds to angiotensinconverting enzyme ace2 using this enzyme as a receptor for invasion and replication in the host cell [ ] causing damage and increasing interpersonal transmission [ ] consequently ace inhibitors have been considered as useful drug alternatives however potential deleterious effects on users of angiotensinconverting enzyme ace inhibitors and angiotensin receptor blockers arbs have tool against potential cardiovascular events inhibition of ace2 enzyme is an important target for treatment against sarscov2 myricetin caffeic acid phenethyl ester hesperetin and pinocembrin rutin interacts with zinc fingers of the active sites of ace2 a metalloprotease that presents the same zinc finger in ace1 emerged as a concern for treatment of covid19 patients an observational study involving patients did not confirm this suspicion and therefore these classes of drugs remain an important infection [ ] güler et al prepared an alcoholic extract of propolis and identified some hydroxycinnamic acids caffeic acid pcoumaric acid tcinnamic acid and cape the flavanons rutin and myricetin and the flavones hesperidin chrysin and pinocembrin using molecular docking evaluations they found that rutin had the highest binding energy to ace2 followed by preproofvarious characteristics including inhibition of ace they found strong inhibition for most of the propolis types they studied with higher than ace inhibition the best results were found in addition to the in silico evidence osés et al evaluated several types of propolis for with the propolis components catechin and pcoumaric acid ace2 and tmprss2 transmembrane serine protease on the surface of host cells are used by sarscov2 via interaction with spike glycoproteins in order to proceed with invasion and replication vardhan sahoo studied several molecules commonly found in medicinal herbs using molecular docking procedures with relevant targets such as rnadependent rna polymerase rdrp ace2 and spike glycoproteins and compared the resulting scores with those of hydroxychloroquine limonin was the most active compound however quercetin and kaempferol also propolis compounds gave high docking scores kaempferol was studied in prostate cancer models and the expression of tmprss2 was reduced showing a potential mechanism of action for an antitumoral effect kaempferol could be an important propolis component for use against covid19 since it is involved in the inhibition of tmprss2 0c potentially interacting with ace2 rdrp and spike glycoprotein sgp besides its antiviral activity table propolis blocks pak1 potentially avoiding lung fibrosis and restoring a normal immune response among the possible targets for controlling covid19 damage the major pathogenic contributes to their suppression pak1 inhibitors can both help combat the virus and restore a normal immune response kinase pak1 is key it is an essential component in malaria and viral infections but it is also involved in a wide variety of other diseases and disease conditions including cancer inflammation and immunosuppression when abnormally activated consequences of pak1 activation include lung fibrosis which is an aggravating factor in covid19 pak1 is activated by rac xu et al demonstrated that caffeic acid and its ester cape components of propolis can inactivate rac consequently inhibiting pak1 the inactivation of pak1 directly or upstream can potentially attenuate coronavirus pathogenesis bcells and tcells are lymphocytes that produce specific antibodies against viruses and other intruders and pak1 preproofimprove the immune response its components increase neutralizing antibody titers activate phagocytosis and increase ifnγ levels and the number of lymphocytes an increase in ifnγ levels was also detected by shimizu et al who evaluated the mechanisms involved in the cape caffeic acid phenethyl ester is a potent inhibitor of activation of nfkb in myelomonocytic cells anse et al demonstrated that propolis cape quercetin hesperidin and some other propolis flavonoids can inhibit the cytokine production of th1 and th2 type t cells while increasing tgfbeta an important antiinflammatory cytokine moreover cape can attenuate oxidative stress and inflammation through downregulation of jak2stat3 signaling propolis from europe and temperate asia usually made by bees from resins collected from poplar trees has predominantly flavonoid compounds while green propolis from baccharis dracunculifolia a propolis exclusively found in brazil has various kinds of flavonoids and prenylated phenylpropanoids such as artepellin c baccharin and drupanin these and all other types of propolis can inactivate pak1 artepillin c selectively inhibits pak1 table some studies have shown that propolis can act as an immunostimulant with the ability to effects of some types of propolis in a herpes simplex animal model 0c as well as having an immunomodulatory effect in which cape inhibits il6 phosphorylation and stat3 which are important for proinflammatory th17 development besides the antiinflammatory effect of cape and kaempferol paulino et al evaluated the antiinflammatory effect of artepellin c in rat paw edema and in cell cultures demonstrating that the activity was at least in part mediated by prostaglandin e2 and no inhibition through nfkb modulation artepillin c is an important biomarker of brazilian green propolis botanical source baccharis dracunculifolia immune modulation is desirable since coronavirus infection dysregulates the immune response in the initial phases of infection which facilitates viral replication however in later stages of covid19 the body develops an exaggerated inflammatory response which can greatly damage the lungs and other ans propolis different from typical immunosuppressants can help avoid immunosuppression during the initial phases of disease and in later stages reduce an exaggerated host inflammatory response inhibiting excess il6 il2 and jak signaling cape a propolis component is also known as an immunemodulating agent and should be considered as an alternative to help reduce an exaggerated inflammatory response in a mouse model propolis had immunomodulatory action in vivo on tolllike receptor expression and on preproofreplication and infectivity potentially decreasing lung inflammation due to antiinflammatory properties while promoting immune system fortification these are useful properties that could there is ample evidence for interference of propolis andor its components with viral propolis has been tested against various viral disease anisms initial successes have prompted research to determine the most useful components which may be modified to produce more active and specific pharmaceuticals viruses that were controlled by propolis in animal models with suggestion for control in humans include influenza [ ] herpes simplex virus type and hiv [ ] shimizu et al evaluated three different types of propolis in ethanol extracts using a murine model of herpes simplex virus type despite the chemical differences proinflammatory cytokine production help minimize the symptoms and deleterious effects of covid19 figure figure propolis as an antiviral substance 0cdue to the different plant origins of the resins the bees used to produce the propolis baccharis dracunculifolia baccharis eriodata and myrceugenia euosma all three propolis extracts not only had direct antihsv1 effects but also stimulated immunological activity against intradermal hsv1 infection in mice antiviral activity of propolis has been reported for dna and rna viruses poliovirus herpes simplex virus and adenovirus in an in vitro model cultured cells the best results were obtained against poliovirus and herpes virus with inhibition of the latter at a propolis concentration of ugml the propolis components chrysine and kaempferol caused a concentrationdependent reduction of intracellular replication of herpesvirus strains when host cell monolayers were infected and subsequently cultured in a drugcontaining medium quercetin another propolis component had the same effect but only at the highest concentrations tested ugml against various human herpes simplex virus strains with a intracellular replication reduction of approximately while it reduced the infectivity of bovine herpes virus human adenovirus human coronavirus and bovine coronavirus about the reduction was in the preproofimpairment and pulmonary failure inflammatory response to infection sarscov2 infection is associated with increased levels of chemokines and activated proinflammatory cytokines that lead to the development of atypical pneumonia with rapid respiratory immunologicalinflammatory phenomena such as cytokine release syndrome have been shown to be important mortality factors in sarscov2 infection higher serum levels of proinflammatory cytokines such as il6 il1 and tnfα are found in patients with severe covid compared to those of individuals with mild disease molecular mechanisms involved in this immune process are the targets of various synthetic medicines being tested in patients including ciclesonide hydroxy chloroquine ivermectin and ketorolac which are pak1 blockers pak1 raccdc42activated kinase is overexpressed in the lung in response to sarscov2 infection and is a critical mediator of the cytokine storm that frequently results in mortality the most critical cases of covid19 which require ventilatorassisted intensive care and often result in prolonged ventilator dependency and death are a result of an exaggerated case of rotavirus antiinflammatory and immunomodulatory properties of propolis 0cin hospitalized patients fortuitously propolis components are effective pak1 blockers table there is considerable evidence that propolis can reduce and alleviate the symptoms of inflammatory diseases and has immunomodulatory properties [ ] however these properties can vary according to the plant origin of the propolis as well as the extraction processsolvent used and the inflammatory protocol cell culture animal models induction by lipopolysaccharides when the propolis extracts are tested tests with animal models have shown that propolis can reduce the levels of il6 and tnfalpha which are key proinflammatory mediators and increase the levels of the regulatory cytokine il10 kaempferol a propolis component reduces il6 tnfalpha and vegf vascular endothelial growth factor via the preproofifnγ in serum fernandes et al found that propolis exerted a positive adjuvant effect on vaccines that were developed against canine coronavirus they assayed ifnγ which is an effective way to measure the cellular response induced by a vaccine in a mouse model propolis added as an adjuvant to inactivated swine herpesvirus type vaccine stimulated increased cellular propolis is considered a safe immunostimulant and a potent vaccine adjuvant propolis has been widely tested as a vaccine adjuvant because it induces an earlier immune response and provides a longer protection period it is also included as an adjuvant ingredient in traditional chinese medicine propolis flavonoids have potential as adjuvants enhancing igg il4 and propolis has potential as a vaccine adjuvant erknfkbcmycp21 pathway table tests on macrophage cell cultures also demonstrated that propolis inhibits the production of il1 beta an important component of the inflammasome inflammatory pathway in diseases such as rheumatoid arthritis lupus and other autoimmune diseases although the mechanisms of action are not well elucidated these propolis components have potential as complementary supplements in the preventive treatment of chronic inflammatory diseases and humoral responses increasing ifnγ [ ] propolis enhanced the immune response to inactivated porcine parvovirus vaccine in guinea pigs when added to a trichomonas vaginalis protein vaccine propolis increased the igg antibody response times in mice 0ccancer is considered a relevant comorbidity factor for covid19 cancer patients have a patients cancer patients compared to the protein alone propolis was also effective as an adjuvant in the immunization of cattle with bovine herpesvirus it improved the humoral and cellular responses in mice inoculated with inactivated virus vaccines propolis as an adjuvant gave a similar immune response increasing ifnγ levels to alum and freunds adjuvant in mice vaccinated with an hiv1 polytope vaccine candidate with less risk of undesirable side effects comorbidities and evidence of how propolis can help reduce their impact in covid19 to risk a visit to a clinic or hospital to determine if they have cancer alternative therapies could help retard cancer or reduce the impact of cancer and cancer treatment in covid19 times higher risk of progressing to severe covid19 disease than patients without comorbidities also the hospital environment during the coronavirus pandemic can interfere with or delay the treatment that cancer patients should receive patients with symptoms may choose not preproofacid cape quercetin and chrysin propolis and its components normally have little impact on normal cells displaying differential cytotoxicity in liver cancer melanoma and breast cell carcinoma cell lines [ ] propolis enhances the activity of tumor various types including bladder blood brain breast colon head and neck kidney liver pancreas prostate and skin cancers propolis could help prevent cancer progression in various parts of the world it is considered an alternative therapy for cancer treatment propolis extracts have been found to inhibit tumor cell growth both in vitro and in vivo including inhibition of angiogenesis demonstrating potential for the development of new anticancer drugs various components of propolis have been shown to inhibit cancer cell growth including cinnamic propolis has potential as a complementary therapy for cancer it has shown efficacy against necrosis factor related apoptosis inducing ligand trail in cancer cells hypertension and cardiovascular disease 0c hypertension and cardiovascular disease are considered relevant comorbidities for covid19 propolis has demonstrated antihypertensive effects in rat models [ ] in cameroon it is used in popular medicine to treat various ailments including high blood pressure propolis has been widely used as a dietary supplement for its health benefits including cardiovascular protective effects [ ] in a human trial consumption of propolis improved critical blood parameters including hdl gsh and tbars levels demonstrating that obesity a natural antiobesity agent it could contribute to a reduced risk for cardiovascular disease obesity is a major comorbidity and predictor of increased mortality in covd19 patients obesity and sarscov2 both induce an inflammatory process exacerbating sarscov2 infection in the obese propolis reduced inflammation and prevented hyperlipidemia and metabolic syndromes in highly caloric diet induced obesity in mice body weight gain visceral adipose tissue liver and serum triglycerides cholesterol and nonesterified fatty acids were all reduced in the propolis fed mice [ ] caffeic acid phenethyl ester a propolis component is preproofplatelet aggregation was reduced by propolis in tests on human blood in vitro and in other in vitro tests caffeic acid phenethyl ester cape a wellstudied bioactive propolis component inhibits collagen induced platelet activation thromboembolism thrombosis and microthrombosis microthrombosis disseminated intravascular coagulation and consequent multian failure are common in severely affected covid19 patients with associated high mortality rates anticoagulants are sometimes prescribed to such patients because they can reduce mortality tang et al an elevated level of plasminogen activator inhibitor1 pai1 is a biomarker and risk factor for thrombosis and atherosclerosis [ ] various types of evidence demonstrate that propolis can reduce platelet aggregation and other thrombosisrelated parameters propolis decreased thrombotic tendencies in mice by suppressing lipopolysaccharideinduced increases in pai1 levels [ ] propolis downregulated plateletderived growth factor and platelet endothelial cell adhesion molecules in lowdensity lipoprotein knockout mice 0c old age the elderly are more often affected by chronic inflammation characterized by systemically increased levels of proinflammatory cytokines which can contribute to development of a cytokine storm a major cause of covid19 mortality propolis has antioxidant properties which could help retard o | Colon_Cancer |
"pd1pdl1 blockade therapy is a promising cancer treatment strategy which has revolutionized the treatmentlandscape of malignancies over the last decade pd1pdl1 blockade therapy has been trialed in a broad range ofmalignancies and achieved clinical success despite the potentially curelike survival benefit only a minority ofpatients are estimated to experience a positive response to pd1pdl1 blockade therapy and the primary oracquired resistance might eventually lead to cancer progression in patients with clinical responses accordingly theresistance to pd1pdl1 blockade remains a significant challenge hindering its further application to overcomethe limitation in therapy resistance substantial effort has been made to improve or develop novel antipd1pdl1based immunotherapy strategies with better clinical response and reduced immunemediated toxicity in thisreview we provide an overview on the resistance to pd1pdl1 blockade and briefly introduce the mechanismsunderlying therapy resistance moreover we summarize potential predictive factors for the resistance to pd1pdl1blockade furthermore we give an insight into the possible solutions to improve efficacy and clinical response inthe following research combined efforts of basic researchers and clinicians are required to address the limitation oftherapy resistancekeywords pd1pdl1 blockade cancer immunotherapy resistance immunotherapy is a validated and significant cancertreatment strategy which eliminates tumors by normalizing the antitumor immune responses [ ] over thelast decade cancer immunotherapy has revolutionizedthe treatment landscape of malignancies and achievedclinical success especially in immune checkpoint inhibitors correspondence 189whueducn lschrjjs163com jinyu sun and dengke zhang are cofirst authors4department of general surgery the first affiliated hospital of nanjingmedical university nanjing china2key laboratory of imaging diagnosis and minimally invasive interventionresearch lishui hospital of zhejiang university the fifth affiliated hospitalof wenzhou medical university clinical medicine of center hospital of lishuicollege lishui chinafull list of author information is available at the end of the signalsandprogrammed death1 pd1 is a class of receptorexpressed on the t cell surface which could downregulate the immune system by abrogating t cellreceptorinducedantigenmediated t cell activation the interaction betweenpd1 and its ligand programmed deathligand pdl1 plays an essential role in maintaining selftoleranceand avoiding autoimmune diseases however pd1pdl1 could also prevent the activation of t cells in thetumor and thus result in immune resistance preventingpd1pdl1 blockade is a breakthrough in cancerimmunotherapy and it has been trialed in a broadrange of malignancies in the preclinical or clinicalincluding melanoma hodgkins lymphomastage breast cancer [ ] nonsmall celllung cancer as well as hepatocellular carcinomansclc the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0csun biomarker research page of [ ] despite the longterm potentially curelikeclinical benefits therapy resistance remains a significant challenge for the further application of pd1pdl1 blockade therapy only a minority of patientsin general are estimated to experience apositive response to pd1pdl1 blockade therapy[] and the primary or acquired resistance mighteventually lead to cancer progression in patients withclinical response [ ]in this review we provide an overview on the resistance to pd1pdl1 blockade and its underlying mechanisms moreover we summarize potential predictivefactors for the resistance to pd1pdl1 blockade furthermore we give an insight into the possible solutionsto improve efficacy and clinical response of pd1pdl1blockade therapyresistance to pd1pdl1 blockade therapycheckpoint inhibitors targeting pd1 or pdl1 coulddisturb the interaction between pd1 and pdl1 whichwould preserve antitumor properties of t cells withdraw immune escape and normalize their ability to induce tumor cell death currently pd1pdl1 blockadehas shown sustained survival benefits in multiple malignancies and is at the forefront of cancer immunotherapy howeverjust as tumor cells can avoid immuneevasion several cancers may evolve to resist pd1pdl1 blockade therapy clinical evidence indicated thateven for patients with tumors highly positive for pdl1more than of them might not respond to pd1pdl1 blockade due to tumor heterogeneity and manyother reasons clinical responses vary largely across different tumor entities the objective response rate was in melanoma in nsclc in head and neck carcinoma and in kidneycancer besides for most patients experiencing initial clinical response acquired resistance remains another problem which would lead to cancer progressionor relapse after a few years [ ]many studies have demonstrated that antipd1therapy can significantly improve survival outcomes forpatients with metastatic or unresectable melanoma however only a small number of patients approximately could achieve a complete response in arecent phase i trial of atezolizumab antipdl1 involving patients with metastatic melanoma the overall response rate was among efficacy evaluablepatients and the median response duration was months moreover in another study on the longtermoutcomes of melanoma patients receiving antipd1therapy complete responses were only observed in of patients after a median followup of months of patients were alive without additional melanoma therapy additionally in the retreatedpatients after disease progression the response was onlyobserved in retreated patients receiving singleagent pd1 blockade therapy and of patientsescalated to pd1 blockade plus ipilimumab therapy inthis cohort most complete responses were durable withthe treatment failure rate of at three years whilethe response to retreatment remained relatively infrequent with a response rate of for patients withsingleagent pd1 blockade therapy moreover in aphase ii study of pembrolizumab on patients withadvancedobjectiveresponse was observed in of patients with a diseasecontrol rate of after a median followup of months adrenocorticalcarcinomatheinterestingly the response rate of some malignanciesis relatively high in hematological malignancies for example for patients with relapsed or refractory classicalhodgkin lymphoma tislelizumab antipd1 achievedan objective response rate of and a completeresponse of in a phase ii singlearm multicenterstudy similarly the complete response rate of camrelizumab antipd1 was with a partial remission rate of mechanisms underlying the resistance to pd1pdl1 blockadesince therapy resistance remains a significant limitationof pd1pdl1 blockade in clinical practice interest isgrowing in understanding the mechanisms underlyingthe resistance the response to pd1pdl1 blockaderelies on a preexisting immune response and determinants of adaptive immunity currently multiple factorshave been discovered to be involved in the efficacy ofpd1pdl1 blockade therapy such as tumor immunogenicity t celltumormicroenvironment and so forthfunction pdl1 expressionthe lack of tumor antigensthe genetic alterations are centralin the oncogenicprocess which could lead to tumor immunogenicity andprovide an opportunity for cancer immunotherapy tumor immunogenicity is positively associated with theability of the t cell to recognize tumor cells which isessential for the antitumor effect of pd1pdl1 blockade however the lack of tumor antigen will significantlyimpede the recognition ability of t cells and eventuallyresult in the failure of immunotherapymicrosatellites are prone to dna replication errorswhich will usually be repaired in normal cells however in tumors with mismatch repair mmr deficiencythese errors will accumulate which eventually result in alarge number of mutations and lead to microsatellite instability msi importantly high msi positivelycontributes to increased neoantigen production greater 0csun biomarker research page of immunogenicity and a more robust immune response moreoverthe resultant high tumor mutationburden would contribute to tumor immunogenic andenhance the response to pd1pdl1 blockade therapy[ ]multiple studies have demonstrated that the tumormutation burden is positively correlated with neoantigenburden as well as response to immunotherapy [ ]for example in colorectal cancer with mmr deficiencywhich usually exhibits a high tumor mutation burdenantipd1 therapy showed a higher response rate andbetter survival outcome compared to other subtypeswith mmr proficiency [] yarchoan analyzed the objective response rates of pd1pdl1blockade therapy for the corresponding tumor mutationburden in various cancers and their results showed thatthe mutation burden was closely associated with the objective response rate moreover pancreatic cancer generally exhibits a lowermutation load compared with other solid tumors andtherefore pd1pdl1 blockade is usually ineffective forthose patients and fails to improve their survival outcomes nevertheless in pancreatic cancer patients harboring an mmr deficiency they appear to be responsiveto pd1pdl1 blockade therapy mmr deficiency significantly increases the somatic mutation rate whichcould be translated into neoantigens and recognized bythe immune system thus making these patients responsive to pd1pdl1 blockade therapy [ ] accordingly pembrolizumab has been approved for selectedcancer patients with mmr deficiencyt cell dysfunctioneffective pd1pdl1 blockade therapy relies on the tcell function and any disruption in the processes of tcell immune function will result in the failure of pd1pdl1 blockade therapy a recent review by ren has provided an indepth insight into the mechanisms underlying the t cell dysfunctionmediated resistance with a focus on t cell recognition activationdifferentiation infiltration depletion as well as chemotaxis identification byantigen presentation is a critical process for the tumorantigensinitial t cells beta2microglobulin b2m is a significant hla1 moleculewhose mutation will hinder tumor antigen presentationand result in therapy resistance [] zaretsky analyzed biopsy samples from patients with metastatic melanoma receiving pembrolizumab who exhibited disease progression after an initial tumor regressionand they found a truncating mutation in the b2m genein the following research gettinger identifiedacquired homozygous loss or downregulation of b2m inlung patients with resistance to pd1pdl1 blockadeto further explore the role of b2m in mediating resistance they knocked out the b2m gene in immunocompetent lung cancer mice by crispr technology and theloss of b2m resulted in the resistance to pd1pdl1blockade additionally b2m mutationinducedresistance primarily occurred in an environment ofactivated pd1 positive t cellinfiltration whichresistance to pd1pdl1 blockadesuggested thattherapy might be particularly common in patients withhigh pd1 positive t cell for example t cellmoreover t cell activation is another critical processfor pd1pdl1 blockade therapy after blocking pd1pdl1 tumor cells can still counteract the activity ofimmune checkpoints and activate additional inhibitorypathways via expression of other immune checkpointsand their ligands within the tumor immune microenvironmentimmunoglobulinmucin3 tim3 is another type of immune checkpointreceptor expressed on tumorinfiltrating lymphocytes inhuman head and neck squamous cell carcinoma tumorinfiltrating lymphocytes pd1 blockade was demonstrated to upregulate tim3 expression which inhibitedt cells activation and contributed to tim3mediatedescape from pd1 blockade in the tumor microenvironment via pi3kakt pathway pd1 or pdl1physiologicallyinteractions between pd1 and pdl1block t cell activation pathways related to the immuneresponse against specific antigens and the expression ofpd1 or pdl1 has gained importance as a significantplayerin regulating the response to pd1pdl1blockade therapy pd1 and pdl1 are upregulated inthe tumor immune microenvironment of various malignancies which is considered as a strategy to evadeimmunosurveillance and imposes a significant barrier ofthe antitumor immune response importantly pdl1 primarily exhibits two distinct expression patternson tumor cells or on tumorinfiltrating immune cellspdl1 expression on immune cells reflects the adaptiveregulation meditated by ifnγ which is accompanied byincreased effector t cells as well as tumorinfiltratinglymphocytes effector t cells differently the expressionof pdl1 on tumor cells is less prevalent and it indicates the epigenetically dysregulated pdl1 gene whichis correlated with reduced immune infiltration scleroticor desmoplastic stroma as well as mesenchymal molecular features multiple studies have revealed a significantly higherobjective response rate in tumor pdl1 positive patientsthan pdl1 negative subgroups together with an improved progressionfree and overall survival [ ]kowanetz observed that atezolizumab antipdl1 achieved an objective response rate of in 0csun biomarker research page of patients with high pdl1 levels on tumor cells alone andof in those with a high expression on immune cellsalone although these observations indicated that thefunctional importance of pdl1 expression in regulatingpd1pdl1 blockadeinduced t cellthemechanistic significance of pdl1 on tumor cells or immune cells remains vagueresponsenoncoding rnasa large amount of micrornas mirnas and some longnoncoding rnas lncrnas have emerged as players inregulating tumor immunity [] and resistance topd1pdl1 blockade therapy recently huber identified a panel of circulating mirnas mir146a mir155 mir125b mir let7e mir125a mir146b mir99b which wereassociated with phenotypic and functional features ofmyeloidderived suppressor cells mdscs in melanomapatients importantly mdscs are a subclass of immature myeloid cells pathologically associated with cancerand play an inhibitory role against antitumor t cell immunity the transcriptional analysis showed thatthese mirnas could facilitate the conversion of monocytes into mdscs by melanoma extracellular vesiclesand the expression level ofthese mirna was upregulated in circulating cd14 monocytes and tumorsamples which was associated with myeloid cell infiltration and could predict the resistance to pd1 blockadetherapy moreover hu revealed the role of oncogeniclncrna for kinase activation linka in losing antigenicity and evading immune checkpoints and demonstrated lncrnadependent antigenicity downregulationsuppression for patients withand intrinsic tumortriplenegative breast cancer and resistantto pd1blockade therapythey showed upregulated linkalevels and downregulated peptideloading complex components the analysis suggested that linka expressioncould attenuate protein kinase amediated phosphorylation of the e3 ubiquitinprotein ligase trim71 via facilitating the crosstalk between phosphatidylinositol []trisphosphate and inhibitory gproteincoupled receptor pathways consequently linka could contribute to the degradation of the antigen peptideloadingcomplex and upregulate intrinsic tumor suppressors gut microbiomethe gut microbiome is a complex system composed ofmore than trillion microanisms which has beendemonstrated to regulate the efficacy and toxicity ofcancer immunotherapy many studies have reported theinfluence of the gut microbiome on cancer immunotherapy and the therapeutic response of pd1pdl1blockade therapy can be improved or diminished via gutmicrobiome modulationin mice models with distinct microbiome a significantly different response to pd1pdl1 blockade therapy was observed for example melanoma mice with anincreased bifidobacterium species in the gut microbiomeexhibited an effective response to pd1 blockade therapy similarly antibiotic administration was reported toreduce the diversity and aggravate dysbiosis of the gutmicrobiome thus influencing the clinical response topd1pdl1 blockade in tumorbearing mice as well ascancer patients [] compared to patients withoutantibiotic treatment the oral antibiotic application couldsignificantly diminish the clinical benefit of pd1pdl1blockade therapy and decrease progressionfree survivaland overall survival therefore dysbiosis of the gut microbiome is considered as one of the putative mechanisms underlying poorresponse to pd1pdl1 blockade therapy and thedualdirectional modulation of the gut microbiome oncancer immunotherapy is increasingly revealed howeverit is still unclear how gut microbiome regulatestherapy response and whether a specific bacterial taxaor gut microbiome as a whole plays a primary role remains largely unclear further research is required toprovide a more indepth understanding of the underlying mechanismspredictive factors for pd1pdl1 blockadetherapydespite the clinical success achieved in pd1pdl1blockade across multiple cancers the knowledge concerning therapy selection criteria is relatively limitedconsidering the potential adverse events and high costof immune checkpoint inhibitor agents there is a substantial need to identify predictive factors to select patients likely to benefit from this therapy currently apartfrom the functional status of immune cells [] ortumor infiltrating lymphocytes multiple factorshave been identified to predict the response to pd1pdl1 blockade therapy such as pd1pdl1 expression antigen recognition gut microbiome and so forthtable pd1 or pdl1 expressioninhibiting the pd1 pathwaymediated immune suppression is the basis and premise of pd1pdl1 blockadetherapy accumulating research has suggested that pdl1 is a biomarker to predict therapeutic response to pd1pdl1 blockade across multiple tumor types forexample atezolizumab achieved overall survival benefitacross all pdl1 expression subgroups in nsclc patients while those with high pdl1 expression experienced a more substantial survival benefit currently 0csun biomarker research page of table predictive factors for pd1pdl1 blockade therapytumor typenonsmall cell lung canceragentatezolizumabmultiple cancerscolorectal cancerurothelial carcinomaurothelial carcinomaurothelial cancermelanomamelanomamelanomapembrolizumabnivolumabatezolizumabatezolizumabatezolizumabantipd1 therapyantipd1 therapyantipd1 therapymmr mismatch repair msi microsatellite instability tmb tumor mutation burdenpredictive factorpdl1pdl1mmr msitmbtmbtmbgut microbiomegut microbiomegut microbiomereference pdl1 testing is recommended as a predictive test fornsclc urothelial carcinoma or head andneck cancers and so forthott assessed the predictive value of pdl1expression in patients with advanced solid tumors receiving pembrolizumab and the analysis showed that tumors with higher pdl1 expression and tumor mutationburden were significantly associated with higher response rate and more prolonged progressionfree survival heat map analysis revealed a close correlationbetween pdl1 expression and a broader pattern ofcoregulated gene expression which involved cytokine recruitment of t cells t cell activation markers as well asantigen presentation also the regression metaanalysisdemonstrated that pdl1 expression level was positivelyassociated with objective response rate p aswell as progressionfree survival p moreover nct02853305 and nct02807636 evaluated the efficacy of pembrolizumab or atezolizumab asfirstline treatment and the current data showed reduced survival in patients with low expression of pdl1accordingly it is advised that pembrolizumab or atezolizumab should be used for adult patients with a relativelyhigh pdl1 expression pdl1 expression of ¥ foratezolizumab and a combined positive score of ¥ forpembrolizumab however the efficacy of pd1pdl1blockade therapy as firstline therapy for advancedurothelial carcinoma still remains unclear [ ]importantly pdl1 positive only is not a predictivefactor for the response to pd1pdl1 blockade sincemultiple factors are involved in the pd1pdl1 blockade therapy in a study on patients with metastaticmelanoma receiving pembrolizumab preexisting cd8t cells were demonstrated as a prerequisite for thetumor regression after pd1pdl1 blockade therapy besidesin advanced adrenocortical carcinomatumor pdl1 expression status was not associated withtherapy response additionally it was reported thatpdl1 expression on tumor cells was not associatedwith therapy response in resected head and necksquamous cell cancer additionalinvestigation isrequired to illustrate the mechanisms accounting for thedifferenceantigen recognitionantigen recognition plays a vital role in initiating theadaptive immune response while the lack of tumor antigens significantly impedes the response to pd1pdl1blockade therapycurrently the fda has approved pembrolizumab totreat unresectable solid tumors with high msi or mmrdeficiency in a study on recurrent or metastaticcolorectal cancer patients with mmr deficiency or highmsi nivolumab showed an objective response rate of and of the patients had a disease control rateof ¥ weeks which indicated that patients with highmmr deficiency or high msi might exhibit better responses to pd1pdl1 blockade therapy [ ] interestingly the responses of tumors with mmrdeficientare highly variable and approximately half are resistantto pd1pdl1 blockade therapy mandal revealed that msi and the resultant mutation load wereresponsible for the variable response to pd1 blockadetherapy in mmrdeficiency tumors and the responsedegree was significantly correlated with the degree ofinsertiondeletion mutation loadseveral studies have revealed the association betweentumor mutation burden and the response to pd1pdl1blockade therapy [ ] mariathasan examined samples from patients with metastatic urothelial cancer receiving atezolizumab treatment and identified highneoantigen and tumor mutation burden as major determinants of clinical outcome their results showed that thetumor mutation burden was closely correlated with the response in the excluded and inflamed phenotypes 0csun biomarker research page of gut microbiome compositionclinical experiments on the human gut microbiomehave identified several specific bacteria genres that playimportant roles in human immunity and can be used asprognostic biomarkers for clinical response to pd1pdl1 blockade therapy based on the gut microbiome analysis of melanomapatients receiving pd1 blockade gopalakrishnan found that patients with prolonged progressionfree survival showed a higher multiplicity of bacteriaand clostridiales ruminococcaceae and faecalibacterium were abundant in therapy responders moreovermatson evaluated the baseline stool samplesfrom patients with metastatic melanoma before pd1pdl1 blockade treatment and the results showed thatcommensal microbial composition was significantly associated with the clinical response bifidobacteriumlongum collinsella aerofaciensand enterococcusfaecium were more abundant in responders similarlyin patients with epithelial tumors routy revealed that akkermansiacea muciniphila and enterococcus hirae were significantly abundant in those withbetter clinical response progressionfree survival months all these results indicate that gut microbiomecomposition may be a potential determinant of therapyresponse and might be used as a predictive factor inthe following research more studies are needed to validate the predictive value of gut microbiome in largercohorts and explore their efficiency in the context ofvarious types of tumorsstrategies and it hasfuture perspectivesimmunotherapy is one of the most promising cancertreatmentrevolutionized thelandscape of cancer management over the last decadehowever together with the costly and timeconsumingtrialanderror approach the limited therapy responseremains a tricky problem which hinders the furtherapplication of pd1pdl1 blockade to overcome therapy resistance and potential adverse events substantialeffort has been made on developing novel antipd1pdl1 based immunotherapy strategies with better clinical response and limited immunemediated toxicityfigs tobetterclinicallikelyachievesystem issince the interaction between cancer and the immunecomplex and involves multiplefactors strategies in combination with multiple agentsareoutcomescompared with singleagent administration a largenumber ofcombinedtherapy is an effective therapeutic strategy againstcancers for example transforming growth factor βtgfβblocking agents concomitantly with combinedpd1pdl1 blockade combined provides a clinicallyrevealed thatstudies haveexperimentson mice withfeasible strategy to improve efficacy and reduce toxicity mariathasan revealed that metastaticurothelial cancer with upregulated tgfβ signalingbefore treatmentresponded poorly to pd1pdl1blockade therapy the tumors with dense collagenfibrils could trap t cells in the stromal compartmentthus preventing them from playing their functions inpreclinicalimmuneexcluded phenotype they demonstrated that the coadministration of pdl1 blockade and tgfβblockingagents could reduce tgfβ signaling facilitate t cellinfiltration and achieve active antitumor immunityand tumor regression similarly the combination ofpd1pdl1 blockade with tumor necrosisfactorinhibitor [ ] metformin antivegf agents or otherinhibitors egcxcr4 has been demonstrated as a clinicallyfeasible strategy with improved antitumor efficacyand reduced toxicityimmune checkpointinhibitor agentspd1pdl1 blockade usually acts on the whole hostimmune system instead ofsitespecifically targetingtumorspecific immune cells while nanomedicine technology provides a powerful tool to selectively deliverimmune checkpointto tumors orlymphoid ans using drugloaded nanops usually to nm in diameter recent studies suggest that the pd1pdl1 antibody could be conjugatedor modified on the surface of nanops which couldmaintain their stability enhance efficiency and minimizethe toxicity of pd1pdl1 blockade [ ] forexamplein gastric cancer cells the pdl1 blockadeconjugated nanops contributed to significantlyhigher cellular uptake and achieved more effective inhibition of pdl1 expression compared with the controlgroups moreover in patients with metastatic triplenegative breast cancer the coadministration of nabpaclitaxelatezolizumabprolonged progressionfree survival owing to thesuccess in previous research clinical trials on nanoimmunotherapysuch asnct03589339 and nct03684785 these clinical trialsshould provide substantial evidence for the combinationof nanomedicine and pd1pdl1 blockade in the nextfew yearscurrently underwayblockadepdl1plusarethe manipulation offurthermore accumulating evidence has demonstrated that gut microbiome significantly impacts theefficacy of cancer immunotherapy which in turn indithe gut microbiomecates thatcould latently affectthe response to pd1pdl1blockade therapy [] currently antibiotic applicationfecal microbiota transplantation fmt anddiet regulation are considered as practical approachesto manipulate gut microbiome for example fmtfrom patients with a positive response to germfree or 0csun biomarker research page of fig overview on the strategies to improve the resistance to pd1pdl1 blockade therapy multiple strategies have been proposed toimprove the resistance to pd1pdl1 blockade therapy including combined therapy nanoimmunotherapy gut microbiome manipulation andso forthin contrastantibiotictreated mice could improve tumor controlaugment t cell responses and ameliorate the antitumor effects of pd1 blockadethetransplantation from resistant patients did not resultin improvement similarly responses to pdl1blockade are distinctin mice with different commensal microbes and the positive response of micewith advantageous gut microbiome can be transplanted to mice with negative responses by fmt orcohousing conclusionsdespite the success across multiple types of cancersonly a minority of patients are estimated to exhibit apositive response to pd1pdl1 blockade therapy andthe primaryacquired resistance might eventually leadto progression in patients with clinical responses thelimitation in clinical response impairs the efficacy andhinders its further application since the understandingof the mechanisms underlying therapy resistance remains vague only a few therapeutic options areavailable for those patients currently illustrating thedeterminants of response or resistance is significant toaccelerate improving survival outcomes and developingimproved treatment options for cancer patients tobetter realize the therapeutic potential of pd1pdl1blockade therapyit is essential to identify predictivebiomarkers for therapy response develop novel therapeutic strategies and improve therapeutic strategies incombination with other agents in the following research combined efforts of basic researchers and clinicians are required to address the pd1pdl1 blockadetherapy resistanceabbreviationspd1 programmed death1 pdl1 programmed deathligand nsclc nonsmall cell lung cancer mmr mismatch repair msi microsatelliteinstability b2m beta2microglobulin tim3 t cell immunoglobulin mucin3mirnas micrornas lncrnas long noncoding rnas mdscs myeloidderived suppressor cells tgfβ transforming growth factor β fmt fecalmicrobiota transplantationacknowledgmentsnot applicableauthors contributionsjys dk z mx and xz wrote original draft preparation sq w jsj and xjprovided critical revision all authors read and approved the final manuscriptfundingthis study was supported by national key research and developmentprojects intergovernmental cooperation in science and technology of chinano 2018yfe0126900 to jiansong ji the key research and developmentproject of zhejiang province no 2018c03024 to jiansong ji the nationalnatural science foundation of china to xl 0csun biomarker research page of availability of data and materialsnot applicableethics approval and consent to participatenot applicableconsent for publicationnot applicablecompeting intereststhe authors declare that they have no competing interestsauthor details1the first college of clinical medicine the first affiliated hospital of nanjingmedical university nanjing medical university nanjing china 2keylaboratory of imaging diagnosis and minimally invasive interventionresearch lishui hospital of zhejiang university the fifth affiliated hospitalof wenzhou medical university clinical medicine of center hospital of lishuicollege lishui china 3college of medicine lishui college lishui china 4department of general surgery the first affiliated hospitalof nanjing medical university nanjing china 5department of radiologyaffiliated lishui hospital of zhejiang university lishui chinareceived april accepted august referenceshellmann md pazares l bernabe caro r zurawski b kim sw carcerenycosta e nivolumab plus ipilimumab in advanced nonsmallcell lungcancer n engl j med niglio sa jia r ji j ruder s patel vg martini a programmed death1or programmed death ligand1 blockade in patients with platinumresistant metastatic urothelial cancer a systematic review and metaanalysis eur urol sun jy lu xj cancer immunotherapy current applications and challengescancer lett andrews lp yano h vignali daa inhibitory receptors and ligands beyondpd1 pdl1 and ctla4 breakthroughs or backups nat immunol prestipino a zeiser r clinical implications of tumorintrinsic mechanismsregulating pdl1 sci transl med betof warner a palmer js shoushtari an goldman da panageas ks hayessa et | Colon_Cancer |
curative therapeutic options for a number of immunological disorders remain to be established and approaches for identifying drug candidates are relatively limited furthermorephenotypic screening methods using induced pluripotent stem cell ipscderived immunecells or hematopoietic cells need improvement in the present study using immortalizedmonocytic cell lines derived from ipscs we developed a highthroughput screening htssystem to detect compounds that inhibit il1 secretion and nlrp3 inflammasome activation from activated macrophages the ipscs were generated from a patient with neonatalonset multisystem inflammatory disease nomid as a model of a constitutively activatednlrp3 inflammasome hts of compounds including fdaapproved drugs and compounds with known bioactivity identified compounds as predominantly il1 inhibitorssince these compounds are known inflammasome inhibitors or derivatives of these resultsprove the validity of our hts system which can be a versatile platform for identifying drugcandidates for immunological disorders associated with monocytic lineage cellsintroductionone of the main cell types affected by immunological disorders are white blood cells such aslymphocytes monocytes and neutrophils although our understanding of the cellular pathophysiology of immunological disorders has greatly benefited from in vitro studies usingpatientderived primary hematopoietic cells or in vivo animal models these approaches haveseveral limitations patientderived hematopoietic cells cannot be obtained in sufficient quantities and their in vitro phenotypes can be affected by the in vivo conditions of the patient suchas the cytokine milieu or the administration of therapeutic agents while animal models haveprovided important insights into these disorders species differences in the immunologicala1111111111a1111111111a1111111111a1111111111a1111111111open accesscitation seki r ohta a niwa a sugimine y naitoh nakahata t induced pluripotentstem cellderived monocytic cell lines from anomid patient serve as a screening platform formodulating nlrp3 inflammasome activity one e0237030 101371 pone0237030editor xiaoping bao purdue university unitedstatesreceived march accepted july published august peer review history recognizes thebenefits of transparency in the peer reviewprocess therefore we enable the publication ofall of the content of peer review and authorresponses alongside final published s theeditorial history of this is available here101371 pone0237030copyright seki this is an openaccess distributed under the terms of thecreative commons attribution license whichpermits unrestricted use distribution andreproduction in any medium provided the originalauthor and source are crediteddata availability statement all relevant data arewithin the manuscript and its supportinginformation files one 101371 pone0237030 august one 0cipscderived monocytic cell lines serves a platform for drug screeningfunding this work was supported by the grant forthe core center for ips cell research of researchcenter network for realization of regenerativemedicine from the japan agency for medicalresearch and development amed [tn and mks] the program for intractable diseases researchutilizing diseasespecific ips cells of amed and [tn and mks]practical research project for allergic diseasesand immunology research on allergic diseasesand immunology of amed [mks]practical research project for rareintractablediseases of amed [mks] and thedevelopment causes discrepancies in the function and phenotype of the immune cells []overall highthroughput screening hts of therapeutic compounds using patientderivedcells or animal models is usually not feasiblethe establishment of disease or patientspecific induced pluripotent stem cells ipscs has led to the development of a new field of disease modeling owing to their pluripotencyand capacity for selfrenewal ipscs can function as an unlimited source of patientderivedsomatic cells and progenitor cells ipscs have also been used as a source of phenotypebasedhts [] however several roadblocks remain for ipscbased hts as follows obtaining alarge number of differentiated progenies from pscs is cost and laborintensive and theyield and function of the differentiated cells often vary among clones and experimentalbatchesjapan society for the promotion of science jspswe have established ipscs from patients with autoinflammatory syndromes including neokakenhi grant number [mks]nippon shinyaku co ltd provided support inthe form of salaries to the authors [rs and hn]the specific roles of rs and hn are articulated inthe author contributions section the funders hadno role in study design data collection andanalysis decision to publish or preparation of themanuscriptcompeting interests rs and hn are employeesof nippon shinyaku co ltd this employmentdoes not alter our adherence to one policieson sharing data and materialsnatalonset multisystem inflammatory disease nomid also known as chronic infantile neurological cutaneous and articular [cinca] syndrome nakajonishimura syndrome and blau syndrome for disease modeling in these studies ipscderived myeloid cells wereimmortalized by transducing lentiviral vectors that encoded myc bmi1 and mdm2 anddisease phenotypes were recapitulated in vitro thus ipscderived immortalized myeloid celllines ipsmls can be expanded from one experimental batch with reduced financial and laborcosts they also can be stored and differentiated into terminally differentiated progenies therefore ipsmls can overcome the roadblocks associated with ipscbased hts nomid is the most severe form of cryopyrinassociated periodic syndrome caps anautoinflammatory disease caused by heterozygous mutations in the nlrp3 gene [ ]nacht lrr and pyd domainscontaining protein nlrp3 is expressed mainly in myelomonocytic lineage cells and acts as a sensor of cellular stress induced by various pathogens andsterile stimuli in normal macrophages a priming stimulus such as lipopolysaccharidelps induces the expression of nlrp3 and prointerleukin il1 an inactive form of theproinflammatory cytokine il1 then an activating stimulus such as adenosine triphosphate atp enhances the assembly of a protein complex known as nlrp3 inflammasomethis inflammasome contains the protease caspase1 which processes proil1 to the matureform on the other hand lps stimulation of monocytic cells obtained from untreated capspatients induces robust il1 secretion without secondary activating signals due to autoactivation of nlrp3 inflammasome indeed antiil1 therapy for caps patients has beenproven effective [ ] however antiil1 therapy has several weak points the efficacy ofantiil1 therapy is often inadequate for patients with severe phenotypes il1 maturation is mediated not only by nlrp3 inflammasome but also other inflammasomes and proteases [ ] thus a complete blockade of il1 may result in excessive immunosuppressionmoreover the cost and lifelong injection of biologics worsen the patients quality of life therefore other therapeutic approaches such as the direct inhibition of nlrp3 inflammasomeactivity are under considerationnlrp3 inflammasome is an attractive drug target because nlrp3 inflammasome activation is associated with the pathogenesis of various chronic inflammatory conditions recently several selective nlrp3 inhibitors entered the clinical phase their chemicalstructures are undisclosed but presumed to be sulfonylureas or their derivatives mcc950 asulfonylureabased potent selective inhibitor of nlrp3 inflammasome activation wasalso recently identified as a direct nlrp3 inhibitor by binding to the walker b atphydrolysismotif of the nacht domain [ ] given that capsrelated mutations frequently occursin the nacht domain it is not surprising that most capsrelated nlrp3 mutantsescape efficient mcc950 inhibition therefore novel nlrp3 inflammasome inhibitorseffective for diseases including caps are sought one 101371 pone0237030 august one 0cipscderived monocytic cell lines serves a platform for drug screeningin the present study we developed an hts system to identify compounds that regulate theactivity of nlrp3 inflammasome using ipscs generated from a nomid patient we established ipsmls which we used as a prototype of nlrp3activated immune cells by immortalizing ipscderived monocytic progenitor cells we conducted hts of approximately compounds and validated their inhibitory effect on the secretion of il1resultsfunctional monocytic cells on feeder and serumfree monolayer culturewe previously reported that macrophages derived from ipscs carrying diseaseassociatedmutations in the nlrp3 gene showed excessive secretion of il1 without secondary signals these macrophages were considered functional based on compatible appearances withprimary macrophages under an electron microscope the secretion of proinflammatory cytokines and the phagocytosis of listeria monocytogenes macrophages in that study wereobtained via a differentiation protocol using op9 feeder layers recently we have updated ourmonocytemacrophage differentiation system to a defined condition without feeder cells andserums which can also produce functional macrophages we therefore examined whethermonocytes obtained with the feeder and serumfree monolayer differentiation system exhibited a similar in vitro phenotypefor this a nlrp3mutated ipsc clone derived from a nomid patient with a somaticnlrp3y570c mutation was differentiated into monocyticlineage cells since thenlrp3 mutation was a somatic mosaicism the nlrp3nonmutated wildtype clonederived from the same donor was used as an isogenic control ipsc line the differentiatedcells showed a mononuclear and slightly foamy appearance consistent with the appearance ofin vitro differentiated monocytes fig 1a and expressed the hematopoietic cell marker cd45the myeloid cell marker cd11b and the monocytic cell marker cd14 fig 1bwe collected floating monocytic cells from the culture supernatant and evaluated theircytokine production we used lps as a priming signal and silica as a secondary inflammasomeactivating signal as expected the wildtype ipscderived monocytes required twosequential signals to secrete il1 fig 1c while the monocytes carrying the nlrp3 mutationshowed excessive il1 secretion without a secondary signal fig 1d both clones showedrobust secretion of il6 confirming the appropriate downstream signal transduction of lpsstimulation fig 1c and 1d overall the monocytic cells differentiated under feeder andserumfree condition showed the abnormal il1 secretion associated with the in vitro phenotype of nomidestablishment of ipsmls from monocytic progenitor cellswe next established an ipsml line from the ipscderived monocytic cells for this we recovered monocytic lineage cells and introduced lentiviral vectors encoding myc bmi1 andmdm2 after days culture in the presence of macrophage colonystimulating factormcsf the cells started to continuously proliferate in an exponential manner fig 2a theipsmls showed a small mononuclear appearance consistent with the appearance of monocyticlineage cells fig 2b the integration of transgenes into the genome of ipsmls was confirmed fig 2c the ipsmls expressed cd45 cd11b and cd14 similar to the originalipscderived monocytic cells fig 2d karyotype analysis of the ipsml demonstrated thatmost of the cells maintain normal karyotype except for some normal variations s1 tablebecause monocytes are heterogeneous progenitor cells that can differentiate into macrophagesand dendritic cells [ ] they might be composed of a heterogeneous population potentiallymaking their cytokine production insufficient we therefore compared the cytokine profiles of one 101371 pone0237030 august one 0cipscderived monocytic cell lines serves a platform for drug screeningfig functional monocytic cells on feeder and serumfree monolayer culture a a representative maygiemsa staining image of monocytic cells derived fromipscs b a flow cytometric analysis of monocytic cells the staining profiles of specific antibodies thick lines and isotypematched controls gray areas are showncd il1 and il6 secretion from monocytic cells with wildtype c and mutant d nlrp3 cells were stimulated with lps ngml for hours and silica μgml for an additional hour bars show the mean ± sem of four experiments � p paired ttest101371 pone0237030g001ipsmls and terminallydifferentiated ipsmlderived macrophages the ipsmls carrying anlrp3 mutation nomidmls were differentiated into macrophages mlmps fig 2esince mlmps produced an increased amount of il1 and il6 compared with nomidmlsfig 2f and 2g and showed less variability than ipscderived monocytes fig 2h wedecided to use mlmps to construct our htsestablishment of an hts platform using mlmpsto establish our hts system we differentiated nomidmls into mlmps and disseminatedthem onto 384well plates we added the compounds at the same time as when we disseminated the cells into the culture wells then the mlmps were cultured with appropriate concentrations of compounds for hours in order for the cells to firmly adhere to the plate surfaceand they were stimulated with lps for another hours fig 3a after incubation the supernatant was collected and the relative concentrations of cytokines were measured using ahomogeneous time resolved fluorescence htrf assay in this protocol a caspase1 inhibitor one 101371 pone0237030 august one 0cipscderived monocytic cell lines serves a platform for drug screeningfig establishment of ipsmls from monocytic cells a cumulative growth curve of ipsmls the number of cells was counted and cumulated in every passageculture the day of lentiviral transduction was regarded as day b a representative maygiemsa staining image of ipsmls c a reverse transcriptionpcr analysisof ipsmls transgenespecific primer pairs were used my myc b bmi1 md mdm2 as a positive control lentiviral expression vectors were used d a flowcytometric analysis of ipsmls the staining profiles of specific antibodies thick lines and isotypematched controls gray areas are shown e a phase contrastimage of mlmps f g il1 f and il6 g secretion from nomidmls and mlmps cells were stimulated with lps ngml for hours the bars show themean ± sem of four experiments �� p paired ttest h the coefficient of variation cv of lpsstimulated ipscderived monocytes fig 1c and 1dnomidmls and mlmps f g101371 pone0237030g002acyvadcho specifically inhibited the secretion of il1 in a dosedependent mannerfig 3b in contrast the proteasome inhibitor mg132 which inhibits the nfκb pathway byblocking the degradation of iκb decreased the production of both il1 and il6 in a one 101371 pone0237030 august one 0cipscderived monocytic cell lines serves a platform for drug screeningfig establishment of an hts platform using mlmps a schematic illustration of the compound screening b dosedependent inhibition of il1 closedcircles and il6 open circles secretion by acyvadcho mg132 and mcc950 bars show the mean ± sem of five experiments c the quality of the assaysystem was evaluated signaltobackground ratios and zfactors of plates for il1 closed and il6 open are shown d criteria and number of hitcompounds [il1] and [il6] indicate the percent inhibition of il1 and il6 secretion respectively e doseresponse curves of the hit compounds bars showthe mean ± sd of four wells101371 pone0237030g003dosedependent manner fig 3b these data proved the specificity of the hts system wealso evaluated the inhibitory effect of mcc950 mcc950 inhibited the secretion of il1 withan approximate ic50 at μm without inhibiting the secretion of il6 fig 3b overall theipsmlbased cytokine assay in combination with the htrf system successfully detected the one 101371 pone0237030 august one 0cipscderived monocytic cell lines serves a platform for drug screeninginhibitory effect of previously reported compounds proving the application of our system tohtshts using annotated compoundsto identify compounds that inhibit the activation of mutant nlrp3 we next performed htsusing compounds including fdaapproved drugs and compounds with known bioactivity to evaluate the quality of the assay system the zfactor and signalbackground sb ratio of each plate were calculated using the data from dmso controls with or without stimuli s1 fig regarding il1 the zfactor and sb ratio were consistently high for each384well plate zfactor ± and sb ratio ± mean±se fig 3c since a zfactor over indicates an excellent assay these data proved the appropriateness of our hts system the zfactor and sb ratio for il6 were slightly lower than those for il1 zfactor± and sb ratio ±in the first screening we assessed the inhibitory effects of all compounds influencing cytokine secretion at μm s2 table and s2 fig we first selected the compounds thatinduced a more than reduction in il1 secretion fig 3d of these the compoundsthat also nonspecifically inhibited or enhanced il6 secretion were excluded we adopted amore relaxed criterion for il6 modulators because the zfactors of the il6 assay were lowerthan those of the il1 assay we also excluded duplicate compounds and known steroidswith broad antiinflammatory effects for the remaining compounds we evaluated thereproducibility of the inhibitory effects at different dose twelve compounds showed ic50values at less than μm and predominant il1 inhibition at least at two different dosesfinally seven compounds consistently showed predominant il1 inhibition fig 3e allseven compounds have previously been reported to inhibit nlrp3 inflammasome []indicating their inhibitory effects on both wildtype and mutant cellsvalidation of hit compounds with primary human cellswe also wondered if the effect of these compounds could be recapitulated in primary humancells we used primary peripheral blood mononuculear cells pbmcs obtained from healthyvolunteers for the validation to obtain prompt il1 secretion from nonmutant cells westimulated the pbmcs with lps and atp compounds modulating the activity of hsp9017aag and herbimycin a showed a selective inhibitory effect on il1 secretion comparable to the effects seen in mlmps fig on the other hand the remaining compoundsaside from the pancaspase inhibitor zvadfmk inhibited both il1 and il6 fig indicating that they had offtarget effects or their il1 inhibitory effects were nonspecificdiscussionwe established a diseaseassociated phenotypic hts system for nomid using ipsmls ourhts platform showed a stable zfactor and sb ratio for the amount of il1 to avoid falsepositives such as nonspecific inhibitors and cytotoxic compounds we also measured theamount of il6 an inflammasomeindependent cytokine we identified compounds as predominant inhibitors of il1 secretion from among candidates the compounds identified here have already been reported as inhibitors of nlrp3 inflammasome proving thevalidity of our strategy screening a larger number of compounds may help identify novel andmore specific compoundswe could efficiently immortalize monocytic progenitor cells derived from human ipscsmoreover these ipsmls could differentiate into terminally differentiated macrophagesmlmps as previously reported [ ] mlmps showed enhanced cytokine secretion and one 101371 pone0237030 august one 0cipscderived monocytic cell lines serves a platform for drug screeningfig hit validation with pbmcs dosedependent inhibition of il1 closed circles and il6 open circles secretion from pbmcs of a healthy donor cellswere stimulated with lps ngml for hours and atp mm for an additional hour bars show the mean ± sd of four wells101371 pone0237030g004therefore were deemed suitable for hts in this manner the financial and labor costs of htscan be dramatically decreased our strategy can be applied to other immunological disordersin which monocytemacrophagelineage cells play critical pathological roles in addition theimmortalization of ipscderived hematopoietic cells for hts can be expanded to otherhematopoietic disorders one concern associated with the immortalization of monocytic cellsis that the immortalization can affect the characteristics of the cells especially regarding thecell cycle and death which may affect the immunological functions to avoid any potentialchanges in the cellular phenotype we first expanded the ipsmls generated a larger numberof frozen stocks and then used the same passage number of the stocks for a series of htssessionsmodulating the function of the inflammasome by small compounds is a promising frontierfor controlling diseases associated with inflammasomemediated chronic inflammation somecompounds such as mcc950 have already been shown to be markedly effective without anysignificant side effects in animal models nevertheless identifying more candidates to modulate nlrp3 inflammasome is desirable as mcc950 inefficiently inhibits capsrelated nlrp3mutants interestingly we identified several compounds effective in mutant cells that were previously reported to inhibit the activation of nlrp3 inflammasome indicating that the activation of mutant nlrp3 shares activating signals to some degree with wildtype nlrp3however several compounds that exerted predominantly il1 inhibitory effects on mutantcells were nonselective on pbmcs which could be explained by offtarget effects or false positives offtarget effects including cytotoxicity could be the result of a higher sensitivity bypbmcs to the true positives the clear reason is difficult to elucidate however because of theheterogeneity of the pbmc population on the other hand ipsmls consist of relativelyhomogenous cell populations and may therefore be more useful than primary cells in in vitro one 101371 pone0237030 august one 0cipscderived monocytic cell lines serves a platform for drug screeningexperiments as for nonspecific inhibitors and false positives in hts the upregulation of certain signaling pathways irrelevant to the inflammasome in ipsmls might be one cause cytotoxic compounds may also suppress cytokine secretion in a nonspecific manneradditionally our hts system relies on the amount of secreted cytokines which can varybetween cells and only indirectly describes the inflammasome activity thus a combination ofmeasuring the cytotoxicity and direct detection of the inflammasome activity should improvethe accuracy of our hts systemto conclude although phenotypic screenings have been performed with various cell typesdifferentiated from human pscs the number of screenings with immune cells is still limitedour hts system provides a large number of functional monocytic lineage cells and a versatileplatform for compound screening to modify diseaseassociated phenotypes therefore it canbe used to screen candidate compounds for the treatment of congenital immunological disorders associated with monocytic lineage cellsmaterials and methodsstudy approvalthis study was approved by the ethics committee of kyoto university r0091g0259 andwritten informed consent was obtained from the patients guardians in accordance with thedeclaration of helsinkimaintenance and differentiation of human ipscswe previously established ipscs from a nomid patient cira188ai with nlrp3 somaticmosaicism y570c undifferentiated ipscs were maintained on mitotically inactivatedsnl feeder cells with primate es cell medium reprocell japan supplemented with ngml bfgf wako pure chemicals industries japan feeder and serumfree monocytic celldifferentiation was performed in accordance with previously described protocols with somemodifications [ ]lentivirus productionlentiviral constructs encoding myc bmi1 and mdm2 in the csiiefrfa vector and twoplasmids for lentiviral vector packaging pcmvvsvgrsvrev and pcaghivgp werekindly provided by dr satoru senju kumamoto university kumamoto japan and hiroyukimiyoshi riken bioresource center tsukuba japan plasmids were transfected into 293tcells crl3216 atcc usa by lipofection lipofectamine ltx thermo fisher scientificusa and days later viral ps in the culture supernatants were concentrated by centrifugation at rpm for hours at Ëcgeneration of ipsmlsipsmls were generated as previously described with some modifications [ ] briefly onday of the monocytic cell differentiation from ipscs floating cells were collected andinfected with lentiviruses the cells were cultured in stempro34 serumfree medium thermofisher scientific containing mm lglutamine in the presence of gmcsf ngml andmcsf ngml rd systems usa after approximately days proliferating ipsmlsappeared for macrophage differentiation ipsmls within passages after thawing were cultured in rpmi1640 medium sigmaaldrich usa containing fetal bovine serum equitechbio usa and mcsf ngml for days with a medium change on day adherent one 101371 pone0237030 august one 0cipscderived monocytic cell lines serves a platform for drug screeningcells were collected by treatment with accumax innovative cell technologies usa and usedfor the subsequent experimentsmaygiemsa stainingcells were seeded onto glass slides by cytospin thermo fisher scientific and stainedwith maygrunwald and giemsa staining solution merck kgaa germany in accordancewith the manufacturers instructions the slides were examined using a biorevo bz9000keyence japan a planapo à objective nikon japan and the bzii viewer software program keyence were used for the image acquisitionflow cytometrycells were treated with fcr blocking reagent miltenyi biotec germany and stained with primary antibodies cd45fitc becton dickinson and company usa cd11bpe andcd14apc beckman coulter usa at dilutions of for negative controls primary antibodies were replaced with mouse igg1 bd dapi sigmaaldrich was used to exclude deadcells the flow cytometric analysis data were collected using a macsquant analyzer miltenyibiotec and analyzed using the flowjo software program treestar usareverse transcriptionpolymerase chain reaction pcrrna samples were prepared using an rneasy mini kit qiagen germany total rna ng was reverse transcribed into cdna using a primescript rt master mix kit takarajapan pcr was performed on a veriti thermal cycler thermo fisher scientific withtakara ex taq hs polymerase using approximately ng cdna the forward primers targeting the coding region of the genes were gatcagcaacaaccgaaaat3 mycccattgaattctttgaccagaa3 bmi1 and gctgaagagggctttgatg3mdm2 the reverse primer targeting wpre was gttgcgtcagcaaacacagt3measurement of cytokinescells were seeded at à cellswell onto a 96well cell culture plate in rpmi1640 mediumcontaining fbs the cells were stimulated with ngml lps from e coli k12 invivogen usa for hours and μgml silica us silica usa for an additional hour aftercentrifugation the supernatants were collected the concentrations of cytokines in the culturesupernatants were analyzed using a flowcytomix kit ebioscience usa and a macsquantanalyzer in accordance with the manufacturers instructioncompound screeningcells were seeded at à cellswell onto 384well cell culture plates in rpmi1640 mediumcontaining fbs and compounds four hours later the cells were stimulated with ngmllps for hours after centrifugation the supernatants were transferred to 384well smallvolume white plates using a biomek nxp beckman coulter the relative cytokine levels weremeasured using an htrf kit cisbio bioassays france and a powerscan4 microplatereader ds pharma biomedical japan with excitation at nm and emission at and nm the htrf signals were calculated as the htrf ratio Ãem nmem nmand then the percent inhibition was calculated using dmso controls cell seeding and reagentdispensation were performed with a multidrop combi thermo fisher scientific thereagents purchased are as follows acyvadcho mg132 merck kgaa mcc950sigmaaldrich fdaapproved drug library iccb known bioactives library enzo life one 101371 pone0237030 august one 0cipscderived monocytic cell lines serves a platform for drug screeningsciences usa usdrug collection international drug collection microsource discoverysystems usa lopac1280 sigmaaldrich tocriscreen mini tocris bioscience uk andkinase inhibitor libraries enzo life sciences merck kgaa selleck chemicals usahit validation with pbmcscryopreserved human pbmcs were purchased from cellular technology limited usa thecells were thawed in accordance with the manufacturers instructions and seeded at Ãcellswell onto 384well cell culture plates in rpmi1640 medium containing fbs andcompounds four hours later the cells were stimulated with ngml lps for hours and mm atp sigmaaldrich for an additional hour the relative cytokine levels were measuredusing the htrf kit as described abovestatistical analysesstatistical analyses were performed using the excel and graphpad prism software programsgraphpad software usa statistical significance was evaluated using the paired ttestp was considered statistically significant the zfactor and sb ratio were calculated asfollowsz0 ¼ 00 ð3shc þ 3slcþðmhc 00 mlcþsb ¼ mhcmlcwhere Ï is the standard deviation sd μ is the mean hc is the high control and lc is the lowcontrolsupporting informations1 fig htrf ratios of the assay controls each of the plates contained high stimulated and low not stimulated controls treated with dmso values falling outside of themean ± 3sd were excluded as outliers red crosses except when zfactors and sb ratios werecalculatedtifs2 fig scatter plot of the compound screening percent inhibitions for il1 xaxis and il yaxis within a range of to are shown seven hit compounds are markedtifs1 table karyotype analysis of ipsmlspdfs2 table a total list of compounds with the assay dataxlsxs1 raw imagepdfacknowledgmentswe thank drs takayuki tanaka mitsujiro osawa and yohei nishi cira kyoto universitykyoto japan for fruitful discussions and technical assistance dr peter karagiannis cirakyoto university kyoto japan for proofreading the paper ms harumi watanabe cira one 101371 pone0237030 august one 0cipscderived monocytic cell lines serves a platform for drug screeningkyoto university kyoto japan for providing administrative assistance and dr akitsu hottacira kyoto university kyoto japan for providing 293t cellsauthor contributionsconceptualization ryosuke seki megumu k saitodata curation akira ohta megumu k saitofunding acquisition megumu k saitoinvestigation ryosuke seki akira niwa yoshinori sugiminemethodology akira ohtaresources akira ohtasupervision akira niwa haruna naito tatsutoshi nakahata megumu k saitowriting original draft ryosuke sekiwriting review editing megumu k saitoreferences mestas j hughes cc of mice and not men differences between mouse and human immunology jimmunol epub 104049jimmunol17252731 pmid holsapple mp west lj landreth ks species comparison of anatomical and functional immune system development birth defects res b dev reprod toxicol epub 101002bdrb10035 pmid geissmann f manz mg jung s sieweke mh merad m ley k development of monocytes macrophages and dendritic cells science 101126science pmid takahashi k tanabe k ohnuki m narita m ichisaka t tomoda k induction of pluripotent stemcells from adult human fibroblasts by defined factors cell 101016jcell200711019 pmid takahashi k yamanaka s induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors cell 101016jcell200607024pmid lo cicero a jaskowiak al egesipe al tournois j brinon b pitrez pr a high throughput phenotypic screening reveals compounds that counteract premature osteogenic differentiation of hgpsipsderived mesenchymal stem cells sci rep epub 101038srep34798 pmid pubmed central pmcid pmc5064407thorne n malik n shah s zhao j class b aguisanda f highthroughput phenotypic screening of human astrocytes to identify compounds that protect against oxidative stress stem cellstransl med epub 105966sctm20150170 pmid pubmed central pmcid pmc4835244 darville h poulet a rodetamsellem f chatrousse l pernelle j boissart c human pluripotentstem cellderived cortical neurons for high throughput medication screening in autism a proof ofconcept study in shank3 haploinsufficiency syndrome ebiomedicine epub 101016jebiom201605032 p | Colon_Cancer |
coronaviruses covs belong to a family of envelopedviruses with a positive sense singlestranded rna genomecovs cause illness ranging from upper respiratory tractinfections urtis resembling the common cold to lowerrespiratory tract infections lrtis such as bronchitis pneumonia and even severe acute respiratory syndrome sarswith most serious disease outcomes in the elderly immunocompromised patients and infants [ ] hcovoc43oc43 hcov229e 229e hcovnl63 nl63 andhcovhku1 hku1 were the ï¬rst documented humancovs hcovs which usually cause urtis and less frequently are associated with ltri diseases in the lastdecades two human coronaviruses created great concernfor the world medical community due to significant diseaseand mortality [ ] in severe acute respiratorysyndromecoronavirus sarscov was characterized byacute atypical pneumonia and diï¬use alveolar damagedad in roughly patients and with almost deathsrepresenting a nearly mortality rate more recentlyin a new human coronavirus designated as middle eastrespiratory syndromecoronavirus merscov was identiï¬ed and the global ongoing outbreak of mers with over oï¬cial cases and deaths represented approximately case fatality rate to date in humans over the last few months a new strain of human coronavirus sarscov2 also known as 2019ncov hascaught the worlds seven continents attention with its rapidglobal spread aï¬ecting at least countries and territoriesinfecting more than and claiming more than lives worldwide the coronavirus pandemichas promoted isolation and uncertainly fear and panicworldwide in additionlikely lead to changes inpolitical and economic power in ways that can be determined only later it willit is important to note that there are many similaritiesamong diï¬erent coronavirus species but not in all aspects 0coxidative medicine and cellular longevitydepending on the molecular mechanism of viral inhibitionpromoted by an antiviral agent the analysis of the data andcomparison between animal and human covs must be donevery carefully in fact it is important to note that there arediï¬erences between human and animal cov receptorswhich will likely result in diï¬erent aï¬nities or unlikely interactions of an antiviral agent with the diï¬erent cov receptors howeverif the antiviral agent interferes with thereplication andor assembly of the covs there is a higherprobability of obtaining similar antiviral activity results inhuman cov tests [ ] following this line our searchin specialized literature was focused mainly on studies thatinvestigated the anticoronavirus eï¬ects of natural antioxidants by inhibiting proteases for viral replication materials and methodsthe present study was carried out based on a search of the literature of natural antioxidants and coronavirus the searchperformed in the pubmed database included studies published until march and used the following keywordscoronavirusstressmerscov sarscov 229e nl63 oc43 hku1merscov virus infection and middle east respiratorysyndrome virus the scientiï¬c publications were selectedfrom studies published in the english languageantioxidants ï¬avonoids oxidative pathogenic mechanism of coronavirusinduced cell damagethe high mortality rate associated with the three pathogenichcovs has been mainly attributed to the development ofdigestive and respiratory tract injuries observed followinginfection acute atypical pneumonia and diï¬use alveolardamage that progress to deposition of ï¬brous tissue denudedairways haemorrhage and elevated macrophage ltrationare sometimes accompanied by watery diarrhoea dehydration and vomiting [ ]despite the molecular mechanisms of coronavirusinduced intestine and lung pathogenesis not fully elucidatedand still unclear studies have suggested that lateterm diseaseprogression is unrelated to viremia it is now believed morelikely to be associated with the immunopathological mechanism [ ] viral clearance and subsequent recovery frominfection require activation of an eï¬ective host immuneresponse however many immune eï¬ector cells may alsocause injury to host tissues together with ammatoryand immune response signaling the presence of oxidativecompounds such as reactive oxygen species ros playsimportant roles in the pathogenic mechanism of cell damageinduced by covs through oxidative stress oxidative stress is deï¬ned as an interruption andorderegulation of the signaling and redox system that can becaused by an imbalance in the production of oxidant andantioxidant species among the main oxidant agentsros and reactive nitrogen species rns stand out in orderto counterbalance the oxidant species there is an antioxidantsystem formed by enzymes and nonenzymatic molecules however during pathological events such as viral infections there may be an increase in the production of oxidantspecies not neutralized by the antioxidant system resultingin oxidative stress that promotes cellular damage throughprotein denaturation changes in the functions of nucleicacids lipid peroxidation and cell death []in addition during viral infection oxidative stress contributes to viral pathogenesis through stimulating ammation loss of immune function and increased viral replicationthat may occur due to the activation of the nuclear factorkappa b nfκb transcription pathway [] currentevidence suggests that cytokine dysregulationalso calledcytokine stormcontributes to severe disease caused by thepathogenic covs [ ] the exact mechanisms are notclear yet but research on uenza a virus shows that infection causes a rapid ux of ammatory cells this isfollowed by an increase in reactive oxygen species productionand cytokine expression and release which ultimately leadsto acute lung injury in general rna viruses promotechanges in the bodys antioxidant defense system aï¬ectingenzymes such as superoxide dismutase sod and catalasecat in addition to reducing the levels of antioxidantmolecules such as ascorbic acid carotenoids and reducedglutathione gsh [] wu reported that glucose6phosphate dehydrogenasean important antioxidantenzyme that produces nadph knockdown cells were moresusceptible to infection by hcov229e than normal cells interestingly ye and colleagues have reported that theinhibition of ros production alleviates ammation causedby uenza a virus infections in an experimental model of sarsinduced acute lunginjury in mice it was noted that phospholipid oxidationdue to oxidative stress is one of the main triggering factorsof acute lung injury this happens through the activation ofthe innate immune response culminating in the activationof pulmonary macrophages via tlr4triftraf6nfκbsignaling furthermore hypoxia caused by acute lunginjury can cause myocardial injury due to the production ofros aggravating infections caused by coronavirus disease covid19 mitochondria have an essential function in energy generation and for this reason their function and integrity arestrictly regulated in order to respond to varying energyrequirements and environmental conditions mitochondria are known to function as the control point in apoptoticpathways releasing proapoptotic factors mainly ros whichfunction as a signaling molecule that may result in cell death[ ] some studies have shown a relationship betweencoronavirus infection and dysfunctional or damaged mitochondria leading to the release of ros and other proapoptotic substances [ ] in a recent study xu reportedthat ros and p53 play key roles in regulating many kindsof the cell process during coronavirus infection in vero cellsaccording to the authors coronavirus infection appears toinduce a timedependent ros accumulation which in turnis linked to regulatory mechanisms of p53 activation andapoptosis in infected cells antioxidant substances promote improvement in casesof disease caused by coronaviruses such as apolipoproteinda lipocalin that promoted a neuroprotective eï¬ect against 0coxidative medicine and cellular longevityencephalitis induced by human coronavirus oc43in micethis protective eï¬ect occurred through the reduction of oxidative stress cerebral lipid peroxidation and regulation ofammation [ ] also the treatment with antioxidantssuch as pyrrolidine dithiocarbamate or nacetylcysteine significantly inhibits moreover melatonin promotes downregulation of acute lungoxidative injury due to its antiammatory and antioxidantactions making it a possible compound in the treatment ofcovid19 based on these studies compounds thathave antioxidant actions can be helpful in the treatment ofinfections promoted by coronaviruscoronavirusinduced apoptosisin general antioxidant properties of polyphenolic compounds such as some ï¬avonoids have been associated withthe presence of aromatic phenolic rings that promote theelectron donation and hydrogen atom transfer to free radicals acting as free radical scavengers reducing agents andquenchers of single oxygen formation thus the aimof this study was to investigate the antioxidant capacity andantiviral activity of natural antioxidants against coronavirusthe compounds are illustrated in figure occurrence and antioxidant properties ofanticoronavirus compoundsquercetin can be found in plants such as rubus fruticosus land lagerstroemia speciosa l pers [ ] also quercetinshows antioxidant activity at a concentration of μmoll inhepg2 cells inhibiting oxidative stress promoted by h2o2 promotes an increase in sod cat and glutathioneperoxidase gpx and reduces lipid peroxidation in rats withchronic prostatitischronic pelvic pain syndrome moreover quercetin improves sepsisinduced acute lung injury inrats by reducing lipid peroxidation and ammation andincreasing sod and cat levels in addition quercetin glycosides with antioxidant activity such as quercetin 3βglucoside have already been isolated from plants such as passiï¬ora subpeltata ortega andchamomilla suaveolens pursh rydb [ ] the administration of quercetin 3βglucoside mgkg poinstreptozotocininduced diabetic rats promotes an increasein the levels of antioxidant enzymes sod cat andgpx and nonenzymatic antioxidants vitamins c and eand gsh and a reduction of lipid peroxidation quercetin 3βgalactoside hyperoside is found mainly in plantsof the hypericum genus such as hypericum perforatum l[ ] moreover it showed cardioprotective activity inhigh glucoseinduced injury of myocardial cells throughdecreased apoptosis and ros production and increasedsod levels quercetin 7ramnoside is also found inplants of the hypericum genus such as hypericum japonicum thunb ex murray this ï¬avonoid shows hepatoprotective activity against carbon tetrachloride in mice bydecreasing lipid peroxidation and increasing cat andgsh levels in addition to presenting values of μmand22diphenyl1picrylhydrazyldpph and ²azinobis3ethylbenzthiazoline6sulphonic acid abts assays respectively μm intheepigallocatechin gallate is present in parkia roxburghii gdon and is one of the main metabolites found in green teaand liubao tea camellia sinensis lo kuntze also gallocatechin gallate can be found in this plant [] literaturedata reveal that the administration ip of mg100 g ofepigallocatechin gallate in rats with streptozotocininduceddiabetes mellitus promotes a reduction in oxidative stressthrough reductions in parameters such as indirect nitricoxide synthesis and status total oxidative as well as anincrease in levels of cat and total antioxidant capacity ofplasma furthermore it promotes cardioprotection byantioxidant mechanisms green tea has a high antioxidant capacity due to the highlevels of catechins present he and collaborators compared the antioxidant activities of catechins and reported thatepigallocatechin gallate has greater antioxidant activity viaradical scavenging activity μm with values of ± ± and ± compared to its epimergallocatechin gallate with values of ± ± and ± in the dpph abts and ferric reducingantioxidant power frap respectively amentoï¬avone is a biï¬avonoid present in leaves ofginkgo biloba l garcinia brasiliensis l and nandinadomestica l [] this biï¬avonoid has a high antioxidantcapacity demonstrated in scavenging tests ofdpph abts superoxide and hydroxyl radicals moreover amentoï¬avone prevents acute lung injury induced bysepsis in rats by decreasing thiobarbituric acid reactive substance tbars levels and by increasing levels of sod andgsh apigenin is mainly present in ï¬owers and leaves beingabundantly found in apium graveolens l petroselinum crispum mill fuss and matricaria chamomilla l sánchezmarzo and collaborators evaluated the antioxidantcapacity of apigenin using the trolox equivalent antioxidantcapacity teac oxygen radical absorbance capacityorac and frap assays the results show that apigeninhas good antioxidant activity with values of ± μmol teammol ± μmol teammol and ± μmol fe2mmol respectively in addition oraladministration of apigenin mgkgday for days in anexperimental model of cardiotoxicity induced by doxorubicin in rats promoted cardioprotection by reducing levels ofmalondialdehyde mda increasing sod levels and preventing cardiomyocyte apoptosis luteolin is present in foods such as carrot cabbage teaand apple and is found in ugni molinae turcz [ ] datashow that luteolin μgml increases the levels of gsh theexpression of gsh synthetase and the activity of sod andcat in human colon cancer cells ht29 furthermoreluteolin attenuates the sepsisinduced acute lunginjury in mice by reducing lipid peroxidation and increasingsod and cat activity in addition to suppressing the nfκbpathway herbacetin is ubiquitous in plants of the genus rhodiolasuch as rhodiola rosea l herbacetin glycosides are alsopresent in the roots of r sachalinensis a bor and show antioxidant activity veeramani reported that theadministration of herbacetin mgkg po in mice with 0coxidative medicine and cellular longevityohohohohhoohooohohhooohoh oquercetinohohoooohohohquercetin 3ð½galactosideohohooohoohoohohhoohohohoh oquercetin 7ramnosideohooohohoohoohoohohohquercetin 3ð½glucosidehooohooohohohohohohepigallocatechin gallateohgallocatechin gallateohhooohoh ohooluteolinhooohoooohoohohoohrhoifolinhohohoohohohohohohoohoohooh oamentoflavoneohhooohohohoherbacetinhohoooohooohohohoapigeninooohooohhopectolinarinooopsoralidinhooohohohohcatechinohhoohoooh ohelichrysetinohohomyricetinohohohhohoohoohoisobavachalconeohhooohscutellareinohresveratrolfigure chemical structures of bioactive antioxidants against coronavirusobesityassociated insulin resistance promotes an increasein the activity of the enzyme glucose6phosphate dehydrogenase which is directly related to the production ofnadph pectolinarin is present in plants of the genus cirsiumsuch as cirsium setidens nakai and cirsium japonicum dcthe administration of pectolinarin and mgkg pofor two weeks in rats promotes antioxidant eï¬ects in hepatic 0coxidative medicine and cellular longevitytable antioxidant properties of natural inhibitors of coronaviruscompoundtestedassaysexperimentalconcentration doseantioxidant eï¬ectreferencetype of cells μmoll mgkg po mgkg poinhibiting oxidative stress promoted byh2o2promoted an increase in sod cat andgpx and reduced lipid peroxidationreduces lipid peroxidation and increasessod and cat levelsincreases levels of sod cat gpx mgkg povitamins c and e and gsh and reduces nmoll mgkgic50 μm dpphec50 μm abtsrats with streptozotocininduced diabetes mellitus mg100 g iprats with streptozotocinnicotinamideinduceddiabetes mellitus mgkg po μm mgkg μgml ± μmol teammol ± μmol teammol and ± μmol fe2mmolrespectively mgkg pomodelshepg2 cellsrats with chronicprostatitischronic pelvicpain syndromesepsisinduced acute lunginjury in ratsstreptozotocininduceddiabetic ratshigh glucoseinducedinjury of myocardial cellsccl4induced liver damagemodel in micedpphabtsquercetinquercetin 3βglucosidequercetin 3βgalactosidequercetin ramnosideepigallocatechingallateepigallocatechingallatedpphabtsfrapgallocatechingallateamentoï¬avoneacute lung injury inducedby sepsis in ratsdpph abts superoxideand hydroxyl radicalsteacoracfrapcardiotoxicity induced bydoxorubicin in ratshuman colon cancer cellsht29acute lung injury inducedby sepsis in micemice with obesityassociated insulinresistance inductionhepatic injury induced bydgalactosamine in ratsoracapigeninluteolinherbacetinpectolinarinrhoifolincatechinlipid peroxidationdecreases apoptosis and ros productionand increases sod levelsdecreases lipid peroxidation and increasescat and gsh levelsscavenging of free radicalsreduces indirect nitric oxide synthesis andtotal oxidative statusincreased levels of cat and totalantioxidant capacity of plasmaincreased levels of cat sod and gshreduced levels of superoxide and proteincarbonyl pco and prevented dnadamage ± ± and ± ± and ± respectively ± respectivelydecreases tbars levels and increaseslevels of sod and gshscavenging of free radicalsscavenging of free radicalsreduces levels of mda increases sodlevels and prevents cardiomyocyteapoptosis μgmlincreases levels of gsh expression of gshsynthetase and the activity of sod and mgkg ip mgkg po and mgkg poapproximately troloxequivalents μmcatreduces lipid peroxidation increases theactivity of sod and cat and suppressesthe nfκb pathwayincreases the activity of glucose6phosphate dehydrogenaseincreases levels of sod gsh glutathionereductase and glutathione stransferasescavenging of free radicalsscavenging of free radicals 0coxidative medicine and cellular longevitytable continuedtype of cellscompoundtestedassaysexperimentalconcentration doseantioxidant eï¬ectreferencemodelsabtsfrap ± mol troloxequivalentsmol ± mol trolox equivalentsmoldihydrorhodamine oxidation assayandic50 ± μmisobavachalconedpph sc50frapabts sc50psoralidinelectron spin resonancemyricetinhelichrysetinscutellareinresveratroldpphchinese hamster lungï¬broblast cells v794treated with h2o2oracdpphabtssuperoxide radicalsdpph sc50r2rats with obstructive lungdiseasehypercholesterolemicapoeko mouserespectively μm ± mmic50 μmequivalent to feso4 mm respectively·7h2o and μgml and μgml μgml ± trolox equivalents ± μm ± μm and ± μm respectivelyscavenging of free radicalsscavenging of free radicalsscavenging of free radicals and respectivelyprevents dna damage and lipidperoxidationincreases the activity of sod cat andgpxscavenging of free radicalsscavenging of free radicals μmoldmscavenging of free radicals mgkg mgkgincreases sod activity and reduces mdalevelsinhibits the activity and expression ofnadph oxidasesincreases sod gpx and cat levels injury induced by dgalactosamine by increasing levels ofsod gsh glutathione reductase and glutathione stransferase [ ]rhoifolin is found in citrus fruits such as citrus limettarisso studies have indicated that its radical peroxyl scavenging capacity is higher than trolox in orac assays approximately trolox equivalents μm [ ]meanwhile the catechin is a ï¬avonoid present inleaves of green tea wine and fruits [ ] grzesik investigated the antioxidant action of catechinthrough the abts scavenging activity and frap teststhe results show values of ± mol troloxequivalentsmol and ± mol trolox equivalentsmol respectively in addition catechin shows greaterprotective properties in the dihydrorhodamine oxida ± μm than gsh and ascorbiction assay ic50acid and μm respectively psoralidin is a prenylated coumestan which is found inplants of the fabaceae family such as psoralea corylifolia lxiao and collaborators investigating the antioxidant potential of compounds isolated from p corylifolia observed thatpsoralidin shows the best antioxidant activity by the methodof electron spin resonance spectroscopy with an ic50 value of μm the compound isobavachalcone has been isolated fromplants of the fabaceae and moraceae families [ ] isobavachalcone shows a strong antioxidant activity in dpphsc50 frap and abts sc50 assays with values of μm ± mm equivalent to feso4·7h2o and mm respectively in addition the compound has beenreported to inhibit the nfκb pathway in sephadexinducedlung injury in rats [ ]helichrysetin is a chalcone that is found in plants of thehelichrysum genus such as helichrysum odoratissimum l in a study investigating the antioxidant activity of natural and prenylated chalcones vogel found that helichrysetin is the substance that shows the highest antioxidantactivity in the orac test with values of ± troloxequivalents myricetin is widely found in the plant families myricaceae and anacardiaceae and is widely used as health foodsupplement due to its antioxidant properties [ ]bennett demonstrated that myricetin reacts withoxygencentered galvinoxyl radicals more than timeshigher than vitamin e dalphatocopherol furthermoremyricetin was able to scavenge and on the dpphassay μgml and μgml respectively interestinglythe compound prevents dna damage by lipid 0coxidative medicine and cellular longevity2h2o2 2gsh2o2h2h2o2vit evit cgshnadphnonenzymatic antioxidantsgpxsodcatsemyzne tnadixoitna2h2o gssgo2 h2o2o2 h2omdapcotbarsbio m arkers of oxidative stressgeneration of rosoxidative stresscell damagenaturalantioxidants sesadixohpdan2o2 nadphnadp 2o2 hfigure the main antioxidant mechanisms of natural compounds reported in this review dashed line inhibition full line activationperoxidation and increasing the activity of sod cat andgpx in chinese hamster lung ï¬broblast cells v794treated with h2o2 scutellarein is found in scutellaria barbata d don andpolygonum viscosum buchham [ ] liu investigated the antioxidant activity of scutellarein through thedpph abts and superoxide scavenging assays they notedthat the compound shows good antioxidant activity withvalues of ± μm ± μm and ± μmrespectively while the trolox a standard antioxidant compound presented ± μm ± μm and ± μm respectively resveratrol is found in grapes peanuts and blueberriesand can be isolated from veratrum grandiï¬orum o loes literature shows that resveratrol has good antioxidantvalues of μmoldmactivity with dpph sc50r2moreover it is able to reduce the production of ros by inhibiting the activity and expression of nadph oxidases byeliminating oxidant agentsincluding radical hydroxylsuperoxide hydrogen peroxide and peroxynitrite the treatment of resveratrol mgkg po in ratsreduces oxidative stress in obstructive lung disease byincreasing sod activity and reducing mda levels indicatinga decrease in lipid peroxidation table shows themain actions of natural antioxidants discussed in this studyand figure illustrates these activities effect of natural antioxidants incoronavirus infectionsthis review focused on studies reporting on the anticoronavirus activity of natural antioxidants based on exclusioncriteria data from nineteen compounds were discussedthe oxidative stress pathway could potentially be a keyelement in coronavirusinduced apoptosis and pathogenesis for this reason it is interesting to investigate the useof antioxidants as potential therapeutic toolseither as analternative or as an adjuvant to conventional therapiesinthe treatment of coronavirus infections among the antioxidant compounds evaluated as for coronavirus infectionsare the ï¬avonoids which are compounds widely found infruits vegetables and certain beverages in fact researchgroups have reported that antioxidant ï¬avonoids includingcatechin luteolin apigenin quercetin and quercetin rhamnosideinhibit ros accumulation and apoptosis ofcells infected with diï¬erent coronavirus including porcineepidemic diarrhoea coronavirus pedv and transmissiblegastroenteritis coronavirus tgev []as shown with the recent covid19 pandemic thesearch for alternative or new antiviral therapies for theremainstreatment of coronavirus diseasesimportantbased on the literature antioxidanttherapies oï¬er anattractive option 0coxidative medicine and cellular longevitytable natural antioxidants tested in in vitro coronavirus infection models and their main results and mechanism of actionantioxidanttype of cells testedconcentrationic50antiviral eï¬ectmechanism of actionreferencecatechintgevinfected st cellscatechin μminhibition of tgevinduced apoptosissuppression of the tgevinducedbcl2 reduction baxredistribution cytochrome crelease and caspase3 activation resveratrolmersinfected vero e6cellsresveratrol μmquercetinepigallocatechingallategallocatechingallate gcgquercetin rhamnosideq7rrecombinant 3clprowas expressed in pichiapastoris gs115quercetin μmepigallocatechingallate μmgallocatechingallate μmpedvinfectedvero cellsq7r μminhibition ofmersinducedreduction of the expression ofinfectionapoptosis andnucleocapsid n protein essential prolonged cellular survivalfor merscov replicationafter virus infectioninhibition of coronavirusreplicationreduction of the formationof a visible cytopathiceï¬ect cpe without dnafragmentationgcg displayed a binding energyof kcal mol1 to the active siteof 3clpro and the galloyl moietyat 3oh position was required for3clpro inhibition activitynot speciï¬city amentoï¬avoneapigeninluteolinquercetinquercetin3βgalactosideherbacetinrhoifolinpectolinarinsarscov 3clproinhibition usingï¬uorescence resonanceenergy transfer analysismolecular dockingsprfretbasedbioassays andmutagenesistryptophanbasedï¬uorescence methodherbacetinisobavachalconequercetin3βdglucosidehelichrysetintryptophanbasedï¬uorescence methodamentoï¬avone3βgalactoside μmapigenin μmluteolin μmquercetin μmquercetin μmherbacetin μmrhoifolin μmpectolinarin μmherbacetin μmisobavachalcone μmquercetin3βdglucoside μmhelichrysetin μminhibition of sarscovreplicationflavonoids exhibited sarscov3clpro inhibitory activity[ ]inhibition of merscovreplicationflavonoids exhibited merscov3clpro inhibitory activity isobavachalconepsoralidinlineweaverburk anddixon plotsmyricetinscutellareinsprfretbasedbioassaysisobavachalcone μmpsoralidin μmmyricetin μmscutellarein μminhibition of sarscovreplicationisobavachalcone and psoralidinexhibited sarscov papainlikeprotease inhibitory activitymyricetin and scutellareininhibition of sarscovpotently inhibit the sarscovreplicationhelicase protein in vitro byaï¬ecting the atpase activity the high number of deaths and clinical complicationsobserved in sars and merscov epidemics motivatedthe search for eï¬ective therapeutic agents this was necessitated when many of the tested conventional drugs andtherapies proved ineï¬ective in treating sarsantiviralcov infections for exampletreatment ofthe initial 0coxidative medicine and cellular longevitycell culture coronavirusnaturalantioxidants suppress bcl2 reductionsuppress bax redistributionsuppress cytochorome c releasesuppress caspase3 activationreduce formation of a visiblecytopathic effect without dnafragmentationreduce nucleocapsid n protein expressioninhibit 3clike proteaseinhibit 3clike proteaseinhibit papainlike proteaseinhibit helicase protein by affectedatpase activitytgevpedvmerscovsarscovfigure inhibitory actions of natural antioxidants against coronavirussarscov with antiviral agents such as ribavirin and corticosteroids did not achieve very satisfactory resultsmainly because corticosteroids exertimmunosuppressoreï¬ects on the humoral and cellular immune systems[ ] other drugs such as pentoxifylline were considered for the treatment of sars due to its interestingtherapeutic propertiesantiammatoryantiviral immunomodulatory and bronchodilatory eï¬ectshowever it too was not successful in the clinical treatment of sarscov infection includethatinhibition ofmany antioxidant compounds show antiviral activityagainst sarscov the antiviral activity has been mainlyattributed to thethe 3clike protease3clpro of sarscov a vital enzyme for sarscov replication as an example multiples studies havereported that quercetin and quercetinderived compoundssuch as quercetin 3βgalactoside display potent 3clproinhibitory e5ï¬ect and consequent reduction of sarscovreplication other antioxidants such as epigallocatechin gallate gallocatechin gallate amentoï¬avone apigeninluteolin herbacetin rhoifolin and pectolinarin are alsofound to eï¬ciently block the enzymatic activity of sarscov 3clpro [ ]moreover some natural antioxidants exhibit promisingantiviral activity against sarscov infection by interferingwith diï¬erent targets involved in sarscov replication inparticular the sarscov papainlike protease plpro andsarscov helicase protein kim reported that isobavachalcone and psoralidin inhibit plpro in a dosedependentmanner with ic50 ranging between and μm previously yu reported that myricetin and scutellareinpotently inhibit the sarscov helicase protein in vitro byaï¬ecting the atpase activity merscov is another zoonotic coronavirus transmitted between animals and human beings that causes severemorbidity and mortality no antiviral medicines with satisfactory eï¬cacy for the treatment of merscovinfectedpatients have been identiï¬ed to date similar to sarscov natural antioxidant libraries have been probed forpotentialinhibitory compounds against merscov 3clike protease jo showed that herbacetin isobavachalcone quercetin 3βdglucoside and helichrysetinfourcompounds with recognized antioxidant activity can blockthe enzymatic activity of merscov 3clpro using atryptophanbased ï¬uorescence method furthermore theexperimental and computational studies show that ï¬avonoland chalcone are favourite scaï¬olds to bind with the catalytic site of merscov 3clpro in a study performed by lin the antiviral activitiesof resveratrol were investigated in mersinfected vero e6cells the authors reported a significantinhibition ofmerscov infection and prolonged host cell survival aftervirus infection which they speculate was promoted by resveratrol in addition they also found that the expression ofthe nucleocapsid n protein which is essential for merscov replicationis decreased after resveratrol treatment it is important to mention that in vitro models of coronavirus infection also show antiviral activity of ï¬avonoidsextracted from ï¬owering cherry cultivars and black tea[ ] finally antioxidants such as resveratrol alsoare able to block infection produced by herpesvirus the discovery of antiviral compounds from a bioactivecompound against other viruses is an interesting strategy forobtaining new antiviral drugs table shows the mainactions of the natural antioxidants against the coronavirusand figure summarizes these activities 0c conclusionsin conclusion this review shows that antioxidant compounds prominently ï¬avonoids exhibit antiviral action inmodels of coronavirus infections in general the antiviralactivity might be attributed at least in part to the inhibitoryeï¬ect on the enzymatic activity of targets involved in coronavirus replication including sarscov 3clpro sarscovpapainlike protease plpro sarscov helicase proteinand merscov 3clpro in addition some studies provideevidence that the reduction of ros accumulation retardsthe coronavirusactivated apoptotic signaling thereforethe mechanisms of oxidative stress could be the key elementto be studied in coronavirus infectionsincluding thoserelated to ammatory processes arising from the action ofthis virus obviously further investigations are needed toelucidate other pharmacological mechanisms by which natural antioxidants play an antiviral eï¬ect despite the ï¬ndingsreported in this reviewthey cannot be generalized tocovid19 however the data provided support to the investigation of natural antioxidants as a potential therapeuticapproach in the treatment for covid19 and its severe clinical complications either as an alternative or as an adjuvantto conventional therapies and contribute to the search fornew prototypes in the development of drugs against coronavirus infectionsabbreviations229e3clproabtshuman coronavirus229e3clike protease²azinobis3ethylbenzthiazoline6sulphonic acidcoronavirusescatalasediï¬use alveolar damage22diphenyl1picrylhydrazylferric reducing antioxidant powerreduced glutathioneglutathione peroxidasehuman coronaviruseshuman coronavirushku1lower respiratory tract infectionsmalondialdehydecovscovid19 coronavirus disease catdaddpphfrapgshgpxhcovshku1lrtismdamerscov middle east respiratory syndromecoronavirusnfκbnuclear factor kappa bhuman coronavirusnl63nl63human coronavirusoc43oc43oxygen radical absorbance capacityoracprotein carbonylpcoporcine epidemic diarrhoea coronaviruspedvplpropapainlike proteasereactive nitrogen speciesrnsreactive oxygenated speciesrossarssevere acute respiratory syndromesarscov severe acute respirator | Colon_Cancer |
" fasassociated factor faf1 has been implicated in parkinsons disease pd and activates the celldeath machinery in the cytosol however the presence of extracellular faf1 has not been studiedmethods serumfree conditioned medium cm from faf1transfected shsy5y cells was concentrated andanalyzed by western blotting exosomes were isolated from cm by ultracentrifugation and analyzed by westernblotting electron microscopy and nanop tracking analysis soluble faf1 from cm was immunodepleted usingantifaf1 antibody transmission of secreted faf1 was examined by transwell assay under a confocal microscopecminduced cell death was determined by measuring propidium iodide pi uptake using a flow cytometerresults faf1 was secreted from shsy5y cells via exocytosis and brefeldin a bfaresistant secretory pathwaysfurthermore faf1 was secreted as a vesiclefree form and a genuine exosome cargo in the lumen of exosomes inaddition faf1 increased the number of exosomes suggesting a regulatory role in exosome biogenesis extracellularfaf1 was transmitted via endocytosis to neighboring cells where it induced cell death through apoptotic andnecrotic pathwayss this study presents a novel route by which faf1 induces neuronal death through celltocelltransmissionkeywords faf1 secretion exosome vesiclefree form celltocell transmission cell death cells secrete proteins harboring signal peptides throughthe classical secretory pathway via the endoplasmicreticulum ergolgi complex from which vesicles withcargo proteins move toward and fuse with the plasmamembrane and subsequently export cargos to the extracellular space however proteins lacking signal peptides are secreted via alternative nonclassical secretorypathways these pathways are classified as vesicularand nonvesicular secretory pathways some proteins correspondence eunheecnuackr gyeongrin park and bokseok kim are considered cofirst authorsdepartment of biological sciences chungnam national university daehakro yuseonggu daejeon south koreaare secreted via extracellular vesicles including exosomesand other vesicles of various sizes [ ] alternativelyother proteins are secreted through membrane poresand atpbinding cassette abc transporters althoughthe exact mechanisms of nonvesicular secretory pathways are elusive [ ]exosomes which are nanosized membrane vesicles nm in diameter secreted into the extracellular environment by various cell types are associated with intercellular communication with neighboring cells and play arole in a myriad of pathological functions in diseases including cancer cardiovascular and neurodegenerative diseasestheextracellular matrix and promote metastasis angiogenesisin particular exosomes[]remodel the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cpark cell communication and signaling page of thrombosis and tumor cell proliferation in cancer exosomes in cardiovascular disease display proangiogenesis procoagulant and proinflammatory effects on thevessel walls in neurodegenerative diseases exosomesare potential sources of key pathogenic proteins such astau amyloid prion and αsynuclein []inflammation triggersin addition to their vesiclemediated secretion manyproteins are secreted through nonvesicular mechanismsfor instance the proteins such as tau and fibroblastgrowth factor are recruited to the plasma membrane toform lipidic pores and are subsequently secreted [ ]in additionthe secretion ofinterleukin1 and transglutaminase through pores fibroblast growth factor translocates across the plasmamembrane via poorly understood mechanisms includingthe membrane release complex upon stress proteinssuch as hydrophilic acylated surface protein b are secretedby abc transporters proteins secreted via nonvesicular secretory pathways are advantageous over cargo proteins in vesicles as immunotherapeutic targets because ofthe antibody accessibility of the extracellular spacefasassociated factor faf1 is involved in diverse biochemical processes including cell death inflammation cellproliferation and proteostasis [] consistent with itsdiverse functions faf1 has been implicated in certain diseases first faf1 plays a tumor suppressive rolethrough activation of the apoptotic machinery and nfκbsuppression [ ] faf1 also suppresses tumor metastasis via tgf signaling moreover faf1 expressionis downregulated in various cancers including lung colonliver prostate brain ovarian and breast cancers second faf1 is involved in parkinsons disease pd as it iscolocalizes with αoverexpressed in pd patientssynuclein and acts as a substrate of parkin furthermorefaf1 mediates the degeneration of dopaminergic neuronsthrough apoptosis and parthanatos []faf1 is an intracellular protein present mainly in thecytosol to date faf1 secretion has not yet been reportedherein we uncovered for the first time that faf1 is secreted via an ergolgiindependent pathway specificallyfaf1 is secreted through exosomal and nonvesicular pathways in shsy5y cells in addition faf1 augments thenumber of exosomes suggesting the involvement of faf1in exosome biogenesis furthermore extracellular faf1moves into neighboring cells via pinocytosis and clathrinmediated endocytosis transmitted faf1 induces cell deathvia apoptosis and necrosis collectivelythese resultspresent a novel measure by which faf1 propagates itsdeath function through celltocell transmissionmethodsreagents and antibodiesthe following reagents and antibodies used in this studywere purchased commercially tnfα from abfrontierseoul south korea zietdfmk caspase8 inhibitormouse antiha antibody and rabbit antigm130 antibody from abcam cambridge uk ²²dichlorofluorescin diacetate dcfhda hydrogen peroxide h2o21methyl4phenylpyridinium mpp propidium iodidepi polydlysine brefeldin a bfa gw4869 monensin cycloheximide chx necrostatin1 nec1 dpqproteinase k pk dynasore heparin heparinase iiimouse antiactin and mouse antiflag antibody fromsigmaaldrich saint louis mo usa ²6diamidino2phenylindole dapi horseradish peroxidase hrpconjugated antimouse antibody and hrpconjugatedantirabbit antibody from thermo fisher scientific incrockford il usa mouse antiflotillin1 from bd biosciences san jose ca usa bafilomycin a1 mouseantialix antibody mouse antigalactosidase antibody401a rabbit anticalregulin antibody mouse anticd63 antibody mouse antiparkin antibody and mouseantifaf1 antibody from santa cruz biotechnologydallas tx usa mouse antihsc70 antibody andmouse antihsp90 antibody from enzo life sciencesfarmingdale ny usa and zvadfmk zvad fromcalbiochem darmstadt germanycell culture and transfectionshsy5y mef hek293 raw2647 hela panc1mia paca2 and mcf7 cells were maintained in dulbeccos modified eagles medium dmem welgenedaegu korea containing fetal bovine serum fbsatlas biologicals fort collins co usa and antibioticantimycotic gibco brl grand island neusa unless otherwise specified cells were transfectedwith the indicated plasmids using bio t manvillescientific inc manville nj usa following the manufacturers protocol rat midbrain cultures derived frompostnatal day were prepared using standard procedures briefly material dissected form the ventral portion of the midbrain was cleaned free of meningealtissue minced and enzymatically dissociated in a mixture of papaindnase sigmaaldrich dissociated cellswere plated onto aminecoated 6well plates bd biosciences cells were maintained in neurobasal mediumgibco with b27 serumfree supplements gibco mm lglutamine after days of culture the cells wereinfected using aav1 adenoassociated virus 1hfaf1viral vectors moi of and sitedirected mutagenesisthe sirnaresistant parkin construct was generatedusing quikchange sitedirected mutagenesis kit stratagene la jolla ca usa the primers were as follows²gagctgagaaacgactggactgtgcagaattgtg3²²gggaaggagctgagaaacgattgcactgtgcaga ² 0cpark cell communication and signaling page of rna interferenceall small interfering rnas sirnas against parkin andscrambled rna scrna were purchased from bioneerdaejeon south korea the sequences of the sirnasused in this study were as follows sirna against parkin²ugaggaaugggacugu3² thescrna orsirna were transfected into shsy5y cells using lipofectamine rnai max thermo fisher scientific according to the manufacturers instructionspreparation of conditioned mediumto prepare conditioned medium cm cells transfectedwith the indicated plasmids were cultured in mmdiameter dishes in dmem containing fbs after h the cells were switched to serumfree dmem forthe indicated times here we used serumfree mediumto avoid interference from albuminenriched fbs thenthe cm was collected and centrifuged at ¨¯g for min and ¨¯g for min to remove cellular debrisfor western blot analysis the cm was concentratedusing kda or kda cutoff amicon ultra filtersmillipore billerica ma usa at ¨¯g for min or minwestern blot analysiscells were harvested washed twice with pbs and lysedwith mammalian lysis buffer [ mm triscl ph mm nacl mm edta nonidet p40 mmphenylmethylsulfonylfluoride] then the protein concentrations were quantified by using a biorad proteinassay kit biorad hercules ca usa after quantification samples were boiled in à protein sample buffer[ mm triscl ph glycerol sds mercaptoethanol] then samples were electrophoresedby sdspage and transferred to nitrocellulose membranes ge healthcare maidstone uk the membranes were blocked with skim milk in pbs with tween20 pbst and incubated with the indicatedprimary antibodies overnight after their washing withpbst the membranes were incubated with secondaryantibodies immunoblot signals were measured by usingchemiluminescent detection lab frontier anyangkoreachx chase assayshsy5y cells were transiently transfected with the indicated plasmids for h after transfection the cells wereswitched to serumfree dmem containing chx sigmaaldrich μgml for the indicated of times subsequently the medium was concentrated with kda cutoff amicon ultra filters millipore and analyzed bywestern blot analysispurification of exosomeswe followed a previously described protocol with somemodifications briefly cells were transfected withthe indicated plasmids for h and incubated in dmemcontaining exosomedepleted fbs system biosciences palo alto ca usa for h the cm was thencollected and subjected to sequential centrifugation at¨¯g for min ¨¯g for min and ¨¯g for min at °c to remove cellular debris using a beckmancoulter optima l90 k ultracentrifuge with a type 41tirotor the supernatant was then spun down at ¨¯gfor min the pellet was resuspended in pbs and thenspun again at ¨¯g for min at °c finally thepellet was resuspended in pbs or radioimmunoprecipitation assay ripa buffer sigmaaldrichin addition to their isolation via ultracentrifugationwe isolated exosomes using exoquicktc system biosciences according to the manufacturers instructionstreatment of vesicles with na2co3to separate integral membrane proteins and luminalproteins purified exosomes were treated with mmna2co3 ph for min at °c as previously described after centrifugation at ¨¯g for minintegral proteins remained in the pellet fraction whileluminal proteins remained in the supernatant fractionthe pellet fractions were resuspended in ripa buffersigmaaldrich and the supernatants were collected ina separate tube for western blot analysisproteinase k digestionpk sigmaaldrich was added to the samples at a finalconcentration of μgml then the samples were incubated at °c for min and mm phenylmethylsulfonyl fluoride was added to inhibit the activation of pkfollowed by the addition of protein sample bufferimmunodepletion of cmfifty microliters of protein gconjugated dynabeadsthermofisher scientific was incubated overnight withmouse monoclonal antibody against faf1 final concentration of or μgml before its addition to cm theantibodydynabeads complex was incubated with mlof cm at °c overnight after the complex was removedusing a magnet the immunodepleted cm was concentrated using a kda cutoff amicon ultra filter andused for western blot analysis for flow cytometryunconcentrated immunodepleted cm was applied to recipient cellsflow cytometryto evaluate cell death we measured pipositive cellstaining by using a guava easycyte flow cytometermillipore briefly cells were switched to serumfree 0cpark cell communication and signaling page of dmem or neurobasal medium for the indicated timeadditionally to measure recipient cell death shsy5yand rat primary neuronal cells were treated with cmfrom donor cells for the indicated times the cells were μgml and evaluatedharvested stained with piusing a guava easycyte flow cytometer following whichthe results were quantified using incyte softwaremilliporeconcentrated cm treatmentshsy5y cells donor cells were plated on mm tissueculture dishes and transfected with gfpvector or gfpfaf1 plasmid at h after transfection the cells wereincubated in serumfree medium for h after the cmwas concentrated by using kda cutoff amicon ultrafiltersthe concentrated cm was dissolved in newserumfree medium that was applied to recipient cellson polyllysinecoated coverslips in 12well plates for hpropagation assayshsy5y cells donor cells were plated on mm tissueculture dishes donor cells were transfected with gfpvector or gfpfaf1 plasmids at h after transfectionthe cells were collected and replated in cell culture inserts polycarbonate membrane μm pore size corning kennebunk me usa at a density of ¨¯ cellsshsy5y cells recipient cells were plated at a densityof ¨¯ cells on polydlysinecoated coverslips in well plates after h the cultures were combined suchthat the donor cells were in the insert and separatedfrom recipient cells plated on a coverslipconfocal microscopycells were fixed with paraformaldehyde for minthe cisgolgi were stained with gm130 after the nucleiwere stained with dapi for min the coverslips weremounted onto microscope slides using fluorescencemounting medium dako carpinteria ca usa andanalyzed using a zeiss lsm laser scanning confocalmicroscope carl zeiss oberkochen germanyelectron microscopyfor transmission electron microscopy tem sampleswere prepared using the exosometemeasy kit containing a formvarcarboncoated em mesh gridwash buffer and em solution bio mountain viewca usa the pellets from the ml of cm vector orfaf1transfected cells obtained by ultracentrifugationwere resuspended in μl of pbs μl of which was applied to the grid all samples were prepared followingthe manufacturers instructions for immunoem thepellets were first fixed with μl of paraformaldehyde and glutaraldehyde sigmaaldrich overnightat °c then the fixed exosome solution was transferredto grids and subsequently treated with m glycinefor min to quench free aldehyde groups after blocking with pbs containing bsa for min the gridswere incubated for h with the indicated antibodies diluted in pbs containing bsa atroomtemperature after three washes with pbs containing bsa the grids were incubated for h with the secondary antibody antimouse igg conjugated to nmgoldroomtemperature three washes to eliminate secondary antibody were followed by incubation with em solution anda wash step samples were viewed under a talos f200xtransmission electron microscope fei hillsboro orusa operated at kv and images were capturedwith a ceta m pixel cmos camera feisigmaaldrichatpsnanop tracking analysisfollowing isolation by differential ultracentrifugation orexoquicktc system biosciences the exosome pelletswere resuspended in μl of pbs then μl of theexosome solution was diluted in pbs to a total volumeof ml the samples were analyzed by nanoptracking analysis using a nanosight ns300 malvernpanalytical ltd malvern uk equipped with a nmlaser to accurately identify the vesicles the detectionthreshold was set at the number of vesicles in eachsample represents the number of ps per ml ofmedium cells were counted using a muse count viability kit millipore on a muse cell analyzer milliporecaspase3 activity assayshsy5y cells were treated with cm from faf1transfected cells plus caspase8 inhibitor or tnfα abfrontier plus chx sigmaaldrich at the indicated concentrations for the indicated times then caspase3 activity was measured by using a caspase3 colorimetricassay kit biovision milpitas ca usa in accordancewith the manufacturers protocol the absorbance at nm was measured with the use of a victor microplate reader perkinelmer norwalk ct usasignalp41we investigated the presence of a signal peptide in faf1using signalp41 httpwwwcbsdtudkservicessignalp41 with secretogranin1 used as a positive controlas it contains a signal peptidestatistical analysesexperiments were independently carried out three timesn all the data are expressed as the mean ± standarddeviation sd statistical comparisons were performedusing students ttest or oneway analysis of varianceanova followed by tukeys hsd post hoc analysis 0cpark cell communication and signaling page of using spss software statistics version ibm incchicago il usa statistical significance was established when the pvalue was lower than resultsfaf1 is secreted via nonclassical exocytosisaccording to the csf proteome resource faf1 is detected in the cerebrospinal fluid and plasma additionalfile this study aimed to determine whether faf1 isalso secreted at the cellular level and investigate faf1secretion in shsy5y human neuroblastoma cellsbecause faf1 is a deathpromoting protein sublethal experimental faf1 transfection conditions wereused to exclude cellular debris due to death additionalfile fig s1a and b the faf1 transfection conditionsin shsy5y cells under which faf1 secretion but notcell death occurs were determined by pi stainingfollowed by flow cytometry cells were transfected with3xflagfaf1 plasmid at a sublethal dose for h andsubsequently moved to serumfree medium faf1 wasdetected in the serumfree medium at h and accumulated henceforth indicating that faf1 is secreted in atimedependent manner fig 1a this finding suggeststhat faf1 is constitutively released from shsy5y cellsto exclude a tagging artifact another epitope taghemagglutinin ha was used hafaf1 was alsodetected in the cm conditioned medium in a dosedependent manner additional file fig s1c furthermore we examined the faf1 secretion with rat primaryneurons using aav1 adenoassociated virus 1mediated faf1 overexpression faf1 overexpression in ratprimary neuronal cells demonstrated consistent resultsin shsy5y cells fig 1b moreover with thatgalactosidase was not detected in the cm of galactosidasetransfected cells demonstrating that therelease of faf1 is not a transfection artifact fig 1c next a pulsechase experiment using chx to inhibit de novo protein synthesis was performed consistently faf1 was secreted and accumulated over timefurther confirming that faf1 is constitutively secretedfrom shsy5y cells fig 1d next we examinedwhether faf1 is also secreted by other cells faf1 wasdetected in the extracellular space of mefs and hek293cells furthermore faf1 was present in the cm of eachof a number of various cancer cell types mcf7 helapanc1 and mia paca2 cells this shows that faf1secretion is notadditional file fig s2afspecific to shsy5y cellsbecause faf1 has been implicated in pd pathogenesisfaf1transfected shsy5y cells were treated with thepdassociated stressors such as mpp h2o2 bafilomycin a1 and αsynuclein overexpression at sublethal dosesadditional file fig s3 there were no significantchanges of faf1 expression in clslysatescelldependent on various pdassociated stressortypeshowever faf1 secretion increased in cms upon allstressors used in this study implying that pdassociatedstressors positively regulate faf1 secretion fig 1eto elucidate the mechanism by which faf1 is secreted two sets of experiments were performed as follows first we examined whether faf1 is released viaexocytosis or passive diffusion as exocytosis is affectedby temperature faf1transfected shsy5y cellswere incubated at either °c or °c faf1 secretionat °c was significantly reduced compared to that at °cindicating that faf1 is released via exocytosisfig 1f second we examined whether faf1 is secretedvia the classical ergolgidependent secretory pathwayusing bfa which generates ros and disassembles golgithrough ergolgi pathway inhibition bfa did not affectfaf1 release fig 1g additional file fig s1d furthermore a signal peptide was not found in faf1when its sequence was analyzed using singalp41 furtherexcluding the possibility of ergolgimediated secretionof faf1 additionalfile fig s4 taken togetherthese data demonstrate that faf1 is released via nonclassical exocytosisfaf1 is secreted via exosomal and nonvesicular pathwaysto determine the mechanism by which faf1 is secreted exosomes were isolated from cm using a differential ultracentrifugation procedure as previouslydescribed and exoquicktc following the manufacturers protocol western blot analysis of exosomesisolated by ultracentrifugation revealed the presenceofthe exosome markers alix cd63 hsc70 andhsp90 but not calregulin an er resident protein a negative exosome control fig 2a exosomes isolated from shsy5y cells by exoquicktc showed anexosome marker profile consistent with that of exosomes purified by ultracentrifugation additional file fig s5a moreover nanop tracking analysisand tem data further confirmed that our purifiedexosomes exhibited a typical size distribution with adiameter ranging from to nm and a typicalmorphology of exosomes fig 2b and cendogenous as well as overexpressed faf1 proteinswere detected in the exosomal fractions isolated by bothmethods indicating that faf1 is secreted as a genuineexosomal cargo fig 2a similarly faf1 was also foundin exosomes isolated from the various indicated cell linesby exoquicktc additional file fig s5bh next weinvestigated whether faf1 is present on the membraneor in the lumen of exosomes exosomes were treatedwith mm na2co3 ph to distinguish betweenthe exosomal membrane and the lumen both alix andflotillin1 were present in the exosomal membrane positive controls whereas faf1 was mainly present in the 0cpark cell communication and signaling page of fig see legend on next page 0cpark cell communication and signaling page of see figure on previous pagefig faf1 is secreted via nonclassical exocytosis a shsy5y cells were transfected with vector control vc or 3xflagfaf1 plasmid at h aftertransfection the culture medium was replaced with serumfree medium for the indicated times se short exposure le long exposure b ratprimary neuronal cells were transduced with aav1hfaf1 viral vectors at days after transduction the culture medium was replaced withserumfree neurobasal medium for h c cells were transfected with lacz or 3xflagfaf1 plasmid at h after transfection the culture mediumwas replaced with serumfree medium and cells were cultured for h d cells were transfected with vc or 3xflagfaf1 plasmid at h aftertransfection the culture medium was replaced with serumfree medium containing chx μgml for the indicated times e left panel cellswere transfected with vc or 3xflagfaf1 plus αsyn plasmid at h after transfection the culture medium was replaced with serumfreemedium containing dmso vehicle mpp mm h2o2 μm or baf a1 nm for h right panel the graph shows the densitometricanalysis of immunoblotting of faf1 in conditioned medium cm shown in the left panel n f upper panel cells were transfected with3xflagfaf1 plasmid at h after transfection the culture medium was replaced with serumfree medium and the cells were cultured at °cor °c for h lower panel the graph shows the result of densitometric analysis of faf1 immunoblotting in cm shown in the upper paneln g upper panel cells were transfected with 3xflagfaf1 plasmid at h after transfection the culture medium was replaced with serumfree medium containing bfa μgml for h lower panel the graph shows the result of densitometric analysis of faf1 immunoblotting incm shown in the upper panel n cell lysate cl and concentrated cm were analyzed by western blotting with the indicated antibodies alllanes were loaded with the same amount of total protein the data are expressed as the mean ± sd of three independent experimentsstatistical comparisons were performed using using anova followed by tukeys hsd post hoc analysis e and students ttest f and g p p p and ns not significanttheexosomeabolishedstructureslumen of the exosomes fig 2d the topology of exosomal faf1 was further examined using pk a nonspecificprotease to digest proteins outside of the exosomesexosomal faf1 but not cytosolic faf1 was protectedfrom pk treatment in the absence of triton x100 txfig 2e tx in contrast pk treatment with tx disintegratingtheexosomemediated protection of faf1 fig 2e txour data demonstrated the presence of faf1 in thelumen of exosomes furthermore the presence of faf1in theconfirmed with theimmunogoldlabeled faf1 under anvisualization ofelectron microscope as shown in fig 2f immunogoldlabeled cd63 was mainly present outside exosomeswhereas immunogoldlabeled faf1 was present in thelumen of exosomes collectively these results consistently showed that faf1 is located in the lumen ofexosomeslumen wasexosomalbecause certain exosomal cargo proteins are alsosecreted via the nonvesicular secretory pathway [] the possibility that faf1 is also secreted viaa nonvesicular route was investigated to this endthe cm was fractionated into exosomal pellet andnonexosomal supernatant fractions by ultracentrifugation faf1 from the cm was present in nonexosomal as well as exosomalfractions fig 2gimplying the presence of a soluble form of faf1 tofurther investigate this cm from faf1transfectedshsy5y cells was treated with antifaf1 antibodyone microgram of antifaf1 antibody almost eliminated faf1 from the cmindicating that faf1 ispredominantly secreted in a soluble form fig 2htaken together these results demonstrate that faf1is concurrently released as a bona fide cargo of exosomes and in a soluble form this new finding addsfaf1 to the list of proteins secreted by nonvesicularas well as exosomal routesrespectivelytransfection offaf1 positively regulates exosome numberthe exosomal cargos such as hsp20 hsp90 andstat3 increase exosome number [] here weexamined whether faf1 also participates in regulating exosome number the exosome markers hsc70alix and cd63 were increased by ± 009fold ± 013fold and ± 022foldinthe cm of faf1transfected shsy5y cells compared with control cells fig 3a additional file fig s6a next we further studied exosome numberchanges using sirnamediated depletion of parkina e3ubiquitin ligase of faf1 siparkin treatment elevated secretion as well as expression of faf1 inshsy5y cells moreoversirnaresistant parkin construct reverted the increased secretion and expression of faf1 by siparkin in shsy5y cells the expression levels of alix and cd63in the cm of shsy5y cells in which parkin hadbeen depleted were elevated by ± and ± 040fold respectively fig 3b collectivelythese data imply that faf1 positively controls exosome number for the direct quantification of exosome number nanop tracking analysis wasapplied the vesicle size distribution profile showinga diverse range of sizes showed that exosomes werepresent predominantly fig 3c the exosome numbers were normalized by cell number additionalfile fig s6b the exosomes of faf1transfectedcells were increased by 25fold compared to thoseof control cells hence these data robustly demonstrate that faf1 augments exosome numberinaddition gw4869 an exosome release inhibitor interfered with the ability of faf1 to increase exosome number while monensin an exosome releasepromoter enhanced this ability implying that faf1functions upstream of the exosome release processfig 3a [ ] 0cpark cell communication and signaling page of fig see legend on next page 0cpark cell communication and signaling page of see figure on previous pagefig faf1 is released to the extracellular space via both exosomal and nonvesicular pathways a shsy5y cells plated on mm dishes weretransfected with vc or 3xflagfaf1 plasmid at h after transfection the culture medium was replaced with exosomedepleted medium andthe cells were cultured for h then exosomes were isolated from the cm by ultracentrifugation cl and isolated exosomes exos wereanalyzed by western blotting with the indicated antibodies calr calregulin b purified exosomes were characterized using nanop trackinganalysis c representative tem images of exosomes are shown scale bar nm left or nm right d the purified exosomes were treatedwith na2co3 after centrifugation at ¨¯g the integral membrane proteins were pelleted memb and nonintegral and luminal proteinsremained in the supernatant sol these fractions were analyzed by western blotting with the indicated antibodies e the purified exosomeswere incubated with pk μgml in the absence or presence of triton x100 tx tx with tx tx without tx f immunogold labelingof purified exosomes with anticd63 antibodies left and antifaf1 antibodies from vctransfected middle or 3xflagfaf1transfected rightcells scale bar nm g cells were transfected with vc or 3xflagfaf1 plasmid at h after transfection the culture medium was replacedwith serumfree medium and the cells were cultured for h after the cm was isolated by ultracentrifugation the supernatant sup and pelletexo were analyzed by western blot with the indicated antibodies h after immunoprecipitation of faf1 from cm with antifaf1 monoclonalantibody the immunodepleted cm was concentrated and analyzed by western blotting with the indicated antibodies all lanes were loaded withthe same amount of total proteinsecreted faf1 is transmitted to adjacent cellswe wondered whether extracellular faf1 is internalizedby neighboring cells donor cells were transfected withgfp or gfpfaf1 plasmid for h subsequently thecm which contained gfp or gfpfaf1 was concentrated and nontransfected recipient cells were treatedwith the concentrated cm for h confocal microscopic images showed the presence of gfpfaf1 in thecytoplasm of recipient cells indicating that gfpfaf1had moved from donor cells to recipient cells in contrast gfp from the cm of donor cells was not detectedin recipient cells excluding the effect of tagging ontransmission fig 4a similarly donor cells were transfected with 3xflagfaf1 plasmid as described abovedonor cellderived faf1 also moved into recipient cellsas shown by western blotting fig 4b corroborating theimmunofluorescence results in fig 4a in these data consistently demonstrate that secreted faf1can be internalized by neighboring cellsto further validate the celltocell transfer of faf1 anin vitro coculture system in which donor cells expressinggfpfaf1 were incubated in upper transwellinsertswhile nontransfected recipient cells were incubated inlower compartments was used consistent with theabove data confocal microscopy of recipient cells revealed the presence of gfpfaf1 indicating that gfpfaf1 moved from cells in the upper transwell inserts tocells in the lower compartments fig 4c consequentlythese results show that extracellular faf1 was transferred to neighboring cells without celltocell contactwe investigated the type of extracellular faf1 thatmoves into recipient cells to this end donor cells weretreated with gw4869 an exosome release inhibitorafter which recipient cells were analyzed by confocal microscopy gw4869 failed to inhibit faf1 transmissionto recipient cells to eliminate vesiclefree faf1 cm ofdonor cells was immunodepleted wit | Colon_Cancer |
" mirnas regulate a multitude of cellular processes and their aberrant regulation is linked to humancancer however the role of mir4255p in lung cancer lca is still largely unclear here we explored the role ofmir4255p during lca tumorigenesismethods cell proliferation was evaluated by cell counting kit8 and colony formation assay western blot and realtime pcr were accordingly used to detect the relevant proteins mirna and gene expression luciferase reporterassays were used to illustrate the interaction between mir4255p and ptenresults we demonstrate that mir4255p is overexpressed in lca tissue and enhances the proliferative and colonyformation capacity of the lca cell lines a549 and ncih1299 through predictive binding assays pten wasidentified as a direct gene target and its exogenous expression inhibited the procancer effects of mir4255pthrough its ability to downregulate pten mir4255p activated the pi3kakt axis we conclude that mir4255p promotes lca tumorigenesis through ptenpi3kakt signalingkeywords mir4255p lung cancer pten pi3kakt signaling pathways lung cancer lca is a leading cause of cancer relatedmortality across the globe lca is prevalent in males and asymptomatic during early disease stages as manyas in every cases are at an advanced stage iii or ivwhen diagnosed and the 5year survival rates remainlow particularly for those with metastatic lca improved lca therapeutics is thus urgently requiredmicrornas mirnas regulate many cell processesincluding differentiation metabolism and tumorigenesis[] emerging evidence suggests that mirnas are keyplayers during lca tumorigenesis [] the aberrantexpression of mir4255p is linked to hepatocellular carcinoma hcc gastric cancer gca and colorectal correspondence kjhhev163comcardiothoracic surgery the fourth affiliated hospital zhejiang universityschool of medicine shangchen road no1 of yiwu zhejiang chinacancer crc [] here we report the upregulationof mir4255p in lca and highlight its contribution tolca development we further identify pten as a novelmir4255p target that is inhibited in lca to promoteptenpi3kakt signalingmethodspatient specimenslca samples n and adjacent healthy tissue at least cm from the resection margin were collected from thefourth affiliated hospital zhejiang university school ofmedicine the study was fully supported by the institutional review board of the fourth affiliated hospitalzhejiang university school of medicine no2015009all participants provided consent for sample analysisand anything about their identities will not be includedin the data the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0czhou bmc pulmonary medicine page of lca cell lines cell culture and cell transfectionhuman lung cancer cell lines a549 ncih1299 ncih460 hcc827 and normal human lung epithelial cellline beas2b were obtained from the cell bank of thechinese academy of sciences shanghai china a549 hcc827 cells were cultured in dmem plus fbsand penstrep ncih1299 ncih460 cells weregrown in rpmi1640 plus fbs at °c in co2beas2bs were cultured in clonetics¢ mediathe mir4255p mimics and negative control mirnanc were chemically synthesized by shanghai genepharma co ltd songjiang shanghai china lipofectamine invitrogen eugene or usa was usedfor transfection according to the manufacturers protocol pi3k activity was assayed as previously described pi3k inhibitor ly294002 was obtained from abcamto analyze the effects of mir4255p on pi3kakt indicated lca cells were cultured in the presence or absence ofthe workingconcentrations of ly294002 were μm for experiments with ly294002 treatmentsindicated lca cellswere pretreated with ly294002 for h prior to exposure to proteasome inhibitorsthe drugs for h at °ccell growth assayslca viability was assessed using cell counting kit8from shanghai haling biotechnology co ltd shanghai china as per the manufacturers protocols brieflyafter incubating the transfected cells for one full daythey were collected after trypsinization and seeded cellswell into 96well plates ten microliters ofcck8 solution were added per well and kept for h at °c the absorbance of the mixture was estimated in amicroplate readerinchercules usa at nmfrom biorad laboratoriescolony formation assaythe colony formation assays were performed as previous each group of treated cells per well wasseeded into cm culture dish and cultured for weeksfinally colonies were stained using crystal violet andthe number of cell colonies was countedqrtpcr analysistotal rna was isolated by trizol® reagent from invitrogen thermo fisher scientific inc and a nanodropnanodrop technologies thermo fisher scientific incwas used to estimate its quality and concentration theexpression of mir4255p was done by reverse transcription using the mirx¢ mirna firststrand synthesis kitfrom takara biotechnology co ltd dalian chinaand quantitative evaluation of the synthesized cdnawas done by quantitative pcr rtqpcr using themirx¢ mirna qrtpcr tb green® kit from takaraforwardbiotechnology as an endogenous control the small nuclear rna u6 normalized the expression of mir4255pthe δδcq system was used to evaluate all genes expressions and the primer sequences were shown as fol² ggggagttaglows mir4255pgattaggtc3² reverse ² tgcgtgtcgtggagtc3² u6 forward ²ctcgct tcggcagcaca² reverse ²aac gct tca cga att tgc gt3²pten forward ² tggattcgacttagacttgacc ² reverse ² aggatattgtgcaactctgcaa² gapdh forward ²catcaccatcttccaggagcg3² reverse tgaccttgcccacagcctt3²to getdual luciferase assaysthe design and synthesis of pten fragments containingbinding sites for wt wildtype and mut mutant onmir4255p was done by shanghai genepharma thesewere cloned into the target expression vector pmirglo dualluciferase from promega corporation wiusathe reporter plasmids wtpten andmutpten one night prior to transfection seeding ofcells confluence was done in plates with wells transfection of these cells was done with reporterplasmids harboring wt or mut in the presence ofmirnc or mir4255p mimic post h of transfectionluciferase activity of the cells was estimated as per instructions using the dualluciferase reporter assay system from promega corporation promega fitchburgwi usa the data normalization was done by the activity of renilla luciferasewbcell lysates ripa lysates were resolved on sds pageand transferred to pvdf membranes membraneswere blocked for h in milk plus tbstincubated with primary antibodies against pten dilution cat no ab170941 abcam pi3k dilution cat no ab32089 abcam pakt dilution cat no ab81283 abcam akt dilution cat no ab32505 abcam pakt dilution cat no ab81283 abcam actin dilution cat no ab8226 abcam at °c overnights andlabeled with hrpconjugated secondarys for h atroom temperature bands werevisualized usingchemiluminescent hrp substrate and analyzed usingimage lab tm softwarestatistical analysesdata analysis was performed using spss treatmentgroups were compared using a oneway anova pvalues were taken as significant experiments wereperformed on at least three occasions data representthe mean ± sd 0czhou bmc pulmonary medicine page of resultsmir4255p is upregulated in lcawe compared mir4255p levels in paired lcaand normal lung tissue samples by qrtpcr analysismir4255p was upregulatedspecimensfig 1a and expressed to high levels in a549 ncih1299 ncih460 and hcc827 cells compared tonormal human lung epithelial cellline beas2bfig 1b consistent with previous findings in othercancer types previous results indicated that mir4255p is upregulated in lcain lcamir4255p enhances proliferation and inhibits apoptosisin lca cellsto dissect the role of mir4255p in lca its expressionwas manipulated using mir4255p mimics figure 2acshows that mir4255p upregulation enhanced cell survival meanwhile enhanced cell colony formation abilityfig 2d and e taken together above results indicatedmir4255p is thus an oncogene in lca cellsmir4255p targets ptenfrom targetscan pten was identified as a predictedmir4255p target fig 3a in pten ²utr reporterassays mir4255p suppressed wt pten expressionfig 3bc but had little effect on mutated pten ²utr fragments fig 3bc the levels of pten werelower in cells transfected with mir4255p mimics fig3de mir4255p also negatively related pten mrnalevels in lca tissue p r2 fig 3f thesedata implicate pten as a cellular target of mir4255pmir4255p promotes lca via ptento define whether mir4255p regulate pten in lcawe firstly overexpression pten in lca fig 4a wbanalysis demonstrated that mir4255p reduces ptenlevels which could be recovered by exogenous expression fig 4b cell viability assays showed that mir4255p enhances lca proliferation which could be reversedby pten transfection fig 4cd suggesting mir4255pmediates its activities via pten this indicates that mir4255p targets pten to mediate its protumor effectsmir4255p activates ptenpi3kakt signalingit has been reported ptenpi3kakt signaling wasclosely related to cell proliferation and apoptosis [] next we explored the effect of mirna4255p onptenpi3kakt signaling as shown in fig 5aincomparison to nc groups pten was down regulated inresponse to mir4255p mimics whilst pi3k and paktlevels increased in addition nsclc cells were treatedwith the pi3kakt inhibitor ly294002 or ly294002 mir4255p mimics mimic fig 5b kinase activity assays further showed that pi3k activity in nsclc transfected with ly294002 was significantly lower than thattransfected with mimic control p and pi3k activity in nsclc cells transfected with both ly294002and mir4255p mimic was significantly higher than thattransfected with ly294002 p take together thisimplicates ptenpi3kakt signaling in the protumorigenic effects of mir4255pdiscussionthe poor prognosis of lca highlights the need for urgent therapeutic strategies mirnas are novel targets forfig mir4255p in highly expressed in lca a qrtpcr of mir4255p b qrtpcr of a549 ncih1299 ncih460 hcc827 and beas2b cellsdata mean ± sd p p p 0czhou bmc pulmonary medicine page of fig mir4255p promotes lca a mir4255p expression in a549 and ncih1299 cells b c cck8 assays in mir4255p mimic transfectedcells d e colonyforming assay in mir4255p mimic transfected cells the raw data of all colony formation experiments was listed insupplemental table data mean ± sd p p p fig mir4255p targets pten in lca a predicted binding of mir4255p and pten b c relative luciferase activity of ptenwt ptenmut inlca cells expressing mir4255p mimic d pten mrna levels are significantly lower after transfection with mir4255p mimic e pten expressionassessed by wb fulllength blotsgels are presented in supplementary figure f mir4255p and pten negatively correlate in lca tissue datamean ± sd p p p 0czhou bmc pulmonary medicine page of fig mir4255p promotes lca growth by targeting pten a qrtpcr of pten in indicated lca cell lines b wb analysis of pten expression fulllength blotsgels are presented in supplementary figure c d cck8 analysis of cell viability in the indicated cell lines with empty vector mir4255p mimic andor pten in a549ncih1299 cells data mean ± sd p p p fig effects of mir4255p on ptenpi3kakt a wb analysis of pten pi3k pakt and akt in lca cells fulllength blotsgels are presented insupplementary figure b the pi3k kinase activity was determined in nsclc cells transfected with ly294002 was significantly lower than thattransfected with mimic control and the pi3k kinase activity in nsclc transfected with both ly294002 and mir4255p mimic was significantlyhigher than that transfected with ly294002 data mean ± sd p p p 0czhou bmc pulmonary medicine page of cancer therapies and their dysregulation occurs in lcatissue [] duan showed that mir203 bindsto zeb2 to suppress emt yuan showed thatmir30a inhibits eya2 migration and invasion andli showed that mir1304 inhibits lca cell divisionthrough heme oxygenase1 the cellular roles ofmir4255p in lca are poorly understoodin thepresent work we further explore the underlying mechanisms of mir4255pinduced lca cell progressionin the present study we confirmed that mir4255p isoverexpressed in lca cell lines and tissues implicating arole in lca tumorigenesis upregulating mir4255plevels enhanced the cell survival and colony formationability of lca cells in vitro implicating it as a novel lcaoncogene in the mechanism using the algorithms targetscan website tools we identified pten as the potential target of mir4255p furthermore we performedluciferase reporter assays and the results showed thatmir4255p may directly target pten3utr the resultof qrtpcr and western blot also confirmed that overexpression of mir4255p could suppress the expressionlevel of pten all the above suggested that pten was apotentialtarget of mir4255p moreovermir4255p also negatively related pten mrna levelsin lca tissue rescue experiment indicated that exogenous pten expression inhibited the procancer effects ofmir4255p pten was downregulated in lca tissuepten is a tumor suppressor with wellcharacterizedphosphatase activity pi3kakt promotes cell cycleprogression inhibits apoptosis and is known to be overactive in a multitude of human cancers [ ] ptencan suppress pi3kakt signaling and thus displays anticancer effects upon assessment of the molecularmechanisms of mir4255p in lca cells its procancereffects were found to be mediated through manipulationof the ptenpi3kakt signaling axisfunctionalin we highlight mir4255p as an oncogenein lca that promotes an oncogene phenotype by inhibiting pten these findings enhance our knowledge of therole of mir4255p and reveal new therapeutic strategiesfor the diagnosis and treatment of lca angiogenesisand metastasis biological experiments will clarify thefunctions and roles of mir4255p in lcasupplementary informationsupplementary information accompanies this paper at httpsdoi101186s12890020012610additional file supplement figure s1 uncropped images of blotsand gels related to fig supplement figure s2 uncropped imagesof blots and gels related to fig supplement figure s3 uncroppedimages of blots and gels related to fig table s1 the raw data of allcolony formation experimentsabbreviationslca lung cancer pten gene of phosphate and tension homology deletedon chromsome ten pi3k phosphatidylinositol 3kinase nsclc nonsmallcell lung cancer gca gastric cancer crc colorectal cancerhcc hepatocellular carcinoma emt epithelialmesenchymal transitionacknowledgementsnot applicableauthors contributionsjsz zsy syc and qf did patient recruitment and assessment andperformed the experiments jhy and cjh were responsible for statisticalanalysis jsz zsy and syc interpreted the data and wrote the manuscriptall authors contributed to the subsequent drafts and approved the finalversionfundingthis research was funded by zhejiang provincial natural science foundationof chinaexploration program lq19h020010 core talent program ofdepartment of health of zhejiang province 2013rca018 zhejiangprovincial natural science foundation of chinageneral programly15ho2004 the funders had no role in the design of the study andcollection analysis and interpretation of data and in writing the manuscriptavailability of data and materialsthe datasets used andor analyzed during the current study are availablefrom the corresponding author on reasonable requestethics approval and consent to participateethics approval for this study was obtained from the fourth affiliatedhospital zhejiang university school of medicine no2015009 all patientsgave written informed consent for participation in the studyconsent for publicationnot applicablecompeting interestsall authors declare no conflicts of interestreceived march accepted august referenceschen w zheng r zuo t zeng h zhang s he j national cancer incidenceand mortality in china chin j cancer res aberle dr berg cd black wc the national lung screening trialoverview and study design radiology ni js zheng h huang zp microrna1973p acts as a prognosticmarker and inhibits cell invasion in hepatocellular carcinoma oncol lettzhang tj wang yx yang dq downregulation of mir186 correlateswith poor survival in de novo acute myeloid leukemia clin lab wang b teng y liu q microrna153 regulates nrf2 expression and isassociated with breast carcinogenesis clin lab lin h huang zp liu j mir4943p promotes pi3kakt pathwayhyperactivation and human hepatocellular carcinoma progression bytargeting pten sci rep yang j li j le y zhou c zhang s gong z pfklmir128 axis regulatesglycolysis by inhibiting akt phosphorylation and predicts poor survival inlung cancer am j cancer res wang r chen x xu t mir326 regulates cell proliferation andmigration in lung cancer by targeting phox2a and is regulated by hotairam j cancer res qin q wei f zhang j wang x li b mir134 inhibits nonsmall cell lungcancer growth by targeting the epidermal growth factor receptor j cellmol med hu h xu z li c mir145 and mir203 represses tgfbetainducedepithelialmesenchymal transition and invasion by inhibiting smad3 in nonsmall cell lung cancer cells lung cancer 0czhou bmc pulmonary medicine page of fang f song t zhang t cui y zhang g xiong q mir4255p promotesinvasion and metastasis of hepatocellular carcinoma cells through scaimediated dysregulation of multiple signaling pathways oncotarget cristobal i madozgurpide j rojo f garciafoncillas j potential therapeuticvalue of mir4255p in metastatic colorectal cancer j cell mol med zhang z wen m guo j clinical value of mir4255p detection and itsassociation with cell proliferation and apoptosis of gastric cancer pathol respract huang z xing s liu m deng w wang y huang z huang y huang x wuc guo x pan x jiang j feng f li t mir26a5p enhances cellsproliferation invasion and apoptosis resistance of fibroblastlikesynoviocytes in rheumatoid arthritis by regulating ptenpi3kakt pathwaybiosci rep meng j liu gj song jy chen l wang ah gao xx wang zj preliminaryresults indicate resveratrol affects proliferation and apoptosis of leukemiacells by regulating ptenpi3kakt pathway eur rev med pharmacol sciliu hy zhang yy zhu bl feng fz yan h zhang hy zhou b mir21regulates the proliferation and apoptosis of ovarian cancer cells throughptenpi3kakt eur rev med pharmacol sci yu g wang c song x liu s zhang y fan l yang y huang y song jformaldehyde induces the apoptosis of bmcs of balbc mice via the ptenpi3kakt signal transduction pathway mol med rep sun xh wang x zhang y hui j exosomes of bonemarrow stromal cellsinhibit cardiomyocyte apoptosis under ischemic and hypoxic conditions viamir4865p targeting the ptenpi3kakt signaling pathway thromb res mou x liu s mir485 inhibits metastasis and emt of lung adenocarcinomaby targeting flot2 biochem biophys res commun su tj ku wh chen hy oncogenic mir137 contributes to cisplatinresistance via repressing casp3 in lung adenocarcinoma am j cancer resliu y wang f xu p mir590 accelerates lung adenocarcinoma migrationand invasion through directly suppressing functional target olfm4 biomedpharmacother yang l luo p song q fei x dnmt1mir200agolm1 signaling pathwayregulates lung adenocarcinoma cells proliferation biomed pharmacother duan x fu z gao l direct interaction between mir203 and zeb2suppresses epithelialmesenchymal transition signaling and reduces lungadenocarcinoma chemoresistance acta biochim biophys sin shanghai yuan y zheng s li q overexpression of mir30a in lungadenocarcinoma a549 cell line inhibits migration and invasion via targetingeya2 acta biochim biophys sin shanghai li cg pu mf li cz microrna1304 suppresses human nonsmall celllung cancer cell growth in vitro by targeting heme oxygenase1 actapharmacol sin li dm sun h ptenmmac1tep1 suppresses the tumorigenicity andinduces g1 cell cycle arrest in human glioblastoma cells proc natl acad sciu s a bellacosa a chan to ahmed nn akt activation by growth factors is amultiplestep process the role of the ph domain oncogene li jw wang xy zhang x gao l wang lf yin xh epicatechin protectsagainst myocardial ischemiainduced cardiac injury via activation of theptenpi3kakt pathway mol med rep qin y huo z song x chen x tian x wang x mir106a regulates cellproliferation and apoptosis of colon cancer cells through targeting theptenpi3kakt signaling pathway oncol lett publishers notespringer nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations 0c" | Colon_Cancer |
" accurate detection of patients with minimal residual disease mrd after surgery for stage ii coloncancer cc remains an urgent unmet clinical need to improve selection of patients who might benefit formadjuvant chemotherapy act presence of circulating tumor dna ctdna is indicative for mrd and has highpredictive value for recurrent disease the medocccreate trial investigates how many stage ii cc patients withdetectable ctdna after surgery will accept act and whether act reduces the risk of recurrence in these patientsmethodsdesign medocccreate follows the trial within cohorts twics design patients with colorectal cancercrc are included in the prospective dutch colorectal cancer cohort plcrc and give informed consent forcollection of clinical data tissue and blood samples and consent for future randomization medocccreate is asubcohort within plcrc consisting of stage ii cc patients without indication for act according to currentguidelines who are randomized into an experimental and a control armin the experimental arm postsurgery blood samples and tissue are analyzed for tissueinformed detection ofplasma ctdna using the pgdx elio¢ platform patients with detectable ctdna will be offered act consisting of cycles of capecitabine plus oxaliplatin while patients without detectable ctdna and patients in the control groupwill standard followup according to guidelinethe primary endpoint is the proportion of patients receiving act when ctdna is detectable after resection themain secondary outcome is 2year recurrence rate rr but also includes 5year rr disease free survival overallsurvival time to recurrence quality of life and costeffectiveness data will be analyzed by intention to treatcontinued on next page correspondence mkoopman6umcutrechtnlsj schraa and kl van rooijen are shared first authorrja fijneman gr vink and m koopman are shared last author1department of medical oncology university medical center utrechtutrecht university heidelberglaan cx utrecht the netherlandsfull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cschraa bmc cancer page of continued from previous pagediscussion the medocccreate trial will provide insight into the willingness of stage ii cc patients to be treatedwith act guided by ctdna biomarker testing and whether act will prevent recurrences in a highrisk populationuse of the twics design provides the opportunity to randomize patients before ctdna measurement avoidingethical dilemmas of ctdna status disclosure in the control grouptrial registration netherlands trial register nl6281ntr6455 registered may wwwtrialregisternltrial6281keywords colon cancer circulating tumor dna ctdna adjuvant chemotherapy twics in patients with stage ii colon cancer cc the recurrence rate rr after surgery is approximately disease management after surgical resection in stageii cc is still under debate because the overall survivalos benefit of adjuvant chemotherapy act in thisgroup of patients varies between and only [ ]moreover offering act in a lowrisk population induces an important amount of overtreatment with unnecessary but sometimes severe toxicity and costsseveral prognostic characteristics of stage ii cc havebeen identified to provide better selection of patientsthat might benefit from act patients with presence ofat least one of the following characteristics are classifiedas being at high risk of disease recurrence poorly differentiated histology pt4 lesions inadequately less than sampled lymph nodes lymphovascular or perineuralinvasion or tumor presentation with perforation or obstruction in contrast patients with a deficient mismatch repair dmmr status in stage ii cc have a low risk ofrecurrence and act is not considered beneficial irrespective of the presence of other risk factors [ ]other known prognostic factors in cc like gene expression profiles or braf v600e and ras mutations have been investigated but do not adequatelyidentify the patients that will benefit from act []despite the definition of high and low risk subgroupsof stage ii cc patients retrospective analyses demonstrated that improved survival after administration ofact was not observed in high risk patients or exclusivelyin patients with a pt4 tumor [] therefore in thenetherlands act is currently only recommended in stageii cc patients with a pt4 tumor without dmmrunfortunately also pt4 is not an absolute predictorfor disease recurrence in stage ii patients in a retrospective analysis of stage ii cc patients with pt4tumors the 3year diseasespecific survival rate aftersurgery was in patients who received act and in patients who did not receive act whichmeans that of these patients are exposed to actunnecessarily considering nonpt4 stage ii patients a population registry analysis of patientsshowed that in this group of patients sufferedfrom recurrences these data demonstrate thatusing pt4 as a prognostic factor results in significantunder and overtreatmentminimal residual disease mrd is defined as the presence of tumor cells in the blood bone marrow or lymphnodes not detected by conventional staging procedures patients who have mrd after surgery are not completely cured and therefore at high risk of developingdisease recurrence development of a highly specific andsensitive biomarker testindicative for mrd wouldallow identification of the subset of patients likely to experience recurrence of disease thereby improving the selection of patients who may benefitfrom adjuvanttreatment in adjuvant trials this would solve problemsof high numbers needed for inclusion and dilution of effectiveness of adjuvant treatment by inclusion of manyalready cured participants cellfree circulating tumor dna ctdna has a strongpotential for being this sensitive and yet specific biomarker ctdna consists of smallfragments usually bp of tumorderived dna containing tumorspecific mutations which can be detected in liquid biopsies such as blood samples [] because of the shorthalflife of ctdna estimates ranging from to min the presence of ctdna in blood samples taken several days after surgery presumably reflects a state ofmrd [] patients with mrd have the highest riskfor disease recurrencerecently the presence of ctdna after tumor resection demonstrated a very strong prognostic value fordisease recurrence in stage ii cc with a 2years rrof versus in patients with and without detectable ctdna after surgery respectively in thisstudy the univariate prognostic value of ctdna was muchhigher than that of pt4 status hazard ratio of versus respectively there are several ongoing trials that usectdna in prognostication nct03637686 nct03737539nct03416478 nct03312374 nct02842203 nct0361 and treatment nct03748680 actrn12615000381 nrggi of nonmetastatic cc but to date thereare no results available of randomized controlled trialsrcts that use ctdna for selection of act treatment 0cschraa bmc cancer page of the accumulating evidence for the strong prognosticvalue of ctdna raises an important ethical dilemma forrandomization of patients when designing a conventional rct in which patients with detectable ctdna arerandomized into act treatment or standard of carefollowup while disclosing ctdna status to the controlgroup indeed the knowledge of having a very highchance of disease recurrence will be a big burden for patients with detectable ctdna in the control group andtheir caregivers as they are not being offered any additional therapy this warrants an innovative trial designlike the trialdifferent from the conventional rctwithin cohorts twics design [] the twics design enables an experimental group in which ctdna status is disclosed and a control group that is unaware oftheir ctdna statusthe medocccreate trial is designed as a multicenter twics study with two parallel groups in whichwe will investigate whether stage ii cc patients with detectable ctdna after resection are willing to receiveact and whether act reduces the rr in these ctdnapositive patientsmethodsdesignaimthis study investigates the willingness of patients to receive act after detection of ctdna postsurgery andthe effect of ctdnaguided act on the rr in stage iicc patientsstudy designthe medocccreate trial follows the twics designand is performed within the prospective dutch colorectal cancer cohort plcrc wwwplcrcnl plcrc is set up by the dutch colorectal cancer groupdccg and collects clinical data and patient reportedoutcome measures proms at baseline and at multipletime points during followup fig at enrollment patients give informed consent for use of their clinical dataand optionally for receiving quality of life questionnairesfig schematic presentation of medocccreate using the trial within cohort twics design a plcrc is a nationwide cohort study in thenetherlands with inclusion of crc patients all stages by optional informed consent regarding collection of biomaterials and futurerandomization observational as well as interventional trials can be performed within the cohort b nonmetastatic crc patients are included inmedocc when the patient signs informed consent for plcrc including additional blood sampling blood samples are withdrawn beforeresection days after resection and every months during the first years of followup c eligible stage ii colon cancer patients arerandomized following the twics design in the experimental group informed consent is being asked for immediate ctdna analysis of theblood sample obtained after resection if ctdna is detectable patients are offered adjuvant chemotherapy the control group is not informedabout medocccreate and will receive standard of care 0cschraa bmc cancer page of collection of biomaterials for research additional sequential blood sampling and for being approached forfuture studies conducted within the infrastructure ofthe cohort either in accordance with the twics design or notpatients with pt4 tumors will be offered act therefore we will include eligible patients with pt4 tumors without a recommendation for act according totheir treating physician and use pt4 status as a stratification factorpatient selection and recruitmentpatients will be recruited in both academic and nonacademic hospitals in the netherlands that are participating in plcrc nonmetastatic colorectal cancercrc patients that give informed consent for plcrcincluding consent for additional blood sampling at enrollment will be included in the observational plcrcsubstudy medocc molecular early detection of coloncancer before surgery the participants are eligible forthe current medocccreate trial if they meet the following criteria after surgery histopathological confirmed and radically resected stage ii cc age ¥ years informed consent for plcrc and medoccincluding consent for randomization in future trials anduse of tissue physical condition allows treatmentwith combination chemotherapy consisting of a fluoropyrimidine and oxaliplatin and no indication foract according to the treating physician andor multidisciplinary board patients who are pregnant have hadanother malignancy in the previous years except forcarcinoma in situ or patients with contraindications forfluoropyrimidines andor oxaliplatin will be excludedcurrently the dutch guidelines recommend act forpatients with pt4 tumors however there is large ageand hospital dependent variation in administration ofact in this group and in clinical practice not all stage iiblood sample collectionblood samples are collected before and days aftersurgery for all patients included in the medocc clinicalstudy predominantly comprising stage i ii and iii ccpatients table blood samples two tubes of mlper timepoint are collected in cell free dna streckblood collection tubes for various research purposesamong which the medocccreate trialrandomizationabout week after surgery when the histopathologicalreport is finished medocc patients who are eligiblefor medocccreate will be randomized to theintervention or control arm using slim an onlineplatform to manage patientinclusion including arandomizationgeneratedcomputerservice therandomization schedule is stratified by tstage and usespermuted blocks of random sizes allocation concealment will be ensured as the service will not release therandomization code only patients randomized to theintervention arm will be informed about medocccreate according to the twics design experimental armafter randomization only patients randomized to theexperimental arm will be asked separate informedtable standard protocol items for intervention trials spirit schedule of enrollment interventions and repeated measurementsact adjuvant chemotherapy ctdna circulating tumor dna qol quality of life intervention group only intervention grouponly if ctdna is positive 0cschraa bmc cancer page of consent for the immediate analysis of ctdna status ofthe postsurgery sample a small proportion of patientsestimated approximately will have detectablectdna in their blood these patients will be offeredact patients decide whether they accept or refuse thistreatment patients without detectable ctdna will receive routine standard of careact will consist of months of capecitabine and oxaliplatin capox or months of fluorouracil leucovorinand oxaliplatin folfox treatment starts preferablywithin weeks and not beyond weeks after surgeryduring and after completing act routine followupwill consist of regular visits at the surgical outpatient department blood withdrawals for analysis of carcinoembryonic antigen cea and imaging standard ultrasoundofthe liver according to current guidelines in thenetherlands no additional imaging will be performed toprevent detection biascontrol armin the control arm patients will not be informed aboutthe medocccreate trial and receive routine followup care consisting of cea tests every months for thefirst years and abdominal ultrasound or ct every months in the first year and once a year afterwards oneyear after surgery a colonoscopy is performed postsurgery blood samples will not be tested for ctdna immediately but will be analyzed batchwise after severalmonths without result disclosure to patients and theirtreating physiciansfollowupblood samples will be collected at 6monthly intervalsfor the first years after surgery for both patients in theexperimental arm and the control arm conform themedocc study protocol these samples will not be analyzed for ctdna immediately and results will not bedisclosed to patients and treating physicianstumor tissueinformed ctdna analysisafter surgery the local pathologist will send a formalinfixed paraffinembedded ffpetissue block to thecentral laboratory where dna will be isolated for further analysisthe postsurgery blood sample is drawn between and days after surgery the sample is not withdrawnbefore day to reduce the risk of falsenegative ctdnatests due to the relatively large amount of cell free dnacfdna released due to cell damage after surgery theblood is taken no later than days after surgery to beable to start chemotherapy within weeks after surgerysamples are kept at room temperature and sent by regular mail to the central laboratory within days wherectdna will be isolated for further analysistumor tissue dna will be analyzed by targeted nextgeneration sequencing of a panel of more than genes using the pgdx elio¢ tissue complete assay frompersonal genome diagnostics pgdx baltimore mdusa plasma ctdna will be analyzed by targeted nextgeneration sequencing of a panel of more than genesusing the pgdx elio¢ plasma resolve assay from pgdxbaltimore md usa both panels include the mostcommonly mutated genes in cc including apc tp53kras and braf tumor tissue dna mutations are usedas input information for plasma ctdna mutation callingthereby increasing both sensitivity and specificity of thectdna testprimary endpointthe primary endpoint is the proportion of patients starting with act after detection of ctdna in the postsurgery samplesecondary endpointsthe most important secondary endpoint is 2year rr inpatients with detectable ctdna in their blood expressedas the proportion of patients that experience a recurrence within years after surgery detection of recurrences in months after surgery will occur by standardfollowup investigations including monthly bloodsampling of tumor marker cea and monthly imagingwith ultrasound liver or ct abdomen and when indicated by symptoms radiological andor histopathological evidence is used to confirm the recurrence thedate of the said investigation is considered the date ofrecurrencedata about followup recurrences and survival areroutinely collected within plcrc using the netherlandscancer registry ncr managed by the netherlandscomprehensive cancer anisation iknl to provideinsight in the characteristics and magnitude of cancer inthe netherlands other secondary endpoints include 2year rr in aperprotocol analysis 5year rr intentiontotreatand perprotocol analysis time to recurrence ttr and 5year disease free survival dfs rate and7year diseaserelated os rate and 5year rr inpatients with undetectable ctdna after surgery quality of life qol and costeffectiveness of the ctdnaguided strategytimetoevent outcomesos rate is expressed as proportion of patients that arealive and years after surgery dfs rate is expressed asproportion of patients that did not experience disease recurrence a second primary cc or death within and years after surgery ttr is expressed as time monthsbetween surgery and detection of disease recurrence 0cschraa bmc cancer page of patients will be censored at the last date of followup if adate of death is not recorded and at the date of death ifthe cause of death is not due to ccquality of lifeqol is measured within the cohort at regular intervalsin patients who gave consent to send questionnaires nationally and internationally validated questionnaires areused among which the european anisation for research and treatment of cancer quality of life questionnaire core and the colorectal cancer moduleeortcqlqc30 and cr29 the work ability indexwai the euro quality of life5 dimensions eq5dthe multidimensional fatigue inventory20 mfi20and the hospital anxiety and depression score hadscosteffectiveness of the ctdnaguided treatmentthe costeffectiveness analysis will be carried out from asocietal perspectiveincluding both direct health carecosts as well as indirect costs from productivity loss thehealth outcome measure in the costeffectiveness analysis will be the total quality adjusted life years qalyper group for analysis offactors related to qalysquestionnaires are used provided within plcrcsample size considerationsthe primary endpoint is the proportion of ctdna positive patients starting with act however 2year rr inthe ctdna positive patients after surgery is an importantsecondary endpoint and the power calculation is performed for this secondary endpoint we estimate thatsimilar to effectiveness in stage iii cc patients act inctdnabased highrisk stage ii cc patients will lead toa absolute reduction of recurrences within yearsafter surgery in the observational trial of patientswith detectable ctdna experienced disease recurrencewithin years after resection with a power of and an alpha of patientswith detectable ctdna need to be included in botharms assuming a prevalence of ctdna after surgery of and adjustment for loss to followup and rejection ofadjuvant therapy in the intervention arm of a totalsample size of patients is calculated in eacharm we expect few patients with detectable ctdna inthe intervention group to refuse act because patientsare selected upfront for being in a physical condition toreceive act and the established prognostic value of detectable ctdna is highwe assume that crossover from the control arm tothe intervention arm will not occur because only eligiblepatients randomly selected in the cohort and allocatedto the intervention arm will be informed about the trialand have the opportunity for immediate analysis ofctdna patients in the control group will not be informed about the trial or their ctdna statuswe assume that of patients in the interventionarm with detectable ctdna will be treated with actthe proportion of patients starting with chemotherapythe primary endpoint can in that instance be determined with a margin of error width of the confidence interval of we expect to complete recruitment of patients within years with more than participating dutchhospitalsdata analysisdata will be analyzed according to the intentiontotreatprinciple for the primary endpoint and the secondaryendpoint of 2year rr in patients with detectable ctdnaafter surgery in this analysis we expect to compare patients with detectable ctdna who received act inthe intervention arm with patients with detectablectdna in the control arm ie based on ctdna analysisperformed retrospectively at least months after surgery and not disclosed to patients and treating physicians the proportion of patients that experience arecurrence in both arms will be compared by means of achisquare test in addition for other secondary endpoints and exploratory analyses we will analyze timetoevent outcomes in patients in both arms with detectablectdna after surgery differences in timetoevent outcomes will be analyzed by standard survival methodseg kaplanmeier curves compared by logrank testscoxs proportional hazards models will be used for multivariable analysiscomparison of qol of the ctdna positive patients inboth study arms will be done using repeated measurements methods and including act as factor qol willalso be analyzed for the whole population in both armsof the study treatment differences at each qol assessment time point will be compared by means of thewilcoxon rank sum testa lifetime horizon will be applied forthe costeffectiveness analysis parametric survival functions willbe used to extrapolate dfs and os curves beyond yearsresponsibilitiesprotocol modifications will be submitted as amendmentto the medical ethical committee by the study coordinator the local principle investigator of each participating hospital is responsible for patient inclusion logisticsof biomaterials to the centrallaboratory and patientfollowup to ensure quality of data study integrity andcompliance with the protocol and the various applicableregulations and guidelines a data monitor of the iknlhas been appointed to conductto thesite visits 0cschraa bmc cancer page of participating centers and randomly check patient datathe study coordinator together with the principle investigator will have access to the final dataset and is responsible for publishing study results the results will besubmitted to a peerreviewed journaldiscussionmedocccreate is the first clinical trial using thetwics design to investigate ctdnaguided strategies instage ii cc taking an important step towards clinicalimplementation of ctdna in cancer diagnostics andcarewithctdnadetectablea few other trials with the aim to reduce recurrencesin cc by use of a ctdnaguided approach are in preparation or recently started the improveit trial adanish study started in october uses a classicalrct in stage i and ii crc patients randomizing between months of act or intensified followup for patientspostsurgerynct03748680 four hundred fifty stage ii crc patients are being included in the australian dynamicstudy and randomized to be treated according to thectdna result with to months of act or accordingto standard of care actrn12615000381583 thecobra study in the united states and canada has asimilar rct approach nrggi also several trialsin stage iii crc patients started recently dynamiciii actrn12617001566325 in the near future thesestudies will provide deeper understanding and lead toimplementation of ctdnaguided strategies in clinicalpracticein the current era of rapidly emerging new diagnosticand treatment strategies the classical rct is challengedbecause of inefficient and therefore timeconsuming recruitment of eligible patients main reasons for patientsto refrain from participation in rcts are preference forone ofthe treatment arms anxiety or aversion torandomization and difficulties understanding the concept of an rct resulting in a delay of availability ofpotential beneficial treatments modern trial designsare being adopted to avoid thistimeconsuming and costly way of conducting trials with highrates of unfinished studies therefore the medocccreate trial uses the modern twics design the twicsdesign has shown to have a positive impact on trialefficiency also by enrolling higher proportions of eligible patients generalizability to daily clinical practiceimproves inefficientthisseveralstudy design hasstrengths firstmedocccreate is nested within the large nationwide plcrc cohort study with currently almost included crc patients the infrastructure of this cohortin which clinical data and biomaterials are collected afterbroad informed consent of participating patients allowsinnovative and efficientcomprehensiveresearch incrc using this infrastructure the study can be quicklyimplemented in many participating hospitals savingcosts and complicated logistics several studies accordingto the twics design are performed within this or comparable cohorts therefore experience with this trialdesign has been gained and this will contribute to execution of the medocccreate study [ ]secondly a difficult ethical dilemma in an rct analyzing ctdna presence postsurgery is avoided by thetwics design with the current knowledge about thestrong association with recurrent disease disclosingctdna status to all participants would be a great burdenfor patients with detectable ctdna and their treatingphysicians in the control group because of disappointment bias in the control group we would expect highdropout and contamination due to crossover when aclassical rct design would be applied making accrualand interpretation of results unfeasible in thistwics study all participants already have blood withdrawn after surgery for research purposes and only theeligible patients allocated to the intervention arm willhave the opportunity to obtain a ctdna test result andact if ctdna is detected patients in the control armtreated according to current guidelines will not be informed about randomization and their blood sampleswill be analyzed at a later point in time beyond the window of act treatmentthis study has also potentiallimitations and challenges the twics design is potentially susceptible tolow statistical power and internal validity biases levelsof participants eligibility and consent should be substantial to achieve valid and reliable results and measurements taken in the control group should be sufficient foradequate comparisons to be made therefore thetwics design is not appropriate for every experimentalintervention in case of the medocccreate studywe argue that eligibility and also consent will be substantial because of the high incidence of cc the large cohortwith high inclusion rates and the assumption that eligible patients in the intervention group are willing toaccept act because of the very strong association of thepresence of ctdna with recurrent diseaseanother limitation is the small sample size for primaryoutcome analysis eventually only patients in botharms of the trial are expected to have detectable ctdnaafter surgery based on previous data relapses areexpected within years and with a high event rate smallnumbers are sufficient capecitabinewe recommend a 6month duration of act consistand oxaliplatin capox oring offluorouracilleucovorin and oxaliplatin folfox forpatients with detectable ctdna after surgery the firstadjuvant cc trials investigating the combination of a 0cschraa bmc cancer page of fluoropyrimidine and oxaliplatin reported results for month duration of act in the idea trialfound a large reduction in toxicity for months treatment compared to months treatment although thistrial could not confirm noninferiority for monthstreatment for all patients treated with capox or folfox in stage iii crc the small difference limits clinicalrelevance besidesit did show noninferiority of theshorter regimen in patients treated with capox consequently dutch guidelines recommend months of actfor cc since however among patients withhighest risk of recurrence t4 n2 or both superiorityof 6month duration of therapy was found additionalideafrance resultsthe esmocongress showed the worst prognosis for ctdnapositive patients who only received months of act therefore in this study we recommend 6monthsact for patients with a very high risk of disease recurrence due to the presence of ctdna after surgerypresentedatliquid biopsy ctdna detection has become a promising technology with multiple putative clinical applications including its potential use as a biomarker for earlydiagnosis prognosis prediction and monitoring of treatment response driven by the excitement of its possibilities the field of technology of ctdna detection andanalysis is rapidly evolving yet the clinical utility ofctdna testing still needs to be proven when to applywhat technology to address which unmet clinical need isa key question that remains to be addressed applying ctdna detection as a biomarker for mrd isa challenging task biologically only a very low amountof ctdna is present in postsurgery patients with mrdstochastically by looking at mutations in a panel ofgenes chances increase that in a given blood sample atleast in one of the genes a mutation can be reliably detected test sensitivity can be further increased by making use of dna mutation information from tumortissue because the stringency in the calling of plasmactdna mutations can be reduced once you know whatmutations to look for tissueinformed ctdna analysisalso increases the ctdna test specificity recent observations showed that ctdna mutation detection can beconfounded by mutations that are present in clonalhematopoiesisincluding mutations in genes that arecommonly affected in cc such as tp53 these confounding mutations can be filtered by applying tissueinformed ctdna analyses as suchtechnically themedocccreate trial makes use of a ctdna test thatis wellsuited for mrd detection clinically howeverthe medocccreate trial needs to resolvewhether a positive ctdna test also allows to select forpatients who truly benefit from act treatment a requirement for clinical implementation to further support clinical implementation of ctdna analyses in thenetherlands the dutch coin initiative aims to providea validation framework for clinicalimplementation ofctdna analyses in the netherlands zonmw projectnumber in conclusion the medocccreate study is thefirst study using the modern and innovative twics design to study ctdnaguided administration of act instage ii cc patients the study aims to answer the important clinical question whether ctdna has prognosticas well as predictive value if this study demonstrates asignificant and substantial difference in disease recurrence in the intervention group compared to the controlgroup ctdna analysis and ctdnaguided treatmentshould be implemented into clinical practice to improvethe prognosis of stage ii cc patientsabbreviationsact adjuvant chemotherapy cc colon cancer cea carcinoembryonicantigen crc colorectal cancer ctdna circulating tumor dnadfs disease free survival dmmr deficient mismatch repair eortcqlqc30 and cr29 european anisation for research and treatment ofcancer quality of life questionnaire c30 and colorectal cancer module eq5d euro quality of life5 dimensions ffpe formalinfixed para | Colon_Cancer |
agar oligosaccharides aos produced by agarase has a good application prospect agar amarine polysaccharide producing from red algae is widely used in food industry pharmaceuticalindustry and biological engineering wonjae as the main component of agaragarose is composed of 36anhydrolgalactose lahg and dgalactose dgal bondededited bybaolei jiachungang university south koreareviewed bychangro leemyongji university south koreago furusawauniversiti sains malaysia usmmalaysiacorrespondencechunsheng lilichunshengscsfriaccnxianqing yangyangxqscsfriaccnyxqgd163comspecialty sectionthis was submitted toevolutionary and genomicmicrobiologya section of the frontiers in microbiologyreceived may accepted july published august citationli c li c li l yang x chen sqi b and zhao y comparativegenomic and secretomic analysisprovide insights into unique agardegradation function of marinebacterium vibrio ï¬uvialis a8 throughhorizontal gene transferfront microbiol 103389fmicb202001934frontiers in microbiology wwwfrontiersinaugust volume 0cli agarase production by vibrio ï¬uvialisalternately by α13 and 14 linkages araki agaroseoligosaccharidesincluding aos and neoagaroligosacchridesnaos are agarose degradation products which exhibit variousphysiological functions such as antioxidant kang antiammatory activity enoki whitening eï¬ectkim immune modulation mussatto and mancilha and antitumor activity bhattarai and kashyap there are two methods to produce agaroseoligosaccharidesincluding acid hydrolysis and agarase biodegradation comparedwith acid hydrolysis agarase biodegradation can be easy tocontrol agaroseoligosaccharide types and produce the productswith ï¬xed polymerization degrees in an environmentally friendlyway without damage of structure xu therefore it isvery important to develop agarases in the industrial productionof agaroseoligosaccharidesmost agarases are produced by marine gramnegative bacteriaincluding agarivorans zhang pseudoalteromonaswang li microbulbifer ma zhu alteromonas seo etc vibrio isalso an important genus of marine gramnegative bacteria toproduce agarases and various strains have been reported abouttheir agarase production characteristics such as vibrio sp jt0107jam vibrio sp cn41 liao vibrio spzc1 su vibrio sp ejy3 yu vibriosp po303 dong and vibrio sp v134 zhang andsun the agarases produced by these strains have been wellcharacterized and classiï¬ed however the genomic informationand speciï¬c taxonomic status of these strains in vibrio remainpoorly understood here we isolated and characterized a marinebacterium vibrio sp a8 with high agar degradation ability froma red algae in the south china sea this strain was identiï¬edas vibrio ï¬uvialis and showed similarity with v ï¬uvialisnbrc103150 by 16s rrna gene sequencing to date only threegenome sequencing projects of v ï¬uvialis have been completedaccording to the ncbi genome database however interestinglyno agarase genes have been found in these genomes based on thegene sequences and annotation data therefore it is significantlyuseful and interesting to study whether vibrio sp a8 belongs tov ï¬uvialis whether this strain can produce agarase and why thisstrain possesses unique agar degradation abilityin this study the complete genome of novel agardegradingbacterium vibrio sp a8 was sequenced using a combinationof nextgeneration sequencing and thirdgeneration sequencingtechnology the microbialfunctions were annotated andanalyzed against the gene databases to investigate the geneticelements related to agar degradation comparative genomicanalysis between vibrio sp a8 and similar strains in vibriowas performed to ascertain the taxonomy position ofthisstrain and to explain whether these bacteria had a commonability of agar degradation secretomic analysis using nanolcmsms was performed to study the molecular propertiesof agarase in the secretome of this strain followed by theanalysis of the biochemical properties of puriï¬ed agarase thisstudy provides information about novel agarase in vibrio andï¬lls in the gap on the genomic information and taxonomyposition of the agardegrading strain in this genus which isvaluable for better understanding the agar degradation pathwayin the bioï¬lm of red algae surface and providing insightsinto biotechnological applications of agarase for production ofagaroseoligosaccharidesmaterials and methodsisolation and biochemical identiï¬cationof strain a8marine bacterium strain vibrio sp a8 with eï¬cient agardegradation ability was isolated from the rotten red algaegracilaria lemaneiformis in the south china sea firstly grotten algae was added into ml sterilized saline water aftersuï¬cient vortex oscillation the solution was spread on lb agerplates peptone yeast extract powder nacl and agar ph using gradient dilution after cultivation at ¦cthe strains with concave around their colonies were selected andinoculated on fresh lb ager plates for h at ¦c the plateswere then stained with lugols iodine solution i2 and ki colonies with a clear zone formed by agar degradation weredetermined as agardegrading strains the strain with the highestagar degradation ability was maintained on lb agar slant peptone yeast extract powder nacl and agar ph and was used for further studybiochemical identiï¬cation of vibrio sp a8 was carried outusing the vitek gn test kit biomrieux according to themanufacturers instructionsagaraseproducing conditionoptimizationvibrio sp a8 was ï¬rst precultured by transferring a loopfulof slant culture to liquid lb medium peptone yeastextract powder and nacl ph and incubated at ¦cand rmin for h thereafter the strain was transferredto ml liquid agaraseproducing medium yeast extractpowder and agar in artiï¬cial seawater ph in mlerlenmeyer ï¬askunless otherwise stated in the agaraseproducing experimentsthe inoculation size of the strain after preculture was theph value of the medium was the culture temperature was¦c and the culture time was h diï¬erent carbon sources galactose glucose sucrose agar agar galactose agar glucose or sucrose agar were ï¬rst added in the agaraseproducingmedium and then the eï¬ects of diï¬erent concentrations of theoptimum carbon source on the agarase activity in the mediumwas studied the optimum nitrogen source for the agaraseproduction was selected by changing the yeast extract powderin the medium into other nitrogen sources peptone nh4no3 nh42so4 nh4cl or nh4cl yeast extract powder the eï¬ects of nacl concentrations ph value temperature ¦c and culture time h on the agarase activity in the medium were studied todetermine the optimum culture conditions of vibrio sp a8the agarase activity was determined by measuring the releaseof the reducing sugars using the 35dinitrosalicylic acid dnsfrontiers in microbiology wwwfrontiersinaugust volume 0cli agarase production by vibrio ï¬uvialisfigure effect of a carbon source with yeast extract powder b agar concentration with yeast extract powder c galactose concentrationwith yeast extract powder and agar d nitrogen source with agar and galactose e yeast extract powder concentration with agarand galactose f nacl concentration with agar galactose and yeast extract powder g ph with agar galactose yeast extract powder and nacl h temperature with agar galactose yeast extract powder and nacl at ph and i culture timewith agar galactose yeast extract powder and nacl at ¦c and ph on the agarase activity in artiï¬cial seawater cultivation with straina8 bars labeled with the same letter are not statistically different p tested by oneway anova and multiple comparison tukey test pearson correlationanalysis was tested between agarase activity and od600 at different culture timemethod miller brieï¬y the medium µl aftercultivation was incubated with melted agar ml at ¦cfor min the solution was then placed in boiling water for min to stop the enzymatic reaction subsequently the dnsreagent ml was added to the reaction solution which washeated in a boiling water bath for min when dropped toroom temperature the mixture was diluted with distilled waterto ml and the released reducing sugars were quantiï¬edspectrophotometrically at nm one unit of agarase activitywas deï¬ned as the amount of enzymes that released µg ofreducing sugars per minute under these experimental conditionswhole genome sequencing assemblyand annotationthe genomic dna from vibrio sp a8 was extracted and puriï¬edusing a wizard genomic dna puriï¬cation kit promega thewhole genome was sequenced using a combination of pacbiors ii smrt and illumina sequencing platforms for illuminasequencing the genomic dna sample over µg was shearedinto bp fragments using a covaris m220 focusedacoustic shearer covaris illumina sequencing libraries weregot from these fragments using the nextï¬extm rapid dnaseqkit bioo scientiï¬c and were then used for pairedend illuminasequencing bp on an illumina hiseq ten machineillumina for paciï¬c biosciences sequencing an aliquot of µg dna was spun in a covaris gtube covaris at rmin for s dna fragments were then puriï¬ed endrepaired and ligated with smrtbell sequencing adapters paciï¬cbiosciences the resulting sequencing library was puriï¬ed threetimes using agencourt ampure xp beads beckman coultergenomics and sequenced on one smrt cell paciï¬c biosciencesusing standard methodsthe complete genome was assembled using both the pacbioand illumina reads feng brieï¬y the pacbio readswere assembled into a contig using hgap and canu thecircular step was checked and ï¬nished manually generating acomplete genome with seamless chromosomes and plasmids theillumina reads was used for the error correction of the pacbioassembly results using pilon the cds trna and rrna wasfrontiers in microbiology wwwfrontiersinaugust volume 0cli agarase production by vibrio ï¬uvialistable genomic features of strain a8featuregenome size bpdna gcchromosomeplasmidproteincoding genestrnasrrnastandem repeatsgenes assigned to gogenes assigned to coggenes assigned to kegggenes with pfam domainsgenes coding for cazymesgenes related to secondary metabolismgenes related to virulent factenes related to antibiotic resistancegenes coding for secretory proteinsgenes coding for transport proteinsgenes coding for transmembrane proteinsgene islandsvaluerespectively predicated by glimmer version trnascanse version and barrnap version the predicted cdsswere annotated from nr swissprot pfam go cog keggand cazyme database using sequence alignment tools such asblast blast2go eggnog diamond and hmmer each set ofquery proteins were aligned with the databases and annotationsof the bestmatched subject evalue were used fene annotation the gene islands were predicted using theisland viewer bertelli the circular genomic plot ofpmtb21 was plotted by circos song h the agarase and naos hydrolase gene sequences werecompared with the gene sequences available from the ncbidatabase phylogenetic tree ofthese gene sequences wereperformed using mega based on the maximumlikelihoodmethod after multiple alignments of the sequences by clustalx larkin numbers represented the frequencieswith which the tree topology wasreplicated after bootstrap iterationscomparative genomic analysisgenomic data of the similar vibrio species analyzed in this studywere obtained from the ncbi the homologous genes wereanalyzed using orthomcl li with the followingparameters evalue 1e percent identity cutoï¬ markovation index phylogenetic analysis was performed byra ml stamatakis after multiple alignments of singlecopy of homologous genes using clustal x larkin to compare the genetic relationship among vibrio species theaverage nucleotide identity ani value was calculated usingjspecies richter and rossellomora saturationsecretomic analysislabelfree quantitative lcmsms was performed to study thesoluble extracellular proteins of vibrio sp a8 the extracellularproteins were isolated by ammonium sulfate fractionation¼ ammonium sulfateredissolved inphosphate buï¬er solution moll ph and thendesalted by dialysis bag the bradford method bradford was used to determine the protein concentration ofsample the protein sample µg was digested using trypsinpromega at ¦c overnight equal volume of formic acidwas mixed with digested sample and centrifuged at gfor min at room temperature the supernatant was slowlyloaded to the c18 desalting column washed by ml of washingsolution formic acid and acetonitrile three times andthen eluted twice using ml of elution buï¬er formicacid and acetonitrile the eluents were lyophilized andreconstituted in µl of formic acid peptide sampleswere injected and analyzed using a q exactive hfx massspectrometer thermo coupled with nanoeasynlc uhplc thermo peptides were ï¬rst trapped onto a homemade c18 nanotrap column cm µm µm and theneluted onto a homemade analytical column cm µm µm via a min gradient of acetonitrile with formic acid at a ï¬ow rate of nlmin min min min min min min ms full scan rangedfrom mz to with a target value of at aresolution of at mz the top precursors of thehighest abundant in the full scan were fragmented using higherenergy collisional dissociation and analyzed in msms with anautomatic gain control target value of and normalizedcollision energy of raw msms data were searched againstthe genome of vibrio sp a8 obtained in this study usingproteome discoverer thermo for abundance calculationmass spectrometric signal intensities of peptide precursor ionsbelonging to each protein were divided by the total abundanceof all detected proteins the mass spectrometry proteomics datahave been deposited to the proteomexchange consortium viathe pride perezriverol partner repository withthe dataset identiï¬er pxd019302phylogenetic tree of agarases based on their amino acidsequences was performed using mega based on the neighborjoining method after multiple alignments of the sequences byclustal x larkin the sequences of agarases thatwere similar to that in vibrio sp a8 were obtained from uniprotdatabase numbers represented the frequencies with which thetree topology was replicated after bootstrap iterationsagarase puriï¬cation and propertiesthe extracellular proteins were isolated using ammoniumsulfate fractionation ¼ ammonium sulfate saturationredissolved in phosphate buï¬er solution moll ph and then desalted by dialysis bag the agarase puriï¬cation wasperformed by deae sephadex anionexchange chromatographythe proteins were eluted by nacl solution at moll theeluent collected from the protein peak was tested for the agarasefrontiers in microbiology wwwfrontiersinaugust volume 0cli agarase production by vibrio ï¬uvialisfigure circular maps of a chr1 and b chr2 of strain a8 the circles represent from inner to outer gc skew gc content rrna and trna cog assignments for cdss on the reverse strand cog assignments for cdss on the forward strand scale in mbfrontiers in microbiology wwwfrontiersinaugust volume 0cli agarase production by vibrio ï¬uvialisfigure phylogeny of vibrio species based on singlecopy core orthologs a core homologous genome genes was computed by the genome of straina8 along with complete vibrio genomes obtained from public database b phylogenetic analysis was performed by ra ml after multiple alignments of singlecopy of homologous genes using clustal x c venn diagram showing homologous and unique genes among v ï¬uvialis strainsfrontiers in microbiology wwwfrontiersinaugust volume 0cli agarase production by vibrio ï¬uvialistable heatmap of the ani value between the conserved regions in the genomes of strains in vibriostrains in vibrio v ï¬uvialis a8 v ï¬uvialis v ï¬uvialis ak 1296a21 v ï¬uvialis atcc v furnissii fdaargos_777 v furnissii nctc v tubiashii atcc v anguillarum v coralliilyticus snuty1species on the ï¬rst column and ï¬rst row of the table are represented in the same numeric order the numbers show the percentage similarity between the conservedregions of the genomes where the colors vary from yellow low similarity to blue high similarityactivity the eluent with the agarase activity was used for thestudy of agarase propertiesunless otherwise stated in the study of agarase propertiesthe diluted enzyme solution µl was incubated with melted agar ml at ¦c and ph for min in whichcondition the agarase activity was set as the agarasesolution was incubated with ml of gl carrageenan alginchitosan cellulose or amylum to study the substrate speciï¬city ofagarase one unit of enzyme activity was deï¬ned as the amount ofenzymes that released µg of reducing sugars per minute underthese experimental conditions using dns method as mentionedpreviously to study the optimal reaction temperature theagarase activity was measured at diï¬erent temperatures and ¦c the optimal ph of the agarase wasassayed in a ph range of in mm na2hpo4citratebuï¬er ph mm phosphate buï¬er ph and mm glycinenaoh buï¬er ph the eï¬ects of diï¬erentmetal ions nacl fecl2 fecl3 cacl2 cuso4 kcl or mgcl2at mmoll on the agarase activity were also studied the sameamount of agarase solution was placed in the water bath at and ¦c respectively for min to study the agarasethermostability the data were ï¬tted to ï¬rst order plots andplots of lnra residual activity versus time were constructedto calculate kd the halflife of the enzyme t12 was calculatedusing the following equationt12 ln kdwhere t12 is the time required for the residual activity reducingto half and kd is the deactivation rate constantresults and discussionagaraseproducing conditionoptimization of strain a8the eï¬ects of medium components and culture conditions onthe agarase production of vibrio sp a8 are shown in figure diï¬erent carbon sources showed obviously diï¬erent eï¬ects onthe agarase activity in the medium figure 1a when glucosegalactose sucrose or agar was added to the medium alonethe agarase activity was the highest in the presence of agarhowever the agarase activity decreased after glucose and sucrosewere added to the medium possibly because the coexistenceof glucose or sucrose reduced the utilization of agar by thisstrain thus inhibiting the secretion of agarase the additionof galactose enhanced the agarase activity with the agar as theonly carbon source probably resulting from the improved straingrowth by galactose therefore the agar and galactose werechosen as the carbon sources of the agarproducing mediumthe eï¬ects of agar and galactose concentrations on the agaraseactivity in the medium were respectively studied as shown infigures 1bc the agarase activity in the medium ï¬rst increasedand then decreased with the increasing agar or galactoseconcentrations the optimum agar and galactose concentrationwas respectively and diï¬erent agarase activities was found in the medium withthe addition of diï¬erent nitrogen source figure 1d anicnitrogen source was more suitable for the agarase production ofvibrio sp a8 than inanic nitrogen source the agarase activitywas highest when yeast extract powder was the only nitrogensource in the medium the agarase activity was not significantlydiï¬erent at various yeast extract powder concentrations figure 1e the highest agarase activity was observed at yeast extract powderbased on artiï¬cial seawater the diï¬erent concentrations ofnacl were added to determine the optimum osmotic pressurefor the agarase production of vibrio sp a8 figure 1f theagarase activity was improved by nacl but was significantlyinhibited by nacl this might be because the excessiveosmotic pressure inhibited the strain growthleading to thedecrease of its agarase production diï¬erent ph values had greatuence on the agarase activity in the medium figure 1gthe optimum ph was at which the agarase activity was uml similar to ph the culture temperature showedsignificant uence on the agarase production of strain a8figure 1h the optimum culture temperature was ¦c atwhich the agarase activity was as high as umlthe eï¬ect of culture time on the agarase production of vibriosp a8 is shown in figure 1i the agarase activity increasedslightly at h markedly rised at h and then reachedequilibrium after h the growth curve was in accordancefrontiers in microbiology wwwfrontiersinaugust volume 0cli agarase production by vibrio ï¬uvialisfigure genes related to agar degradation in the genome of v ï¬uvialis a8 a number of genes in each cazyme class b number of genes related topolysaccharide degradation in ghs genomic islands gi contained genes related to agar degradation c gene3149 coding for α13lna2 hydrolase andgene3151 gene3152 and gene3153 coding for agarase were in gi d gene3109 coding for agarase was in gi e the gc content in the gi gi chr2 and genome of v ï¬uvialis a8 as well as in the genomes of v vulniï¬cus strainswith the agaraseproducing curve pearson correlation analysisshowed that the growth of strain a8 was significantly correlatedwith agarase activity p indicating that the positiveuence of strain growth on the agarase productionthe optimum medium for agarase production of vibrio spa8 was determined as agar galactose yeastextract powder and nacl in artiï¬cial seawater and theoptimum culture conditions were determined as temperatureat ¦c ph at and incubation time for h underthese conditionsthe fermentationbroth was uml which was higher than thatbefore optimizationthe agarase activity offrontiers in microbiology wwwfrontiersinaugust volume 0cli agarase production by vibrio ï¬uvialistable genes related to degradation of agar and relative compounds in the genome of v ï¬uvialis a8gene idlocationstrandstartendgene lengthprotein lengthannotationgh familygene3109gene3151gene3152gene3153gene3149gene0378gene1513gene3148gene4670gene1669gene3155chr2chr2chr2chr2chr2chr1chr1chr2chr2chr1chr2agaraseagaraseagaraseagaraseα13lna2 hydrolasegalactosidasedgalactosidasegalactosidasegalactosidaseαgalactosidaseαgalactosidasegh50gh50gh50gh50gh117gh2gh2gh2gh2gh36gh36genome features of strain a8the genomic features of vibrio sp a8 are presented in table the complete genome of vibrio sp a8 was bp gc in length composed oftwo circular chromosomeschr1 bp gc figure 2a and chr2 bp gc figure 2b and no plasmidwas detected the genome contained proteincodinggenes trna genes and rrna genes and all rrnatable genes involved in degradation of agar and relative compounds in thegenomes of strains in vibrio based on homologous gene analysisstrains in vibrioagar degradation gene numbersagarase neoagarobiosegalacto sidasehydrolasev ï¬uvialis a8v ï¬uvialis v ï¬uvialis ak 1296a21v ï¬uvialis atcc v furnissii fdaargos_777v furnissii nctc v tubiashii atcc v anguillarum v coralliilyticus snuty1 v coralliilyticus re98 v antiquaries ex25 v parahaemolyticusforc_014 v alginolyticus k10k4 v natriegens atcc v diabolicusfdaargos_105 v campbellii atccbaa1116 v owensii v180403 v breoganii ff50 v vulniï¬cus cect v casei dsm v nigripulchritudo sfn1 present absent genes were located in chr1 there were genes assigned to the go terms including genes in biologicalprocess genes in molecular function and genesin cellular component the most abundant go annotationincluded the metabolic process genes membrane genes and catalytic activity genes in respectivecategory supplementary figure s1 there were genes assigned to the cog terms and high percentageof genes was classiï¬ed into the amino acid transport andmetabolism genes transcription genes inanicion transport and metabolism genes energy productionand conversion genes and carbohydrate transport andmetabolism genes as well as the function unknown genes supplementary figure s1 in addition genes were allocated to the kegg pathways in whichthe carbohydrate metabolism genes global and overviewmaps genes amino acid metabolism genes andmembrane transport genes were the predominant pathwayssupplementary figure s1identiï¬cation of strain a8strain a8 was initially identiï¬ed as vibrio sp a8 based on 16srrna gene sequence to determine the exact species of thisstrain the comparative genomic analysis between a8 and similar strains in vibrio was studied among the vibrio strains homologous genes were found and constructed the coregenome figure 3a the genome phylogenetic tree was thenconstructed based on the core homologous genome figure 3bthe phylogenetic tree was capable of grouping various strainsof a single species into a single cluster strain a8 clustered withv ï¬uvialis atcc v ï¬uvialis and v ï¬uvialisak 1296a21 suggesting that these four strains might harborsimilar biological properties the biochemical identiï¬cation andcharacterization observations for strain a8 were made on vitek gn system supplementary table s1 strain a8 was identiï¬edas v ï¬uvialis with of probability on the basis of aboveresults the strain a8 could be determined as v ï¬uvialis andwas renamed as v ï¬uvialis a8 the ani similarity analysis alsoproved that strain a8 belonged to v ï¬uvialis it was reported thatthe strains could be considered as the same prokaryotic specieswhen the ani value was over richter and rossellomorafrontiers in microbiology wwwfrontiersinaugust volume 0cli agarase production by vibrio ï¬uvialis as shown in table intraspecies similarity of v ï¬uvialiswas higher than the highest similarity with v ï¬uvialisa8 was v ï¬uvialis atcc reaching which wasconsistent with the result of phylogenetic tree although homologous genes were found among these v ï¬uvialis strainsthere were still unique genes in v ï¬uvialis a8 figure 3camylumcelluloserecognitiongenes related to agar degradation invibrio ï¬uvialis a8carbohydrateactive enzymes cazymes that are responsiblefor the complex carbohydrate metabolism mainly includethecarbohydratebinding module cbmssynthesis glycosyltransferases gts and degradation glycosidehydrolases ghs polysaccharide lyases pls carbohydrateesterases ces and enzymes of auxiliary activities aasof the carbohydrates huang there were genes coding for cazymes in the genome of v ï¬uvialis a8table this strain had a high fraction of carbohydratedegrading enzymes especially ghs genes which couldincluding agar agarohydrolyze various polysaccharidesoligosaccharidesandchitin figure hemicelluloseagarases are classiï¬ed into αagarase ec andagarase ec according to their cleavage position onagarose αagarase catalyzes the α13 linkage to produce aoswhile agarase produces naos by split of 14 linkage fuand kim agarases are grouped into ghs based on thesequence similarity eg agarase grouping into the gh16gh50 gh86 and gh118 families and αagarase only groupinginto the gh96 family su in this study a total offour genes coding for agarase which all belonged to gh50family were found in the genome of v ï¬uvialis a8 amongthe agarase genes the gene3151 gene3152 and gene3153arranged in order from to on the forward strandof chr2 while the gene3109 was located on the reverse strandof chr2 from to table naos can be furthercatalyzed by αnaos hydrolase ec which has beenclassiï¬ed into gh family and can recognize neoagarobiosena2 neoagarotetraose na4 and neoagarohexaose na6 assubstrate ariga watanabe in thegenome of v ï¬uvialis a8 the gene3149 coding for α13lna2 hydrolase was classiï¬ed into gh family this enzymecan hydrolyze na2 and produce lahg and dgal watanabe it was reported that galactosidase in vibrio spejy3 could act on aos to release dgal lee there were four genes of galactosidase and two genes ofαgalactosidase in the genome of v ï¬uvialis a8 which mightcontribute to its ability of agar degradation the abundant genesof agarase naos hydrolase and galactosidase in v ï¬uvialis a8oï¬er an enormous advantage in the sacchariï¬cation of agar inred algae for its use as a biomass feedstock for the productionof fermentable sugarto study whether the strains closely related to v ï¬uvialisa8 shared the common ability of agar degradation the agardegrading genes of these strains were analyzed by comparativegenomic analysis table interestingly although the vibriostrains exhibited the genes coding for galactosidase the homologsfor agarase and na2 hydrolase were not observed in all othertested strains even in the same species v ï¬uvialis v ï¬uvialis ak 1296a21 and v ï¬uvialis atcc thissuggested that v ï¬uvialis a8 was probably diï¬erent fromother similar strains and possessed the unique agar degradationability although the agardegrading genes were not found inthe tested genomes another vibrio sp strain ejy3 exhibitedsimilar agarolytic system with v ï¬uvialis a8 and also couldproduce four agarases gh50 and one αnaos hydrolasegh117 yu horizontal gene transfer also knownas lateral gene transfer refers to nonsexual transmission ofgenetic material between unrelated genomes oliveira as the most important form of horizontal gene transfergenomic island gi contains numerous genes associated witha variety of biological functions godde it hasbeen reported that horizontal gene transfer in the bacteriaaï¬ected the phenotypes such as thermotolerance and multidrugresistance of closely related strains hegstad matsutani horizontal gene transfer also givesbacteria unique polysaccharide degradation ability alteromonasmacleodii harbored an alginolytic system within a kbgi which comprised ï¬ve putative alginate lyases and othercazymes neumann the marine bacteriumpseudoalteromonas haloplanktis ant505 acquired a gi with afunctional pathway for pectin catabolism including two multimodular pectate lyases pela and pelb hehemann a novel agarase gene aga1 in an inland soil agardegradingbacterium paenibacillussp ssg1 might be horizontallytransferred from marine bacteria via human microbiota song t in the genus vibrio horizontal gene transfer mainlycontributes to the virulence of strains le roux and blokesch deng however few studies of horizontal genetransfer related to agar degradation were reported in the genusvibrio in the genome of v ï¬uvialis a8 there were gisin which genes located accounting for over of totalproteincoding genes interestingly all the gis were located inthe chr2 of this strain the genes coding for agarase and α13lna2 hydrolase were all contained in the gis the gene3151gene3152 gene3153 and gene3149 were in the gi figure 4cwhile the gene3109 were in the gi figure 4d theseresults proved that these genes related to agar degradation wereobtained from other species through horizontal gene transferthe gene sequence similarity was further studied to determine theprobable source using the phylogenetic tree analysis figure 4dthe gene3109 gene3151 gene3152 gene3153 and gene3149in vibrio vulniï¬cus vawere found most similar to thatidentity with v vulniï¬cuswgs18058 percentvawgs18047 percent identity with v vulniï¬cusvawgs18038 percent identity with v vulniï¬cus vawgs18058 percent identity with and v vulniï¬cusvawgs18058 percent identity with respectivelyfurthermore the gc content in the gi and gi was and respectively which was obviously lower thanthat in the genome gc and chr2 gcof v ï¬uvialis a8 but much closer to that in the genome ofv vulniï¬cus strains gc figure 4e thesefrontiers in microbiology wwwfrontiersinaugust volume 0cli agarase production by vibrio ï¬uvialistable proteins with the highest abundance and enzymes belonging to the ghs in the secretome of v ï¬uvialis a8protein idgene idannotationmw [kda]calculated pirelative abundance proteins with thehighest abundanceenzymes belongingto the ghsgene3819gene2266gene2186gene3152gene2187gene4060gene4061gene2267gene3797gene2482gene4295gene3152gene4670gene4149gene3263gene0718gene1292gene0815gene3730zinc proteaseflagellin protein fladflagellin protein flaaagaraseflagellin protein fladhemolysin hlyathermolabile hemolysin precursorflagellin protein flacmetalloproteasemembrane proteinaminopeptidaseagarasegalactosidasechitodextrinasechitodextrinasechitodextrinasepullulanaseglycosidaseglycosyl hydrolaseresults indicated thatv vulniï¬cus strains through horizontal gene transferth | Colon_Cancer |
" variability in the health effects of dietary fiber might arise from interindividual differences in the gutmicrobiotas ability to ferment these substrates into beneficial metabolites our understanding of what drives thisindividuality is vastly incomplete and will require an ecological perspective as microbiomes function as complexinterconnected communities here we performed a parallel twoarm exploratory randomized controlled trial in adults with overweight and classi obesity to characterize the effects of longchain complex arabinoxylan n at high supplementation doses female gday male gday on gut microbiota composition and shortchainfatty acid production as compared to microcrystalline cellulose n nonfermentable control and integratedthe findings using an ecological frameworkresults arabinoxylan resulted in a global shift in fecal bacterial community composition reduced αdiversity andthe promotion of specific taxa including operational taxonomic units related to bifidobacterium longum blautiaobeum and prevotella copri arabinoxylan further increased fecal propionate concentrations p friedmanstest an effect that showed two distinct groupings of temporal responses in participants the two groups showeddifferences in compositional shifts of the microbiota p permanova and multiple linear regression mlranalyses revealed that the propionate response was predictable through shifts and to a lesser degree baselinecomposition of the microbiota principal components pcs derived from community data were better predictors incontinued on next page correspondence jenswalteruccie nguyen k nguyen and edward c deehan contributed equally to this work1department of agricultural food nutritional science university of albertaedmonton ab t6g 2e1 canada13department of biological sciences university of alberta edmonton abt6g 2e1 canadafull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cnguyen microbiome page of continued from previous pagemlr models as compared to single taxa indicating that arabinoxylan fermentation is the result of multispeciesinteractions within microbiomes this study showed that longchain arabinoxylan modulates both microbiota composition and theoutput of healthrelevant scfas providing information for a more targeted application of this fiber variation inpropionate production was linked to both compositional shifts and baseline composition with pcs derived fromshifts of the global microbial community showing the strongest associations these findings constitute a proofofconcept for the merit of an ecological framework that considers features of the wider gut microbial community forthe prediction of metabolic outcomes of dietary fiber fermentation this provides a basis to personalize the use ofdietary fiber in nutritional application and to stratify human populations by relevant gut microbiota features toaccount for the inconsistent health effects in human intervention studiestrial registration clinicaltrialsgov nct02322112 registered on july keywords arabinoxylan dietary fiber gut microbiota interindividual variability overweight adults shortchainfatty acids epidemiologic studies consistently associate dietary fiberconsumption with a reduced incidence of obesityassociated pathologies [ ] in largescale observationalstudies whole grains and cerealderived fibers eg arabinoxylan and glucan showed stronger associationswith reduced risk of developing cardiovascular diseasetype ii diabetes gastrointestinal cancers and of allcausemortality when compared to other fiber sources [ ] asubstantial body of animal research further consolidatedthe mechanisms by which fiber reduces metabolic pathologies despite these convincing associations findings obtained from human dietary intervention trialsaimed to improve metabolic risk markers by supplementing isolated dietary fibers remain inconsistent possibly due to an individualized clinical response [ ]owing to their chemical structure dietary fibers resistdigestion in the smallintestine and reach the colonwhere they become substrates for the gut microbiotathe microbial fermentation of fiber to shortchain fattyacids scfas has been implicated in the prevention ofobesityassociated pathologies propionate and butyrate are two scfas that are especially relevant as theyhave been linked to beneficial immunological and metabolic effects intervention studies with arabinoxylanisolated from wheat endosperm for instance have demonstrated increased fecal concentrations of both butyrateand propionate dietary fibers can further modulategut microbiota composition in a structuredependentway through the enrichment of bacterial taxa that utilizethe substrate and tolerate or benefit from the environmental changes caused by fiber fermentation [ ] forexample dietary interventions with shortchain fractionsof arabinoxylan resulted in an enriched abundance ofbacterial species that can either utilize arabinoxylan oliaxos directly eg bifidobacteriumgosaccharidesadolescentis and bifidobacterium longum or benefitfrom metabolic byproducts released during axos degradation eg anaerobutyricum hallii and faecalibacterium prausnitzii although fiberinduced alterationsto the gut microbiota are significant the effects are alsohighly individualized and this variability might haveclinical ramifications that could explain the individualized clinical responses to understand the individualized response of the gutmicrobiota to dietary fiber an ecological perspective isrequired as fiber fermentation is determined by complexinterspecies interactions between members of the gutmicrobiota the process is often based on a crossfeeding cascade where primary degraders that access thefiber provide breakdown products oligosaccharides disaccharides and monosaccharides to other microbesand metabolites that result from the fermentation ofthese products also serve as substrates interindividual variation in gut microbiota composition mayresult from the absence of keystone species that initiatethe degradation of recalcitrant fibers differences inunrelated species with similar ecological functions thatcompete for the same substrate or variation instrains of the same species that differ in their capacity tometabolize the substrate these compositional variations likely determine both the competitive and cooperative relationships between community membersthat form trophic networks some of which anize intoecological guilds that collaborate to degrade complexfibers although interindividual variation in the response of the gut microbiota to fiber can influence metabolite outputs relevant to health ie propionate orbutyrate this topic and the underlying ecologicalprinciples have received little attentionthe objective of this study was to apply an ecologicalframework to characterize the compositional and metabolic responses of the human gut microbiota to a longchain arabinoxylan isolated from corn bran compared to 0cnguyen microbiome page of a fiber that is not fermented by the gut microbiotamicrocrystalline cellulose mcc we further assessedwhether nutritional and microbiotarelated factors couldexplainamongindividualsresponsesobservedthevariableresultssubject characteristics and protocol adherenceto compare the effects of arabinoxylan and mcc weconducted a 6week parallel twoarm exploratory randomized controlled trial in individuals with overweightand classi obesity where females received gday andmales gday of either fiber fig of the subjectsenrolled and randomized to an intervention arm sevenwithdrew from the study in the arabinoxylan groupthree experienced challenges consuming the supplementand one reported constipation in the mcc group twowithdrew due to personal reasons and one due to constipation and weretherefore excluded from analysesadditional file fig s1 subjects that completed thestudy protocol n included females and males aged ± years with a body mass indexbmi of ± kgm2 no differences in age sex orbmi were detected between the intervention groups atbaseline additional file table s1 overall protocoladherence assessed by the amount weight of returnedsupplement was ± and ± in the arabinoxylan and mcc arms respectivelyeffect on the composition of the fecal microbiotafecal microbiota diversitynonmetric multidimensional scaling analysis of euclidean distances between subjects based on centered logratio clrtransformed operationaltaxonomic unitotu data showed that the two treatment groups harbored bacterialcould not becommunitiesthatdifferentiated at baseline p permutational multivariate analysis of variance [permanova] fig 2aoneweek supplementation with arabinoxylan alteredthe global fecal bacterial community which became significantly different from the fecal microbiota of subjectsreceiving mcc p this effect was maintaineduntil the end of the fiber intervention p thesechanges occurred by arabinoxylan inducing temporalshifts in fecal microbiota composition determined as theaverage diversity between the individuals treatmentand baseline samples which were significantly largerwhen compared to the mcc group p mannwhitney test fig 2b in addition while mcc increasedinterindividual differences diversity between subjectsp generalized estimated equation [gee] modelarabinoxylan reduced it p fig 2canalysis of αdiversity showed that arabinoxylan reduced fecal bacterial diversity shannons index p gee model fig 2d but not richness total otusafter weeks of supplementation overall these findingsshowed that while the nonfermentable mcc had no detectable effects on measures of bacterial diversity arabinoxylan altered the global bacterial community within week inducing temporal shifts in composition and a reduction of both interindividual variation and αdiversityeffect on the relative abundance of bacterial taxa and coabundance response groupsneither arabinoxylan nor mcc altered microbiota composition at the phylum level at lower taxonomic levelschanges in the relative abundance of two bacterial families were detected at weeks of arabinoxylan relative tobaseline and mcc namely an increase in bifidobacteriaceae q wilcoxon test fig 2e additional file table s2 and a decrease in erysipelotrichaceae q fig study design shaded study week blocks indicate a scheduled clinic visit the x indicates the task was completed during the study weekcdhq ii canadian diet history questionnaire ii stool characteristics selfreported stool consistency and bowel movement frequency 0cnguyen microbiome page of fig arabinoxylan alters the global composition of fecal bacterial communities and induces distinct shifts in taxa a nonmetricmultidimensional scaling nmds plot based on euclidean distance metrics of arabinoxylan and microcrystalline cellulose groups at each timepoint intersubject diversity showing changes in the distance between subjects over time euclidean distances b between fecal microbiotas ofsubjects at each study time point intersubject and c between each subjects fecal microbiota at baseline and during w1 and w6 of treatmentintrasubject d αdiversity displayed as shannon index and total otus of the fecal microbiotas of subjects at each time point e absolutechange δw6bl in relative abundance of bacterial taxa affected by the dietary intervention data analyzed using permanova for a gee modelswith bonferroni correction for b and d and mannwhitney tests for c for e data were analyzed using either wilcoxon tests to assess withingroup changes relative to baseline or mannwhitney tests to assess betweengroup changes ie ax vs mcc with fdr correction diversityand compositional data were reported as mean ± sd and centered logratio transformed prior to the statistical analyses bl baseline otuoperational taxonomic unit w1 week w6 week at the genus level arabinoxylan increased thegenera bifidobacterium and prevotella when comparedto both baseline and mcc and enriched blautia whencompared to mcc otu level analysis revealed that otus changed during arabinoxylan treatment relative tobaseline henceforth referred to as significant otusin particular otus related to bifidobacterium longumotu4 prevotella copri otu6 bacteroides plebeiusotu53 bacteroides sp otu56 bacteroides ovatussuccinatutensotu26otu38 blautia obeum otu85 subdoligranulum spotu11 clostridium leptum otu46 mollicutesotu32 and muribaculaceae otu79q phascolarctobacteriumbecame enriched while otus related to ruminococcusbromii otu5 eubacterium oxidoreducens otu41bacteroides uniformis otu7 and faecalibacillus sppotu21 declined in relative abundance supplementation with mcc only increased the family lachnospiraceaegenus parasutterella q numerically the dominant compositional effects of arabinoxylan were to a large degree specific to b longumotu4 and p copri otu6 as these taxa increased inrelative abundance by an average of 46fold and 4fold while other otus increased by and thein an attempt to identify groups of cooperating species that could function as ecological guilds in the 0cnguyen microbiome page of degradation of arabinoxylan we adapted a clustering approach conceptually similar to that described by tong instead of absolute proportions of taxa weused arabinoxylaninduced shifts to identify clusters ofspecies whose responses were intercorrelated this analysis revealed a total of seven coabundance responsegroups cargs fig 3a five of which showed statistically significant responses to arabinoxylan while noneresponded to mcc additional file table s2 thecarg that showed the largest increase in relative abundance was carg1 p wilcoxon test whichconsisted of six out of the eleven otus that increasedthrough arabinoxylan fig 3b among those six otusbshift andshowed significant connections to all but one member ofcarg1 rs q spearmans correlations usingpermutation tests suggesting arabinoxylan may be degraded through cooperative interactions between theselongum otu4 exhibited the largesttaxa in carg6 p copri otu6 exhibited the largestresponse but only showed one strong connection withanother member of the carg bacteroides massiliensisotu98 rs q which suggests that pcopri might act to a larger degree independently to degrade arabinoxylan fig 3b the majority of taxa thatdecreased during arabinoxylan consumption particularlyb uniformisotu7 clustered within carg7 andshowed negative correlations with taxa of carg1carg2 and carg6 suggesting competitive or antagonistic interactionstemporal responses of otus and cargsto determine if short and longterm treatment witharabinoxylan and mcc differed in their effects on thefecal microbiota we compared shifts from baseline toweek w1 with those from baseline to week w6however there were no detectable differences betweenfig identification of coabundance response groups cargs during arabinoxylan supplementation a heatmap shows the change δw6bl inrelative abundance of otus affected by arabinoxylan p wilcoxon test the hierarchical dendrogram shows clustering of centered logratio clr transformed otus rows based on spearmans correlation distances by the completelinkage clustering algorithm and then groupedon the dendrogram into seven cargs by permanova p subjects columns clustered based on euclidean distances colors from blue tored indicate the direction and magnitude of change b coresponse network analysis each node represents an otu where the size isproportional to the change δw6bl in relative abundance the shape indicates direction of change positive circle negative square and thecolor references the respective carg to which it was clustered lines between nodes represent significant positive red line or negative blueline spearmans correlations rs values ¥ or and q values bl baseline otu operational taxonomic unit w6 week 0cnguyen microbiome page of the two time frames q wilcoxon test data notshown in addition comparison of baseline w1 andw6 values by friedmans test indicated that the effectsof arabinoxylan occur rapidly within week with nofurther detectable changes at weeks fig 4a considering these findings analyses on compositional changeswere performed with w6 data unless otherwise statedinterindividual variation in responses to arabinoxylanbacterial shifts in response to arabinoxylan and theirmagnitude were highly individualized fig 4b for instance absolute increases in relative abundance rangingfrom to to 429fold change were detected inseven subjects for the otu classified as blongumotu4 while other subjects showed either a muchsmaller increase a decrease or the species was undetectable otus related to b obeum otu85 subdoligranulum sp otu11 b ovatus otu26 and c leptumotu46 were enriched by arabinoxylan in around twothirds of the subjects less frequently enriched wereotus classified as potu6 b plebeiusotu53 and bacteroides sp otu56 p copri otu6responded in only four subjects but effects were largewith the species expanding beyond to 7foldchange ofthe total bacterial community in threesubjectscoprito determine drivers of these individualized responseswe used multiple linear regression mlr analyses totest if responses in otus that showed numerically thelargest shifts p copri b longum b obeum and subdolifig temporal and individualized responses of the otus and cargs affected by arabinoxylan and microcrystalline cellulose a plots show thetemporal response of the ten most abundant otus detected in of subjects and the seven cargs centered logratio transformed datawere analyzed by friedmans test with dunns correction to assess withingroup changes between time points ie δw1bl and δw6w1 bbubble plot shows individualized differences δw6bl in relative proportions of the ten most abundant otus percentage of total microbiotacomposition and cargs sum of otus detected after weeks of arabinoxylan and microcrystalline cellulose supplementation the size of thebubble is proportional to the change in abundance relative to baseline while the color of the bubble represents the direction of the change redincrease black decrease the x indicates that the otu was either undetected or the change was relative abundance bl baseline cargcoabundance response group otu operational taxonomic unit w1 week w6 week 0cnguyen microbiome page of granulum sp and in cargs with significant responsescargs and could be predicted by baselinediet or microbiota composition baseline microbiota allotus and significant otus and diet variables werefirst reduced in their dimensionality by principal component analysis pca and then treated as predictors thisanalysis revealed that individualized responses of bacterial taxa and cargs to arabinoxylan and mcc could notbe predicted by baseline diet or microbiota compositionq additional file fig s2effect on stool characteristics and bowel movementswhile fecal moisture content was not changed by eitherfiber q wilcoxon test additional file table s3subjects consuming arabinoxylan reported softer stoolconsistencies when compared to subjects consumingmcc treatment effect p gee model additional file fig s3a both arabinoxylan and mcc ledto an increase in bowel movements relative to baselinep gee model additional file fig s3b withno difference detected between treatment groups treatment effect p effect on fecal ph and scfasfecal ph and scfa concentrations did not change after weeks of either fiber treatment q wilcoxon testadditional file table s3 considering that absoluteconcentrations of fecal scfas are affected by their absorption in the gut we additionally assessedchanges in the percentages of acetate propionate andbutyrate relative to total scfa concentrations at w6which has been previously shown to vary little across colonic regions this analysis revealed an increase inthe percentage of propionate produced through arabinoxylan when compared to mcc q mannwhitney test and a reduction in the percentage ofbutyrate relative to baseline q wilcoxon test although differences in butyrate were not detected whencompared to mcc q further investigation ofthe ratio between propionate and butyrate showed an increase in propionate relative to butyrate when comparedto baseline q and mcc q suggestingthat arabinoxylan supplementation directed the outputof scfas in favor of propionatecharacterization of the temporal response in the threeprimary scfas also showed an increase in fecal propionate concentrations by arabinoxylan at w1 p friedmans test fig 5a although propionate concentrations remained elevated at w6 this increase was notstatistically significant when compared to baseline p this loss of significance was caused by an increasein the interindividual variation at w6 fig 5b visualevaluation of the individualized temporal response ofpropionate to arabinoxylan revealed clear separation offig temporal and individualized output of fecal scfas in response to arabinoxylan and microcrystalline cellulose supplementation a line plotsshow the temporal response of acetate propionate and butyrate reported as mean ± sd b individualized temporal propionate response of w6responders red and w6nonresponder black grouped based on δw6w1 data analyzed for a and b using friedman test with dunnscorrection to assess withingroup changes between time points and for b using mannwhitney tests to assess differences betweengroup ateach time point bl baseline carg coabundance response group otu operational taxonomic unit scfa shortchain fatty acid w1 week w6week 0cnguyen microbiome page of subjects into two distinct patterns fig 5b based on thedirection of change from w1 to w6 ie positive ornegative subjects were grouped into w6respondersδ w6w1 and w6nonresponders δ w6w1 in general w6responders showed a higher outputof propionate at w6 p friedmans test butnot at w1 while the opposite isseen in w6nonresponders p the two groups differed bypropionate concentrations at w6 p mannwhitney testw6propionate responders and nonresponders differ intheir microbiota response to arabinoxylanmicrobiota compositionalbaseline and shifts anddiet data were ordinated using pca and then differencesandbetween w6propionaterespondersnonresponders were tested using permanova thisthe bacterial communities ofanalysis revealed thatw6responders wereindistinguishablefrom w6nonresponders at baseline but differed in their response to arabinoxylan δw6baseline fig thiswas detected if analysis was based on all otus p the significant dietresponsive otus p or the seven cargs p in contrastneither baseline microbiota composition fig nordietary factors additional file fig s4a separatedaccording to w6 response p in addition comparing w6respondersinterms of their baseline total grain whole grain andtotal fiber consumption or their stool consistency andbowel movement frequency during treatment did notreveal any differences either p mannwhitneyand w6nonrespondersfig the individualized temporal propionate response to arabinoxylan associates with compositional responses in the fecal microbiota principalcomponent analysis plots based on euclidean distance comparing the relative abundance of fecal microbiota both at baseline and arabinoxylaninduced shifts δw6baseline between w6responders red and w6nonresponders black microbiota variables ie otu or carg thatcontributed the most to intersubject variation were shown as vectors on the plot when statistical significances were determined by permanovap carg coabundance response group otu operational taxonomic unit w1 week w6 week 0cnguyen microbiome page of test additional files and fig s3c and fig s4btogether these findings indicate that the temporal response in fecal propionate concentrations is primarilyassociated with the shifts in the microbiota and notbaseline microbiota composition or dietindividualized scfa responses can be predicted by gutmicrobiota featuresas with compositional responses gut microbiota functional responses to fiber interventions have been shownto be individualized [ ] but what drives this variation is poorly understood we applied mlr to determine whether fecal scfa responses could be explainedby stool consistency and bowel movement frequencydiet or microbiotarelated factors and then comparedthe quality ofthe models using corrected akaikeinformation criterion aicc values where lower valuesmean higher quality these analyses revealed that thew6 scfa response to arabinoxylan could be predictedby the fecal microbiota fig additional file fig s5but not by baseline diet stool consistency or bowelmovement frequency reported during treatment additional file fig s6a and fig s6b the best modelswere achieved for propionate especially when principalcomponents pcs generated from w6 shifts of all otuswere used as predictors fig 7a additional file tables4 models were of lower quality when w6 shifts of significant otus cargs pcs of cargs or single otuswere used suggesting that global community measuresexhibited stronger linear relationships with the propionate response than single or groups of taxa although themodels that used baseline and w1 shifts of otus asfig individualized arabinoxylaninduced propionate responses could be explained by baseline gut microbiota composition and microbiotashifts a heatmap shows the linear associations between the individualized propionate response δw6bl dependent variable columns andmicrobiota profiles bl δw1bl δw6bl predictors rows cells represent individual multiple linear regression models with fdr correction thatassess whether the predictors explain the individualized propionate response multivariate microbiota data were simplified into principalcomponent pc variables pc1 pc2 and pc3 prior to analysis each model contained the best one or two predictors of pcs individual cargs orsignificant otus selected by stepwise regression all models were adjusted by fiber dosesex colors from white to red indicate relative aicchighest aicc value x lower aicc values red indicate higher quality models b scattercorrected akaike information criterion values calculated byplots show the linear relationship between propionate responses δw6bl and either the baseline contribution of all otus to pc1 or the shiftsof carg1 color and size of each point indicate propionate response magnitude and the shaded area specifies the confident interval thetop six otus that contributed the most to either pc1 of all otus or carg1 are further provided ax arabinoxylan bl baseline carg coabundance response group mcc microcrystalline cellulose otu operational taxonomic unit w1 week w6 week aicc value 0cnguyen microbiome page of predictors were of lower quality than those based on w6shifts they are still valid showing q values less than after benjaminihochbergs false discovery rate fdrcorrection linear relationships between propionate responses and significant predictors using baseline pc1 ofall otus and shifts carg1 were further visualizedusing scatter plots fig 7b reaffirming the quality ofthe analysis as a majority of subjects fall within the confidence regionssignificant models could also be designed for acetateand butyrate responses to arabinoxylan additional file fig s5 interestingly in contrast to propionate the bestmodels to predict butyrate responses were achieved usingshifts of a single otu e oxidoreducens otu41 aknown butyrate producer however overallthemodels for acetate and butyrate were of much lower quality than those for propionate in summary while individualized responses in scfas showed no association withdiet they could be predicted by microbiota shifts andbaseline composition in contrast to the analysis of the effects of arabinoxylan not one single mlr model wasfound to be significant for mcc indicating that the statistical approach based on mlr models did not detect anyassociations independent of fiber fermentationdetermining the role of bacterial taxa in propionateresponsemlr analyses were applied to determine connectionsbetween arabinoxylan responding otus within cargs and and fecal propionate concentrations fig 8athis analysis revealed that shifts in p copri otu6 didnot predict propionate responses while blongumotu4 and correlated taxa in carg1 showed strongerlinear relationships the highest quality models were obtained with b obeum otu85 b plebeius otu53and p succinatutens otu38 all of which encodemetabolic pathways for propionate production such analysis provides a potential explanation for themetabolic interactions between proposed primary degraders secondary fermenters and metabolite utilizersthat result in the promotion of propionate in responseto arabinoxylan fig 8bdiscussionin the present study we characterized the impact of a6week highdose corn bran arabinoxylan supplementation on the composition and function of the fecalbacterial community in healthy adults with overweightand classi obesity arabinoxylan treatment changedfig relationship between propionate responses to arabinoxylan and proposed primary degraders secondary fermenters and metaboliteutilizers a individual multiple linear regression models determine otu responses δw6bl that predict the fecal propionate response δw6blyaxis shows the coefficient for each predictor as in the average propionate response when otu relative abundance increases xaxis showsthe p value for each predictor all models were adjusted by fiber dosesex where bubble size represents the adjustedr2 b proposed model ofbacterial crossfeeding in the gut during degradation of complex soluble arabinoxylans otu operational taxonomic unit 0cnguyen microbiome page of showing two distinctcommunity structure and induced specific shifts inthe composition of the gut microbiota that manifestedthemselves after week of treatment without furtherchanges at w6 arabinoxylan induced increases inpropionate output both compositional and functionalresponses were highly individualized with propionateresponsestemporal patternscompositional responses to arabinoxylan could not bepredicted and functional responses were independentof stool consistency bowel movement frequency andbaseline diet however baseline microbiota composition and especially the compositional shifts correlatedwith propionateresponses the nonfermentablemcc showed virtually no effect on gut microbiotacomposition or functionan understanding of compositional and functional responses of the gut microbiota to changes in diet requiresan ecological framework arabinoxylan supplementation provides resources that can be used by microbesthat possess the traits to either access the chemicalstructures directly or utilize public goods released duringarabinoxylan degradation in our study the dominant effects of arabinoxylan were directed toward twobacterial species b longum and p copri while nine additional otus showed smaller increases including threebacteroides species eg b ovatus b plebeius and bacteroides sp this high degree of specificity toward blongum over other bifidobacterium species is in agreement with other studies testing longchain arabinoxylans[] and genomic analyses that showed that genesencoding arabinoxylandegrading glycosidase eg xylosidase and αarabinofuranosidase are conservedonly among b longum strains [ ] in contrast to thespeciesspecific enrichment of b longum arabinoxylanenriched several species within the phylum bacteroidetesthat possess the genetic and functional traits necessaryfor accessing arabinoxylan [] although arabinoxylan utilization is not universally conse | Colon_Cancer |
" a critical barrier to improving the quality of endoflife eol cancer care is our lack of understanding of themechanisms underlying variation in eol treatment intensity this study aims to fill this gap by identifying anizational and provider practice norms at major us cancer centers and how these norms influence providerdecision making heuristics and patient expectations for eol care particularly for minority patients with advanced cancermethods this is a multicenter qualitative case study at six national comprehensive cancer network nccn andnational cancer institute nci comprehensive cancer centers we will theoretically sample centers based upon nationalquality forum nqf endorsed eol quality metrics and demographics to ensure heterogeneity in eol intensity andregion a multidisciplinary team of clinician and nonclinician researchers will conduct direct observations semistructuredinterviews and artifact collection participants will include cancer center and clinical service line administrators providers from medical surgical and radiation oncology palliative or supportive care intensive care hospital medicineand emergency medicine who see patients with cancer and have high clinical practice volume or high local influenceprovider interviews and observations and adult patients with metastatic solid tumors and whom the providerwould not be surprised if they died in the next months and their caregivers patient and caregiver interviewsleadership interviews will probe about eol institutional norms and anization we will observe inpatient andoutpatient care for two weeks provider interviews will use vignettes to probe explicit and implicit motivations fortreatment choices semistructured interviews with patients near eol or their family members and caregivers willexplore past current and future decisions related to their cancer care we will import transcribed field notes andinterviews into dedoose software for qualitative data management and analysis and we will develop and apply adeductive and inductive codebook to the datadiscussion this study aims to improve our understanding of anizational and provider practice norms pertinent toeol care in us cancer centers this research will ultimately be used to inform a provideroriented intervention toimprove eol care for racial and ethnic minority patients with advanced cancertrial registration clinicaltrialsgov nct03780816 december keywords endoflife norms heuristics cancer minority health correspondence amberbarnatodartmouthedu2the dartmouth institute for health policy and clinical practice geisel schoolof medicine dartmouth college lebanon nh usa5department of medicine geisel school of medicine hanover nh usafull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cknutzen bmc palliative care page of the national academy of medicine has identified increasingly aggressive burdensome and expensive endoflife eol treatment as a major public health problem the american society of clinical oncology andnational quality forum nqf define aggressive burdensome and expensive eol treatment in cancer as thereceipt of chemotherapy in the last days of life nqf intensive care unit icu admission in the last days of life nqf and non nqf orlate nqf hospice referral such treatmentadversely affects patient quality of life quality of dyingand caregiver bereavement outcomes [] minoritiesare more likely to receive such eol treatment []potentially in disproportion to their preferences [ ]despite attention focused upon integrating early palliative care into advanced cancer treatment [] icuadmission in the last days of life and late hospicereferral have been secularly increasing yet not allanizations are equal cancer centers vary by morethan twofold in these eol intensity measures [ ]these variations cannot be explained by structural characteristics or casemix since centers serving ahigher proportion of minority patients have systematically higher eol intensity than centers serving a higherproportion of white patients these variations inpractice patterns may contribute to racial disparities inburdensome treatment near death moreover despiteefforts to attribute such variation to differences in patient preferences rather regionlevel analyses suggest that the impact of thesepreferences on variation are likely very small [ ]than racial disparitiesin local anizational and providerwe posit that a critical barrier to improving thequality of eol cancer care in the us and amongminorities in particular is our lack of understandingregarding the mechanisms underlying cancer centervariation in eol treatment intensity the overarching hypothesis driving this study is that differencessocialnorms rules about which there is at least some degree of consensus enforced through social sanctions are a key mechanism underlying this variationwe base this hypothesis on our preliminary work attwo us academic medical center hospitals at oppositeextremes of eol treatmentintensity demonstratingmarked differencesicu and lifesustaining treatment decision making these normswere found to directly and indirectly via influencing patient and family treatment expectationsand provider decision making heuristics affect treatment decisions for minority and nonminority patients with advanced cancer norms are fruitfulforstudy because once understood they are potentiallymalleableandleadership effortsthrough explicitin norms ofimplementation of new forms of positive and negativesanctions via social marketing interventions our study aims to study mechanisms underlying cancer center variation in eol treatment intensity amongminority and nonminority patients using a qualitativecase study design and has two objectives first we willidentify the local anizational and provider practicenorms that influence decisions about laterline chemotherapy hospice and icu use among minorities withadvanced cancer at major us cancer centers second wewill assess the influence of these norms on patient andfamily expectations and provider decision making heuristics for laterline chemotherapy hospice and icu useamong minorities with advanced cancer at major uscancer centers below we describe our qualitative studydesign approach to meet our study objectivesrecommendationsmethodsdesignthe design of the studywe chose a qualitative case study design at six sites toidentify local anizational and provider practice normsthat influence variation in eol treatment intensity particularly for minority patients based on medicareclaims data analyses we will recruit of the ncinccn designated cancer centers serving at least african american advanced cancer patients webased our sample size on recent literature related tosample size sufficiencyto reachmultisite data saturation and qualitative research expertise of our study team first our study is guided by atheory based on previous research and uses data frommultiple sources to test and crosscheck for confirmingor disconfirming evidence of our theory a necessarycomponent of ensuring data sufficiency [ ] inaddition conducting qualitative case studies at six siteseach of which will include over interviews plus multiple observations will produce finegrained and rich descriptive analysis to generate and compare theoreticalinsights across sites as well as across stakeholders egproviders patients within sites [] our targetnumbers for interviews and observations are well withinrecommendationsandsaturation given our well defined study aim lastlyresearch suggests that qualitative research expertise including the quality and depth of interview process is animportant criterion to consider for assessing sample size our site visit team has over years of collectiveexperience conducting qualitative research in healthcaretopics and settings ensuring a rigorous and thoroughprocess at each of the six sitesreaching dataadequacyforwe willtarget national comprehensive cancernetwork nccn and national cancer institute ncicomprehensive cancer centers for our study becausethey set national standards for high quality cancer care 0cknutzen bmc palliative care page of we will engage up to six sites serving a high proportionof african american patients ranging in eol care intensity and theoretically sampled to maximize our ability to compare and contrast anizational and providerpractice norms related to eol care we defined highproportion of minority patients as we measuredeol care intensity based upon riskadjusted metrics ofeol quality using medicare feeforservice claimsdata receipt of chemotherapy in the last days of lifenqf intensive care unit icu admission in thelast days of life nqf and non nqf or late nqf hospice referral our approach tocalculating these eol quality metrics has been publishedelsewhere given the multivariable nature of thesemetrics we use data visualization to purposively selectsites for case study that maximize potential heterogeneity in practice patterns following the principles ofpositive deviance sampling[ ] we will samplehigher versus lower eol quality sites in a ratio of we will employ qualitative case study researchmethods in this study including inpatient observationprocedures and provider patient and leadership semistructured interviews that have been previously developed and piloted [ ] we will augment thesemethods with outpatient and tumor board observationprocedures which we developed and tested at a nonstudy ncidesignated cancer center serving a whiterural population we williteratively revise all datacollection procedures based on researcher experiencesand thematic insights following each sampled case studysite visitqualitative data collectionthe study team will collect types of data field notesfrom direct observation of inpatient and outpatient cancer care and cancer tumor boards transcribed audiorecorded semistructured interviews with cancer centerleadership providers and patients family members andcaregivers and artifacts table we will link all datafrom observations using a unique identification idnumber we will use a data collection form for field observations to capture provider and patient demographicinformation as well as location time and individualspresent at the encounter in addition to relevant clinicaldata observers will note sociolinguistic dimensionssuch as turn taking tone affect body positioning andeye contact artifacts collected during the site visit willinclude workflows marketinginformational materialsorientation guidelines quality reporting and communication documents used in the cancer centersemistructured interview guides for site leadershipfocus on institutional norms including resources programs and policies related to eol care and outcomesas well as sitespecific workflows and scheduling logisticsin preparation for site visits cognitive mental modelssemistructured interview guides for providers whichtable data collection rationaledata collection methoddirect observationrationaleto learn about eol care for minority patients with advanced cancer specifically how it is influenced by anizational and provider practice norms provider decision making heuristics patient and family expectationssemistructured interviewsleadershipto probe anizationlevel norms including resources programs and policies sitespecific workflows and scheduling logisticsprovidersto explore individuallevel motivations decision heuristics andor rationalizations unconscious beliefs and assumptions that structure advanced cancer decision making using casevignettes to prime mental modelspatients family memberscaregiversto probe individuallevel preferences for cancer care past current and future decisions related to cancer careartifact collectionto learn how the anization standardizes workflows marketinginformational materials orientation guidelinesquality reporting and communication documents used in the cancer center and how this impacts local anizational and provider practice norms provider decision making heuristics patient and family expectations 0cknutzen bmc palliative care page of include eol case vignettes explore motivations decision heuristics andor rationalizations six eol vignettes were developed bya medical oncologistradiation oncologist and palliative care providers tohighlight key decision points common to outpatient orinpatient providers see table each vignette has oneversion with a photo of an african american patient andone with a photo of a white patient providers will viewone african american case and one white patient caseto facilitate mental models debriefing and uncover unconscious beliefs and assumptions related to race thatstructure advanced cancer decision making semistructured interview guides for patients family membersand caregivers probe past current and future decisionsrelated to their cancer caresite visit teams will consist of researchers we willidentify a sitespecific principal investigator pi at eachsite to help facilitate support from site leadership andidentify and recruit informants for pre sitevisit interviews and providers for sitevisit observation and interviews up to two months in advance of the site visit thestudy team will conduct leadership interviews by phoneincluding physician and nursing leaders outpatient oncology practice managers and key referral service lineleaders from palliativesupportive care hospital medicine and intensive care we will approach other siteleaders for interviews at the suggestion of the site piwhen necessaryleadership interviews will take placeduring and after site visitsone month in advance of the site visit the study teamwill recruit providers for observation in the inpatientand outpatient setting the observation schedule will involve one researcher assigned to each observed providerfor a halfday observation in outpatient clinics morningtable vignette summariessettingvignette number andpatient racespecialtyinpatient african american white african americaninpatient white african americanmedicaloncology white african american whiteradiationoncology african americansurgicaloncology white african american outpatient whitevignette summary and key questionsummary yearold man with metastatic gastric cancer he was living in a skilled nursing facilityafter a long hospitalization for infection he is now hospitalized with recurrent fever respiratorydistress and anxietykey question how to manage anxiety and respiratory distress in a patient with advanced cancerand high risk for shortterm deathsummary yearold woman with recurrent metastatic pancreatic cancer and mild dementia she isscheduled to start palliative chemotherapy next week she presents to the ed with decliningperformance status decreased appetite and abdominal pain her hospital evaluation demonstratespoor kidney function low blood pressure and rapid breathing all worrisome for rapid constitutionaldeclinekey question how to manage a patient with an aggressive cancer presenting to the emergencydepartment with multiple signs of constitutional declinesummary yearold man with advanced metastatic colon cancer he is married and lives at homewith his wife he presents to clinic with pain weight loss and signs of cancer progression he asksdo you think the chemo is workingkey question how to answer patient questions about prognosis and next steps in treatment ofadvanced cancer with limited treatment optionssummary yearold man with a new diagnosis of metastatic renal cell carcinoma he presents withseizures brain metastases and lung metastases he is unmarried and without children his performancestatus is poor and he is not able to make his own health care decisions his eldest brother is his durablepower of attorney and asks doc what would you do if he was your brotherkey question how to approach surrogate decision making about management approach for a patientwith poor prognosissummary yearold man with newly diagnosed nonmetastatic lung cancer he has severe lungdisease and significant vascular disease from heavy smoking he is a poor surgical candidate hementions that the stress of his cancer diagnosis has caused him to drink alcohol more heavily thanusual and he is coughing up about tablespoons of bright red blood dailykey question how to approach a patient with a new diagnosis of a potentially curable cancer whenthere are a number of red flags that the patient may do poorly with surgical treatmentsummary yearold woman with a recent diagnosis of pancreas cancer she has been hospitalizedwith weight loss pain and declining activity her evaluation shows a borderline resectable pancreaticcancer initial treatment would be chemotherapy or chemoradiation if she could tolerate this she hasbeen unable to eat or ambulate for the last five days due to poor appetite and performance statusshe says im a fighter not a quitter and with jesus anything is possible she then asks what comesnextkey question how to approach a patient who has a treatable diagnosis but who does not have theperformance status to tolerate treatment 0cknutzen bmc palliative care page of or afternoon session the emergency department byshift and inpatient setting by timing of daily service orconsult rounds as well as scheduled tumor boards andfamily meetings selection criteria for providers to observe and interview focus on maximizing our ability toassess provider norms for advanced cancer care withinthe particular institutional context that we will exploreduring leadership interviews provider selection criteriaincludes providers who manage patients with metastatic solid tumors ie we excluded leukemia lymphoma and bone marrow transplant providers and haveeither high volumes of patients andor high peerinfluence as perceived by the site pi or other key informants we seek to recruit medical radiation and surgical oncology providers as well as palliativesupportivecare providers who see cancer patients see table fortarget sampling frame we also seek to recruit providersfrom intensive care hospital medicine and emergencymedicine who care for acutely ill cancer patients wepresent an example observation schedule in fig wewill ask all providersrecruited for observation tocomplete an interview interviews with providers willoccur in person during or by phone after the site visitwe will digitally record all interviews and compensateall providers for participating in an interviewat least weeks prior to the site visit we will sendflyers about the study with photos of the study team tothe site pi who will facilitate posting of the flier in publicsettings such as waiting rooms clinic rooms infusionsuites and inpatient units the purpose of this flyer is toalert nonconsented individuals to our study purposeand to provide instructions for opting outduring patient care observation researchers will directly approach patients and their familycaregivers following introduction by the consented providerifpatients or their familycaregiver verbally consent to beinterviewed the study team member will obtain contactinformation to arrange for a phone interview at thepatient family member or caregivers convenience at alater date selection criteria for patient interviews includes adults aged years or older with metastatic solid tumor whom the provider would not besurprised if they died in the next months and seenby a consented provider we will seek to recruit equalnumbers of minority and nonminority patients we willdigitally record all interviews and compensate each participant for participatingtheoretical modelfindingsqualitative data analysiswe will use a qualitative and mixed methods data analysis platform dedoose to manage and analyze all transcribed field notesinterviews and artifacts and linkrelevant data to contextual information eg patient andprovider race site features sociocultural research consultants llc we will develop a codebook first deductively using ourin theliterature and priorresearch and then inductivelythrough an iterative process of close readings and discussion of the data in order to identify additional codesthree qualitative researchers will apply the codebook tothe data two who will divide and code all the data andone who will assess reliability of coding by reviewing asubset ofthree qualitative researchers will discuss differences in coding and resolveby consensus we will repeat the analysis process aftereach site visit to conduct constant comparative analysisregarding similarities and differences between and withinsites in support of study aims and after completionof each site visit the study team will develop a writtensummary of preliminary quantitative and qualitativefindings specific to the site which will then be sent to allparticipants from that site to assess initial validity of thesitespecific findings after completion of all site visitsand analysis of data we will send final study reports toparticipating sitesthe coded data alltable target sampling frame at each sitedata collection settingoutpatientmedical oncologysurgical oncologyradiation oncologysupportivepalliative careemergency medicineinpatienthospital medicineintensive caresupportivepalliative careoncology consultrigor and reproducibilityour research team is also conscious of conducting purposefully informed and respectful research on the cancerexperiences of racial and ethnic minorities and we havetaken steps to ensure scientific rigor of our approachand results through study design development and willcontinue to do so through data collection and data analysis based in a relatively nonracially diverse geographicregion our team n is comprised of racial andethnic minority researchers as such we seek to incorporate greater diversity of racial and ethnic knowledge aswell as disciplinary perspective through an external advisory board with deep topical expertise in cancer carepalliative care racial and ethnic health equity and socialnorms additionally to address potential researcher biasthe entire study team completed implicit bias trainingn 0cknutzen bmc palliative care page of fig mock onsite observation schedule researchers will ideally observe relevant outpatient clinics during week and inpatient servicesduring week researchers will go to tumor boards attended by consented providers as well as other relevant staff meetings eg fellowsmeetings researchers will observe providers during either am or pm blocks using the alternating daily block to dictate field notes and conductinterviews with providers and patients onsitefocused on unconscious biases related to attitudes aboutrace ethnicity cancer cancer treatment death anddying one researcher participating in data collectionwill remain blinded to sites eol treatment intensityclassification until data collection is completefinally we will employ multiple methods of triangulation to assure comprehensiveness and validity of datatwo to three researchers of a multidisciplinary team willparticipate in each site visit and an additional three researchers will conduct qualitative analysisto satisfyinvestigator triangulation method triangulation will include direct observation semistructured interviews andartifact collection we will achieve data triangulation byobserving and interviewing leadership personnel providers and patients family members and caregivers ateach site of various s specialties and diagnoses respectively qualitative analysis will use both deductive and inductive methodsto achieve theorytriangulationethics approval and consent to participatethe study has been approved by the dartmouth collegecommittee forthe protection of human subjectsstudy00031129 and is considered minimal risk allparticipating sites will waive independent irb approval in favor of acknowledging dartmouths irb rely on dartmouths irb via a smart irb reliance or conduct a local ethical review and approval we willobtain a waiver of informed consent for participant observation all providers will provide written electronicconsent for observation and interview and allinterviewed leadership patients and families will provide oralconsent for interview we have obtained a certificate ofconfidentiality from the national institutes of healthnih for this studystaffwe will not record any identifiable or personal information about providers patients family members caregivers orin field notes except demographicinformation a unique id number will link data fromobservations and interviewsincluding demographicdata to consented participants the key linking the idnumber and identifying information of the consentedparticipants will be maintained on a passwordprotectedserver only the research team will have access to thelinkage file all data collected on individuals will belinked to their id number alone we will audiorecordand transcribe all handwritten field notes without anyidentifiable information we will store all original fieldnotes in a locked filing cabinet and all transcripts on apasswordprotected server to which only the researchteam will have access we will give a discreet lapel pinto all providers staff patientsfamily members and 0cknutzen bmc palliative care page of caregivers who do not wish to be observed as advertisedby the informational flyers posted prior to the studyteams arrival at the site we will not document any individual wearing such a pin in field notes nor will weapproach them for an interviewwe will give all individuals participating in interviewsan information sheet prior to the interview and we willobtain informed consent verbally at the time of theinterview we will obtain informed consent verbally asmany of the interviews will be conducted by phone either before or after the site visit the process of obtaining verbal consent has been approved by the dartmouthcollege committee forthe protection of humansubjects we will record all interviews and later professionally transcribe them without any identifiable information we will store all recordings and transcripts on apasswordprotected server to which only the researchteam will have access additionally we consulted theguidelines for endoflife research putforth in themethods of researching end of life care morecareproject while designing the protocol for this study specifically we considered the risks egintervieweedistress and rewards eg potential therapeutic effectthat qualitative interviews may have for patients familymemberstheseprotocolsgivers while designingand carediscussionour study is the first comprehensive qualitative study oflocal anizational and provider norms at minorityserving nccn and ncidesignated comprehensive uscancer centers if the aims of this study are achieved weexpect to identify targets for institutional change at cancer centers with lower eol quality metric performancethe two main deliverables of this research will be knowledge regarding norms and their impact on eoldecision making at participating cancer centers and identification of potential members of a community research advisory board to oversee future institutionlevelinterventions aimed at improving eol care respectiveto the first deliverable participating cancer centers willreceive a customized report of our findings about theirown center following completion of our site visit aftercompletion of all site visits we will work with theamerican cancer society and participating cancercenters to identify local chapters ofthe americancancer society acs and provider medical societieseg county medical and nursing societies at which wecan discuss our findings and their implications for localpatients and providersrespective to the second deliverable we anticipate theopportunity to develop institutionlevelinterventionsaimed at eol care in the future norms are fruitful forstudy because once understood they are potentiallymalleable through explicit leadership efforts and implementation of new forms of positive and negativesanctions specifically social marketing the use ofmarketing principles to influence human behavior to improve health or benefit society is a promisingstrategy for changing norms interventionists have successfully applied the principles of social marketing tochange hiv risk behaviors [ ] and palliative careconsultation use integrating social marketing interventions in cancer centers with high intensity eol carecould have the effect of improving the quality and costof cancer care particularly for racial and ethnic minorities further by studying norms of decision makingamong groups of physicians this project will overcomethe limitation of past research which uniformly hasneglected this important issueabbreviationsnccn national comprehensive cancer network nci national cancerinstitute eol endoflife nqf national quality forum icu intensive careunit id identification pi principal investigator irb institutional reviewboard acs american cancer societyacknowledgementsthank you to inas kayhal for her assistance with the cluster analysis for sitesampling and associated visualizations and to garrett wasp for his input onthe clinical vignettesauthors contributionskek led coordination of the study participated in design of the protocolcontributed to instrument development and led preparation of themanuscript aeb led design and writing of the grant and protocolcontributed to instrument development and participated in preparation ofthe manuscript kes participated in design and writing of the grant andprotocol led instrument development and participated in preparation of themanuscript gfm and rb participated in design of the protocol contributedto instrument development and participated in preparation of themanuscript gab and nsk created the clinical vignettes contributed toinstrument development and participated in design of the protocol ssacontributed to instrument development and participated in design of theprotocol the authors read and approved the final manuscriptfundingthis research is funded by the american cancer society grant number rsg1801701cphps the funding body had no role in the design of the studyand will have no role in the collection analysis and interpretation of data orwriting of the manuscriptavailability of data and materialsendoflife metrics data can be found at httpswwwdartmouthatlasinteractiveappsendoflifecancercare qualitative data will be deidentifiedand made available to researchers through the ninrfunded palliative careresearch cooperative qualitative data repository after analyses in support ofthe primary aims are complete all materials and instruments developed forthis study are available by request of the authorsethics approval and consent to participatethis study has been approved by the dartmouth college committee for theprotection of human subjects study00031129 this study is consideredminimal riskconsent for publicationnot applicable 0cknutzen bmc palliative care page of competing interestsall authors declare no competing interest with respect to the researchauthorship or publication of this author details1department of behavioral social and health education sciences rollinsschool of public health emory university atlanta ga usa 2the dartmouthinstitute for health policy and clinical practice geisel school of medicinedartmouth college lebanon nh usa 3department of general internalmedicine boston medical center boston ma usa 4evidera pharmaceuticalproduct development bethesda md usa 5department of medicine geiselschool of medicine hanover nh usa 6norris cotton cancer center atdartmouthhitchcock medical center lebanon nh usareceived july accepted august refere | Colon_Cancer |
" vibrio cholerae are oxidasepositive bacteria that are classified into various serotypes based on the osurface antigen v cholerae serotypes are divided into two main groups the o1 and o139 group and the nono1nono139 group o1 and o139 v cholerae are related to cholera infection whereas nono1nono139 v choleraenovc can cause choleralike diarrhea a pubmed search revealed that only cases of necrotizing fasciitis causedby novc have been recorded in the scientific literature to date we report the case of a japanese woman whodeveloped necrotizing fasciitis caused by novc after traveling to taiwan and returning to japancase presentation a 63yearold woman visited our hospital because she had experienced left knee pain for thepast days she had a history of colon cancer stage iv t3n3 m1a and had received chemotherapy she hadvisited taiwan days previously where she had received a massage she was diagnosed with septic shock owingto necrotizing fasciitis she underwent fasciotomy and received intensive care she recovered from the septic shockhowever after weeks she required an aboveknee amputation for necrosis and infection her condition improvedand she was discharged after weeks in the hospitals with the increase in tourism it is important for clinicians to check patients travel history cliniciansshould be alert to the possibility of necrotizing fasciitis in patients with risk factors necrotizing fasciitis caused bynovc is severe and requires early fasciotomy and debridement followed by intensive postoperative carekeywords necrotizing fasciitis vibrio cholerae taiwan massage septic shock polymyxin b correspondence kei610805gmailcom1emergency department minaminara general hospital ooazafukugamiooyodocho yoshinogun nara japanfull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0ctsuruta bmc infectious diseases page of vibrio cholerae are curved gramnegative rod gnrbacteria that are oxidase positive they are classified intovarious serotypes based on the o surface antigen vcholerae serotypes are divided into two main groups theo1 and o139 group and the nono1nono139 group o1 and o139 v cholerae are related to cholera infection whereas nono1nono139 v cholerae novccan cause choleralike diarrhea novc are found as autochthonous microbes in coastal and marine environments outbreaks of choleralike illness caused bynovc have been reported in the united states o141and o75 former czechoslovakia o37 sudan o37peru o10 o12 and mexico o14 [] moreovernovc can cause a range of extraintestinal infectionsincluding bacteremia meningitis pneumonia peritonitischolangitis salpingitis and softtissue infection seafood including oysters fishes shrimps clams musselsand apple snail is the most common source of infection a pubmed search revealed that only cases of necrotizing fasciitis caused by novc have beenreported in the scientific literature to date we reportthe case of a patient who developed necrotizing fasciitisand septic shock caused by novc which necessitatedan aboveknee amputation of her left legcase presentationa 63yearold woman visited minaminara general hospital in nara japan because she had experienced leftknee pain for days prior to her visit she had been diagnosed with colon cancer stage iv t3n3 m1a years and months previously and had undergone surgery and received chemotherapy her most recent doseof chemotherapy was administered days before herinitial consultation she had visited taiwan days previously where she had received a massage after themassage she developed gradually worsening pain in herlower left leg on presentation she was able to walkunaided and she reported her history of colon cancerand recent travel as we suspected that the pain in herleg could be due to necrotizing fasciitis we requestedmagnetic resonance imaging mri of her left lowerleg the images showed a swollen soleus muscle andposterior tibial muscle and the t2weighted imageshowed hyperintensity of the muscle tissue fig after the mri our patients condition deteriorated andthe following vital signs were observed blood pressurebp mmhg heart rate beatsmin respiratory rate breathsmin and temperature °cthe results of arterial blood gas analysis were as fol mmhglows ph paco2 mmhg hco3base excess be meql and lactate mmoll the patients laboratory test results were as followscreactive protein crp mgdl blood ureanitrogen bun mgdl creatinine mgdlprocalcitonin ngml nterminal probrain natriuretic peptide ntprobnp pgml and fibrinfibrinogen degradation products fdp μgmlinitiatedadministeredlowvenovenoushemodiafiltrationsurgery her blood pressure wasintravenous infusion of meropenem and noradrenaline wasand the patient underwentemergency surgery before the surgery the compartment pressure of her left leg was measured by simpleneedle manometry the pressures were as follows mmhg mmhg mmhg and mmhg in theanterior lateral superficial posterior and deep posterior compartments respectively some muscle tissuesin the anterior and deep posterior compartments werenecrotic for double incision fasciotomy a relaxationincision was made on her left knee and theaffected area was irrigated and debrided fig aftertheandtherefore wepolymyxin b directhemoperfusion pmxdhp to trap endotoxins andcontinuoususinghemofeel ch13 w toray medical co ltdurayasu japan as a slightly curved gnr that wasoxidase positive was detected in her blood we diagnosed her with necrotizing fasciitis and septic shockcaused by vibrio species we changed the antibioticsfrom meropenem to ceftriaxonelevofloxacin andminocycline we used the pmxdhp once again andtapered the dose of noradrenalin gradually wediscontinued noradrenalin on day postoperativelyon day postoperatively the anism was identifiedas novc theantibiotics wasconfirmed postoperatively on day and we discontinued levofloxacin table although the patientsgeneral condition improved there was a discharge ofpus from the postoperative wound on day postoperatively a second debridement was performedseveral muscles in the patients left leg including theanterior tibial muscle had become necrotic and thenecrosis had spread to her knee on day postoperatively an aboveknee amputation was performedher vital signs and laboratory data obtained since admission are shown in fig her condition improvedand she was discharged weeks after admissionsusceptibility ofdiscussion and sixteen cases of necrotizing fasciitis caused by novchave been previously reported table [] themajority of patients were exposed to seawater or hadan injury in rare cases vigorous massage is one ofthe risk factors of necrotizing fasciitis howeverthe patient in the present case had a risk of novcinfection because of colon cancer and immunosuppression due to chemotherapy as she received chemotherapy within a month thusthein this case 0ctsuruta bmc infectious diseases page of fig t2weighted magnetic resonance images of the patients lower legs a coronal image b axial image these images show that the soleusand posterior tibial muscles on the left lower leg indicated by red arrows are swollen and inflamedfig photographs of lesions in the patients leg the patients leg before surgery shows multiple large blisters 0ctsuruta bmc infectious diseases page of table susceptibility of antibioticsantibioticsampicillinminimal inhibitory concentrations piperacillinceftazidimeimipenemcilastatinamoxicillinclavulanategentamicinminocyclinechloramphenicolsulfamethoxazoletrimethoprimlevofloxacinfosfomycins s s ¦s s s ¦s ¦s ¦s ¦r the novc remains unknown assource ofthepatient did not report any exposure to sea water oreating seafood the only potential cause of injury toher left leg was the massage she received thereforewe speculate that the massage might have been thesource ofthe novc based on the circumstantialevidence we administered blood purification therapyusing pmxdhp and venovenous hemodiafiltrationfor septic shock although no previous studies havereported the use of pmxdhp for novc a studyreported the use of pmx for v vulnificus thirdgenerationtetracyclineandfluoroquinolone were used for severe vibrio infections tetracycline combined with the fluoroquinoloneorcephalosporinfollowed by oral fluoroquinolones or doxycycline wasrecommended for invasive novc infections [ ]an in vitro study revealed that cefotaxime and minocycline have a synergistic effect in the treatment forcephalosporinsaparenteralthirdgenerationfig change of vital signs and laboratory data during the hospital admission a changes in the patients vital signs during days ofhospitalization b changes in patients blood biochemistry during days of hospitalization atiii antithrombin iii crp creactive proteinfdp fibrinfibrinogen degradation products map mean arterial pressure nad noradrenaline 0ctsuruta bmc infectious diseases page of age sex underlying diseasestable clinical characteristics of patients with nono vibrio cholerae necrotizing fasciitisyear ofreportsourcesurgery amputation multiple debridementand antibiotics ticarcillinclavulanate imipenemgentamicin clindamycinrisk factors mdiabetes mellitussurvived usatreatmentoutcome country oantigen epidemiologicexposureexposure of achronic plantar ulcerto sand in abathhouse mcirrhosissurgery cefotaxime minocycline cefotaximesurvivedtaiwan f mcirrhosis congestiveheart failurecirrhosis diabetesmellitus mhepatitis csurgery ceftriaxonediedtaiwansurgical debridement ceftazidime doxycyclinediedtaiwan o56surgery antibiotics thirdgeneration cephalosporindoxycyclinediedtaiwanhandling seafood mhepatitis steroidssurgery antibiotics thirdgeneration cephalosporindoxycyclinesurvivedtaiwan mcirrhosissurgery clindamycin ceftazidime tetracyclinesurvivedtaiwansurgery antibioticssurgery antibioticsdiedtaiwandiedtaiwan m m mcirrhosis hepatitis cdiabetes mellituscirrhosis hepatitis bhepatitis c diabetesmellituscirrhosis diabetesmellitusexposure to seawaterprobable woundinfectionconsumption of rawseafoodseawater exposureinsect bite woundinfectionminor abrasionexposed to seawaterseawater exposuresurgery antibioticsdiedtaiwanseawater exposure mcirrhosissurgery antibioticssurvivedtaiwanseawater exposure mcopdsurgery antibioticssurvivedtaiwan mhiv hepatitis ccirrhosis mdiabetes mellitus michthyosis cellulitisnone mcopd chronic constructive pulmonary diseasesurgical debridement daptomycin levofloxacinsurviveditalyo137surgical debridement piperacillintazobactamfosfomycinsurgical debridement piperacillintazobactamtigecycline metronidazolesurgical debridementpenicillin gentamicin metronidazolesurvived austriadiedaustriaseawater exposuresurvived croatia o8seawater exposurev cholerae infections as patients with novcbacteremia require antibiotic treatment for at least month we administered ceftriaxone and minocycline for month necrotizing softtissue infectionscaused by novc are more lethal than those causedby v vulnificus to conclude we treated a woman with necrotizingfasciitis and septic shock caused by novc this caseillustrates that early fasciotomy and debridement arenecessary forsevere necrotizing fasciitis caused bynovc and prolonged intensive care may be requiredafter surgeryo139 vibrio cholerae ntprobnp nterminal probrain natriuretic peptidepmxdhp polymyxin b direct hemoperfusionacknowledgementsnoneauthors contributionskt tu tw kn and ku treated the patient kt tu hf reviewed the literatureand mainly wrote this report kn tw ku reviewed the literature andmodified this paper based on specialty orthopedics emergency departmentinfectious disease all authors have read and approved the manuscriptfundingnoneabbreviationsbun blood urea nitrogen fdp fibrinogen degradation productsgnr gramnegative rod mri magnetic resonance imaging novc nono1availability of data and materialsall data are included in this published 0ctsuruta bmc infectious diseases page of ethics approval and consent to participatethis case report was approved by the ethics review committee atminaminara general hospital and was conducted in accordance with thedeclaration of helsinki consent for participation is not applicableconsent for publicationwritten informed consent was obtained from the patient for publication ofthis case report and any accompanying images a copy of the writtenconsent is available for review by the editor of this journalcompeting intereststhe authors declare that they have no competing interestsauthor details1emergency department minaminara general hospital ooazafukugamiooyodocho yoshinogun nara japan 2orthopedic departmentminaminara general hospital nara japan 3infectious diseases departmentminaminara general hospital nara japan 4department of emergency andcritical care medicine nara medical university nara japanreceived march accepted august referencesgardner ad venkatraman kv the antigens of the cholera group of vibriosj hyg lond hirk s huhulescu s allerberger f lepuschitz s rehak s weil s necrotizing fasciitis due to vibrio cholerae nono1nono139 after exposureto austrian bathing sites wien klin wochenschr dobroviÄ k rudman f ottaviani d crnek sÅ¡ leoni f Å¡krlin j a rare case ofnecrotizing fasciitis caused by vibrio cholerae o8 in an immunocompetentpatient wien klin wochenschr jain akc varma ak mangalanandan kh kumar h bal a surgical outcome ofnecrotizing fasciitis in diabetic lower limbs j diab foot comp ikeda t kanehara s ohtani t furukawa f endotoxin shock due to vibriovulnificus infection eur j dermatol su ba tang hj wang yy liu yc ko wc liu cy in vitro antimicrobialeffect of cefazolin and cefotaxime combined with minocycline against vibriocholerae nono1 nono139 j microbiol immunol infect tsai yh huang tj hsu rww weng yj hsu wh huang kc necrotizing softtissue infections and primary sepsis caused by vibriovulnificus and vibrio cholerae nono1 j trauma publishers notespringer nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations morris jg nono group vibrio cholerae a look at the epidemiology of anoccasional pathogen epidemiol rev li m shimada t morris jg jr sulakvelidze a sozhamannan s evidence forthe emergence of nono1 and nono139 vibrio cholerae strains withpathogenic potential by exchange of oantigen biosynthesis regions infectimmun dalsgaard a albert mj taylor dn shimada t meza r serichantalergs o characterization of vibrio cholerae nono1 serogroups obtained froman outbreak of diarrhea in lima peru j clin microbiol isaacmárquez ap lezamadávila cm eslavacampos c navarroocaña acraviotoquintana a serotypes of vibrio cholerae nono1 isolated fromwater supplies for human consumption in campeche mexico and theirantibiotic susceptibility pattern mem inst oswaldo cruz hughes jm hollis dg gangarosa ej weaver re noncholera vibrioinfections in the united states clinical epidemiologic and laboratoryfeatures ann intern med deshayes s daurel c cattoir v parienti jj quilici ml de la blanchardière anono1 nono139 vibrio cholerae bacteraemia case report and literaturereview springerplus mubarak sj owen ca doubleincision fasciotomy of the leg fordecompression in compartment syndromes j bone joint surg am wagner pd evans sd dunlap j ballonlanda g necrotizing fasciitis andseptic shock caused by vibrio cholerae acquired in san diego californiawest j med ko w chuang y huang g hsu sy infections due to nono1 vibrio choleraein southern taiwan predominance in cirrhotic patients clin infect dis cheng nc tsai jl kuo ys hsueh pr bacteremic necrotizing fasciitis causedby vibrio cholerae serogroup o56 in a patient with liver cirrhosis j formosmed assoc tsai yh hsu rww huang kc chen ch cheng cc peng kt systemicvibrio infection presenting as necrotizing fasciitis and sepsis a series ofthirteen cases j bone joint surg am changchien ch bacteraemic necrotizing fasciitis with compartmentsyndrome caused by nono1 vibrio cholerae j plast reconstr aesthetic surg maraki s christidou a anastasaki m scoulica e nono1 nono139 vibriocholerae bacteremic skin and soft tissue infections infect dis lond ottaviani d leoni f rocchegiani e canonico c masini l pianetti a unusual case of necrotizing fasciitis caused by vibrio cholerae o137 j clinmicrobiol 0c" | Colon_Cancer |
neprilysin nep is a neutral endopeptidase it is also known by different functional names such as common acute lymphoblastic leukemia antigen calla the cluster of differentiation cd10 endoprotease endopeptidase and membrane metalloen dopeptidase nep is a member of m13 family of zinc peptidase in the body nep cleaves many peptides such as atrial natri uretic peptides btype natriuretic peptides angiotensins i ii ii en ix bradykinin substance p endothelin i ii amyloid dorphin neurotensin vasopressin etc [] the progression of various pathological conditions such as kidney and heart disease obesity diabetes [ ] few malignancies such as colon can a corresponding author email address anoopkishoremanipaledu a kishore 101016jmolstruc2020129073 elsevier bv all rights reserved cer lung cancer and melanomas [] etc is associated with the peptidase activity of nep in the us food and drug ad ministration fda approved sacubitrilvalsartan the combination of a neprilysin inhibitor and an angiotensin receptor blocker arb respectively commonly known as angiotensin receptor neprilysin inhibitor arni for heart failure with reduced ejection fraction further in clinical trials involving sacubitrilvalsartan treatment groups performed well in the renal failure population as compared to treatment with an arb valsartan alone there fore nep has gained considerable attention in the last decade for its peptide degrading property and its inhibition has therapeutic potential in multiple diseases but the known and available nep inhibitors are limited hence drug repurposing using different in silico tools can aid in speeding up the process of drug discovery for the development of new nep inhibitors the role of nep has been extensively studied in various dis eases the study report of the paradigm trial highlighted the role 0c r sankhe e rathi and s manandhar of molecular structure of nep inhibitors in the population of heart failure with reduced ejection fraction in an invivo study of subtotal nephrec tomy the renoprotective effect of sacubitrilvalsartan was found to be stronger as compared to valsartan alone according to the result of the uk harpiii trial the combination of sacubi trilvalsartan is effective and is welltolerated in the chronic kidney disease population similarly various studies are focussed on the importance of nep on chronic kidney and cardiovascular dis eases nep inhibition in streptozotocininduced diabetic mice im proved outcomes of cardiac function for heart failure with reduced ejection fraction in diabetic nephropathy the combination of the nep inhibitor thiorphan with an angiotensin receptor blocker and an angiotensinconverting enzyme ii activator showed significant improvement in the condition by modulating components of the reninangiotensin system and natriuretic peptide system the activation of the leptinaldosteroneneprilysin axis contributes to the pathogenesis of cardiac complications in obese patients in obesity and type diabetes nep inhibition showed improve ment in insulin sensitivity and glycaemic control the inhibition re sults in modulation of several peptides with glucoregulatory prop erties such as bradykinin cholecystokinin glycogen like peptide glucosedependent insulinotropic peptide secretin and vasoactive intestinal polypeptide leading to improved glucose homeostasis and weight loss a study conducted to evaluate the effect of nep on nociception concluded that nep inhibition can be a good strategy for pain management in cancers such as colon cancer [ ] lung cancer [ ] and melanomas the increased levels of nep is correlated with neoplastic progression the peptidase ac tivity of nep and its interaction with akt focal adhesion kinase is assumed to contribute to the pathogenesis of colon cancer in aggressive melanomas cd10 nep is the biomarker for detec tion a recent report has highlighted the role of arni in enhanc ing antiammatory and natriuretic peptide systems in covid patients [ ] additionally the use of arni is also recom mended for patients suffering from covid19 all these ï¬nd ings highlighted the need for designing novel nep inhibitors but de novo drug development is resource intensive and time consum ing hence drug discovery by repurposing the existing drugs can be an attractive strategy with the beneï¬t of reduced developmen tal risk especially in the case of nep inhibitors the computation repurposing is known as insilico drug re purposing in in the us approximately of drugs ap proved was through the drug repurposing approach the con cept of drug repurposing has been already practiced in cardio vascular disorders cancer obesity erectile dysfunction smoking cessation stress psychosis etc drug repurposing using al ready approved drugs reduces the time and money on preliminary screening toxicity studies clinical trials bulk manufacturing and formulation development on the other hand the establishment of new drug candidates requires lots of time and resources a good example is the case of allopurinol which was originally approved for cancer and is now available for the treatment of gout in this context we decided to identify a series of inhibitors for nep using insilico drug repurposing the protein structure of the extracellular domain of nep with sacubitralat the active metabo lite of sacubitril was used in the current study the inhibitor bind ing pocket in the protein structure of the extracellular domain of human nep pdb id 5jmy has already been revealed by schier ing nikolaus the inhibitor binding pocket contains the catalytically essential triad of his583 his587 and glu646 for our drug repurposing study the structures of fda approved drugs were downloaded from the zinc database based on the binding pocket of the nep inhibitor the high throughput virtual screening of existing fda approved drugs was done to ï¬nd out a new se ries of nep inhibitors to the best of our knowledge this is the ï¬rst study based on drug repurposing approach that is being re ported and employed for the development of nep inhibitors using receptorinhibitor complex materials and methods in the current study the maestro molecular platform version by schrodinger llc was used to perform molecular dock ing and simulation studies on an hp desktop system with linux ubuntu lts platform intel haswell graphics card 8gb ram and intel core i34160 processor protein preparation and grid generation xray crystallographic structure of the extracellular domain of human nep pdb id 5jmy was downloaded from the rcsb pro tein data bank the pdb id 5jmy has a resolution of Ëa prior to docking and simulation studies the biological unit of protein was prepared using protein preparation wizard in schrodinger suite during the process of protein preparation the protein was subjected to import and reï¬ne review and modify and minimize processes in protein preparation wizard missing side chains and residues were ï¬lled using the prime tool the active site and cat alytically important residues were retained in the protein structure the water molecules beyond Ëa were deleted and stages were generated for hetero atoms to generate low energy state protein energy minimization was done using opls3e optimized potential for liquid stimulation force ï¬eld and the prepared protein was used for molecular modelling to generate a grid around the lig and the receptor grid generation workï¬ow was used by keeping all functional residues in the grid ligand preparation the structures of fda approved drugs from zinc database were downloaded for ligand preparation the lig prep tool was employed the lowest energy 3d structures with cor ± under the opls3e related chiralities were generated at ph force ï¬eld in this process all the ligands were preprocessed which includes generation of tautomers ionization state at ph ± using epik addition of hydrogen bond charged group neu tralization and ligand geometry were optimized ligand docking all the molecular docking studies were carried out using the ligand docking tool glide gridbased ligand docking with ener getics module the glide module was used for predicting ligand protein binding modes and ranking different scoring functions are involved in glide such as highthroughput virtual screening htvs standard precision sp and extra precision xp first all the drugs were docked with htvs mode but computationally htvs docking does not use descriptor and explicit water technol ogy as used in the xp mode hence to avoid falsepositive results few drugs were reanalyzed using sp and xp modes [ ] free ligand binding energy calculation the prime module was used to determine absolute ligand binding aï¬nities to nep using mmgbsa molecular mechanics energies generalized born and surface area continuum solvation method the mmgbsa assay of top eight xp docked drugs was performed using pose viewer ï¬le of glide xp mode the prime mmgbsa method is dependent on the vsgb solvation model that uses a variabledielectric generalized born model and water as a solvent under the opls3e force ï¬eld to calculate binding energy 0cr sankhe e rathi and s manandhar of molecular structure adme analysis for the assessment of the adme proï¬le the qikprop tool from the maestro modeling platform was used the qikprop tool helps in the prediction of the druggable property of best four hits based on adme analysis during this process various descriptors such as molecular weight cardiotoxicity qplogherg predicted octanolwater partition coeï¬cient qplogpow permeability qp pcaco polar surface area psa human oral absorption oral absorption and lipinski rule of ï¬ve were calculated induced ï¬t docking ifdsp table docking score and prime mmgbsa score of top eight drugs sr no drug dock score xp kcalmol mmgbsa 01g bind sacubitrilat zinc000001533877 zinc000000001427 zinc000001851195 zinc000000402909 zinc000000601283 zinc000000000797 zinc000003831594 zinc000028973441 ifdsp was carried out using the inducedï¬t docking module from maestro molecular modelling platform based on the xp glide docking score binding energy crucial residues involved and adme analysis four zinc0 zinc0 zinc0 and zinc0 drugs were selected for ifdsp docking in ifd based on the bfactor side chains were trimmed with receptor and van der waals scaling of and respectively and a maximum of poses were set for each ligand further prime sidechain prediction and minimization were performed in which reï¬nement of all residues within Ëa of the ligands pose and side chains were performed this pro cess allows the ligand structure and conformation to accommodate nearby reorienting side chains the ligands and residues were min imized in inducedï¬t protein structure all the ligands were rigor ously docked and ifd score for each was calculated using the for mula prime_energy glide score ifd score glide_ecoul molecular dynamics md simulation the ï¬exibility of the receptor is restricted in gridbased dock ing systems like xp and ifd these do not mimic the actual bio logical systems where the protein and drug are solvated in wa ter hence to tackle this problem md simulation was performed based on the glide docking score free binding energy and ifd score four drugs were selected for md simulation for 20ns for md simulation three steps were performed viz system builder mini mization and md simulation the docked complex of protein and ligand were selected and the system model was made by prede ï¬ned spc solvent under orthorhombic boundary conditions next the system model was subjected to energy minimization until a gradient threshold reached kcalmol Ëa balanced at k tem perature and bar pressure via npt ensemble in the ï¬nal step minimized ligandprotein complex were subjected to md simula tion bioisostere replacement for optimization of adme and biological properties of top two selected compounds zinc0 and zinc0 the bioisostere replacement of functional group was performed the bioisosteric replacement tool from maestro molecular modelling platform was employed to create bioisosteric structures of better potency and adme proï¬le further the results of the generated bioisosteres were analysed through interaction of ligands with crucial amino acid residues xp glide docking score free binding energy and adme analysis results nep was prepared at a neutral ph of αhelical α subdomains were present in the extracellular domain both helical subdomains of nep are connected with the linker region ± two and essential catalytic triad are present in the central cavity of both subdomains in the central cavity the catalytically impor tant zinc atom is coordinated with the side chains of amino acid residues his583 his587 and glu646 [ ] in the protein the cocrystallized ligand sacubitrilat is bound to the active site of nep and showed crucial interactions with his583 his587 and glu646 residues a fourth interaction was provided by the car boxylate oxygen adjacent to the p1 methyl group of sacubitri lat to generate a receptor grid receptor grid generation workï¬ow was used and the cubic box of speciï¬c dimensions was generated around sacubitrilat to perform molecular docking studies ligand docking around ligands from zinc database were screened with htvs docking mode of glide panel htvs docking mode utilizes a small period to a large set of drugs by reducing the ï¬nal torsional reï¬nement and comprehensive sampling but during htvs dock ing mode the number of intermediate conformational sampling is limited hence a total of drugs with dock scores less than kcalmole were ï¬ltered and reanalyzed in sp docking mode after performing sp docking around drugs were subjected to an extensive xp docking mode of glide panel xp docking mode is more accurate avoids the possibility of falsepositive results and gives an appropriate correlation between a good pose of drugs and a good dock score finally based on xp dock score and pivotal interactions eight active drugs zinc0 zinc0 zinc0 zinc0 zinc0 zinc0 were identiï¬ed for further screening the docking score of cocrystalized ligand sacubitralat was found to be all the eight selected drugs showed docking scores between to given in table zinc0 zinc0 all the eight drugs showed similar interaction as sacubitri lat schiering nikolaus et al had reported that the hydropho bic interaction of sacubitrilat with phe544 was towards the shal low s1 pocket of nep protein the charge positive interac tion with arg717 and polar interaction with asn542 were found to be common in sacubitrilat and selected eight drugs even in this study all the eight drugs showed hydrophobic interactions with phe544 sacubitrilat also showed interactions with asn542 arg717 arg110 and arg102 our eight selected drugs showed in teractions with atleast two of the aforementioned residues insilico docking studies also showed that all the eight drugs showed in teraction with his711 which then formed a hydrogen bond with zinc causing the stabilization of zinc transition state this in teraction with zinc and its stabilization might result in decreased catalytic activity of nep as it is a zinc dependent endopeptidase nep degrades various peptide substrates at the amino sides of hydrophobic amino acids according to the reports the pro tein structure of nep consists of a large hydrophobic pocket con taining the side chains ala543 ile558 phe563 met579 val580 0c r sankhe e rathi and s manandhar of molecular structure his583 val692 and trp693 the cocrystalized ligand sacu bitrilat showed hydrophobic interaction with ala543 ile558 phe563 met579 val580 val692 and trp693 the eight se lected drugs also showed hydrophobic interaction with ala543 ile558 phe563 met579 val580 val692 and trp693 but the hydrophobic interaction with ile558 met579 and trp693 were missing in interactions of zinc0 zinc0 and zinc0 respectively sacubitrilat and the selected eight drugs showed polar pipi stacking and cation interaction with his583 the interactions with side chains of ala543 ile558 phe563 met579 val580 his583 val692 and trp693 may con tribute to inhibition of the peptidase activity of nep according to previous reports amino acid residue glu584 is important for peptidase activity and residues such as ala543 and asn542 are important for nep inhibition in the current study all eight selected drugs possess interaction with glu584 asn542 and ala543 the 2d interaction diagrams with a summary of all non bonding interactions are given in table free ligand binding energy calculation the primemmgbsa was employed to calculate the binding en ergy of the top eight drugs with selected docked poses all the 01g bind eight drugs showed stability in the docked pose with 01g bind ing energy kcalmol described in table the ing energy of cocrystallized drug sacubitrilat was found to be 9651kcalmol the cocrystalized ligand and the eight drugs were found to be stable with docked pose this ï¬nding indicates that the selected drugs may act as nep inhibitors induced ï¬t docking ifdsp after the virtual docking studies based on the ligand interac tion and binding energy of the eight drugs four ligands showing good values were taken forward for induced ï¬t docking ifd in virtual docking protocol the interactions occur between the bind ing site of the rigid protein and the ï¬exible ligand but this is not the case with the actual ligandprotein interactions in the body where the target protein undergoes backbone or sidechain move ments after binding with ligands this induces alteration in binding sites of the protein also in the body the ligand binding site on the proteins conforms to the ligand shape and binding mode ifd was conducted to resolve the shortcomings of rigid docking pro tocols ifd has two main applications ï¬rst it generates the most accurate active complex structure of ligand which is not possi ble in virtual molecular docking with rigid protein structure sec ond ifd avoids falsenegative results of virtual docking in virtual docking screening of the ligands was done with the single confor mation of ligands however in ifd conï¬rmers were generated for each ligand hence ifdsp was carried for zinc0 zinc0 zinc0 and zinc0 and a maximum of conformers were generated for each ligand based on molecular docking and binding energy further the ifd score and ligand interaction were analyzed for selected drugs the ifd score and 3d ligand interactions are given in fig zinc0 showed similar nonbonding interactions as predicted in xp docking the zinc0 exhibits a new hbond interaction with his711 with similar nonbonding interactions as observed in xp docking in ligand interactions of zinc0 the new hbond interaction was observed with his711 and lost with glu584 the hydrophobic interaction with ala543 val580 met579 phe689 val692 trp693 phe563 and phe106 was also lost similarly new hydrophobic interaction was observed with ile718 and lost with ile558 and phe544 the new pipi stacking interactions were observed with trp693 and phe106 and missing with amino acid residue his583 the pipi cation interaction with arg717 was retained and lost with arg110 as predicted in xp docking zinc0 retained hbond interaction with his711 and glu584 showed new hbond inter action with trp693 and lost hbond interaction with arg717 the new pipi stacking interaction was observed with phe106 zinc0 also showed new hydrophobic interaction with phe689 and met579 and hydrophobic interaction missing with tyr545 it also showed similar hydrophobic interaction patterns with other amino acid residues as predicted in xp docking adme analysis adme properties of the four drugs were analyzed using the quikprop module the adme proï¬le was assessed using vari ous descriptor calculations such as molecular weight qplogherg qplogpow qppcaco human oral absorption psa and lipinski rule of ï¬ve given in table all the selected drugs obey the lip inski rule of ï¬ve molecular dynamics md simulation molecular dynamics is used to simulate ligandprotein com plexes in presence of systems with biological relevance it includes the explicit solvent representation with the entire protein the main advantage of md stimulation is that it represents the actual conditions of the biological system it provides a highly dynamic protein structure and the ligandprotein complex is solvated with water as happens in the biological system ifd however pro vides limited ï¬exibility which is insuï¬cient to mimic the actual conditions of a biological system hence md simulation studies were carried out to get insights into the top four drugs in terms of binding stability and nonbonding interactions with crucial amino acid within the drugbinding pocket of nep protein in a dynamic state in md simulation the frame was captured for 20ps which results in the generation of frames for 20ns stimulation time and saved in a trajectory further rmsd root mean square devi ation for nep protein and lig ï¬t prot for the ligands were com puted to estimate the stability of ligandprotein complex based on molecular docking score binding energy and ifd score the md simulation was carried out for four ligand protein complexes viz zinc0 01427nep docked complex complex zinc0 01533877nep docked complex complex zinc0 0601283nep docked complex complex and zinc0 03831594nep docked complex complex for com plex rmsd values for protein and ligand were found to be Ëa and Ëa respectively the rmsd values were found to be in the acceptable range Ëa but the drift in the ligandprotein complex was observed for a period of 05ns20ns in case of complex the ligandprotein stabilization was observed from 022ns and 59ns respectively and drift was observed for 720ns in complex the rmsd values are Ëa and Ëa for protein and ligand respectively for complex the rmsd values were found to be Ëa for both the complex was initially stable but there was drift for 313ns and eventually stabilization was observed for 1320ns according to the results obtained from md simulation complex is possibly more stable than complex and similarly complex showed rmsd value of Ëa for both the protein and the ligand the com plex showed initial drift from to 13ns but eventually stabi lized from 1320ns overall better stability in protein and ligand was observed in complex and compared to complexes and the rmsd plot of selected ligandprotein complexes are given in fig further the binding pattern and nonbonding interactions were analyzed for all four complexes the binding pattern was found to be different for all four complexes in complex the signiï¬ 0cr sankhe e rathi and s manandhar of molecular structure table 2d interaction diagrams of top eight drugs with a summary of all nonbonding interactions sr no drug 2d ligand interaction diagrams nonbonding interaction sacubitrilat zinc000001533877 zinc000000001427 zinc000001851195 hbond glu584 his711 arg717 arg102 asn542 hydrophobic met579 val580 ile558 phe689 val692 trp693 phe563 phe106 ile718 ala543 phe544 polar his583 his587 asn542 salt bridge zn806 arg102 pipi stacking trp693 his583 charged positive arg102 his711 arg717 arg110 charged negative asp650 glu646 glu584 hbond arg717 glu584 ala543 asn542 hydrophobic ile718 phe689 val692 trp693 ala543 phe544 met579 val580 phe106 ile558 phe563 polar thr721 his587 his583 asn542 salt bridge zn806 his711 arg110 pipi cation his583 charged positive his711 arg717 arg110 charged negative glu646 asp650 glu584 hbond ala543 his711 glu584 hydrophobic ile558 phe544 ala543 val580 met579 ile718 phe689 val680 trp693 phe563 phe106 polar asn542 his583 his587 salt bridge zn806 pipi stacking his583 trp693 charged positive arg717 his711 charged negative asp650 glu646 hbond glu584 his711 ala543 trp693 hydrophobic ile718 ile558 ala543 phe544 phe689 ala690 val692 trp693 met579 val580 phe563 phe106 polar thr721 his587 his583 asn542 salt bridge zn806 pipi stacking trp693 charged positive arg717 his711 arg110 charged negative asp650 glu646 glu584 zinc000000402909 hbond his711 glu584 hydrophobic ile718 ala543 phe544 phe689 val692 trp693 met579 val580 phe106 phe563 polar his587 his583 asn542 pipi stacking phe106 his583 salt bridge zn806 charged positive arg717 his711 charged negative asp650 glu646 glu584 continued on next page 0c r sankhe e rathi and s manandhar of molecular structure table continued sr no drug 2d ligand interaction diagrams nonbonding interaction zinc000000601283 zinc000000000797 hbond his711 glu584 hydrophobic phe544 ala543 trp693 val692 phe689 val580 met579 phe106 ile558 phe563 polar his587 his583 asn542 salt bridge zn806 pipi stacking his583 pipi cation arg717 arg110 charged positive arg102 arg110 his711 arg717 charged negative asp709 glu646 glu584 asp650 hbond asn542 hydrophobic ile718 val580 met579 phe689 val692 trp693 ile558 ala543 phe544 phe563 phe106 polar his587 his583 asn542 salt bridge zn806 pipi stacking his711 phe544 his583 pipi cation his711 charged positive arg717 his711 charged negative glu646 glu584 asp650 zinc000003831594 hbond glu584 his711 arg717 hydrophobic val580 ala543 phe544 tyr545 phe106 phe563 ile558 trp693 val692 polar his587 his583 asn542 salt bridge zn806 charged positive arg717 his711 arg110 arg102 charged negative glu646 glu584 zinc000028973441 hbond glu584 his711 hydrophobic met579 val580 phe544 ala543 phe106 trp693 val692 phe563 ile558 polar his587 his583 asn542 salt bridge zn806 pipi stacking phe106 pipi cation arg110 his711 charged positive arg717 his711 arg110 arg102 charged negative glu646 glu584 asp650 0cr sankhe e rathi and s manandhar of molecular structure fig 3d ifd ligand interactions and scores of the top four selected drugs ligand interaction of a zinc0 b zinc0 czinc0 0601283d zinc0 with different amino acid residues of nep fig rmsd plot of ligandprotein complexes rmsd plot of a zinc0 b zinc0 c zinc0 d zinc0 with the active site of nep 0c r sankhe e rathi and s manandhar of molecular structure table adme analysis of top four selected drugs using qikprop compound id molecular weight qplogp ow qplogherg qplogs qppcaco oral absorption psa rule of ï¬ve sacubitrilat zinc000001533877 zinc000000001427 zinc000000601283 zinc000003831594 fig ligandprotein interaction diagram obtained after md stimulation ligand interaction of a zinc0 b zinc0 c zinc0 d zinc0 with different amino acid residues of nep cant hbond interactions were observed with amino acid residues glu584 ala543 and his711 and pipi interaction with his583 and trp693 as predicted in xp docking the hydrophobic interac tions with ala543 trp693 met579 and phe689 were retained in md simulation on the other hand hydrophobic interactions with ile558 phe544 and phe563 were missing in md simulation the hydrophobic interaction with ala543 val580 ile718 val692 and phe106 was weaker affecting the stability of the ligand protein complex similarly the water bridgetype interaction with glu584 was observed in complex strong hbond interaction was shown by asn542 arg717 glu584 and ala543 additional hbond interactions were also observed with his711 and glu646 the hydrophobic interaction with ala543 ile718 phe689 trp693 met579 val580 ile558 phe106and phe563 were weakly con tributing to the stability of ligandprotein complex and the inter action was lost with the amino acid residue phe544 additional water bridge type of interaction was shown by asn542 glu646 and ala543 the pipi cation interactions were retained with his583 as predicted in xp docking in complex hbond interac tion was retained with glu584 and his711 and new hbond inter action was observed with asp709 and arg110 in md simulation complex showed weak hydrophobic interaction with ala543 phe544 val580 trp693 phe563 ile558 and phe106the hy drophobic interaction was lost with amino acid residues met579 phe689 and val692 the new pipi stacking interaction was ob served with his711 however pipi stacking interaction was missing with his583 the new pipi cation interaction was observed with arg717 and pipi cation interaction was missing with arg110 as compared to xp docking the additional water bridge type of inter action was shown by asp709 and glu584 in complex hbond interaction was retained with his711 and arg717 new hbond in teractions were found with trp693 and ala543 whereas hbond interaction was lost with glu584 complex showed strong hy drophobic interaction with trp693 and ala543 whereas weak hy drophobic interaction with val680 phe106 phe563 ile558 and val692 in contrast to xp docking similarly hydrophobic interac tion was missing with amino acid residues phe544 and tyr545 the additional water bridge type of interaction was observed with ala543 among all four complexes complexes and were found to more stable the additional hbond interactions in complexes and may contribute to the stability of the ligandprotein com plexes the ligandprotein md interaction diagrams and histograms of selected complexes are given figs and bioisostere replacement the zinc0 indomethacin a nonsteroidal anti ammatory drug and zinc0 tyropanoic acid a ra diocontrast agent were found to be more stable in md simulation for 20ns the zinc0 is antiammatory antipyretic 0cr sankhe e rathi and s manandhar of molecular structure fig histogram of ligandprotein complexes histogram of a zinc0 b zinc0 c zinc0 d zinc0 with different amino acid residues of nep and analgesic in nature it is commonly used in rheuma toid arthritis acute shoulder pains osteoarthritis spondylitis and acute gouty arthritis zinc0 is known as sodium tyropanoate which is employed in xray diagnosis and imaging of gallstones though they exhibit good binding aï¬nity for nep one of the major disadvantages of zinc0 is its rapid elimination from the body [ ] therefore bioisostere re placement of zinc0 and zinc0 was per formed to enhance biological activity and surpass rapid excretion bioisosteres are the molecules which are generated by replace ment an atom or a group of atoms from the parent drug with other functional groups two main advantages associated with bioisostere replacement are ï¬rst it will result in generation of new bioisostere molecules with similar biological characteristics of the parent drug second bioisosteres can overcome various prob lems associated with the parent drugs activity pharmacokinetics and toxicity during the bioisosteric replacement and bioisosteric structures of zinc0 and zinc0 respec tively were generated out of these the top two bioisosteres were identiï¬ed based on the ligand interactions with the crucial amino acid residues of nep docking score the binding energy calculated employing mmgbsa and adme parameters the top two selected bioisosteres of zinc0 and zinc0 are il lustrated by fig the docking scores of the bioisosteres of zinc0 structure structure are and with binding en ergies and kcalmol respectively similarly the dock ing scores of structure and of zinc0 were found to be and with binding energies and Ï Ïkcalmol respectively table further assessment was done based on the ligand interactions with crucial amino acid residues of the protein compared to the parent drugs table structure of zinc0 | Colon_Cancer |
bcl9 and pygo are bcatenin cofactors that enhance the transcription of wnt targetgenes they have been proposed as therapeutic targets to diminish wnt signaling output inintestinal malignancies here we find that in colorectal cancer cells and in developing mouseforelimbs bcl9 proteins sustain the action of bcatenin in a largely pygoindependent mannerour genetic analyses implied that bcl9 necessitates other interaction partners in mediating itstranscriptional output we identified the transcription factor tbx3 as a candidate tissuespecificmember of the bcatenin transcriptional complex in developing forelimbs both tbx3 and bcl9occupy a large number of wntresponsive regulatory elements genomewide moreover mutationsin bcl9 affect the expression of tbx3 targets in vivo and modulation of tbx3 abundance impactson wnt target genes transcription in a bcatenin and tcflefdependent manner finally tbx3overexpression exacerbates the metastatic potential of wntdependent human colorectal cancercells our work implicates tbx3 as contextdependent component of the wntbcatenindependenttranscriptional complexintroductionthe wnt pathway is an evolutionarily conserved cell signaling cascade that acts as major drivingforce of several developmental processes as well as for the maintenance of the stem cell populations within adult tissues nusse and clevers deregulation of this signaling pathway resultsin a spectrum of consequences ranging from lethal developmental abnormalities to several forms ofaggressive cancer nusse and clevers most prominently colorectal cancer crc is initiatedby genetic mutations that constitutively activate wnt signaling kahn secreted wnt ligands trigger an intracellular biochemical cascade in the receiving cells that culminates in the calibrated expression of target genes mosimann this transcriptionalresponse is orchestrated by nuclear bcatenin that acts as a scaffold to buttress a host of cofactorsto cisregulatory elements occupied by the tcflef transcription factors valenta among the cofactors the two paralogs bcl9 and bcl9l referred to as bcl99l and pygo12proteins reside within the wntbcatenin transcriptional complex and their concerted action isrequired to efficiently activate wnttarget gene expression kramps parker for correspondencekonradbaslerimlsuzhch kbandreasmoorbsseethzchaemclaudiocantuliuse cc these authors contributedequally to this workpresent address ¡division ofmolecular pathology thenetherlands cancer instituteamsterdam netherlandscompeting interests theauthors declare that nocompeting interests existfunding see page received april accepted august published august reviewing editor roel nussestanford university unitedstatescopyright zimmerli this is distributed under theterms of the creative commonsattribution license whichpermits unrestricted use andredistribution provided that theoriginal author and source arecreditedzimmerli elife 20209e58123 107554elife58123 of 0cshort reportdevelopmental biologyfigure the intestinal epitheliumspecific recombination of pygo12 does not recapitulate the effects of deleting bcl99l a schematic representationof the wntbcatenin transcriptional complex with emphasis on the socalled chain of adaptors components bcatenin bcl99l and pygo wntfigure continued on next pagezimmerli elife 20209e58123 107554elife58123 of 0cshort reportfigure continueddevelopmental biologyresponsive element wre the homology domains hd13 of bcl99l are shown b epithelialspecific pygo12 deletion via vilcreert2 pygo12ko does not lead to any obvious histological or functional defect neither in the small intestine nor in the colon as seen by hematoxylin and eosinstaining left panels the proliferative compartment detected via ki67 right panels seems also unaffected also refer to the count in figure figuresupplement 1de c quantitative rtpcr detecting lgr5 mrna extracted from colonic epithelium of control black bcl99l blue or pygo12 redconditional mutants ko d weekold male mice were treated with five tamoxifen tam injections ip mgday for five consecutive days days later mice were treated with dextran sodium sulfate dss ad libitum in drinking water for days while of control mice n wereseverely affected or died due to the dss treatment red lines of conditional bcl99lko n mice performed poorly in this test deletion ofbcl99l increased significantly the death rate after dss treatment pvalue000013 in fishers exact test no difference between pygo12ko andcontrol mice could be measured of control mice n and of pygo12ko n were affected upon dss treatment pvalue05626 infishers exact test e weekold female mice were exposed to a single dose of the carcinogenic agent azoxymethane aom followed by daysof dss administration in the drinking water this regimen results in the emergence of dysplastic adenomas that are collected for rna extraction andanalysis of the indicated targets via rtpcr wnt target genes and genes expressed during epithelialtomesenchymal transition emt associated withcancer metastasisthe online version of this includes the following figure supplements for figure figure supplement efficient epithelialspecific pygo12 deletion does not lead to obvious defectsfigure supplement intestinal epitheliumspecific recombination of pygo12 does not recapitulate the effects of deletingbcl99l van tienen figure 1a during vertebrate development their requirement in thebcateninmediated transcription appears to be contextdependent cantu li and they also have evolved bcateninindependentfunctions cantu cantu curiously however bcl9 and pygo always seem to act as a duetkennedy importantly bcl99l and pygo proteins were found to significantly contribute to the malignanttraits typical of wntinduced crcs deka gay jiang mani mieszczanek moor talla and brembeck theseobservations provided impetus to consider the bcl9pygo axis as relevant targetable unit in crclyou mieszczanek talla and brembeck zimmerli however here we noticed an apparent divergence between the roles of bcl99l and pygo proteins we found that genetic abrogation of bcl99l in mouse crc cells results in broader consequences than pygo12 deletion suggesting that bcl9 function does not entirely depend on pygo12among the putative bcateninbcl9 interactors we identified the developmental transcription factortbx3 intriguingly we show that also during forelimb development bcl99l possess a pygoindependent role in this in vivo context tbx3 occupies bcateninbcl9 target loci genomewide andmutations in bcl99l affect the expression of tbx3 targets finally tbx3 modulates the expression ofwnt target genes in a bcatenin and tcflefdependent manner and increases the metastaticpotential of human crc cells when overexpressed we conclude that tbx3 can assist the wntbcatenin mediated transcription in selected developmental contexts and that this partnership could beaberrantly reactivated in some forms of wntdriven crcsresults and discussionwe induced intestinal epitheliumspecific recombination of pygo12 loxp alleles pygo12ko thatefficiently deleted these genes in the whole epithelium including the stem cells compartment figure figure supplement 1a and b consistently with recent reports mieszczanek talla and brembeck and similarly to deletion of bcl99l deka mani moor pygo12ko displayed no overt phenotypic defects figure 1b figure figure supplement 1ce we were surprised in noticing that the expression of lgr5 themost important intestinal stem cell marker and wnt target gene barker was heavilydownregulated upon loss of bcl99l but unaffected in pygo12ko figure 1c to address the functionality of the stem cell compartment in these two conditions we subjected both bcl99l andpygo12 compound mutants koregeneration by dss treatmentkim figure 1d while bcl99lko mice showed a defect in regeneration after insultdeka pygo12ko proved indifferent when compared to controllittermatesfigure 1d while we cannot exclude that pygo12 also contributes to the wntbcateninto a model ofintestinalzimmerli elife 20209e58123 107554elife58123 of 0cshort reportdevelopmental biologydependent transcriptional regulation our results highlight that the bcl99l function in the intestinalepithelium homeostasis and regeneration does not entirely depend on pygo12 this was surprising since bcl99l proteins were thought to act as mere bridge proteins that tethered pygo tothe bcatenin transcriptional complex figure 1a fiedler mosimann both bcl9 and pygo proteins have been implicated in colorectal carcinogenesis gay jiang mieszczanek talla and brembeck we tested if the consequence of the deletion of bcl99l and pygo12 genes was also different in the context of carcinogenesis specifically we looked at the contribution to gene expression in chemicallyinduced aomdsscolorectal tumors figure 1e as previously observed bcl99lko tumors exhibit a massive decreasein wnt target gene expression epithelialtomesenchymal transition emt and stemness traitsdeka moor which was not observed in pygo12ko tumors figure 1efigure figure supplement 2a and b this phenotypic difference is consistent with a recentstudy in which bcl99l but not pygo12 loss reduced the activation of wnt target genes induced byapc lossoffunction mieszczanek we interpret this as an independent validation ofour observation all these experiments open up the question of how bcl99l imposes its functionindependently of pygosurprisingly the intestinespecific deletion of the homology domain hd1 of bcl99lfigure 2a that was previously annotated to interact only with pygo12 cantu kramps i suppressed the metastatic phenotype of the aomdss tumors while deletion of pygo12 did not figure 2b and ii induced a strong downregulation of wnt target emt andstemness genes figure 2c figure figure supplement the discrepancy between the geneexpression changes induced by recombining pygo12 or deleting the hd1 domain of bcl99l impliesthat currently unknown proteins assist bcl99l function we set out to identify new candidate bcl9partners that might be responsible for the different phenotypes to this aim we performed a pulldown of tumor proteins expressing either a fulllength or a hd1deleted variant of bcl9 followedby mass spectrometry figure 2d among the proteins differentially pulled down by control but notby mutant bcl9 we detected tbx3 figure 2d and e and selected it for further validation the invivo deletion of the hd1 domain in bcl99ldhd1 embryos leads to severe forelimb malformationswhile pygo12ko embryonic forelimbs are unaffected figure 2falso see schwab limb development thus represents another context where bcl99l appear to act independently ofpygo of note tbx3 plays a fundamental role in the development of this structure frank we confirmed cytological vicinity between transfected tagged versions of bcl9 and tbx3 byproximity ligation assay pla figure figure supplement 2ab however overexpressionbasedin vitro coimmunoprecipitation experiments could not detect any stable interaction between thesetwo proteins suggesting absence of direct binding or a significantly lower affinity than that betweenbcl9 and pygo figure figure supplement 2c hence we aimed at testing the functional association between tbx3 and bcl9 in a more relevant in vivo context to this aim we collected ca forelimbs from dpc wildtype mouse embryos and subjected the crosslinked chromatin toimmunoprecipitation using antibodies against bcl9 salazar or tbx3 followed bydeepsequencing of the purified dna chipseq figure 3a by using stringent statistical parameters and filtering with irreproducible discovery rate idr we extracted a list of high confidencebcl9 and tbx3 peaks figure 3b and c surprisingly we discovered that bcl9 occupies a largefraction ca 23rd of the tbx3bound regions figure 3d suggestive of a role for tbx3 within thewntdependent transcriptional apparatus motif analysis of the common tbx3bcl9 target loci identified statistical prevalence for tcflef and homeobox transcription factor consensus sequencesbut not for any tbx transcription factor figure 3e this suggests that tbx3 interacts with the dnain these locations via affinity to the wntbcatenin cofactors rather than via direct contact with dnaaccordingly tbxspecific motifs were detected within the group of tbx3 exclusive peaks which donot display bcl9 binding figure figure supplement notably tbx3 and bcl9 occupancywas detected at virtually all previously described wntresponsiveelements wre within known wnttarget genes figure 3fto test whether the in vivo abrogation of the simultaneous interactions mediated by bcl99lwould influence the expression of genes associated with tbx3 peaks we set out to mutate the bcl99l interaction domains while leaving tbx3 protein unaffected we combined different bcl99l allelesin which the hd2 bcatenininteracting and hd1 pygonew cofactorinteracting motifs arezimmerli elife 20209e58123 107554elife58123 of 0cshort reportdevelopmental biologyfigure identification of tbx3 as a putative bcl9 cofactor a the deletion of the hd1 pygointeracting domain of bcl9 and bcl9l induces avariation in the chain of adaptors causing the loss of pygo association with the wntbcatenin transcriptional complex cantu bimmunofluorescence staining of tumors collected from control or conditional pygo12ko and bcl99lko mice prox1 red and dapi blue are shownin in the top panels vimentin green and laminin red in the bottom panels c quantitative rtpcr of selected groups of targets compare it withfigure continued on next pagezimmerli elife 20209e58123 107554elife58123 of 0cshort reportfigure continueddevelopmental biologythe same analysis of pygo12ko in figure 1e of rna extracted from control or bcl99ldhd1 tumors d experimental outline of the tumor proteinspulldown and massspectrometry tbx3 was identified among the proteins potentially interacting with bcl9 but not with bcl9dhd1 e the ipproteins analyzed by mass spectrometry were in parallel subjected to sds page electrophoresis and probed with an antitbx3 antibody upper panelthe expression of tbx3 in control compared to bcl99ldhd1 tumors n was evaluated via qrtpcr bottom panel to exclude that differentialpulldown was due to lost expression in mutant tumors f dpc bcl99ldhd1 embryos display forelimb malformations and absence of digitsemphasized by dashed white lines a characteristic tbx3mutant phenotype upper panels the limb defect is absent in pygo12ko embryosbottom panels underscoring that bcl99l act in this context independently of pygo12the online version of this includes the following figure supplements for figure figure supplement qrtpcr of target genes associated with intestinal stem cell functionfigure supplement cytological proximity of bcl9 and tbx3deleted cantu double heterozygous animals for the hd1 bcl9dhd1 bcl9ldhd1 orthe hd2 bcl9dhd2 bcl9ldhd2 deletions are viable and fertile the cross between them leads to atransheterozygous genetic configuration in which both domain deletions are present bcl9dhd1dhd2bcl9ldhd1dhd2 referred to as bcl99ld1d2 figure 1a as in these mice bcl99l retain both thehd1 and the hd2 domains in heterozygosity this allelic combination is a way of testing the consequences of abrogating the tripartite complex mediated by the two interacting motifs of bcl99lwithout causing a full lossoffunction of these proteins bcl99ld1d2 embryos also display forelimbmalformations the cause of which cannot be due to pygo figure 2f but must be caused by thefailure of recruiting the new hd1interacting partner by bcl99l onto the bcatenin transcriptionalcomplex we collected forelimbs from control and bcl99ld1d2 mutant embryos at and measured gene expression via rnaseq figure 3g we found a significant enrichment hypergeometrictest p14e6 of tbx3 targets among the genes differentially expressed in bcl99ld1d2 mutantsfigure 3h the enrichment was particularly significant when considering downregulated genes inbcl99ld1d2 mutants indicating that the bcl9tbx3 partnership sustains transcriptional activationfigure 3h of note the design of our experiment directly implicates that these tbx3 transcriptional targets are also bcatenindependent the overlap list includes several regulators of limbdevelopment such as meis2 capdevila irx3 li and eya2 grifone figure 3h heat map on the right despite being of correlative nature this analysis supportsa model in which bcl99l and tbx3 cooperate to the activation of target genesso far we have presented genetic evidence that bcl9 proteins require additional cofactors andthat tbx3 associates with the bcateninbcl9 bound regions on the genome possibly influencing theexpression of target genes however the similarity of genomic binding profiles between tbx3 andbcl9 might be due to their binding in different cells and the decreased expression of genes withnearby enhancers bound by bcl9 and tbx3 might imply a requirement for bcl99l but not necessarily for tbx3 we reasoned that our hypothesis in which bcl9 functionally tethers tbx3 to the bcatenin transcriptional complex raises several testable predictions that will be addressed belowfirst our model implies that tbx3 could impact on wnt target gene expression and its activityshould be dependent on the main constituents of the wntbcatenin transcriptional complex second if tbx3 is tethered by bcl9 to its targets mutations in bcl99l should influence the ability oftbx3 to physically associate with wres finally as for bcl9 tbx3 should be capable of enhancingthe metastatic potential of colorectal cancer cellsto test our first prediction implying a potential role of tbx3 in the transcription of wnt targetgenes we overexpressed it in hek293t cells and monitored the activation status of wnt signalingusing the transcriptional reporter supertopflash stf consistent with its role as repressor tbx3led to a moderate but significant transcriptional downregulation that was importantly specific tothe stf but not the control reporter plasmid figure 4a upon wnt signaling activation achievedvia gsk3 inhibition tbx3overexpressing cells exhibited a markedly increased reporter activity whencompared to control cells in particular at nonsaturating pathway stimulating conditions figure 4aleft panel importantly tbx3 proved transcriptionally incompetent on the stf if the cells carriedmutations in tcflef d4tcf or ctnnb1 encoding for bcatenin dbcat doumpas strongly supporting the notion of its cellautonomous involvement in the activation of canonical wnttarget gene transcription figure 4a central and right panels respectively endogenous wnt targets showed a similar expression behaviour to that of stf upon tbx3 overexpression figure zimmerli elife 20209e58123 107554elife58123 of 0cshort reportdevelopmental biologyfigure tbx3 and bcl9 occupy wnt responsive elements wre in vivo a artistic representation of the chipseq experimental outline bc barplots showing the genomic distribution of highconfidence bcl9 peaks b total and tbx3 peaks c total d overlap of the highconfidence peak groups between bcl9 and tbx3 e selected result entries from motif analysis performed on the bcl9tbx3 overlapping highconfidence peaks significant enrichment was found for tcflef and homeobox motifs no tbx consensus sequence was detected in this analysis ffigure continued on next pagezimmerli elife 20209e58123 107554elife58123 of 0cshort reportfigure continueddevelopmental biologyselect genomic tracks demonstrating occupancy of bcl9 and tbx3 within the wnt responsive element wre of known wnttarget genes axin2ccnd1 nkd1 and lef1 and genes important in limb morphogenesis hand1 and hand2 the scale of peak enrichment is indicated in the topleftcorner of each group of tracks in light blue the bcl9 salazar and in orange the tbx3 replicates and in green the control track igggenomic tracks are adapted for this figure upon visualization with igv integrative genomic viewer igv two independent replicates forbcl9 and tbx3 chipseq experiments are shown g volcano plot displays all the differentially expressed genes degs in developing forelimbs uponmutation of bcl99l bcl99ld1d2 vs ctrl degs were a total of p005 with upregulated and downregulated n of individualmouse embryos for each condition were used for this analysis h a significant portion of degs exhibited overlap with tbx3 chipseq peaksthe overlap with tbx3 chipseq peaks appeared statistically significant in particular when the downregulated genes were considered hierarchicalclustering of samples ctrl versus bcl99ld1d2 right panel based on genes overlapping between degs and genes annotated for tbx3 chipseqpeaks normalized rnaseq read counts wards clustering method euclidian distance annotation added for genes associated by gene ontology townt signaling fgf10 ptk7 kremen1 zfp703 bmp2 and gli3 and genes known as regulators of limb development meis2 irx3 and eya2the online version of this includes the following figure supplements for figure figure supplement overlap of the highconfidence bcl9 and tbx3 peaks in developing murine limbs reveals the existence of bcl9 exclusive oneexample displayed in the genomic tracks on the left and tbx3 exclusive peaks one example in the genomic track on the rightfigure supplement while our experiments show that tbx3 can influence the expression of wnttarget genes the mechanisms by which this occurs remain to be elucidatedwe then addressed our second hypothesis in which bcl99l are required for tethering tbx3onto wres we performed chip of tbx3 in hek293t cells followed by quantitative pcr to detectenrichment on the wre present in the axin2 promoter jho consistent with an effecton transcription in the absence as well as in the presence of chir99021 chir figure 4a tbx3 wasbound to this region both in off and in on conditions figure 4b we then exploited ahek293t clone devoid of both bcl9 and bcl9l db99l van tienen and tested iftbx3 was capable of physical association with the wre of note enrichment of tbx3 in db99 l cellswas dramatically reduced to background levels figure 4b while we cannot exclude that tbx3might act independently of bcl99l on several of its targets this observation supports the notionthat bcl99l are responsible for tbx3 recruitment on classical wres figure 4c this also suggeststhat the previously identified targets of both bcl9 and tbx3 figure 3df must display simultaneous cooccupancy of these two factors in agreement with the notion that bcl99l are themselvesrecruited by the tcfbcatenin axis the physical association of tbx3 with the axin2 promoter wasalso lost in d4tcf and dbcat cells figure 4bfinally we evaluated the effects of tbx3 overexpression oe on growth and metastatic potentialof hct116 human colorectal tumor cells a representative model of crc driven by activating mutations in ctnnb1 mouradov using a in vivo zebrafish xenograft model rouhi approximately labelled control or tbx3oe hct116 cells were implanted in theperivitelline space of hours postfertilization hpf zebrafish embryos figure 4d three days afterinjection tbx3oe cells displayed a marked increase in number in the caudal hematopoietic plexusfigure 4ef the main metastatic site for cells migrating from the perivitelline space rouhi of note tbx3oe hct116 cells maintained consistently high expression of tbx3 within fishembryos throughout the experiment and this was accompanied by increased wntbcatenindependent transcription as measured by axin2 expression figure 4g while this experiment does notallow to exclude that tbx3 might also act independently of bcl9bcatenin in this context it showsthat increased expression of tbx3 enhances proliferation and migratory capability of human crccells bearing constitutively active wnt signaling and this is associated with simultaneous enhancement of the wntbcatenindependent transcription figure 4gtaken together our experiments show that in specific developmental and disease contexts thetranscription factor tbx3 can take active part in the direct regulation of wnt target genes by functional interplay with the bcateninbcl9dependent transcriptional complex our study suggests anew paradigm in which tissuespecific cofactors might be the key to understand the spectrum ofpossible transcriptional outputs observed downstream of wntbcatenin signaling nakamura moreover tbx3 has been linked to different cancer types willmer our observations suggest that tbx3 or its downstream effectors could be considered as new relevant targetsto dampen crc progressionzimmerli elife 20209e58123 107554elife58123 of 0cshort reportdevelopmental biologyfigure tbx3 controls the expression of wnt target genes a bcatenintcf luciferase reporter stf assay in parental left bcatenin knockout dbcat center and tcf knockout d4tcf right hek293t cells cells were treated with the indicated concentration of chir or dmso overnightoverexpression of tbx3 oe black bars compared to control ev empty vector white bars showed that tbx3 acts as a repressor on a wnttcfpathway reporter but switches to activator upon pathway induction only significant pvalues p005 are indicated three independent experimentsfigure continued on next pagezimmerli elife 20209e58123 107554elife58123 of 0cshort reportfigure continueddevelopmental biologyn are shown note that the logarithmic scale on the yaxis of the histogram on the left is different from the linear scale of the central and middlepanels b chip followed by qpcr in hek293t cells treated with dmso wntoff or chir wnton enrichment was identified on axin2promoter and the downstream enhancer note that the enrichment on the enhancer is only present upon pathway stimulation we interpret this asevidence for the enhancer looping onto the promoter occurring when the wntdependent transcriptional regulation is active the data are normalizedto immunoprecipitation performed in cells transfected with an empty vector ev and presented as the mean ± standard deviation of independentexperiments the fold enrichment of tbx3flag on axin2 promoter and enhancer n is lost upon mutations in bcl99l db99l n cnttb1dbcat n and tcflef d4tcf n c schematic representation of the axin2 locus indicates the position of the primers used black arrowsto test the binding of tbx3 despite the apparent absence of direct physical interaction between tbx3 and bcl99l the data support a model of tbx3recruitment by bcl99l onto the bcatenintcf transcriptional complex d schematic diagram of the human crc zebrafish xenografts model parentaland tbx3 overexpressing hct116 colorectal tumor cells were harvested and labeled with dii dye red the stained cells were injected into theperivitelline space of day old zebrafish embryos zebrafish were visualized with fluorescent microscopy at day post injection dpi and three dpi andprimary tumor cell invasion and metastasis were counted e representative images of hct116 tumor invasion and dissemination at and dpi inzebrafish xenografts for both control and tbx3 overexpressing cells the red asterisks indicate the position of the primary tumor red arrowheadspoint at clusters of disseminatingmetastatic cells f scatter plot representing the quantification of primary tumor growth and metastasis after hct116xenograft horizontal bars represent the mean value only significant pvalues p005 are displayed g quantitative rtpcr confirmed continuedincreased expression of tbx3 while hct116 disseminate through zebrafish tissue and that this is accompanied by enhanced wntbcatenintranscription as seen by axin2 expression each datapoint represents the extraction of total rna from pools of zebrafish embryos figure legendof figure supplementsthe online version of this includes the following figure supplements for figure figure supplement axin2 and nkd1 are here considered as representative wnt transcriptional targetsmaterials and methodstreatment of mice and histological analyseshomeostasis weekold male and female mice bcl9floxfloxbcl9lfloxflox vilcreert2 and bcl9floxfloxbcl9lfloxflox no cre littermates pygo1floxfloxpygo2floxflox vilcreert2 and bcl9floxfloxbcl9lfloxflox no cre were treated with five tamoxifen injections ip mgday sigma for five consecutivedays and the small intestine and colon were analyzed at different time points thereafter mouseexperiments were performed in accordance with swiss guidelines and approved by the veterinarianoffice of vaud switzerlandinduction of dss colitis weekold male mice were treated with five tamoxifen injections ip mgday for five consecutive days days later dss mg mp biomedicals catno was administered ad libitum in the drinking water for daysinduction of tumors weekold female mice were treated with five tamoxifen injections ip mgday for five consecutive days days later they were injected ip with mgkg body weightdmh 2hcl nn dimethylhydrazine dihydrochloride after another days later dss was administered ad libitum in the drinking water for daysmice were monitored clinically for rectal bleeding prolapse and general signs of morbidityincluding hunched posture apathetic behavior and failure to groomthe relative body weight in was calculated as follows x weight at a certain dayweight atthe first day of dss treatment epithelial damage of dss treated mice was defined as percentage ofdistal colon devoid of epitheliumto determine proliferation rates mice were injected ip with mgkg brdu sigma hr priorto sacrifice small intestines and colons divided into three equal segments to be named proximalmiddle and distal colon were dissected flushed with cold pbs cut open longitudinally and fixed in paraformaldehyde for hr at rt and paraffin embedded sections mm were cut and used forhematoxylineosin and alcian blue staining and for immunohistochemistry the primary antibodiesused were rabbit antisynptophysin dako rabbit antilysozyme dako mouse antiki67 novocastra mouse antibrdu sigma antibcatenin bd pharmigenantiactive caspase cell signalingthe peroxidaseconjugated secondary antibodies used were mouse or rabbit envision dakoor mouse antirat hrp biosourcezimmerli elife 20209e58123 107554elife58123 of 0cshort reportdevelopmental biologyrealtime pcr genotypingto determine the deletion rate the intestinal epithelium was separated from the underlining musclethe intestine was dissected flushed with pbs cut open longitudinally and incubated in mm ethylenediamine tetraacetic acid edta and mm dithiothreitol dtt in pbs for hr at rt on arotor the tubes were shaken vigorously the muscle removed and the epithelium centrifuged andused for genomic dna extraction sybr green realtime pcr assays were performed on each sample analyzedchromatin immunoprecipitationforelimb buds were manually dissected from ca rjorlswiss outbred dpc mouse embryoschromatin immunoprecipitation was performed as previously described cantu brieflythe tissue was dissociated to a single cell suspension with collagenase 1mgml in pbs for hr at Ëc washed and crosslinked in ml pbs for min with the addition of mm ethylene glycolbissuccinimidyl succinatethermo scientific waltham ma usa for proteinprotein crosslinkingsalazar and formaldehyde for the last min of incubation to preserve dnaprotein interactions the reaction was blocked with glycine and the cells were subsequently lysed in ml hepes buffer sds tritonx m nacl mm edta mm egta mmhepes chromatin was sheared using covaris s2 covaris woburn ma usa for min with the following set up duty cycle max intensity max cyclesburst max mode power tracking the sonicated chromatin was diluted to sds and incubated overnight at Ëc with mg of antibcl9abcam ab37305 or antitbx3 santacruz sc17871 or igg and ml of protein ag magneticbeads upstate the beads were washed at Ëc with wash buffer sds deoxycholate triton x100 m nacl mm edta mm egta mm hepes wash buffer sds sodium deoxycholate triton x100 m nacl mm edta mm egta mmhepes wash buffer m licl sodium deoxycholate np40 mm edta mmegta mm hepes and finally twice with tris edta buffer the chromatin was eluted with sds m nahco3 decrosslinked by incubation at Ëc for hr with mm nacl extractedwith phenolchloroform and ethanol precipitated the immunoprecipitated dna was used as inputmaterial for dna deep sequencing the pull downs | Colon_Cancer |
colorectal carcinoma crc as one of the most commongastrointestinal malignancies has developed the worldsfourth most deadly cancer with a high rate of incidence andmortality [ ] liver metastasis which is the most commonform for crc metastasis is the leading cause of high mortality for this severe malignancy however few clinical prevention and treatment measures could be available for tumormetastasis therefore it is really urgent to develop new biomarkers and explore the underlying mechanism for crcmetastasis and eventually develop new therapeutic strategiesfor crc patientsduring cancer progression metabolic reprograming withincreasing glucose utilization is termed as warburg aï¬ectwhich is accompanied by altered pyruvate and mitochondrialmetabolism the fate of pyruvate is the core manifestationto distinguish normal cells and tumor cells through metabolism in normal cells pyruvate was used for eï¬cient atpproduction directly into mitochondria however pyruvatewas converted into lactate in cytosol despite of normoxicand hypoxic conditions in cancer cells this may be dueto the impaired process of pyruvate from the cytosol intothe mitochondrial matrix which is a critical metabolic steplinking glycolysis and mitochondrial oxidative phosphorylation the mitochondrial pyruvate carriermpc a 0c of immunology researchmultimeric complex containing two distinct proteins mpc1and mpc2 which is located in the inner mitochondrialmembrane is responsible for eï¬cient mitochondrial pyruvate uptake loss of mpc expression or activity blockspyruvate entry into the tca cycle which results in a metabolism switch to increase glycolysis and the compensatoryusage of glutamine [ ]existed studies have reported that mpc1 was related withimmunoregulation stemness metabolism cellular morphology etc [ ] currently the important role of mpc1was uncovered in several tumors in crc and esophagealsquamous cell carcinoma decreased mpc1 results in accelerated aerobic glycolysis and malignant progression [ ] inlung adenocarcinoma mpc1 deï¬ciency accelerates lung adenocarcinoma progression through the stat3 pathway in prostate cancer mpc1 was reported to be involved instemness and metabolism which regulated by couptfii[ ] in renal cell carcinoma hypoxiainduced loss ofmpc1 enhanced the expression of mmp7 and mmp9 to promote cell invasion collectively these data suggestedthat mpc1 maybe serves as a suppressor to disrupt tumormalignancy however whether mpc1 is involved in crcmetastasis and the underlying mechanisms remain to beillustratedin the present study we ï¬gured out the relationshipbetween the mpc1 expression and crc liver metastasiswe identiï¬ed that decreased mpc1 was closely correlatedwith patients metastasis as well as led to poor prognosisfunctionally mpc1 overexpression could attenuate themigration and invasion capacities of crc cells both in vitroand in vivo mechanically mpc1 suppressed crc metastasisthrough mediating the wntcatenin signaling thus ourï¬nding ï¬rstly revealed a critical role of mpc1 in crc livermetastasis materials and methods data mining seven geo datasets gse21510 gse5206gse20916 gse9348 gse89393 gse67675 and gse4183and tcga were used to analyze the mpc1 expression pattern in crc the primary data for tcga datasets weredownloaded wwwcancergov the primary data feo datasets were downloaded at wwwncbinlmnihgovgeo the oncolnc database httpwwwoncolnc was used to detect the prognostic value of mpc1 inthe tcga cohort patients and clinical specimens in our study a total of patients containing paired crc tissues and adjacentnontumor tissues were enrolled from the department of gastrointestinal surgery renji hospital school of medicineshanghai jiao tong university among them cases ofliver metastases were collected and only cases wereavailable with complete followup data for survival analysisinformed consents were signed by all patients the researchwas approved by the research ethics committee of renjihospital and carried out in accordance with ethical standardsas formulated in the helsinki declarationtable sequences of primers used for realtime pcrprimermmp7 forwardmmp7 reverseecadherin forwardecadherin reversesnail1 forwardsnail1 reversemyc forwardmyc reversegapdh forwardgapdh reversesequence ²²gagtgagctacagtgggaacactatgacgcgggagtttaacatatgagtgtcccccggtatcttcacgagcagagaatcataaggcggtatccagagctgtttggaaacattttcctcccaggccatcacagccctcactcacacagattccacaaggtgcctgggctacactgagcaccaagtggtcgttgagggcaatg cell culture and cell transduction human crc celllines hct116 ht29 sw620 rko sw480 and lovoand mouse crc cell line mc38 were gained from the cellbank of the chinese academy of sciences shanghai chinaall cells were cultured in dmem medium supplemented°with fetal bovine serum and antibiotics at c in ahumidiï¬ed incubator with co2mc38 cells were transfected with lentivirus containing aluciferase reporter plasmid stable transfection cells werescreened with μgml blastisidin for days which termedmc38luc in addition one short hairpin rna shrnasequence against mpc1 was packaged as lentivirus andtransfected into mc38luc cells lovo and sw480 were transfected with lentivirus containing fulllength human mpc1cdna or empty vehicle control stable transfection cells werescreened under μgml puromycin for days and veriï¬edby western blot in those assays all lentiviral transfectionswere performed in the presence of μgml polybrene immunohistochemical ihc the protocol of this assayand quantify the mpc1 protein expression level were performed according to previously reported primary antibodies used as follows mmp7 yt2663 immunoway ecadherin cst snail1 ab53519 abcam and mycyp0861 immunoway cellular immunoï¬uorescence assays were performedaccording to the previous description brieï¬y cells wereincubated with antibodies against catenin ab32572abcam and incubated with alexa 488conjugated secondaryantibody the nuclei were stained with ²6diamidino2phenylindole dapi western blotting wholecell lysates or nuclear proteinwas extracted using a protein extraction buï¬er beyotimeshanghai china or nucleoprotein extraction kit sangonbiotech c500009 respectively proteins were resolved bysdspage and transferred onto nitrocellulose nc membranes using standard methods primary antibodies used asfollows mpc1 ab74871 abcam catenin ab32572abcamlamin ac ab8984 abcam and gapdhab9485 abcam speciesspeciï¬c secondary antibodies used 0c of immunology researchtable related enzyme and carrier of pyruvate analyzed in this studygenepdha1pdhbpdk4pdhxmpc2pdk2pdk1pdk3mpc1pcpdp1pdprpdha2pdp2pklrdescriptionpyruvate dehydrogenase lipoamide alpha pyruvate dehydrogenase lipoamide betapyruvate dehydrogenase kinase isozyme pyruvate dehydrogenase complex component xmitochondrial pyruvate carrier pyruvate dehydrogenase kinase isozyme pyruvate dehydrogenase kinase isozyme pyruvate dehydrogenase kinase isozyme mitochondrial pyruvate carrier pyruvate carboxylasepyruvate dehydrogenase phosphatase catalytic subunit pyruvate dehydrogenase phosphatase regulatory subunitpyruvate dehydrogenase lipoamide alpha pyruvate dehydrogenase phosphatase catalytic subunit pyruvate kinase liver and rbclocationmitochondrion matrixmitochondrion matrixmitochondrion matrixmitochondrion matrixmitochondrion inner membranemitochondrion matrixmitochondrion matrixmitochondrion matrixmitochondrion inner membranemitochondrion matrixmitochondrion matrixmitochondrion matrixmitochondrion matrixmitochondrion matrixmitochondrionas follows irdye goat antimouse igg licor andirdye goat antirabbit igg licorttest was used for comparison between groups p wasconsidered as statistically significantsynthesized using the primescript realtime pcr total rna was extracted using trizoltakara according to the manufacturersinstructionscdna wasreversetranscriptionpolymerasechain reaction rtpcr kittakara the qpcr was performed using sybr green masct method was used to analyze theter mix roche the data and gapdh was used as a loading control the primersequences are listed in table liver metastasis model this study was performed inaccordance with the recommendations in the guide for thecare and use of laboratory animals and relevant chineselaws and regulations the protocol was approved by theinstitutional animal care and use committee iacucof shanghai jiao tong university the mc38 cells transplanting luciferaseexpressing were injected into the spleensof c57bl6n mice n with a concentration of cellsmouse two weeks later the mice were killed andthe liver metastasis tissues were harvested luciferase reporter assay the protocols of this assaywere operated in accordance with previous reported ng top reporter plasmid wntcatenin signalingor ng fop reporter plasmid negative control ofwntcatenin signaling and ng renilla were mixedand transfected into crc cells using lipofectamine dualluciferase reporter assay system promega was usedto detect the ï¬reï¬y and renilla luciferase activities statistical analysesdata are shown as means ± sd spss chicago il usaand graphpad prism software were used to manipulate statistical analyses kaplanmeier method was used to calculatecumulative survival time the chisquare test or students results mpc1 expression was aberrantly decreased in crcpyruvate is a pivotal intermediate in the process of cellmetabolism which connects glycolysis and the tca cycleto determine the potential maladjustment genes involvedin pyruvate metabolism which is located in mitochondriaas shown in table we analyzed the tcga dataset containing crc and their normal counterparts the resultsshowed that multiple genes are significantly upregulated ordownregulated in crc t tissues compared to normal colonn notably mpc1 had a log2 fold change less than figures 1a and 1b as known to us pyruvate translocation from the cytoplasm to the mitochondria is the ï¬rst stepinto the tca cycle which needs mpc1mpc2 heterodimerin the analysis no significant change was found in mpc2therefore mpc1 was selected for further study the expression pattern of mpc1 was further analyzed in ï¬ve independent geo datasetsgse21510 gse5206 gse20916gse9348 and gse4183 consistently we found thatmpc1 was downregulated with statistical diï¬erence in crctissues figure 1c and inï¬ammatory tissue supplementary figure in comparison to their normal counterpartsmeanwhile we found decreased mpc1 expression inhuman crc tissues compared to their normal counterpartsfigure 1d in addition a similar phenomenon wasrevealed in aomdss induced mouse crc modelsfigure 1e then we evaluated the protein level of mpc1used a tissue microarray containing matching cancerand corresponding adjacent nontumortissues whichsubjected ihc staining the expression of mpc1 wasscored as based on the staining area andintensity figure 1f we found that more lower mpc1expression score as and was presented in crc 0c of immunology researcheulavp goltcga t nmpc1mpc2log2 fold changenoisserpxe cpm evitalertcgan normaltumornoisserpxe cpm evitalergeo datasetsgse21510 gse5206 gse20916 gse9348normaltumorabhuman samplecrcnormalaom dssnormalccrcesacesacesacesacdempc1 lower expressionmpc1 higher expressionf egatnecrepromutlamrongfigure expression pattern of mpc1 in crc a volcano plot showed fold changes xaxis and corresponding p values log10 yaxis ofpyruvate metabolismrelated genes located in mitochondria analyzed in the tcga dataset between paired normal and crc samplesstudents ttest b the comparison of mpc1 expression in tumor and matched normal tissues using the tcga dataset students ttest p c expression analysis of mpc1 in tumors and corresponding normal tissue using four independent geo datasetsgse21510 gse5206 gse20916 and gse9348 students ttest p d ihc staining of mpc1 expression in matched crctumor and nontumor tissues scale bar μm e ihc staining of mpc1 expression in the aomdss induced crc mouse models andcontrol animal scale bar μm f representative ihc staining of mpc1 expression in tissue microarray from the ren ji cohortwhich contained crc patients and paired adjacent normal tissue n the tumor tissues were divided into two groups based on theexpression level which scored as scale bar μm g the percentage of tissue displaying diï¬erent expression level ofmpc1 in crc tumor and adjacent nontumor tissues fishers exact test p tissues figure 1g overall these results revealed thatmpc1 was disrupted during crc decreased mpc1 enhanced tumor metastasis capabilityand predicated poor prognosis in crc it is well known thatcrc is one of the most malignant tumors given its strongmetastasis ability so we tried to ï¬gure out whether thempc1 expression was correlated with metastasis we foundthat lower mpc1 expression was closely correlated withmetastasis p lymph node invasion p and tnm stage p which revealed by the analysisbetween the clinical signiï¬cance and mpc1 expression incrc table next to illuminate the expression patternof mpc1 in diï¬erent process of crc data mining was carried out by two independent geo datasets gse21510 andgse89393 which contained normal tissue primary crcand metastatic lesion in the liver mcrc as shown infigures 2a and 2b mpc1 expression was gradually downregulated in patients with an increase in metastasis ability asimilar result was found in mice cells ct26 with high livermetastasis hmct26 or poor liver metastasis pmct26figure 2c which isolated by in vivo selection in an orthotopic mouse model of colon cancer metastasis to the liverfurther analysis showed that mpc1 protein expression was 0c of immunology researchtable clinicopathological correlation of mpc1 expression in theren ji crc cohortclinicopathological featureexpression ofmpc1lowhighp value Ï2testage years¥gendermalefemalemetastasisyesnolymph node invasionyesnotumor size cm¥ cmtnm stageiiiiiiivkras mutationmutationno mutationgradually downregulated in normal tissue primary crc andliver metastasis crc crlm tissues figure 2d furthermore survival analysis showed that patients with lowermpc1 expression had a worse outcome compared to thepatients with higher mpc1 expression using the tcgacohort figure 2e and ren ji cohort figure 2f additionally among patients with metastasis worse prognosiswas emerged in the mpc1 low cases figure 2g while nosignificant association was observed in patients withoutmetastasis figure 2h mpc1 overexpression impaired crc cells motility bothin vitro and in vivo to evaluate the role of mpc1 on themotility of crc cells the transwell assay was performedfirstly we examined low mpc1 protein expression in humanand high mpc1 protein expression in mouse mc38 crccells by western blot figure 3a mpc1overexpressingstable cell lines were established using a lentivirus carrying the mpc1 gene in lovo and sw480 cells and theoverexpression eï¬ciency was conï¬rmed by immunoblotsfigure 3b mpc1 knockdown in lucmc38 cells wereestablished and veriï¬ed by wb figure 3c mpc1 overexpression exhibited significantly weaker migration and invasion ability than the control cells in both lovo figure 3dand sw480 figure 3e cells following the liver metastasismodel of crc was established by spleen orthotopicallyinjecting mc38 cells transplanting luciferaseexpressingwhich would simulate mc38 metastasis to the liver throughsplenic veinportal vein the results revealed that the mpc1knockdown promoted mc38 cells metastasis to the liverthrough detecting the luminescence intensity monitoredby bioluminescence imaging figure 3f notablythenumber of metastatic liver nodules in the mpc1 silencinggroup wasin the control groupfigure 3g histological examination also proved thatmpc1 knockdown decreased the metastatic potential ofcrc in vivo figure 3gthan thatsmaller decreased mpc1 activated the wntcatenin pathwayby promoting nuclear translocation of catenin to furtherexplore the underlying mechanism of mpc1mediated inhibition of crc metastasis the tcga database was used toperform gsea analysis the results indicated that mpc1was involved in the wntcatenin signaling when set themrna expression median as a cutoï¬ figure 4a anddualluciferase reporter gene assay revealed that mpc1 overexpression obviously inhibited the activity of wntcateninpathway figure 4b which conï¬rmed the result abovewe then tested the distribution changes of catenin innuclear and cytoplasmic which was a crucial step inwntcatenin pathway as shown in figure 4c no significant diï¬erence was emerged in the total amount ofcatenin between mpc1 overexpression cells and the control cells figure 4c however obviously decreased distribution of nuclear catenin was presented in mpc1overexpression cells compared to that in the control cellsas revealed by the stronger gray corresponding to cateninfigures 4c and 4d consistently immunoï¬uorescenceif staining showed that mpc1 overexpression weakenednuclear catenin localization in both lovo and sw480 cellswhen compared to control cells figures 4e and 4f asimilar phenomenon was observed in mouse liver metastasistissues by ihc figure 4g following qpcr analyses displayed some downstream target genes of catenin such asmmp7 ecadherin snail1 and myc obviously increasedin ecadherin and decreased in mmp7 snail1 and mycwere observed after the mpc1 overexpression in both lovoand sw480 cells compared to that in the control cellsfigures 4h and 4i meanwhilethe opposite trendwas observed in mc38 cell after mpc1 knockdownfigure 4j and similar results were observed in mouseliver tissue detected by ihc figures 4k4n takentogether the data above indicated that mpc1 mediatedcrc cell metastasis through the wntcatenin pathway discussionaccumulating evidences have shown that reprogrammedenergy metabolism conduces to the tumor malignant propertiesincluding enhanced crc liver metastatic capacity[] mitochondria as the primary site of energy production regulate the pyruvate metabolism under both physiologic and pathologic conditions the ï¬rst step of the tcacycle is mediated by mpc which transports pyruvate into 0cnoisserpxe cpm evitalergse21510normal crc mcrcnoisserpxe cpm evitalernormalacrccrlmesacesac of immunology researchgse67675pmct26 hmct26ctcgan n p hr time daysgse89393normal crcmcrcbnoisserpxe cpm evitaler egatnecreplavivrus llarevolamroncrcmlrclavivrus llarevoren ji cohortn p hr n time monthsfdlavivrus llarevolavivrus llarevocrc with metastasis n n p hr n time monthsmpc1 higher expressionmpc1 lower expressiongecrc without metastasis n n n p hr time monthshfigure decreased mpc1 enhances tumor metastasis and predicts poor prognosis in crc a b data mining showed mpc1 expressionwas gradually decreased in normal tissue primary crc and liver lesion of metastatic crc mcrc from two independent geo datasetsoneway anova a gse21510 p b gse89393 p c data from gse67675 revealed that mpc1 expressionwas lower in high liver metastasis ct26 cells hmct26 than that in poor liver metastasis ct26 cells pmct26 students ttestp d gradually decreased mpc1 expression was presented in normal tissue primary crc and liver metastasis crccrlm tissue scale bar μm n fishers exact test p e kaplanmeier overall survival os curves in the tcgadataset of crc patients according to the mrna expression of mpc1 the lower quartile value of expression was utilized as a cutoï¬logrank test p f kaplanmeier os curve for the mpc1 expression in the ren ji cohort logrank test p gkaplanmeier os curve for the mpc1 expression in patients with metastasis logrank test p h kaplanmeier os curve forthe mpc1 expression in patients without metastasis logrank test p mitochondrial from cytoplasm in the beginning tcgawas used to analyze the relative genes involved in pyruvatemetabolism which is located in mitochondria the resultsrevealed that mpc1 expression was significantly downregulated in crc tissues meanwhile the geo datasets analysisas well as ihc staining on crc patients tissue and mousemodels conï¬rmed this trend these phenomena may indicate that loss of mpc activity enhanced tumorigenic glucoseutilization by blocking mitochondrial pyruvate uptake andoxidation interestingly in the course of data analysis wefound that the expression of mpc1 was decreased at thestage of intestinal inï¬ammation which was not diï¬erentfrom that in tumor tissue mpc1 has been reported to beinvolved in immune regulation of peripheral t cell homeostasis through metabolic regulation and a decrease inmpc1 was found at the earliest stages of crc hence 0c of immunology researchhumanmouselovosw480lovosw480mpc1gapdhmpc1mpc1gapdhmpc1mpc1gapdhwsthwsovolokrcmtchablovorotcevitnelcpmitnelrotcevitnelcpmitneldlefi rep sllec edavnidlefi rep sllec edavnirotcevitnelcpmitneldsw480rotcevitnelcpmitnelcncpmcncpmdlefi rep sllec detargimdlefi rep sllec detargimccncpmcncpmmc38shnc shmpc1¨¯ sp xufl latotcnhscpmhscnhscpmhsradiancepseccm2srfemc38suledon sisatsatemcnhscpmhsgfigure mpc1 overexpression inhibits the motility of crc cells in vitro and in vivo a mpc1 expression in human and mouse crc celllines examined by western blot gapdh serves as loading control b mpc1 overexpression in lovo and sw480 cells c mpc1 silencing by shrnampc1 in mouse mc38 cells d e transwell assays showed that upregulated mpc1 suppressed the invasion and migration ability of lovod and sw480 e cells quantiï¬cation of invaded and migrated cells was performed for ï¬ve randomly selected ï¬elds values are means ± sd frepresentative bioluminescence photograph of mice spleen implanted with luciferaseexpressing mc38 cells treated with shmpc1 or controlvector total ï¬ux was quantiï¬ed by the ivis system to verify the ability of liver metastasis g representative image of liver metastases andquantiï¬ed by the nodules in mice inoculated with mc38 cells treated with shmpc1 or control vector as well as representative images ofhe staining of the liver metastatic lesions scale bar μm students ttest p p p 0c of immunology researchse erocs tnemhcirnewnt signaling pathwaynes p mpc1 highmpc1 lowoitar pof pot evitalerð½cateninlamin acð½cateningapdhmpc1alovosw480csuelcunlatot icanmalnnetacð½ilovoncmpc1bsw480lovosw480ncmpc1dð½catenindapimergeð½catenindapimergeð½catenincpmcnovolnoisserpxe evitaleregnahc doflelovolianscympmmncmpc1nirehdacehcpmcnwsfsw480lianscympmmncmpc1nirehdaceifigure continuedcmcnhscpmhsmc38glianscympmmnirehdaceshncshmpc1jnoisserpxe evitaleregnahc doflnoisserpxe evitaleregnahc dofl 0c of immunology researchmmp7ecadherinsnail1myccmcnhscpmhscmcnhscpmhscmcnhscpmhscmcnhscpmhsklmnfigure decreased mpc1 activates the wntcatenin pathway by promoting nuclear translocation of catenin a gsea analysis ofmpc1 expression in crc using the tcga dataset nes normalized enrichment score b luciferase reporter gene assay of crc cellstreated with mpc1 overexpression or not c the expression of total catenin and nuclear catenin was detected in control and mpc1overexpression crc cells respectively gapdh and lamin ac were used as the loading control of total and nuclear protein respectivelyd the gray value analysis of nuclear catenin in mpc1overexpression cells and control cells e f mpc1 overexpression could inhibitthe nuclear translocation of catenin in crc cells scale bar μm g ihc staining of catenin in mouse liver metastatic lesionsinoculated with mc38 cells treatment with shmpc1 or control vector scale bar μm h i relative mrna expression level of catenin target genes in crc cells with mpc1 overexpression or control vector j relative mrna expression level of catenin targetgenes in mc38 cells with shmpc1 or control vector kn relative protein expression level of catenin target genes in mouse livertissue detected by ihc scale bar μm students ttest p p we suspect that mpc1 is involved in bowel inï¬ammation totumorigenesis and more studies need to be devised to illustrate this processfollowing in the analysis between the clinical signiï¬cance and mpc1 expression in crc we found that mpc1expression was especially correlated with metastasis inspiredby this we detected the mpc1 expression pattern in normaltissue primary crc and metastasis crc by geo datasetsand patients tissue all results revealed that gradually downregulated mpc1 was in patients with an increase in metastasis ability survival analysis indicated that worse outcome waspresented in patients with lower mpc1 expression especiallyin patients with metastasis additionally function assays veriï¬ed that mpc1 overexpression could attenuate the migration and invasion capacities of crc cells in vitro andmpc1 knockdown could enhance the metastasis capacityin vivo and existing studies have revealed that the mpc1was participated in metastatic dissemination of pgc1αtransduced cholangiocarcinoma through elevating reactiveoxygen species ros production besides mpc inhibitor uk5099 treatment could trigger strong invasive capacitythrough blocking pyruvate translocation into the mitochondria so as to attenuate mitochondrial oxidative phosphorylation and trigger aerobic glycolysis these phenomenatogether with our results indicate that mpc1 could act as atumor suppressor through inhibiting tumor metastasis andexisting studies have shown that mpc1 could alter the maintenance and fate of stem cells through regulating cancermetabolism in crc however the relationship betweenthe metabolism and tumor metastasis regulated by mpc1was not being mentioned thus further evidence should bereceived to conï¬rm thissubsequently the underlying mechanism of mpc1 inregulating metastasis was explored for this purpose gseaanalysis was performed the results indicated that mpc1was involved in the wntcatenin signaling the next seriesof experiments also conï¬rmed that mpc1 could mediate thewntcatenin pathway by redistribution of catenin previous reports showed that cytoplasmic catenin phosphorylated in nterminally localized to sites of cellcell contact isassociated with ecadherin and was required for intact cellcell adhesions without any change detected in the levels oftotal catenin [] simultaneously cellcell adhesionbased on cadherin binding with catenin limited wnt signals in addition catenin was reported to interact withusp9x to inhibit the degradation of catenin through thedeubiquitination of catenin in breast cancer a constitutive irs1 and catenin protein interaction activated mycexpression in acute lymphoblastic leukemia cells inhcc catenin was reported to interact with yap1 to leadto rapid tumorigenesis hence it is reasonable to guessthat accumulated cytoplasmic catenin maybe crosstalkwith other genes or involved in other biological processeswhat is more some downstream target genes of cateninsuch as mmp7 ecadherin snail1 and myc were changedin expression as known to us mmp7 is a member of theproteolytic enzyme family which promotes the invasionand metastasis of tumor cells by degrading the basementmembrane and extracellular matrix and previous studies had evidenced for involvement of mmp7 activation incolorectal cancer liver metastases [ ] ecadherin andsnail1 were considered as the epithelialmesenchymal transition emt marker which was involved in metastasis ofmalignant tumor moreover ecadherin was reportedto be involved in cellcell junction to regulate cancer invasionand metastasis [ ] and the gsea analysis also revealedthat mpc1 could aï¬ect the cellcell contacts data notshown as described previously mmp7 could facilitatemorphological transition by cleaving ecadherin thecommunication between the cells is disrupted when ecadherin was shredded leading to destructed cell adhesionand induction of emt followed by increased cell migration inspired by this we assumed that mpc1 could mediatetumor cell motility through aï¬ecting mmp7 activity cellcell 0c of immunology researchcontacts and emt however further studies need to be performed to clarify the detailed underlying mechanisms conclusionsin conclusion we ï¬rstly demonstrated that decreased mpc1was closely correlated with patients metastasis as well as ledto poor outcome moreover mpc1driven nuclear translocation of catenin contributed to crc cell motility thismeans that mpc1 has the potential to be a diagnostic biomarker and therapeutic target for metastasis patientsabbreviationscrc colorectal carcinomampc mitochondrial pyruvate carrieremt epithelialmesenchymal transitiongsea gene set enrichment analysisdata availabilitythe data used to support the ï¬ndings of this study are available from the corresponding author upon requestconflicts of interestall authors declare no conï¬icts of interestauthors contributionsyahui wang and guangang tian conceived and designedthe study chunjie xu and kaixia zhou obtained and anized the data guangang tian analyzed the data zhigangzhang and jianren gu contributed reagentsmaterialsanalysis tools xueli zhang and guangang tian wrote themanuscript guangang tian chunjie xu and kaixiazhou contributed equally to this workacknowledgmentsthis work was supported by the national natural sciencefoundation of china no to zhigang zhangand the natural science foundation of shanghai no18zr1436900 to xuelili zhangsupplementary materialssupplementary figure expression analysis of mpc1 innormal ibd and tumor tissues using the gse4183 datasetoneway anova was used to analyze the statistical diï¬erences between ibd adenoma and crc tissues ns no significance students ttest p p p supplementary materialsreferences h brody colorectal cancer nature vol no r l siegel k d miller s a fedewa et al colorectal cancerstatistics ca a cancer for clinicians vol no pp a j rauckhorst and e b taylor mitochondrial pyruvatecarrier function and cancer metabolism current opinion ingenetics development vol pp m g vander heiden l c cantley and c b thompsonunderstanding the warburg eï¬ect the metabolic requirements of cell proliferation science vol no pp s herzig e raemy s montessuit et al identiï¬cation andfunctional expression of the mitochondrial pyruvate carrierscience vol no pp c yang b ko c t hensley et al glutamine oxidationmaintains the tca cycle and cell survival during impairedmitochondrial pyruvate transport molecular cell vol no pp t bender g pena and j c martinou regulation of mitochondrial pyruvate uptake by alternative pyruvate carrier complexes the embo vol no pp a g ramstead j a wallace sh lee et al mitochondrialpyruvate carrier promotes peripheral t cell homeostasisthrough metabolic regulation of thymic development cellreports vol no pp 2899e6 x zhou z j xiong s m xiao et al overexpression ofmpc1 inhibits the proliferation migration invasion and stemcelllike properties of gastric cancer cells oncotargets andtherapy vol pp d li c wang p ma et al pgc1α promotes cholangiocarcinoma metastasis by upregulating pdha1 and mpc1 expression to reverse the warburg eï¬ect cell death diseasevol no j c schell k a olson l jiang et al a role for the mitochondrial pyruvate carrier as a repressor of the warburg eï¬ectand colon cancer cell growth molecular cell vol no pp y li x li q kan et al mitochondrial pyruvate carrierfunction is negatively linked to warburg phenotype in vitroand malignant features in esophageal squamous cell carcinomas oncotarget vol no pp h zou q chen a zhang et al mpc1 deï¬ciency accelerateslung adenocarcinoma progression through the stat3 pathway cell death disease vol no l wang m xu j qin et al mpc1 a key gene in cancermetabolism is regulated by couptfii in human prostatecancer oncotarget vol no pp y zhong x li d yu et al application of mitochondrialpyruvate carrier blocker uk5099 creates metabolic reprogramand greater stemlike properties in lncap prostate cancer cellsin vitro oncotarget vol no pp x p tang q chen y li et al mitochondrial pyruvatecarrier functions as a tumor suppressor and predicts theprognosis of human renal cell carcinoma laboratory investigation vol no pp g a tian c c zhu x x zhang et al ccbe1 promotesgist developmentthrough enhancing angiogenesis andmediating resistance to imatinib scientiï¬c reports vol no article c xu g tian c jiang et al nptx2 promotes colorectalcancer growth and live | Colon_Cancer |
"introduction a diet low in fermentable oligosaccharides disaccharides monosaccharides and polyols fodmap is an effective way to reduce gut symptoms in people with irritable bowel syndrome ibs this diet reduces the intake of fermentable fibres leading to changes of the gut microbiota and insufficient fermentation in the large bowel resulting in reduced production of short chain fatty acids scfas such as butyrate which has unfavourable implications for gut health sleep and mental health this study will examine the effect of fibre fix a supplement containing a mix of dietary fibres on the human gut microbiome composition fermentative capacity sleep quality of life qol and mental health of people with ibs who consume a low fodmap diet lfdmethods and analysis a randomised double blind placebo controlled study design is proposed to examine whether fibre fix added to an existing lfd may help modulate gastrointestinal function improve markers of sleep mental health and promote qol in patients with ibs participants will provide stool and blood samples daily bowel symptoms diaries and day diet records additionally they will complete validated questionnaires relating to fodmap intake sleep mental health and qol before and after a week intervention gut health will be assessed via faecal microbiome composition faecal ph and scfa levels alteration of sleep will be recorded using an actigraphy device worn by all participants over the whole study multivariate analysis will be used to examine the gut microbiome and repeated measures analysis of variance anova will be used for dependent variables from questionnaires related to bowel symptoms stool type sleep mental health and qol to assess the differences between intervention and control groups after adjustment for confounding variablesethics and dissemination ethics approval was obtained from the human research ethics committee of edith cowan university yan results will be disseminated in peer review publications and conference presentations participants will be provided with a summary of findings once the study is completed if fibre fix is shown to result in favourable changes in gut microbial composition scfa production sleep and mental well being without exacerbating symptoms this will provide additional dietary management options for those with ibs following an lfdtrial registration number actrn12620000032954introductionirritable bowel syndrome ibs is one of the most frequently diagnosed functional gastrointestinal disorders affecting approximately of the global adult population1 diagnosis of ibs is challenging due to the subjective nature of digestive symptoms and is currently based on the rome iv criteria3 this functional disorder typically presents with recurrent abdominal pain with alterations in bowel habits namely stool consistency and frequency coexisting bloating flatulence and abdominal distention the syndrome is subtyped into four patterns ibs with predominant constipation or predominant diarrhoea mixed ibs and un subtyped4 due to the dominance of chronic symptoms and frequently present comorbidities somatic and psychological ibs imposes a heavy burden on individuals and communities both economically and socially5 of those employed patients with ibs reported absenteeism and presentism due to their syndrome7 in two independent studies it were estimated that yan a0r et a0al bmj open gastro 20207e000448 101136bmjgast2020000448 0copen access ibs related absenteeism and presenteeism cost industry ££ or to ¬¬ annually9a diet that is low in fermentable oligosaccharides disaccharides monosaccharides and polyols fodmap10 alias a low fodmap diet lfd is an effective dietary intervention for ibs a blinded and placebo controlled trial found that approximately three quarters of patients with ibs benefit from an lf11 the lfd reduces food fibre compounds that are poorly absorbed in the small intestine rapidly fermentable in the proximal colon and thereby contribute to the gastroenterological symptoms the lfd includes a selective elimination diet for weeks followed by a reintroduction phase of fodmap containing foods followed by personalisation of a diet that minimises symptoms12 despite the positive effects of the lfd in reducing gut symptoms and improving quality of life qol in those with ibs it only treats the symptoms of ibs studies suggest potentially negative impacts of long term adherence to an lfd including nutritional inadequacy potential increased risk of gastrointestinal complications and imbalance of the gastrointestinal microbiome12 evidence from both animal and human studies has demonstrated that a low intake of dietary fibres can reduce microbiota diversity leading to increased cancer risk16 reintroduction or restoration of dietary fibres to an lfd diet can be difficult with whole food due to the coexistence of a range of fibres in individual foods this study therefore reintroduces dietary fibre using a supplement however this process should be done gradually and continuous otherwise unwanted symptoms such as gas flatulence and cramps may impact adherenceresearch suggests a low fodmap intake rapidly and negatively changes the gut microbial community abundance and diversity15 in healthy people a week lfd resulted in an alteration of the gut microbiota reduced beneficial bacteria such as actinobacteria predominantly bifidobacterium and a lower overall total bacterial count22 after a week lfd bennet 23 observed an increased dysbiosis manifested as altered gut microbial fermentation leading to lower total short chain fatty acid scfa concentrations24 in patients with ibs additionally a randomised cross over trial comparing lfd with a standard australian diet15 found a marked reduction in butyrateproducing clostridium cluster xiva and cluster iv favourable mucus associated akkerkmansia muciniphila and an increase in mucus detrimental ruminococcus torques in another study bifidobacterium and faecalibacterium prausnitzii associated with butyrate production were significantly decreased following a week lfd25 taken together these results suggest the lack of fibre associated with lfd may explain the microbial changes human gut microbiota is able to recognise and degrade different forms of complex carbohydrate26 a diet rich in dietary fibres with different extents of fermentability and solubility is recommended which means more varied and complex dietary fibres in the diet leads to a more dynamic diverse and stable gut microbiota29 various purified dietary fibres are capable of nourishing specific gut bacteria such as bifidobacteria f prausnitzii and eubacterium hallii26 dietary fibre improves the human gut microbiota by providing a substrate for fermentation and subsequently increases production of scfa it is well established in the literature that higher levels of scfa can be obtained from a higher intake of fermentable dietary fibres19 butyrate one of the major scfa throughout the colon provides the primary fuel for colonic cells to maintain growth and integrity and thereby improve gut health35 furthermore research suggests that butyrate can positively affect circadian rhythm regulation38 and enhance sleep via interplay between gut and brain40 therefore this study will increase the amount of fibre in the diet of patients with ibs to restore the gut microbiome and its metabolite profile to potentially prevent increasing the risk of patients developing other more severe gastrointestinal diseasesthirty three per cent of patients with ibs reported they had sleep problems such as sleep fragmentation poor sleep quality reduced sleep time and frequent awakening41 disordered sleep or sleep disturbances are also recorded with a greater prevalence in ibs sufferers compared with healthy individuals43 despite unknown causal relationship between impacted sleep and ibs the close association between gastrointestinal symptoms and sleep disturbances has been identified by others45 the gut microbiota is suggested to play a pivotal role and affect multiple mechanisms in this complex relationship between human sleep disturbances and gastrointestinal disease48 smith 50 found that gut microbial diversity was positively associated with total sleep time as well as sleep efficiency which also were positively correlated with phyla richness of bacteroidetes and firmicutes their findings indicate that an diet intervention represent a promising way to improve sleep by manipulating the gut microbiota to promote sleep related phyla and taxa in the human gut microbiome50 in addition it has been suggested that gut microbial composition could be altered by sleep fragmentation resulting in a reduction in actinobacteria and in bacteroidetes but a increase in firmicutes which is similar to the microbial profile of obese individuals51 many research studies have demonstrated the significant association between ibs and mental health even though the causation relationship is still unclear a meta analysis of guillaume fond et al53 concluded that patients with ibs were more likely to develop depression and anxiety than healthy volunteers groups whereas sibelli 54 found that depression and anxiety doubled the risk of ibs onset it is estimated that of patients with ibs have associated mental health conditions such as depression and anxiety55 patients with ibs have been observed with gut microbial alterations related to depression including greater rates of kynurenine a deleterious metabolite of tryptophan an elevated kynureninetryptophan ratio56 and declined lactobacillus and bifidobacterium which are also less abundant in patients with major yan a0r et a0al bmj open gastro 20207e000448 101136bmjgast2020000448 0cdepressive disorder57 clostridia a major class within the phylum firmicutes appears at increased abundance in patients with ibs58 this link to an animal study showing abundance of clostridia were significantly higher after stress related stimuli in stress vulnerable rats compared with stress resilient rats60 demonstrating that gut microbial communities respond to stress differently among animals with distinct stress vulnerability furthermore the researchers suggest a bacilli to clostridia ratio can reflect stress effects with a higher value indicating less stress derived inflammatory reactions60some inflammatory markers in human blood are associated with both human gut health and mental health and provide a potential mechanism for the role of dietary fibre in mitigating mental health a randomised controlled trial in patients with serious depression demonstrated serum concentration of high sensitivity c reactive protein hs crp and scores of beck depression inventory questionnaire significantly decreased after taking a probiotic supplement lactobacillus spp and bifidobacterium bifidum61 additionally proinflammatory cytokines like tumor necrosis factor alpha tnfα interleukin il6 and il1β are able to cross the blood brain barrier bbb and their entry and following influences can have a negative effect on mental health62 the entry of the cytokines however can be reduced by improving the integrity of blood brain barrier the permeability of bbb can be decreased by the scfa butyrate which is produced in the gut via gut bacterial fermentation of fermentable carbohydrate residue escaping from small intestinal digestion63in summary a healthier gut microbiota altered by a dietary fibre intervention or supplement in patients with ibs may not only improve gut health but also sleep mental health and qol the objective of this research study is to determine in patients with diagnosed ibs and on an lfd whether fibre fix compared with a placebo control improves gut microbial composition faecal scfa levels sleep quality qol markers inflammation and of mental well being without exacerbation of ibs symptoms this study will be the first to explore the bidirectional relationship between dietary fibres supplement and sleep modulated by the gut microbiota in patients with ibs following the lfdmethods and analysisstudy designthe study is designed as a randomised double blinded placebo controlled trial with a week intervention period figure the total time required for participant involvement is weeks including a week baseline and a week interventionsample sizethe a priori sample size for the proposed study was determined based on the results obtained by mcorist 64 where a sample size of participants per group open accessfigure flow chart showing study design overviewwas required to detect a change in log scfa concentration of at power and level of significance allowing for a drop out rate the total sample size of subjects per group is required this sample size is sufficient to detect at least a medium between within group interaction effect cohens f025 in sleep improvement at power and significance level whereby the corresponding sample size requirement is participantsfifty eight people n58 with ibs on an lfd will be recruited participants must be between and years old and have been clinically diagnosed with ibs using the rome iv edition diagnostic criteria65 by a gastroenterologist or other medical professional participants will be on an lfd for month prior to the intervention additionally participants will need to be available to attend the local clinic visits and be willing to consume the fibre fix supplement or matched placeboparticipants will be excluded if they are current smokers pregnant or planning to become pregnant have a known diagnosis of other gastrointestinal illness eg inflammatory bowel disease malabsorption of any macronutrients bowel resection coeliac disease have had previous abdominal or gastrointestinal surgeries severe mental health and sleep related conditions eg insomnia renal or hepatic diseases and major medical illness currently use pharmaceutical agents that could modify or treat ibs eg probiotics antibiotics eluxadoline lubiprostone and linaclotide or sleep conditions follow other restrictive dietary patterns or therapies eg low carbohydrate ketopaleo diet take any prebiotics yan a0r et a0al bmj open gastro 20207e000448 101136bmjgast2020000448 0copen access have any other disease condition or habit that may interfere with completion of studyrecruitmentparticipants will be recruited through networks of registered western australian based dietitians and gastroenterologists who will be provided with information flyers to promote the study to potential participants information flyers will be posted on websites including social media groups relating to ibs or fodmap a university webpage will advertise study informationallocation blinding and compliancea computer generated list of random numbers provided by a statistician will be used to randomly assign participants to either the intervention or control group the participants will receive a resealable snap lock bag labelled a or b containing sachets of either fibre fix or placebo both participant and researcher will be blinded from the group allocation bag labelling will be completed by an independent person the participants will be required to return unused sachets to calculate compliance an online daily checklist together with a weekly textemail reminder will be provided to participants to record time of consuming the intervention a daily tick listcalendar will be created for participants to follow consumption of of the sachets sachets during the week period will be considered compliantinterventionfibre fix consists of one soluble dietary fibre and one insoluble fermentable fibre which will be provided to participants in separate sachets with gradually increasing amount table after baseline data collection participants will be required to consume fibre fix as per the labels on the sachets one sachet per day for the first days and two for the remaining days according to the schedule table for participants convenience all sachets have been labelled with day and time am or pm on the package for example day am and will be provided orderly in resealable plastic bags the placebo sachet contains a combination of the same soluble dietary fibre and highly digestible fibre and will be delivered in the same way as the interventionprimary outcomesfaecal scfa and gut microbiotafaecal scfa levels and gut microbiota will be assessed through hours stool samples which will be obtained at baseline and at the end of the intervention participants will be provided with the stool collection kit including a portable cooler bag frozen icepacks and an instruction sheet all stool samples collected with the hours period will be pooled and homogenised if the number of individual samples is more than one on receipt stool samples will be immediately weighed and stored at °c individuals samples will be thawed at °c and kept at this temperature during homogenised and aliquoting for all planned analysis and re frozen at °c until analysesthe concentrations of bacterial metabolites in faeces such as scfa acetate propionate and butyrate will be determined by gas chromatography66 in brief an acidified aqueous methanol solution will be used to extract scfa from faecal samples followed by separating scfa by gas chromatography with a fatty acid column using a thermo scientific tg wax column30 m x mm x μm the scfa level will be qualified via internal standardsmicrobial analyses will be performed at the wa human microbiome collaboration centre curtin university western australia dna will be extracted using the qiaamp powerfecal dna kit qiagen using qiacube extraction platform microbiome signatures are generated using the illumina miseq platform using uniquely barcoded 16s rrna gene primers 515806v4 for bacterial and its2 primers for fungal profiling pending on funding following pcr inhibition assessment of each dna extract pcr free ligation protocol is thereafter deployed for the process of library building samples will be sequenced to a depth of minimum reads which is sufficient to identify microbes to a genusspecies level quality control and mock community samples are included in the analysis from sample collection to sequence analysis sequence read quality is initially assessed with fastqc before demultiplexing and preprocessing by ghapv2 an in house tool cutadapt67 is used for removal of all non biological sequences dada268 is then used for quality filtering error correction amplicon sequence variants asvs picking a trained naïve bayes classifier then assigns asvs to genusspecies against a curated database of microbial reference sequences such as the ribosomal database project rdp69 or genome taxonomy database70 for fungal classification the unite database71 will be usedsecondary outcomesobjective measures of sleepparticipants will be provided with the readiband v5 readiband fatigue science canada a wrist activity monitor that has been validated and objectively assesses sleep using accelerometery72 compared against polysomnographythe gold standard of sleep measurement the wrist activity monitor does not require laboratory setup table grams of dietary fibre supplement in each sachet provided to participants during week interventional perioddayam pm \\\\yan a0r et a0al bmj open gastro 20207e000448 101136bmjgast2020000448 0ctable definitions of sleep measures as extracted from the readiband fatigue science canada device based on dunican 72sleep measuresabbreviated measurement descriptionacronym unitsopen accesstime at lights outtalotime of dayhhmmsleep onset latencysolminutesmintime at sleep onsettasotime of dayhhmmtime at sleep onset variancesleep durationtasovminutessdminutesminminwake after sleep onsetfragmentation indexwasominutesminfifrequencynumbertime at waketawtime of dayhhmmtime in bedsleep efficiencytibseminutesminpercentage deriveddirectly measuredderiveddirectly measuredderivedderiveddirectly measureddirectly measureddirectly measuredderivedtime at sleep onset minus sleep onset latencynumber of minutes from time at lights out to time at sleep onsettime of day when the first epoch of sleep occurs between time at lights out and time at waketime at sleep onset consistency relative to mean time at sleep onsetnumber of minutes from time at sleep onset to time at wake minus number of minutes awake wasonumber of minutes awake after time at sleep onsetnumber of awakenings between time at sleep onset and time at waketime of day when awake with no further sleep durationthe total time spent in bed from time at lights out to time at wakesleep duration divided by time in bed multiplied by nor trained personnel72 moreover the readiband can automatically identify time at lights out using a proprietary algorithm which not only eases the burden of sleep diary but also avoids the potential bias from self reported data of recalling time for bed72 this technology has been widely applied to the sleep related research73 participants will be required to wear the monitor on the non dominant wrist for hours per day during the study the monitor derived sleep measure data will be downloaded via the automated readiband sync software table subjective measures of sleepparticipants will be required to complete five validated questionnaires related to sleep at baseline and at the end of the interventionpittsburgh sleep quality indexthe pittsburgh sleep quality index psqi retrospectively assesses sleep quality and relevant disturbances over the previous month this self administrated questionnaire has been validated in a population based setting to measure sleep quality the summary score is calculated as the summation of items grouped into seven components ranging from better to worse a score indicates poor sleep quality77epworth sleepiness scalethe epworth sleepiness scale ess is a self rated eight item questionnaire designed to measure daytime sleepiness78 participants score each question from high chance of dozing to would never doze which yield a global score of ess ranging from to scores higher than nine reflect excessive daytime sleepiness and severe problems with daytime somnolence79insomnia severity indexinsomnia will be assessed through the self reported insomnia severity index isi comprising seven items participants are required to rate each question on a point likert scale as per their own experience over the past weeks the total score ranges from to and represents clinical insomnia when it is higher than sleep hygiene indexthe sleep hygiene index shi is a self reported instrument for assessing individual behaviours in sleep hygiene the items that comprise shi are rated on a point likert scale and produce a total score ranging from to with higher scores representing poorer status of sleep behavioural hygiene81restorative sleep questionnaire weekly versionthe restorative sleep questionnaire weekly version rsq w is composed of nine questions completed on restorative aspects of the sleep during the past week and whose reliability and validity has been published82 each item scales from one to five the first two items and the last item are reversed scored the total score ranging yan a0r et a0al bmj open gastro 20207e000448 101136bmjgast2020000448 0copen access from to calculates as rsq w average score across completed itemsà the higher total scores indicate better restorative sleepmental health and qol assessmentthe condition of mental health and qol in both groups will be assessed using in total four validated questionnaires at baseline and at the end of the interventiondepression anxiety stress scalethe depression anxiety stress scale dass21 is a validated self reported questionnaire designed to measure three subscales which are depression anxiety and stress with seven items for each dimension83 higher scores are indicative of poorer mental condition and severity of symptoms but the dass21 is not a clinically diagnostic instrument nonetheless dass21 has broad applicability and free availability and has been validated among the general population and for patients with chronic disease84 scores above and in the three dimensions indicate severe depression anxiety and stress respectively in such instances participants will be referred to their medical practitioner for clinical carevisceral sensitivity indexthe visceral sensitivity index vsi is a validated self reported questionnaire and will be employed to measure gastrointestinal specific anxiety the total vsi score is generated from all items each defined on a point likert scale patients with a higher score will be regarded as experiencing severe gastrointestinal symptom specific anxiety86 ibs quality of lifefor assessment of participants qol ibs qol questionnaire will be used at the stages of baseline clinic prior to and after intervention the ibs specific questionnaire is a validated measurement tool generating one total and eight subscale scores with items covering dysphoria interference with activity body image health worry food avoidance social reaction sexual activity and relationships88who five wellbeing indexwho five well being index who5 is a validated self reported questionnaire consisting of five items that measures mental well being in relation to the past weeks responders rate each item on a point likert scale the result will be calculated by multiplying the raw total score ranging from to by four the higher scores represent those with a better imaginable well being condition89all questionnaires will be collated in the software qualtrics and administered online to reduce participant burdendemographic informationparticipants will complete a demographic questionnaire which requires personal information gender age nationality marital status area of residence mobile number email address smoking history alcohol consumption birth delivery mode dietary pattern and physical activityanthropometric measurementsparticipants height cm and weight kg will be measured to the nearest cm and kg respectively by an seca digital column scale seca usa where circumferences of waist and hips will be measured in accordance with international operating procedures for anthropometric assessment90 body mass index bmi and waisthip ratio will be calculated91 percentage of lean and fat mass will be obtained using the bod pod cosmed rome italy an air displacement plethysmograph using whole body densitometry to determine body composition fat vs lean fat mass92 and conducted following manufacturer protocols for measurement this will require subjects to fast for hours prior to testing and wear tightly fitted gym clothes for measurement blood pressure mm hg will be measured using an omron ia1b automated blood pressure device omron healthcare japan all measurements will be carried out at baseline and end of interventionblood biomarkersthe venous blood samples will be collected after an overnight fast at baseline and at the end of the intervention blood samples will be centrifuged and processed within half an hour after collection for separating plasma and serum and frozen at °c after being aliquoted into ml vials analysis of fasting lipids glucose and glycated haemoglobin will be performed by a pathology laboratory in accordance with the protocols from the national association of testing laboratories the outcome measures of hs crp tnfα il6 and il1β will be analysed if funding is made availablegut symptomsparticipants will complete a bowel symptom questionnaire at baseline and postintervention to assess changes in symptom severity the questionnaire consists of gastrointestinal symptom rating scale for ibs gsrs ibs93 and the ibs severity scoring system ibs sss94 which have been validated in clinical trials the item gsrs ibs uses a point likert scale for severity of symptoms characteristic of ibs including abdominal pain diarrhoea constipation and bloating satiety this instrument is short and simple and will assist researchers to determine the specific symptoms encountered by patients93 the ibs sss point with a maximum of for each item is also used for classification of patients as remission mild moderate or severe participants will use a visual analogue scale to score each of the five questions regarding symptom severity which include pain severity and duration abdominal distention yan a0r et a0al bmj open gastro 20207e000448 101136bmjgast2020000448 0copen accessbowel dysfunction and qol for this study a reduction of more than points of ibs sss is defined as symptoms improvement13daily symptoms checklisteach participant will be provided with an online daily bowel symptom checklist to report the sachet consuming time and daily symptoms throughout the entire study period adapted from the not for profit international foundation for functional gastrointestinal disorders iffgd personal daily diary https aboutibs org symptom diary html the checkbox include bristol stool chart type time and amount of fibre added to meals bowel movement number of motions stress level and menstrual cycle adverse symptoms monitoring scoring symptoms will be reported by participants daily and include abdominal pain constipation diarrhoea bloating flatus eructation headache nausea and vomitingitems food recordsdietary intake will be assessed using a day weighed food record preintervention and postintervention this will be completed by participants via a free downloaded smart phone application research food diary xyris queensland australia participants will be provided with a set of scales propert supertex industries and instructed on the correct recording methods for weighed diet records the monash university comprehensive nutrition assessment questionnaire will be used to specifically quantify individuals fodmap intaketable schedule of primary and secondary endpoints that will be measured over the studystatistical analysesbaseline participant demographics and primary and secondary outcome variables will be described and compared for differences by group descriptive statistics in the form of mean±sd will be used to describe continuous variables and frequencies and proportions for nominal and ordinal variables all continuous outcome and demographic variables will be examined normality using the shapiro wilk test median±iqr range will be presented instead for non normal continuous variableschange in individual and total scfa levels and faecal ph will be examined using mixed model analysis of variance anova between groups and within groups covariates including gender and age will be entered into the model as confounders to analyse the gut microbiota profiles multivariate analysis primer7 and permanova primer e plymouth and various r packages will be used principal coordinates analysis will be deployed to visualise data distance based linear table study assessment schedulestudy itembaseline period week dietary interventionw1w2w3demographic informationbmi body fat waisthip ratiogut symptoms questionnaire ibs sssgsrs ibs pittsburgh sleep quality indexepworth sleepiness scalesleep hygiene indexinsomnia severity indexrestorative sleep questionnaire weekly versiondepression anxiety stress scalevisceral sensitivity indexibs quality of lifewho five well being indexmonash university comprehensive nutrition assessment questionnaireblood samplestool samplethree day diet record vi | Colon_Cancer |
subcutaneous hydration and medicationsinfusions effectiveness safety acceptability asystematic review of systematic reviews one e0237572 101371 pone0237572editor jose´ das neves university of portoportugalreceived november accepted july published august copyright broadhurst this is an openaccess distributed under the terms of thecreative commons attribution license whichpermits unrestricted use distribution andreproduction in any medium provided the originalauthor and source are crediteddata availability statement all relevant data arewithin the manuscript and its supportinginformation filesfunding this project was partially supported viaan unrestricted project grant provided by bectondickinson canada a portion of the work of dswas funded as salary by the sponsor grant db asa former employee of medical pharmacies duringthe conduct of this research reports the researchas work independent of her employer with noemployer contributions to the research db is soleobjectiveto synthesize the current evidence for subcutaneous hydration and medication infusionsfrom systematic reviews and to assess their methodological qualityintroductionperipheral intravascular cannulacatheter insertion is a common invasive procedure foradministering fluids and medications venous depletion is a growing concern for severalpatient populations subcutaneous access for the administration of isotonic solutions andmedications is an alternative however vascular access assessment and planning guidelines rarely consider this routemethodssystematic review of systematic reviews prospero crd42018046504 we searched databases published in english language from to june identifying subcutaneousinfusions an alternate route for fluids or medication methodological quality was evaluatedusing amstar criteria and data for mechanisms of infusion and outcomes related toeffectiveness safety efficiency and acceptability extracted the johanna briggs institutesgrades of recommendation informed the strength of recommendationresultsthe search yielded potential systematic reviews were excluded through and duplicate screen of the remaining s were excluded and were includedoverall evidence is strong for recommending subcutaneous hydration infusions for olderadults weak for pediatric patients and inconclusive for palliative patients there is strongevidence for medications weak evidence supporting medications however there areeight medications with inconclusive evidence to make a recommendation and four medications not appropriate for subcutaneous delivery one 101371 pone0237572 august one 0cproprietor of infusion excellence consulting whoseservices are unrelated to this research bg ownsand is employed as senior consultant and directorof education by clinical pharmacy partners aneducational and consulting firm for infusiontherapy providers and clinicians the funders hadno role in study conception design data collectionand analysis decision to publish or preparation ofthe manuscript the specific roles of these authorsare articulated in the author contributions sectioncompeting interests db reports personal andconsulting fees ie travel and honoraria and nonfinancial support from 3m including researchgrant in unrelated field angiodynamicscardiomed medical supplies covalon freseniusand excelsior medical dbs former employermedical pharmacies provides infusion serviceshowever db affirms research was conductedindependent of her employers servicesproductsand of her current consulting business mc reportsgriffith university has received unrestrictedinvestigator initiated research or educational grantson mcs behalf from product manufacturersbaxter becton dickinson and company centurionmedical products and entrotech lifesciencesunrelated to this research ds reports no conflictsbg reports personal and consulting fees ieconsultancy travel and honoraria from nutrishareand covalon bg affirms no relationshipspresenting competing interests or influences thisdoes not alter our adherence to one policieson sharing data and materialssubcutaneous hydration and medication infusionssubcutaneous access should be considered alongside intravenous therapy for hydration inolder adults and several medications there are additional benefits in terms of ease of useand costeffectiveness of this mode inclusion of subcutaneous access in clinical guidelinesmay promote uptake of this route to help preserve vessel health of vulnerable patients further highquality research is needed to inform subcutaneous infusion therapy in a variety ofpopulations including pediatrics and palliative care and medications and clarifying themechanism of deliveryintroductioninfusion therapy is a common treatment modality to deliver medications and fluids in theacute and home care settings and is gaining prevalence in the longterm care setting traditionally these parenteral therapies have been delivered via the intravenous route howevervenous depletion is a growing concern with an increasing aging population and patients withlongterm complex comorbidities [] the problems of venous depletion are compoundedby unnecessary peripheral venipuncture which add to patient physical and psychologicaltrauma compromised intravenousrelated outcomes suboptimal use of healthcare resourcesand increased costs [ ] additionally infusion therapy is moving beyond the boundariesof the acute care and home care sector to hospices and longterm care facilities recentlypublished vascular access planning tools are either oriented primarily to the acute care settingor do not address the option of subcutaneous access [ ]the infusion therapy standards of practice recommend consideration of subcutaneousaccess for the administration of isotonic solutions and for continuous opioid and other infusion therapiesmedications eg immunoglobulin therapy subcutaneous access isachieved through the placement of a small catheter in the subcutaneous tissue with the infusate absorbed from this space into the circulatory system this route has the advantages ofrequiring a small catheter and less technical insertion skills than that used for intravenousaccess with its ease of application lending to its use in multiple settings additionallytime to place the catheter is less and fewer complications are likely resulting in cost benefits[ ] although practiced since there has been a slow uptake in part due to a lack offamiliarity with the technique among physicians and healthcare professionals and perceivedsuboptimal outcomes eg hypovolemic shock due to inappropriate use of hydration solutions [ ] in our scoping literature search to prepare for a systematic review of primarystudies numerous systematic reviews were identified however each either had a narrowscope addressing one treatment eg fisher and colleagues review of iron overload management or provided limited literature search or limited evidence eg duemsnoreiga andblascos review of subcutaneous fluid and drug deliverygiven the worlds aging population health care systems rapidly changing diversity in practice setting and complexity of care and patient health conditions the uptake and use of subcutaneous continuous infusions may help address these issues and challenges the aim of ourstudy is to determine the effectiveness safety acceptability and efficiency related to the use ofsubcutaneous infusion sci as an alternate route to intravenous for the management of conditions or treatments such as dehydration and palliation for children and adults in all care settings through a synthesis of systematic reviews of studies a secondary objective is to identify one 101371 pone0237572 august one 0csubcutaneous hydration and medication infusionsthe mechanisms of subcutaneous fluid and drug delivery that facilitated achieving these outcomes this will provide an uptodate and rigorous review of subcutaneous infusion therapymaterial and methodswe adopted the systematic review of systematic reviews methodology from the joannabriggs institutes to conduct this review the study protocol was registered with theprospero database registration number crd42018046504 and we report the reviewaccording to the preferred reporting items for systematic review and metaanalysisprisma guidelines s1 table eligibility criteriawe considered all types of systematic reviews which included the following characteristics¢ a clearly stated set of objectives with an explicit reproducible methodology¢ a systematic search that attempts to identify all studies that would meet the eligibilitycriteria¢ an assessment of the validity of the findings of the included studies eg assessment of riskof bias and confidence in cumulative estimates and¢ systematic presentation and synthesis of the characteristics and findings of the includedstudies metaanalyses where some of the primary studies in the reviews were duplicated we did not exclude thereviews as they had different aims and objectives and thus added to the overall understandingof sci to meet the aims of this current systematic review we did however only include the primary studies findings once in our analysis and synthesis expert opinionconsensus andbench research s and editorialscorrespondence were excluded no restrictionsregarding age gender diagnosis geographical location or healthcare setting were appliedwe included reviews that assessed interventions that used subcutaneous infusion for aduration of around hours or more as an alternate route for fluid or medication therapy subcutaneous infusion is defined as the delivery of fluids or medication into the subcutaneousspace for absorption into the circulation via perfusion diffusion balance between hydrostaticosmotic pressure and lymphatic drainage p118 an infusion of around hoursduration was determined from considering the studies in a review by caccialanza where the range of infusion was hours to greater than days during the full textscreening numerous intermittent sc insulin vs continuous sc insulin systematic reviewswere identified and excluded because they did not meet our inclusion criteria of sci as analternate route reviews that included other routes as comparators such as intravenous andintraosseous were excluded if data on subcutaneous infusions could not be extractedseparatelyoutcome measuresthe primary outcomes of interest investigated included¢ effectiveness defined as clinical response to therapy eg cureimproved clinical failure orno change completion of therapy within prescribed time frame¢ safety defined as medication or vascular access adverse event eg abscess erythema bruising electrolyte imbalance edema infection pain fluid overload vascular collapse and one 101371 pone0237572 august one 0csubcutaneous hydration and medication infusionsroute failure survival status eg died of underlying condition other causes lost to followup or status unknown and complications related to treatment eg unplanned hospitalreadmission related to treatment¢ acceptability defined as patient andor health care provider preference satisfaction or perceived benefits of subcutaneous therapy¢ efficiency defined as healthcare resource utilization including costs of infusion therapy supplies and treatment timesecondary outcomes included indications for subcutaneous infusion therapy medicationsolution type infusion rates volumes and duration subcutaneous access sites dwell timesand infusion control devices usedsearch strategya systematic search was conducted november and updated in june of reviews from as recommended by aromataris from the following databases excerpta medica database embase pubmed cumulative index to nursing and allied health literaturecinahl cochrane database of systematic reviews joanna briggs institute of systematicreviews and database of s of reviews of effects dare search terms included combinations of medical subject headings mesh and key word terms [subcutaneous infusionor hypodermoclysis or subcutaneous therapy] and the term systematic review with theassistance of a university librarian study resources limited inclusion to only english languageas listed in s2 table one author of a relevant was contacted to establish the status ofthe review however the review had not been completedthe title and of each were scanned independently by one reviewer ds andfull copies of s of potentially eligible reviews were obtained full texts of these reviewswere then screened independently by two reviewers mc and db against the review selectioncriteria disagreements were resolved by discussion between these reviewers and with consultation by a fourth reviewer and subject expert bgdata extraction and quality appraisaldata were extracted and assessed independently by the reviewers in pairs [bg db] [mcds] with anomalies reconciled by agreement data were obtained primarily from the systematic reviews although primary studies were consulted for critical missing data eg missinginfusion properties data extracted included authoryear aim design and number of studiesincluded search strategy population interventioncomparator quality appraisal of primarystudies infusion characteristics outcomes as previously defined key findings study limitations funding and smethodological rigour of each review was independently assessed by four reviewers in pairsas above using the amstar tool this is a 16item tool used to appraise the quality ofsystematic reviews and is frequently used in cochrane overviews a score of overall confidence high moderate low and critically low was assigned by the reviewers to depict the accuracy and comprehensiveness of the data summary and critical methodological flaws thisassessment informed the final grading of the recommendationsdata analysis and synthesisdue to the different interventions outcomes and outcome measures a metaanalysis was notfeasible quantitative outcome data are provided and synthesized where possible only data one 101371 pone0237572 august one 0csubcutaneous hydration and medication infusionsfrom randomised controlled trials rcts and prospective cohort studies were included todetermine subcutaneous hydration outcomes however reviews exploring medications alsoincluded retrospective studies and case reports the jbi grades of recommendation were usedto derive the grading score to inform the strength of recommendation for the interventionjoanna briggs institute levels of evidence and grades of recommendation working party data synthesis for hydration and medications are provided as a final summary ofrecommendations grade a indicates the interventions desirable effects outweigh undesirableeffects with adequate supporting evidence a weak recommendation grade b depictsdesirable effects outweigh undesirable effects although this is not as clear or evidence not ofhigh quality where there was insufficient evidence we determined that a recommendation isinconclusiveresultsthe search strategy yielded potential systematic reviews fig of which wereexcluded through and duplicate screen of the remaining s were excludedafter full text examination against inclusion criteria in the full text screening for reasonsdescribed as above as listed in s3 tablecharacteristics of included studiestable details the systematic reviews of subcutaneous infusion of medications andor fluidswith focused on medications only on fluids and on both types of therapies the publication dates of reviews ranged between and [ ] most reviews focused onadult populations generally or specifically for example pregnant women and adults withamyotrophic lateral sclerosis als two reviews however are specifically paediatric [ ]eight all ages [ ] two focused on older adults and paediatrics [ ] andone where age was not reported the systematic reviews incorporated a diversity of study designs and quality levels fromcase studies to systematic reviews and randomised controlled trials however only reviewsincluded only randomised controlled trials [ ] similarly sample sizes varieddepending on the number of sci studies included in the review synthesise of findings withvery few metaanalyses performed due to varied study designs outcomes and sizemethodological qualitythe amstar quality scores of the included reviews are described in s4 table while reviews achieved an overall confidence rating of high sixteen of the reviews had critical methodological weaknesses studies with moderate low and studies rated as criticallylow the main deficiencies noted were failure to report a priori protocol study design selection full details of excluded studies funding sources of primary studies and results reflectingrisk of bias assessment the cochrane risk of bias tool or an adaptation was the most commonly used tool by systematic review investigators to determine quality of the primary rctstudies n while only provided a narrative discussion of quality most reviewauthors noted that results should be interpreted with caution due to study limitationsmechanisms of subcutaneous accesslimited data were reported on the materials and techniques used to achieve subcutaneousaccess for hydration or medication therapy table describes the characteristics of sc devicedesign and dwelltime subcutaneous anatomical sites and insertion techniques and infusion one 101371 pone0237572 august one 0csubcutaneous hydration and medication infusionsfig prisma flow chart the diagram details our literature search and screening process for selection of systematic reviews included in thesystematic review adapted from moher d liberati a tetzlaff j altman dg the prisma group preferred reporting items for systematicreviews and metaanalyses the prisma statement med e1000097 101371 pmed1000097101371 pone0237572g001delivery systems in relation to studies reporting on hydration therapy no studies comparedthe safety and efficacy of the various characteristics including mode of delivery the findingsfor hydration and medication sci are presented separatelysubcutaneous hydrations5 table describes the indications and contraindications for sci for hydration as described inthe studies the most common indicator was mild to moderate dehydration with the mostcommon contraindication being rapid high fluid volume requirements one 101371 pone0237572 august one 0ctnatropmiyllacinilcmumnmiidecneirepxedahohwstneitaphtlaehdnaefilfoytilauqsuhtgivinoseacimetsyslatipsohnehwnoitpecrepfostroperdesaercnievahyamemohotdegnahcgividesabelihwseacimetsysdnalacolseidutsgicsdesabdellorneevahyamseiduts¢stneitapnitnemevorpmi¢gicsnostneitaperomderreferpdnadetarelotdnacirtaidepybgivinahtsnoitcejnigicsdesabemoh¢stneitaptludamuresggielbatpeccaseveihcayparehttnemecalpergics¢sleveldnaytienegoretehlaitnatsbusseidutsssorcanosirapmocsnoitatimilllacigoodohtemsaibfoksiretaredomecnediveytilauqwol¢mrettrohsnoylnoatadloq¢puwollofnotcefferettebaevahyamlanoitnevnocnahtloqyparehtdesisehtnysseidutsylno¢suoenatucbussuounitnoc¢icsottnavelerenositrocordyhfonoisufnicsotviderapmocstcron¢derosnopsyrtsudni¢daehotdaehecnereffidehtniecnacifingislacitsitatssawerehthguohtla¢aenpsydecuderdnafoesuehthtiwdwmnillarevoehtsnilcycatsorpehtteemtondidtceffenoitarudpuwolloftrohsllasedulcnisisylanaatemsnoitnevretnifosepyt¢¢caidracgenoitcnufcaidracerusserplairtathgirxednievorpmiyamlanitsorpertcs¢gnitarehtgnitagitsevnirofairetircfoytilauqllacigoodohtemytilauqwolseidutsytilauqetaredomytilauqhgihrnsgnittesnodesabairetircstcrnerdlihcdnastludatrohocborenarhcocyranibnodesabseidutsydobitnayramirphtiwseicneicifedgnisuselbairavonseysegnareziselpmassesabatadotpuetadyraunajotelbacilppaseidutsseidutslatotweiveremoctuo]noitatic[snoitacidemfonoisufnicsseidutsginilubolgonummiinassahobalslairtlacinilcevitcepsorp¢¢strohocevitcepsorter¢strohocytefasdnassenevitceffevisusrevcs][saibfoksirhgihmuidemseidutslanoitavresboelacsawattoeltsacwenseidutslanoitavresborofytilauqriafstcrnielpmasycneiciffusniylujotpuetadsuounitnocegnarezisrnsgnittesdnaegacsenositrocordyhstcrretnecitlumlacinilclebalnepo¢ydutsrevossorc¢noisufnissenevitceffestnemtaertlootborenarhcocstcrlanerdahtiwstneitapsesabataddedulcniseidutsdiocitrococugl][lafonlagnitroperdnanoitcelessisongaidsraeysehcraesdnahicsottnavelerycaciffelinitsorpertfoksirwolborenarhcocegaegaeziselpmassesabatadstcrtcrnilcycatsorp][senrabsaiblairetrayranomuplotpusetadnoisnetrepyhrebmetpesytefasdnasnoitatimilnoisulcnocllarevosrohtualootlasiarppaytilauqnoitalupopygetartshcraesforebmundnangisednoitnevretnimairohtuadaelsweivercitametsysfoscitsiretcarahcelbatsubcutaneous hydration and medication infusionsdeunitnocelbaileraedivorptonnacdnataienmatekfotceffeylekilemoctuoylekilfonoitacidniottnavudanajsaesodynawolsiecnediveytilauqwol¢ehtfonoitacidnielbaileron¢stceffeebnacniaprecnacnisdioipostroperoslatubnoitacidemssenderdnanoitatirrietissertemfodohserhtldetroperstneveesrevda¢ehtotdetaleryltsomevitingocdnasnoitanicullahrofdetroperecnabrutsidienmatekfosesodrehgihebotthguohttcrnidetroperydutsehtotdetalerylbissopstneveesrevdasuoiresowt¢gurdgestneveesrevda¢dedivorpborraelcnudahniaprecnaclairtlacinilcpuienmateksraeynaemegaeracevitaillapgnittespuobecalpeziselpmasotsetadceddnenajignnnigebdesiversretsigerweiverenarhcoctcr¢ytefasdnassenevitceffeicssdioipoottnavudajtcrlootborenarhcocyrotcarferhtiwstneitaptludalsupsesabatadottnavelerseidutsnasaienmatek][lleb one 101371 pone0237572 august one 0cdnadetimilsawsemoctuomretgnolfognitroper¢evisulcnocnisawecnedivefoytilauqllarevotaslairtoteudwolyrevotwolesicerpmisetamitseemoctuoborsuoiresyrevrosuoiresesuairetircnoisulcxeevitcirtserfokcaldnastnemerusaemloqdetadilavnonfoatadfognitroperaimeassalahthtiwelpoepsiofdnehwrojamrodetacidniartnocetauqedanifoesuroetuorsuonevartnirosrotalehcnorilarorehtorosuoenatucbusrehtieybdehsilbatseehtsniamerhtobcaidracesreverottnemtaertnorioteudnoitcnufsydtnemtaertofddeifisnetni¢daolrevohtiwderapmocenorpirefeddetaertstneitapnidnaofdenorpirefeddenibmochtiwhtiwderapmocofddnahtiwdetaertstneitapnienolaofddesaercnierastneveesrevda¢ecnerehdasevorpmiyparehtofdfoicsotderapmocnoitalehclarofiniatrecnu¢foksirehtsecuderyparehtnoitalehclarofiniatrecnu¢ofdfoicsotderapmocstceffeesrevdasuoiresytilatromsecuderyparehtofdfoicsotderapmocnoitalehclarofiniatrecnu¢otdesulootbordetadilavon¢elbaliavaivdehcraessesabatadylno¢niaprecnacyrotcarfertnemtaertrofnoitpollamsfonoitatimilgehtiwseidutsforebmunwefseziselpmasllamsesreviddnastcrdednilblseigoodohtemegnarseidutsseidutsfoytilauqssessaelbaivaebyamenmateki¢ecnediveehtfoytilauqehtcirtaideaprofeziselpmasicsrofonoitaredisnocasedulcniegnarstludascirtaidepseidutsydutslebalnepolairtdellortnocnustroperesacweiverevitcepsortertcr¢¢¢¢¢atadlevelhgihdetimli¢erasegasoddednemmocer¢loottnemssessaonhtiwnerdlihcdnastludasesabatadicsottnavelerseidutstnemtaertienmatek][ualderbsnoitatimilnoisulcnocllarevosrohtualootlasiarppaytilauqnoitalupopygetartshcraesforebmundnangisednoitnevretnimairohtuadaelgnitarotelbacilppaseidutsseidutslatotweiveremoctuo]noitatic[deunitnocelbatihgkgmotmorfnoissucsiddetroperelpmasniapdetalerrecnacrnsetadstludaytefasdnassenevitceffeweivernidaolrevonorignitaertrofksirwolksirraelcnutnednepednoisufsnartlacidemrehtoenorpirefedderapmocnidedulcnislairticsylno¢detacidnillitssienorpirefed¢lootborenarhcochtiwnerdlihcdnastludalsupsesabatadseidutstcrsusrevenorpirefed][rehsifrofpunidnaspuehtrofsraeydnanaemhtiwsrydnaydutsydutsyadcinilctneitaptuognittescirtaideapertneceraclatipsohelpmasaimeassalahtdnasesabatadnienolaofddnaenolaegnaregaezisseirtsigerlairtslairtesehtfosryydutsrnydutsotpusetadsuoenatucbusdetropernisryfonaemhtiwhcramofdfonoisufniienmaxoirrefseddnaytefasofdssenevitceffesubcutaneous hydration and medication infusionsdeunitnocdnaytidilavehtniecnedifnoc¢lduohsyparehtpmupfoesu¢siecnedivefoytilibicudorperhcraeserehtotdetimilebwolgnittesfoytefasgnidragersnrecnoc¢tsisrepyparehtsmrahfoelacstnemssessalanoitanadnasetisretnecsgnitarsaibfoksirllarevomargorplatanireptneitaptuoehtmorfsmetidetcelesdessessasaibnoitirttaytilauqretsammcelgnissgnittes±egarevaegnarrneziselpmasmuidemhgihwolesabataddnanoitcetedecnamrofrepsnoitcartnocmreterpsetadtrohocevitcepsortertrohocevitcepsorpseiresesacdezimodnarnonlairttcr¢¢¢¢¢ssenevitceffednaytefassisylocotrofpmupnoitcelesgnidnuofnochtiwnemowtnangerpsesabatadseidutsybenilatubretfoics][teduagegaeziselpmasgnittesegnarcinorhccinilctneitaptuolatipsohcirtaidepscinilcertnecitlumertneceraclootborenarhcocrostnecselodanerdlihclsupsesabatadksirhgihraelcnuesaesidllecelkcishtiwwolraelcnustludadnasrevigeracriehtlacidemrehtodnasesabatadstcrseidutsotecnerehdaevorpmitcrnoitnevretninoitacidemotsnoitnevretninoitacidem][nitrofaimeassalahtyraurbefyparehttnednepednoisufsnartnonotpusetadnoitalehclarohtiwssenevitcefferotnednepednoisufsnartseirtsigerlairtofdfoicsderapmocyparehtnoitalehcnori one 101371 pone0237572 august one 0csubcutaneous hydration and medication infusionsehtetacilpmocseirtnuocstluserfonoitazilarenegtnereffidnistcudorpgicshtiwloqhdevorpmiotsanoisulcnocmrifon¢emohsawtirehtehwtahtesreptnemtaertdetadtuosiseidutsredolehtnistsocfonoitamitse¢wolhtiwderewoprednudnahtiwelpoepnitceffelaicifenebdengisedylrooperewseiduts¢foecnedivetnacifingison¢llasedulcnisisylanaatem¢enietsyclytecafostifenebon¢snoitnevretnifosepytshtnomtalavivrusnidnastnapicitrapfosrebmunnoitarudtrohsfoslathgieemitforeptrohsdetareloteradebircsedstceffeataddetimilsuhtskeewstneitapybllewylbanosaerrevostceffeedisrolavivrusnoefildnasuoiresyllaitnetoparevodemrofreperewseiduts¢edisehtfoemoshguohtla¢repregnolanacsnoitacilpmocignnetaerhtecnediveytilauqdetimliderohtuadnaderosnopsstroperdesimodnarnonyrtsudniseidutsllahtiwniaprecnacfotnemtaertehtnistifenihpronerpuberehwyasottluciffidecnediveytilauqwol¢¢¢¢ehtotdetalerylniamruccohtiwmetsysyreviledgurdnoitartsinmdaisuonevartnierevestoneraohwsmotpmyssuonevartnieriuqerothguonegnitaertnielortnatropmietaredomhtiwstneitapnaevahyamlinitsorpertcs¢nilcycatsorpdetimillebduohsicssngisedsisemerepyhelbatcartniotydutsytilauqwoloteud¢eromotevisnopsernutnemtaertlanoitnevnocenihpronerpublaugnilbus¢feilerniapfotesnoretsaflamredbusotderapmocenihpronerpubdeunitnoccseraseidutsrehtofiraelcnu¢tneitapseiduts¢icsrosnoitcejnivirevocsrofecnereferpsdioipognortsfeilerniapfonoitarudralimisnisecnereffidtnacifingisonsea¢¢emitdnaemohtacsesuacebgnivasnoitcafsitasecneirepxetneitapdnaecnerehdaecneulfnilliwtnemtaertderreferptneitap¢viseliforpytefasdnaycaciffeelbarapmocdewohsyduts¢acpcsehtfoseicilopecirptnereffidesodnoitutitsbuscsehtlwocimonocehtlaehgnirusaemfosdohtemesrevidgicsdnagivineewtebseahtiwwolecnedivefoytilauq¢niecnereffidfoecnediveelttil¢snoitcefnignitroperdnanoitutitsbusecnarusnidnahtlaehlanoitansatluciffidnoitartsinmdaviidnaseirtnuocfosmetsysetaluclacotdesusdohtemowtneewtebsecnereffidrojam¢dnacsneewtebsnosirapmoc¢etaredomlanoitavresbowefmorfegnarlwolanoitavresbodetropertahtseidutsetaredomtcrtnemssessarneziselpmasycneicifedlicnuochsidewsedargroyramirphtiwstneitaplygoonhcethtlaehnoenummiyradnocessesabatadrnsetadgnitarsnoitatimilnoisulcnocllarevosrohtualootlasiarppaytilauqnoitalupopygetartshcraesforebmundnangisednoitnevretnimairohtuadaeldeunitnocelbattubertnecitlumsgnittesdnadehsilbupeganaematadrnscificepsdehsilbupnuenilesabemoctuoairetirccitsongaidksiretauqedapuwolloffossenetelpmocdnasecnereffidgnidnilbdnasraeynaemegaeziselpmasrngnittesseidutslatnemirepxeisauqrostcr¢ssenevitceffetnemlaecnocksirraelcnusisorelcslaretalsetadicsobecalpsvtnemtaerthcaorppaborenarhcoccihportoymahtiwstludasesabatadottnavelerseidutstnadixoitnacs][llerroelacstniopdadajehtdesueziselpmaslairttnavelerrofstnemssessalanoitiddahppinoisnetrepyhdetcatnocerewnoitacollafotnemlaecnoclyranomupyramirpseinapmocgurdobecalpsvlinitsorpertniseugolanastissenevitceffe][othcaorppaenarhcoccihtapoidihtiwstludasesabataddeidutsstcrfoenoronilcycatsorpnayahtomarapdetroperecnedivefolevelaesuanhtiwnemowtnangerpesabatadseidutsedimarpolcotemcsnnamhcieriiieveliiileveleziselpmasroetadonsetadgnitimovdnaralohcselgoogdehctamdezimodnarnonevitcepsorter¢otnortesnadnodnadnaaesuantaert][egnarrngnittessnoitcirtseregaugnalesacevitcepsorter¢seiresydutstrohocssenevitceffegnitimovairetircsetadrofksirhgihotraelcnuniaprecnacsecruoserrehtoicsytefasdnassenevitceffe][nesnahborenarhcochtiwnerdlihcdnastludasesabatadottnavelerstcrenihpronerpubtdimhcswoleegnarnaemegaroilanmodbaevitceleyregrusytimertxelatipsohgnittesytefasytilibatpeccavisvcsacpeziselpmassetadicsottnavelergenoitartsinmdaiborenarhcocstludarnsesabatadstcrseidutsnoitacidem][renotsegaynaegarngnittesotelbacilppaseidutsseidutslatotweiveremoctuo]noitatic[seidutsnilubolgonummicsnamgnlilanoitavresboseidutstcr¢¢cimonocehtlaeh¢seidutsycneiciffeytefasssenevitceffevisusrev][emmarf one 101371 pone0237572 august one 0csubcutaneous hydration and medication infusionsdeunitnocsaibfoksirregralrofylisaedeveihcaotderapmocdiulffosemuovlsisihtfitubsetuorrehtonahtsuoessoartniehtelbissoptonerasetuorsuoenatucbusdnasevitanretlaelbaiveraohwstneitaprofelbatiustubdetardyhedylerevestonsessoldiulfgniognonehwekatnilaroybtemebtonnacebyametuorsuoenatucbus¢eraatadytefasdetimli¢tcrderosnopsyrtsudni¢elbaliavagnittesdeehtnitahttseggussdiulfcserapmocseidutson¢vitsniagaenolalaitnetopevahslairtgnitsixe¢ynapmoclacituecamrahpaybderosnopssaibfhrgnicudorpotroirefninonerasdiulfcshtiwnoitardyhedetaredomenilrofsemitnaemrekciuqotdlimfosesacniivitnerapretaergdnatnemecalpnoitcafsitasrevigerachcihwsnoitcaeretisnoisufnidecneirepxestneitaptsom¢rehtruftuohtiwdevorpmisuoiresonnoitnevretnistneveesrevdalufsseccusdetroperseiduts¢csfhrhtiwnoitardyherenolasdiulfsraeyhtmcirtaideapsrynaemhtiwsraeytonsetaddetimilelgoogfodeepsdnaesufoesaeytilibailersdohtemnoitresnihgihraelcnutaslairtderewopfitsebsisseccasuonevartnisaibfoksirhgihraelcnustludastcregatnangilamnonniylralucitrapedometismsinahcemmrofniotsnoitalupopybemuovdnalycneuqerfecnedivetsuboron¢ehttroperrednuseiduts¢ecitcarpsinoitardyhlaicifitrahcihwfoyticuapagnitaercdedivorpybecnadiugdesabecnediveecitcarpothcihwemmargorplatsopeziselpmaswolnsrotcodfoyevrusmuidemevitaillapsgnittesegahgihytinummocredolctlgnittesllaweivercitametsyslairtdellortnoctcrevitcepsorpyevrusevitcepsorterevitarapmocevitpircsed¢¢¢¢¢¢¢noitarudsnoitacolepytdnaytitnauqsllikslasiarppalacitircnoitalupopssenllidecnavdasetadsesabatadseidutssmsinahcemics][tabrofgnitarotelbacilppaseidutsseidutslatotweiveremoctuo]noitatic[cdhsisylcomredopyhsdiulffonoisufnicssnoitatimilnoisulcnocllarevosrohtualootlasiarppaytilauqnoitalupopygetartshcraesforebmundnangisednoitnevretnimairohtuadaeldeunitnocelbatsnoitadnemmocerevitinifeddetsissayllacidemfoesumrofniotecnedivedetimli¢ehtnitifenebtnacifingison¢eracevitaillapninoitardyhstneitapksirraelcnuhgihscpksirhgihtcrksirwolscprofelacseracevitaillapgnittesrnegakcnrilootborenarhcocetadpusihtrofstcreziselpmashcramrecnacsetadydutsevitarapmocevitcepsorpscptcr¢¢dnaloqnoitardyhefilfohtgnelrofelacsytilauqdrofxodecnavdahtiwstneitaptludasesabatadicsottnavelerseidutsdetsissayllacidem][doogecnedivefoytilauqdetimli¢otecnedivetneiciffus¢yletauqedafokcaloteudlarehpireptahtedulcnocyltsomdahstcrllascirtaideapdnasetebaiddnastsilecnereferstcrsseccalaretnerapborenarhcochtiwstneitapstludaredolsesabatadtnavelerseidutstnereffidfonoitresni][rekroopdnaseiresesacesacdnatrohocytilauqseidutslortnoctrohoclaudividnibeziselpmasdnalacidemrehtoytilauqwolgnidulcniycnegremelatipsohsgnittesseirtsigerslairttcrtnemtrapedrnsetadtolipertnecitlumseiresesactcr¢¢¢noitardyhedetaredomslevelmbeccirtaideaplsupesabatadseidutsrofivisvics][rakiram one 101371 pone0237572 august one 0chtiwlyamedirohcmuissatopotdeddaylefasebnoituacstseggusecnedivedetimli¢icsrofdetiusyllaediebyamcdh¢noitardyhedfotnemtaertehtodsecnalabmicilobatemsadetcerrocebotevahtonyletaidemminodesabetuorrehtiegnisuylralimisdebrosbasidiufl¢stcrgnitaertrofnoitponasicdh¢stneitapnehwnoitardyhedekatnilaroetauqedanievahstceffeesrevdasnoituosleerfetylortcelediova¢etomorpotesadinorulayh¢sniamernoitprosbadevlosernufonoitaredisnocasedulcniecnediveehtfoytilauqehtroopsaegnaregarngnittesseidutselbaliavafoytirojam¢etylortcelehtiwcdh¢ytilauqroopfoeraefassisdiulfignniatnocnoissucsiddetropereziselpmassetadloottnemssessaonnoitardyhedhtiwstludaesabatadseidutsytefasnoitardyhcs][nohcorseiresstroperesa | Colon_Cancer |
it is hard to comprehend how much of their lives poor people spend waiting in the emergency room dealing with indifferent bureaucracies that are supposed to address basic needs in the laundromat where people must scramble for available machines and keep a close eye on their laundry waiting adds yet more pressure and pain on feet that may already be swollen ¦ many poor people spend years of their lives on waiting lists for a public housing unit a section voucher a bed in rehab a hearing in landlordtenant court they wait for erratic buses food at a food pantry or a bed in a shelter their names fill long lists of people in dire need williams p waiting is a pervasive aspect of poverty just as poor people are most likely to wait for laundry machines housing food so too they are most likely to wait for their healthcare and this can have especially grave consequences andaya 2018b oostrom the link between the timing of treatment and health outcomes is well documented in health disparities research it has become axiomatic that timely adherence to and receipt of medications screenings and followup health services is vital for decreasing inequities in health outcomes bickham and lim dickman as a result much public health intervention work aims to identify and rectify the sources of protracted times to diagnosis andor treatment diamant koopmanschap similarly healthcare anizations are increasingly attentive to time elements identifying time as a critical quality metric shorter wait times particularly for appointments or between diagnosis and the initiation of treatment reflect greater efficiency and improved patient satisfaction and outcomes bleustein michael nevertheless anizationlevel effort to reduce wait times is unevenly distributed bureaucratic interventions disproportionately advantage wealthier and privately insured patients the task of waiting corresponding author email address jeanhunlethwustledu jm hunleth 101016jsocscimed2020113296 received in revised form august accepted august socialsciencemedicine2642020113296availableonline19august2020027795362020publishedbyelsevierltd 0caa lee in and for healthcarein waiting rooms for future appointments procedures and treatmentsis performed overwhelmingly by people who are poor kennedy oostrom in this we focus on what we are calling active waiting a concept that we suggest more accurately describes the lived experience of gaps in healthcare action than does the more frequently used word delay when contemporary health disparities researchers examine patient behavior and action as it relates to the timing of treatment they often focus on patients delaying diagnostic tests or treatments while understanding why a person might delay a diagnostic procedure or treatment regimen can identify barriers eg long wait times on phones or for followup procedures a lack of transportation or money that discourage individuals from taking more immediate actions diamant thinking in terms of delays can also place excessive responsibility and blame on individuals for nonaction and nonadherence hunleth why did she delay scheduling her followup appointment is not the same as why did she wait to schedule her appointment and though waiting can lead to assumptions about passivity or nonaction social scientists have demonstrated that waiting is not a passive state auyero mulcahy we derive our own focus on active waiting as opposed to delay from narrative interviews we carried out with people who have chronic illnesses and who have difficulties affording their medications studies of chronic illness in the us offer important insights into waiting people living with illness and in poverty wait a lot during the many appointments needed to manage their conditions ie foron health service and treatment providers and for disability insurance and paychecks ie foron bureaucratic actors of key importance active waiting unlike delay remains true to the linguistic preferences of the individuals with whom we have worked participants in the study we describe below spoke of their experiences of temporality in healthcare as of waiting and not as of delaying further the concept of active waiting allows us and others working in and studying healthcare to move beyond analyses that either locate responsibility for wait times solely with the individuals ng the waiting or perpetuate overlydeterministic views of systemic barriers that erase experience and social action to frame our conceptualization of active waiting we bring together two scholarships one on the political economy of healthcare which includes the elucidation of bureaucratic time and the other on waiting as a feeling and an experience which gains meaning within power relations and through social interactions bureaucratic time and the agency of waiting patients who then comply with bureaucratic state demands auyero waiting may be imposed but these researchers show that it always entails a response understanding the experience of waiting in the us requires recognizing that the actions people take while waiting are interpreted using neoliberal ideologies that privilege among other things individual responsibility mulligan rylkobauer and farmer this focus on individual responsibility can lead to the blaming and shaming of people who time healthcare inappropriately or do not act appropriately within others expected timeframes holmes scholars have shown that this privileging of individual responsibility reinforces racialized and classed hierarchies of deservingness horton notions of individual responsibility and thus of individual failure place pressure on patients to do and in ng they avoid or mitigate the likelihood of blame and shame while they wait as we have previously shown the focus on individual responsibility can lead people to blame themselves and their loved ones for waiting too long to seek care hunleth sociologist andreas g¨ottlich suggests that waiting is an interaction that depends on the mutual interpretations of the actorsthose who wait and who are waited uponas well as others who behold the scenes of waiting g¨ottlich p that is people wait in relationship to and with others researchers who center the interpretive acts and emotions of waiters in healthcare settings such as waiting rooms offer several insights into what we refer to as active waiting in a study of interactions in a waiting room strathman and hay suggested that when patients are told that they cannot get an appointment within the desiredeven physician recommendedtimeframe it is like being told that their health and by extension they as persons are less important than bureaucratic schedules strathmann and hay p that is waiting can add to a sense of being devalued or neglected andaya sj¨oling and it can negatively influence peoples perceptions of their quality of care and confidence in their care provider bleustein given the above work we understand active waiting to be the experiences and responses that people devise to navigate shorten or otherwise survive waiting and also to anticipate and craft possible futures within the relationality and power dynamics of bureaucratic time resisting wait times by seeking information and other resources is part of active waiting as is delaying and complying active waiting is composed of such discrete actions that people might take as patients in one place or time but is irreducible to those actions and must be situated in how people manage multiple wait times at various scales further we include not only action but also how waiting on bureaucratic time feels carr teucher and casson have used the phrase lived wait time to communicate the fact that feelings of time differ by circumstances researchers have shown that chronic illness lends itself to particular feelings of time and of life including those of time as suspended eg feelings that life is on hold brown mulcahy sj¨oling we expand this by focusing on how chronic illness feels while waiting on powerful others to make life or death decisions while wait time is lived people also live during their wait times daily lives are not easily paused sj¨oling this means that the circumstances of individuals days and lives often change while they wait on healthcare less has been said about the waiting that goes on outside of clinical settings yet is ultimately tied to health in fact waiting is an implicit and normalized mechanism of bureaucratic systems in what follows we consider how these various aspects of time and waiting combine for people experiencing illness and financial strain we use active waitingconceptualized as an action orientation relationship and feeling that is irreducible to a singular time and place and which shapes how individuals approach their health in particular times and places we do so in order to best examine how various aspects of time and waiting interweave in the lives of people who are chronically ill and poor though healthcare bureaucracy in the us requires most people to wait for care how long people wait how waiting is structured and the consequences of waiting vary people who do not have access to insurance or other resources must endure state and federal evaluations of their lowincome statuses abilities to work severities of illness citizenship and more to qualify for assistance tickamyer as funding for social programs like medicaid food stamps and disability are rolled back and work requirements become stricter demonstrating qualifications to obtain support is increasingly difficult and time consuming whittle while social welfare programs offer access to health services they often require long waits in waiting or exam rooms at facilities that accept medicaid or offer services on a sliding fee scale becker oostrom researchers have identified how people deal with the bureaucratic schedules that shape the course of their treatment mulcahy described how some people with cancer resisted the assumptions that they must be patient patients by seeking information and resources to shorten their waiting periods still other researchers have shown that long waittimes for healthcare personnel in clinics and waiting rooms can lead to other forms of action waiting can push individuals out of the healthcare system or lead them to delay care becker and it can induce feelings of pervasive uncertainty and powerlessness among socialsciencemedicine26420201132962 0cthis research comes from a mixed methods study investigating cost related issues that affect adherence to medications recruitment occurred in and amidst political uncertainty about changes to healthcare legislation on the national stage a new and controversial presidential administration was beginning its term this administration had goals to repeal the affordable care act aca and introduce block grant funding to medicaid which remained a possibility throughout our data collection period for people with chronic illnesses the aca was significant it mandated that preexisting conditions be covered by insurance plans and that insurers offer at least a minimum prescription drug benefit while also creating platforms for uninsured individuals to purchase plans additionally national conversations about changes to prescription drug pricing were prominent in news coverage most of our narrative interviews were conducted in st louis missouri though a few occurred in east st louis illinois missouri did not expand their medicaid program with the rollout of the aca illinois did st louis is a metropolitan area with significant racial and economic health inequities to address the health needs of those without insurance in light of not expanding medicaid the gateway to better health program was created which provided basic coverage to people living below the poverty line this program was set to end in december during our study but its extension was later approved beyond that date many of the people who participated in this study were using or had used this program recruitment interviews our research team recruited people between the ages of and who had one or more chronic illnesses for which they were prescribed medication and who identified as having difficulties affording their prescriptions the study had a mixed methods design participants in the survey n were recruited from newspaper ads federally qualified health centers and a multispecialty clinic a subsample of the survey participants n also participated in a narrative interview focused on financial strain while we have presented the mixed methods results elsewhere this focuses solely on the narrative interviews we initially aimed to recruit interview participants stratified evenly based on prescription insurance coverage age and gender however this was adjusted based on the availability of participants the final number of interviews was table the interview guide was based on the mcgill illness narrative interview mini groleau we revised the guide and refer to it as the modified financial and illness narrative interview it is structured similarly to the mini to elicit different types of narratives but with an added focus on financial history two local nonprofit community anizations were selected as interview locations due to their proximity to and rapport with the participant population nonclinical environments available private interview spaces and anizational relationships with study team members trained research team members conducted interviews and a note taker was present in most cases the interviewer obtained informed consent and interviewees were compensated with a gift card interviews were audiorecorded and lasted about minutes immediately after the interview the interviewer and note taker wrote detailed field notes using a structured template that included their observations about body language any conversations not recorded a summary of the financial and illness narrative and reflective memos analysis interviews were professionally transcribed verbatim deidentified and checked for accuracy before being uploaded with their respective fieldnotes to nvivo we describe the codebook development below which was designed with the mixed methods study in mind during that codebook development the team identified the repetition of comments about waiting across the interviews the team developed a codebook using deductive and inductive codes deductive codes were based on questions of interest from the quantitative survey these focused primarily on medication cost coping access to basic needs and related concepts the team identified inductive codes while conducting interviews and refined and added to these codes while reviewing transcripts during the analysis and throughout the coding process we took note of a crosscutting theme in the narratives when asked to describe what it was like to deal with chronic illness participants said that chronic illness was about waiting we coded all discussions of waiting in the interviews defined as any mention of waits or delays and in the fieldnotes while waiting is a broad concept and was used to reference different processes we chose to include in our analysis all aspects of waiting described by participants rather than focus on just one eg waiting for health insurance coverage we did so to acknowledge that people may experience waiting that is interrelated and different from the temporal distinctions made by many healthcare practitioners four team members coded the interviews using the final codebook all transcripts were coded by at least two team members and discrepancies were reconciled through consensus between the two coders al memoed on the waiting code throughout this process focusing on issues such as what kinds of things people waited for how this influenced their decisions and emotions the outcomes of their waiting on their health and healthcare and how waiting shaped their aspirations for the future the team including coders interviewers and investigators compared how waiting was discussed within and across the transcripts and narrative types elicited by the guide as well as the intersection of waiting with other codes interpretations and exemplar quotes were evaluated by multiple team members to guard against selective use and bias in interpretation of the data and to encourage reflexivity all study activities were approved by washington universitys institutional review board results aa lee methods setting table interview participant characteristics raceethnicity nonhispanic black or african american nonhispanic white other gender female male had continuous health coverage in the past year had a gap in coverage in the past year age mean range health coverage n we anize our results to make clear the meanings and experiences of active waiting in the first section we outline the breadth of waiting that shapes how people wait and answers the question waiting for what the second section addresses the question waiting for whom and describes how participants ascribed meaning to waiting according to their social and economic positions and in relation to those in power what some participants referred to as the waiting game finally and building on the previous sections we focus on waiting as an active process that people manage through a variety of tactics in ng so we detail the ways in which people actively wait living their lives within structures of power that make them wait socialsciencemedicine26420201132963 0caa lee waiting for what big and small waits because we cover multiple forms of waiting we wish to be specific about what participants referred to when they talked about waiting the list was long and it included waiting in waiting rooms for transportation on the phone for surgery for paychecks and welfare checks for housing and for approval for disability and insurance benefits the things participants waited for varied according to their own unique health and social circumstances these waits were often predictable and included the spaces in which people wait eg waiting rooms the bureaucracies that enforce waiting eg disability claims the conditions that create waiting eg strained social safety net and the financial demands that when unmet lengthen wait times eg a ride when one does not have transportation a paycheck to afford a prescription the variation in chronic illnesses and in life circumstances of participants in the study however meant that they discussed the structures of waiting in different ways and put emphasis on different aspects within the predictability and variability there was an overarching theme of waiting for one or a succession of big things to happen what we term big waits big waits were for things that had a hopedfor endpoint ones that might change an individuals health situation for example many participants talked about waiting for disability assistance to come through or for health insurance coverage to start or for stable housing and they often centered their discussions of waiting around this primary event the endpoints could be a decision made by a social service agency or could be an anticipated and desired life change one woman told us that her big wait for insurance coverage was almost over because she would soon be eligible for medicare which multiple others also mentioned that they were awaiting the good news for me is a year from this august i turn but um i always feel like its all pending there is nothing concrete in any of the healthcare while participants talked about big waits in terms of their endpoints they also talked about how such endpoints led to other big waits take kate for example kate is a black woman in her early fifties who previously worked in home healthcare she has osteoarthritis graves disease hepatitis c and depression after both her mother and husband died a few years ago she struggled to afford housing and continue working through her arthritis pain when we interviewed kate she had been homeless for several months and was recently accepted off a waiting list for transitional housing kate had several big waits that came in succession and were contingent upon one another she waited for housing assistance she waited for medical release to work after sustaining injuries in a car accident she waited to consult with a doctor about surgeries she waited on a disability hearing scheduled months away though she had been diagnosed with hepatitis c her doctor would not begin treatment until her disability application was approved because of the high costs of the medication which would be covered if she were on disability kate worried that all of these big waits would have longterm repercussions on her health until the disability [benefits are approved] or something [else comes through] and september [the date of her disability hearing] comes they aint going to even give me the medicine ¦ we will just wait and see so i am just in limbo until until my hearing ¦ i know i aint going to die right away but i do want to get the medication kate found herself waiting a lot on a lot the interdependence of her waiting meant that one wait exacerbated other waits while taking care of her sick family members she put off her own health needs and bill payments leading to her eventual housing instability her lack of a consistent mailing address delayed her ability to submit a disability application even after she found more stable housing and was able to see a doctor the disability application delays led to postponed medical treatments without getting her health and injuries under control she could not work and get out of transitional housing for kate each big wait led to another big wait with worsening and compounding repercussions on her health and wellbeing other participants described having waited years for their disability applications to be approved or often denied in the meantime they incurred greater debt hired lawyers relied on family for support postponed treatments and endured these big waits waiting for disability benefits and for other significant changes in their financial or familial situations also created a number of smallerscale daytoday situations in which study participants waited one uninsured woman described waiting to fill her prescription and waiting to see her doctors while she searched for a full time job to afford both you wait to the extreme before you do anything about whatever the problem is because you dont want to go to the hospital it costs too much many participants missed appointments or went without medications while waiting for new insurance benefits or payday or financial assistance paperwork to go through holding off on care was just one way to wait actively available to those whose actions were constrained by chronic illness and financial strain while big waits figured strongly into participants imaginations of their futures small waits were not insignificant allens diabetes had damaged his kidneys so severely prior to getting medicaid coverage that he needed dialysis he described the cycle of waiting while on dialysis allen whose big wait had been first getting medicaid coverage and then once covered getting on a kidney transplant list did not have a car or a steady way to get to his weekly dialysis appointments a few times a week he waited to be picked up by a medicaidcovered transportation company he had little control over when they would arrive and their arrival was often sporadic when they arrived early he had to wait at the clinic when they were late he either had to pay for his own cab or push back his dialysis appointment to later in the day and wait even more optimistic allen expressed gratitude that he was able to use medicaid and live with his brother while waiting for a kidney transplant his own big wait within multiple small waits while enduring the small waits he imagined a better future after the transplant describing to us what he would write in a book about his experiences kidney transplant got him back to a better life now hes back in the work force he hasnt looked back to the sickness yet ¦ thats my story the daytoday waiting was frustrating but the promise of this story along with having family able and willing to assist him an unmarried man without children shaped how he waited and his feelings while waiting big and small waits contribute to the dynamic aspect of active waiting where social ties and financial resources regulate how one is able to navigate and survive waiting waiting was drawn out for those without the economic resources to avoid institutions such as slidingscale clinics welfare agencies and financial assistance programs throughout the interviews participants described waiting as chronic but they also held onto a sense that once a waiting period passed then their future goals of health and financial stability would be that much closer for kate waiting was incremental and each waiting period required different tactics the big waits participants described both promised and suspended the future people grappled with the present while anticipating what might happen when and if the waiting ended some people imagined a time when they could afford healthcare holding off certain appointments until that time while others imagined a future in the work force and attended appointments diligently even in the face of smaller or shorterterm waits such small waits too structured participants responses in how they waited based on what they were waiting for waiting for whom the waiting game many participants identified feeling like the systems in place to assist chronically ill and financially strained people were playing games with their lives by making them wait they were made to wait by social service programs appointment schedules hospital and clinic waiting rooms insurance company call centers the waiting punctuated their socialsciencemedicine26420201132964 0caa lee daytoday lives and left them guessing about the reasons behind their extensive chronic waiting they guessed at answering why mewhy they had to wait they also guessed at a course of action to shorten or otherwise deal with waiting the waiting game they described was one in which the odds were stacked against them the rules were opaque and endurance was necessary to win some study participants searched for ways to get around the game or to play it to their advantage how they played the waiting game which we recognize as active waiting depended on both their interpretations of the meaning of waiting and on the resources to which they had access in our interviews some people brought up suspicions about being forced to wait for appointments and in waiting rooms due to their economic status age race and other discriminatory factors one participant talked about how once in the examining room she felt as if her time waiting was not reciprocated by providers they do not want to spend a lot of time with you you know theytheyve got to make a living too so they move on some speculated that doctors reserved time for people of a higher economic status these patientprovider dynamics that waiting exemplified left people feeling slighted by the medical system ah i sat there in that waiting room for hours and and they still wasnt ready to see me ¦ no one had the respect ah even the courtesy to come out and say well the doctor is running a little bit behind people felt that their lives were undervalued and unimportant when their time was not acknowledged or respected that bureaucratic systems seemed to withhold resources by imposing waiting suggested to some a denial of their belonging and deservingness as patients and as humans worthy of care experiences of medical racism and sexism frequently underpinned participants feelings that their time was being wasted rachel a black woman told us that she had been working and paying taxes since she was a kid and still the welfare system did not support her when she needed it recognized that high blood pressure was black peoples number one killer and explained that her waiting also stressed her body contributing to her anger these indignities played out in the clinic adding to the daily stresses she said she already felt as a black woman and grandmother living near the ferguson neighborhood where an officer murdered michael brown in and was not indicted for that murder participants also talked about waiting as a game that required endurance not just because of a lengthy office wait or the months or years it could take to get a disability application approved or denied but because of the opacity of the process they questioned application processes wondering if the wait to hear back about welfare and assistance services was a test of endurance it is just a waiting game [for disability] until they get tired tired of you being in front of them and they decide to give it to you that is what they hope for is [you] giving up on it but if you keep with it sooner or later you will get it while a number of participants expressed optimism for a better future once a big wait was over waiting out bureaucratic processes wore down that optimism i dont believe i can keep going through this too much longer erica said she was exhausted from the strain of balancing utility payments with medical costs while waiting for her new insurance plan to begin and while coping with illness unexplained waiting on bureaucratic systems whether for an application to be approved or for insurance to kick in or to be seen by a busy doctor in a slidingscale clinic felt like an unfair game designed to keep them from receiving necessary medical care by framing waiting as a game the participants explained that there were rules that shaped the length and types of waiting and that they did not write them the rules were far from transparent and enforced differently based on race and social status as sherry made evident in her frustration with being made to wait waiting is not a passive state that happens without mental and physical consequences when people responded with patience and endurance through the game just like when they responded with anger and confrontation their waiting was active put differently participants described fashioning tactics in part based on their perceptions of the intentions of those for whom they waited they kept turning me down for disability and they kept saying i was too young to have problems i had you know but my body was breaking down and the doctors would never [help with the application] which was really so unfair and now thats the part that really makes me feel a little sad like there is so much i hate to say racism but just unfair treatment because im a woman because of my age and then because of my race and so were talking like years [voice breaking] ¦ it made me be really poor rachels doctors held the power to demonstrate that she qualified for disability but as she recounted they were unwilling to fill out the documentation she needed to apply for disability she felt that the us had enough resources to | Colon_Cancer |
this study hypothesizes that bromelain bl acts as radiosensitizer of tumor cells and that it protects normal cells from radiation effects in vitro and in vivo studies have been carried out to prove that assumption in vitro mtt cell proliferation assay has shown that the irradiated ehrlich ascites carcinoma eac cell line could be sensitized by bl pretreatment in vivo animals were randomly divided into groups group control pbs ip for days group ehrlich solid tumor est bearing mice group est Îradiation fractionated dose gy group est bl mgkg ip daily for days group est bl for days followed by Îirradiation gy the size and weight of tumors in gammairradiated est bearing mice treated with bl decreased significantly with a significant amelioration in the histopathological examination besides bl mitigated the effect of Îirradiation on the liver relative gene expression of poly adp ribose polymerase1 parp1 nuclear factor kappa activated b cells nfκb and peroxisome proliferatoractivated receptor α pparα and it restored liver function via amelioration of paraoxonase1 pon1 activity reactive oxygen species ros content lipid peroxidation lpo and serum aspartate transaminase ast alanine transaminase alt and albumin alb it is concluded that bl can be considered as a radiosensitizer and radioprotector suggesting a possible role in reducing radiation exposure dose during radiotherapykeywordsbromelain tumor Îradiation radiosensitizer radioprotectorsubmitted april revised july accepted july introductionradiotherapy has been used for a long time in treating cancer1 however from the clinical perspective radiotherapy provides inadequate success due to the radioresistance of many tumors as well as the high risk of recurrence and effects on normal cells may occur23 radioresistance occurs as the microenvironment of solid tumors is hypoxic compared with normal tissue4 in addition some tumors have either an intrinsic resistance to ionizing radiation or can attain this property through accumulation of genetic mutations causing an increased survival and proliferation5 thus strategies to improve radiation therapy could include increasing resistance of normal tissues to radiation andor increasing sensitivity of the tumor cells6radiosensitizing agents increase the sensitivity of tumor cells via enhancing the generation of reactive oxygen species ros increasing lipid peroxidation depletion of glutathione which leads to dna damage inhibition of dna repair inhibition of dna synthesis induction of cell cycle arrest induction of apoptosis and inhibition of proliferation7 numerous nutritive cancer chemopreventive compounds having antioxidant properties have been recognized to potentiate radiation therapyinduced cytotoxic 1drug radiation research department national centre for radiation research and technology egyptian atomic energy authority nasr city cairo egypt2biochemistry department alazhar university cairo egyptcorresponding authorhanan a fahmy drug radiation research department national centre for radiation research and technology atomic energy authority p o box nasr city cairo egypt email fahmyhananyahoocomcreative commons non commercial cc bync this is distributed under the terms of the creative commons attributionnoncommercial license creativecommonslicensesbync40 which permits noncommercial use reproduction and distribution of the work without further permission provided the original work is attributed as specified on the sage and open access pages ussagepubcomenusnamopenaccessatsage 0c integrative cancer therapies effects on cancer cells inversely decreasing its toxicity on normal adjacent tissues89 in this regard much research has aimed to develop numerous antioxidant drugs of both natural and synthetic origin tested in both in vitro and in vivo models and also human clinical trials to overcome injuries caused by ir exposure and to induce killing of cancer cells at the same time previous studies have reported that phytochemical soy isoflavones genistein daidzein and glycitein which exhibit anticarcinogenic properties through their antioxidant activities could be used as potent radiosensitizers to enhance the efficacy of radiotherapymediated suppression of the growth and metastatic ability of cancers1011 along parallel lines resveratrol and piperine which possess antitumor activity have been shown to augment ionizing radiation irinduced apoptosis and loss of mitochondrial membrane potential in murine colon carcinoma and melanoma cells via enhancing irinduced ros generation12 moreover pentoxifylline ptx a methylxanthine that possesses antioxidant properties is known for improving tumor tissue oxygenation in murine hypoxic tumors and inhibiting post radiation induced normal tissue injury in mice1314 consequently searching for a natural product possessing anticancer activity that increases radiosensitivity of tumor cells and radioresistance of normal cells may lead to a potential future drug in cancer therapyamong the natural products bromelain bl extract attracts interest due to its anticancer antioxidant as well as antiinflammatory effects1517 bl an extract from pineapple stem ananas comosus belongs to a group of protein digesting enzymes it is a mixture of diï¬erent thiol endopeptidases and other components like phosphatase glucosidase peroxidase cellulase escharase calcium and several protease inhibitors1819 the anticancer activity of bl has been examined in various types of gastrointestinal and breast cancers cell lines in in vivo models bl has shown antimetastatic effect and reduction in local tumor growth2023 it is also used for reducing the severity of such radiation therapy side effects as mucositis skin reactions and dysphagia in patients24 hence this study was aimed to evaluate the radiosensitizing and radioprotective effect of bl using in vivo and in vitro approachesmaterials and methodin vitro studiesmtt cell proliferation assay the growth and viability of ehrlich ascites carcinoma eac cell line were tested in vitro by 345dimethylthiazol2yl25diphenyltetrazolium bromide mtt assay according to freimoser and buch 2526 to verify the antitumor and radiosensitizing effect of bl two plates were designed for this study the first one contained eac cells maintained by serial subculturing at the national cancer institute egypt incubated for hour before irradiation irr gy alone and with different concentrations of bromelain bl in phosphate buffer saline pbs the second one contained eac cells serving as a control and eac with different concentrations of bl each test was seeded in triplicate into a plate at concentration of cellswell containing rpmi media with fbs nahco3 uml penicillin and µgml streptomycin and each plate was incubated for hours at °c in co2 and humidity atmosphere then μl mtt reagent bio basic inc canada was added over the cells in each well and the plate was incubated in the dark for to hours until a purple precipitate was seen and the absorbance was measured at nm the amount of color produced was directly proportional to the number of viable cells viable cell a samples a blanka control a blank the inhibitory concentration ic50 is the dose of a drug which reduces the viability to and was calculated using nonlinear regression analysisfree radical scavenging assay the antioxidant activity of bromelain was evaluated by 1diphenyl2picrylhydrazyl dpph radical assay and its scavenging power was compared with some antioxidants naringin polyphenolic antioxidant garlic oil and glutathione sulfur containing antioxidants about µl of samples mgml dissolved in dist water was added to µl of a solution of dpph g100 ml dissolved in vv methanol after minutes incubation at room temperature in the dark the absorbance was read at nm against a blank µl dist water µl dpphmethanol solution the experiments were done in triplicate according to the method of braca 27 glutathione mgml was used as a standard antioxidant the scavenging percentage of dpph was calculated according to the followscavengin ing equation where b was the absorbance of the blank and a was the absorbance of samples or standard ec50 is defined as concentration of sample that causes dpph loss there values were calculated using nonlinear regression analysisb ab\uf8ee\uf8ef\uf8f0\uf8f9\uf8fa \uf8fbin vivo studiesradiation processing whole body Îirradiation of mice was carried out using gamma cell40 137cesium manufactured by the atomic energy of canada limited ontario canada installed in the national center for radiation research and technology ncrrt cairo egypt the dose rate was gymin during the experimental period daily correction for humidity barometric pressure and temperature were madeanimals adult female swiss albino mice weighing to g obtained from the breeding unit of ncrrt cairo egypt all animal procedures were performed in accordance with the committee of scientific ethics at faculty of 0cmekkawy table sequences of primers for realtime quantitative pcrgeneparp1 nm0074152nfκb nc0000696pparα nc0000816βactin nc0000716forward primerreverse primer²ccatcgacgtcaactacga3²²caatggctacacaggacca3²²actccacctgcagagcaacca3²²gcgtggggacagccgcatctt3²²gtgcgtggtagcatgagtgt3²²cactgtcacctggaaccaga3²²tagatctcctgcagtagcggg3²²atcggcagaaggggcggaga3²pharmacy alazhar university egypt following the guidelines for animal use the animals were housed in colony cages micecage under proper environmental conditions that is hours darklight cycle good ventilation condition and temperature to humidity at the ncrrt animal house fed with standard diet pellets and provided with water ad libitum animals were left week for acclimatization on the lab environment before starting the experimenttumor transplantation the eac cell line was supplied by serial subculturing at the national cancer institute cairo university egypt it was implanted in each donor female swiss albino mice by ip injection of cells22 g b wt and allowed to multiply28 the ehrlich solid tumor est was obtained by the intramuscular inoculation of ml of viable eac in the right lower limb of each mouse29 mice with a palpable solid tumor diameter mm3 that was maintained within to days after inoculation were used in the studyanimal grouping animals were randomly divided into groups mice each group control not bearing tumor received pbs ip for days group ehrlich solid tumor est bearing mice received pbs ip for days group est Îirradiation gy fractionated doses starting days after tumor appearance mm3 and lasting for days group est bearing mice receiving freshly prepared bl dissolved in pbs mgkg ip daily for days according to pilot study starting once est becomes mm3 bl was purchased from merck kgaa co darmstadt germany group est bearing mice received bl as in group hours before Îirradiation as in group mice were anesthetized days after last irradiation dose using urethane mgkg30 blood samples were collected through cardiac puncture and divided into parts edta coated and plain vials at that time they were euthanized by cervical dislocation liver and tumor tissues were dissected out rinsed with icecold saline dried on a filter paper and weighed then homogenized in icecold pbs ph and stored at °c until used for subsequent biochemical analysisestimation of total body tumor and liver weights animals in each group were checked daily for any adverse clinical symptoms and deaths after to days post inoculation with eac body weights were recorded so body weight change could be estimated tumor and liver weights were measured during sample collection and then the tumor inhibitory ratio was calculated by the following formula inhibition ratio aba where a is the tumor weight average of the control and b is that of the treated group also relative liver weight was calculated as liver weighttotal body weight histopathological examination three tumors of each group were collected and fixed in neutral buffered formalin the specimens were dehydrated in ascending grades of ethyl alcohol cleared in xylene and embedded in paraffin wax four micron thick paraffin sections were mounted on clean slides stained with ehrlichs hematoxylineosin he31 and examined using an olympus microscope bx41 hamburg germany histopathological evaluation was done by assessment of necrosis and calculation of tumor area percentage using image analysis software image j 146a nih usa through the following equation of tumor area area of tumortotal area of the field molecular analyses the mrna levels of poly adpribose polymerase parp1 nuclear factor kappa b nfκb and peroxisome proliferatoractivated receptors pparα genes and of the housekeeping gene βactin were measured by real time polymerase chain reaction rtpcr total rna was isolated from liver tissues using qiagen tissue extraction kit qiagen usa in accordance with the manufacturers instructions the extracted rna μg was used for cdna conversion using high capacity cdna reverse transcription kit fermentas usa and μl reaction volume sybr chemistry in applied biosystems thermal cycler usa to amplify pcr under the following conditions °c for denaturation then °c to °c for annealing using primers mentioned in table and °c for elongationresults were expressed using the comparative ct method for relative mrna quantification of target genes normalized to an endogenous reference βactin and a relevant control equal to ct ct is the difference between the mean ctsample and mean ctcontrol where ctsample is the difference between the mean ctsample and the mean ctβactin and ctcontrol is the difference between the mean ctcontrol and the mean ctβactin 0c integrative cancer therapies estimation of lipid peroxidation lpo reactive oxygen species ros and paraoxonase pon1 in liver homogenate liver lipid peroxidation was estimated by measurement of malondialdehyde mda formation using the thiobarbituric acid method of yoshioka 32 a modified technique of vrablic 33 was used to measure the generation of ros by the intracellular conversion of nitro blue tetrazolium nbt to formazan by the action of superoxide anion paraoxonase activity was estimated by using fluorometric assay enzchek® kit invitrogen uk for the anophosphatase activity of paraoxonase based on the hydrolysis of a fluorogenic anophosphate analog34hematological and biochemical analyses whole blood was immediately analyzed for complete blood count with platelet count using the fully automated analyzer abx cobas micros roche germany estimation of serum alanine aminotransferase alt aspartate aminotransferase ast and albumin alb assays follow the recommendations of the international federation of clinical chemistry ifcc but were optimized for performance and stability using the rochehitachi cobas c 311systemstatistical analysis the statistical analysis was performed using oneway analysis of variance anova and the groups were compared by tukeykramer test viability percentage at different concentrations and body weight change analyzed by twoway anova followed by bonferronis posttest graphs were sketched using graph pad prism isi® software usa version software data were presented as mean ± standard error se and p values considered significantresultsin vitro studieseffect of bromelain and gammairradiation blirr on tumor cell growth and viability the radiosensitizing effect of bl on eac cells was determined by performing mtt assay eac cells exposed to gy Îradiation showed high cell viability percentage reflecting a radioresistance of eac cell line while bl treatment showed in vitro cytotoxic activity with ic50 value of mgml however the maximum cytotoxic effect appeared when the eac cells were subjected to bl then Îradiation gy compared to control or irradiated group with ic50 mgml table effect of bromelain and some natural compounds as free radical scavengers the inhibitory percentage of each compound is shown in figure the ec50 value concentration of sample that causes dpph activity loss is a reliable way for estimation of the radical scavenging activity the ec50 value of glutathione referenced antioxidant is mgml while table cytotoxic activity of blirr against eac cell line bromelain concentration mgmleac bl mgmlic50 mgmlviability non irradiated eac irradiated eac48ab59ab60ab69a787a10ab158ab18ab27ab52ab ± ± each value indicates the mean of records statistical analysis carried out by twoway anova followed by bonferroni posttests a significant versus control ehrlich ascites carcinoma eac group where b significant versus irradiated eac group at p ic50 ± se values were calculated by using nonlinear regression analysisbromelain and garlic oil ec50 are almost equal and mgml respectively however the naringin phenolic antioxidant is the least potent one ec50 mgml in this comparisonin vivo studieseffect of bromelain and gammairradiation blirr on tumor weight and volume table shows a significant decrease in tumor weight in groups treated with bl andor Îirradiation as compared to the est nontreated group the more drastic decrease in the tumor weight ratio observed in the combined therapy group bl irr compared with the estirradiated group as well as est group indicates that combination therapy is more significantly effective than single agent therapy the photograph of est xenografts at the time of sacrifice shows the synergistic effect of bl and irr on tumor volume figure effect of bromelain and gammairradiation blirr on tumor histopathological features of est bearing mice histopathological examination of solid tumor sections revealed typical malignant features including sheets of malignant cells infiltrating adjacent muscular tissue the malignant cells show pleomorphism hyperchromatism and mitotic activity while the necrotic cells appear as nonviable homogenous structureless material with degenerated or karyorrhectic nuclei untreated est bearing group shows a deeply stained tumor cells arrow head and areas of necrosis arrow figure 3a also it displays intact cancer cells arrow and giant cells arrow figure 3b and c the estirradiated group shows muscle fibers invaded by deeply stained tumor cells arrow head and large areas of necrosis arrow figure 3d also displays a notable necrosis of cancer cells n figure 3e the bl treated group shows a 0cmekkawy figure dpph 1diphenyl2picrylhydrazyl reduction curve for glutathione bromelain naringin and garlic oil each value represents mean ± se all experiments were replicated timestable tumor weight and inhibition ratio of ehrlich solid tumor estbearing mice treated with gammairradiation irr gy andor bromelain bl mgkggroupsestest irrest blest bl irrtumor weight g ± ± 004a ± 005a ± 005abtumor inhibitory ratio ± 24a ± 14ab ± 204ababbreviations est ehrlich solid tumor est irr ehrlich solid tumor irradiation est bl ehrlich solid tumor bromelain est bl irr ehrlich solid tumor bromelain irradiationthe values shown are mean ± se of data a significant versus est group where b significant versus estirradiated group at p figure a photograph of ehrlich solid tumor est xenografts at the time of scarification showing the effect of bromelain and gammairradation blirr on tumor volume e ehrlich solid tumor e ir ehrlich solid tumor irradiation e br ehrlich solid tumor bromelain e ir br ehrlich solid tumor bromelain irradiation 0c integrative cancer therapies figure photo micrograph of ehrlich solid tumor est xenografts in different animal groups est sections show the degree of tumorogenesis necrosis n regression of tumor by appearance of muscle fibers m and a b c est ehrlich solid tumor d e est irr ehrlich solid tumor irradiation f g est bl ehrlich solid tumor bromelain h i est bl irr ehrlich solid tumor bromelain irradiationwide area of necrosis arrow and n few groups of cancer cells arrow head and muscle fiber m figures 3f and 4g however combined treatment bl irr displays muscle fiber m significant regression of tumor or wide areas of necrotic cancer cells n and few groups of intact cancer cells arrow figure 3h and 3i the tumor area percentage per total tissue area could determine the degree of proliferation as seen in figure there is a great regression of tumor area in the group treated with bl alone or bl and irr compared with untreated est or estirradiated group indicating that combination therapy significantly more effective than single agent therapy 0cmekkawy bl irr shows nonsignificant group change additionally bl irr group significantly upregulated pparα expression compared with est and estirradiated groups indicating that bl might have a hepato as well as radioprotective effect figure effect of bromelain and gammairradiation blirr on the hepatic lipid peroxidation lpo level reactive oxygen species ros content and paraoxonase1 pon1 activity of ehrlich solid tumor est bearing mice lpo in liver tissues significantly increased in all est bearing groups compared to the control group except the combined treated group irr bl succeeded in returning mda lpo measured as mda malondialdehyde level to the normal level however liver ros significantly increased only in untreated and Îirradiated est bearing groups when compared to the control group while a significant decrease in liver ros showed in estirradiated mice treated with bl in comparison with both est untreated and estirradiated groups pon1 activity in liver homogenate was significantly decreased in est untreated and estirradiated groups when compared with the control group bl treated groups revealed significant increases in pon1 when compared with both est untreated and estirradiated groups showing that bl might have a hepato and radioprotective effect figure effect of bromelain and gammairradiation blirr on hematological measurements wbcs and plts were significantly elevated while hgb and hct significantly decreased in the untreated estbearing mice in comparison with control mice however Îirradiation resulted in a significant decrease in wbcs rbcs plt hgb and hct compared with the control mice treatment of the estbearing mice with bl shows a significant amelioration in wbcs plt and hct compared to est untreated mice combined treatment bl irr shows an enhancement in wbcs plt and hct compared to est untreated and gammairradiated est bearing mice table effect of bromelain and gammairradiation blirr on the serum alanine transaminase alt aspartate transaminase ast and albumin alb to investigate the cytoprotective effects of bl against irradiation the levels of serum alt ast and alb were measured figure it was found that alt and ast significantly increased and conversely alb significantly decreased in est untreated and estirradiated groups compared with the control group however estbearing mice treated with bl alone show nearly the same result of alt and alb as control values estbearing mice treated with bl in combination with irradiation initiated a significant decrease in ast and alt as compared with estirradiated group which may reflect a potential hepatic radioprotective effect of blfigure percentage of tumor areatotal tissue area of ehrlich solid tumor est bearing mice treated with gammairradiation irr gy andor bl mgkg the values shown in the plotted area are mean of records from animals ± se significant versus est group where significant versus estirradiated group at p effect of bromelain and gammairradiation blirr on body weight change and relative liver weight regarding the day by day documented recording of body weight bwt illustrated in figure there is almost no change in bwt of bl treated group while it increases significantly in the untreated est group conversely estirradiated groups with or without bl treatment show a significant decrease in bwt when compared with the control group table relative liver weight was compared after normalization to mg body weight untreated estbearing group shows a significant increase in liver weight by hepatomegaly while nonsignificant changes were observed in bl treated groups compared to the normal group table effect of bromelain and gammairradiation blirr on the poly adpribose polymerase parp1 nuclear factor kappa b nfκb and peroxisome proliferatoractivated receptors pparα relative gene expression of ehrlich solid tumor est bearing mice to test the possibility that bl reduces radiation damage to the liver mrna gene expression of parp1 nfκb and pparα was measured in the liver homogenates of est bearing mice and compared to control pbs treated mice the results illustrated in figure show that irr causes significant increases in parp1 and nfκb expression compared to the control group however combined treatment bl irr shows a significant increase in parp1 and nfκb expression compared to control group and a significant attenuation compared to estirradiated groupmoreover all est bearing groups show significant decreases in hepatic pparα relative gene expression compared to the control group except the combined therapy 0c integrative cancer therapies figure effect of bromelain and gammairradiation blirr on body weight during experiment period each value represents the mean of records ± se significant versus control group where significant versus est group and significant versus estirradiated group at p est ehrlich solid tumor est irr ehrlich solid tumor irradiation est bl ehrlich solid tumor bromelain est bl irr ehrlich solid tumor bromelain irradiationtable change in body weight and relative liver weight of control mice and ehrlich solid tumor est bearing mice treated with gammairradiation irr gy andor bromelain bl mgkggroupscontrolestest irrest blest bl irrbody weight change ± ± 101a ± 217ab ± 201b ± 341abrelative liver weight ± ± 026a ± 022a ± 026a ± 026ababbreviations est ehrlich solid tumor est irr ehrlich solid tumor irradiation est bl ehrlich solid tumor bromelain est bl irr ehrlich solid tumor bromelain irradiationeach value represents the mean ± se a significant versus control group where b significant versus est group at p body weight changes percent are related to the initial weight of animalsdiscussionresistance of tumor cells to chemoradiotherapy as well as the damaging effects to nearby normal tissues remains a major obstacle to successful cancer management therefore the current study has been conducted to estimate the effect of bromelain bl as a tumor radiosensetizer and to show to what extent it can protect normal tissue from radiation hazardsradiosensitizers are compounds that when combined with radiation therapy achieve greater cytotoxicity they can be determined in vitro by the mtt assay2635 the present study has found that the radioresistant eac cells could be sensitized when incubated with bl before irradiation it was known previously that in vitro treatment with bl on mouse tumor cell lines resulted in inhibition of cell growth and invasion capacities3637 the anticancer property of bl has been mainly attributed to the protease component through digestion and diffusion in tumor cells38 it may also be due to the bl enhancement of p53 expression as well as another activator of apoptosis eg bax39 in addition it decreases the activity of cell survival regulators such as akt and erk it also deactivates aktdependent proapoptotic regulator foxo3a thus promoting apoptotic cell death in tumors40it is well known that during cancer and radiotherapy excessive energy is used from the host41 ultimately contributing to mechanisms that promote loss of weight as shown in the present study which also showed that bl could return body weight to a normal level by decreasing tumor weight and volume currently the combined therapy bl irr has been shown to be more effective than single agent therapy in reducing tumor volume and weight indicating that bl could possess a radiosensitizing effect in addition the combined therapy has revealed a drastic decrease in tumor area percentage wide areas of necrotic cancer cells and presence of muscle fiber in the histopathological examination compared with the control est and estirradiated groups this seems to be in agreement with other findings of the role of bl in reducing metastasis and local tumor growth2342 in chemically induced mouse skin papillomas topical application of bl reduced tumor formation tumor volume and caused apoptotic cell death39 bl is a hydrolytic enzymatic complex which shows an efficient digestion and diffusion in tumor cells through attacking the glycosidic linkages and hence denatures glycoproteins thus it protects against tumor growth37 another study has demonstrated the use of controlled proteolytic activity on tumor as a successful strategy to increase therapeutic efficacy43 0cmekkawy figure effect of bromelain and gammairradiation blirr on relative gene expression of liver a parp1 b nfκb and c pparα each value represents the mean of records ± se significant versus control group where significant versus est group and significant versus estirradiated group at p est ehrlich solid tumor est irr ehrlich solid tumor irradiation est bl ehrlich solid tumor bromelain est bl irr ehrlich solid tumor bromelain irradiationthe aim of the radiotherapy protocols is to achieve the maximum curative effect on tumor cells with minimal damaging effect on normal cells hence antioxidants and other nutrients which do not interfere with therapeutic modalities for cancer may enhance the killing property decrease side effects and protect normal tissue44for estimation of the antioxidant ability of bl dpph assay was conducted in vitro and the free radical inhibitory action of bl was compared with some antioxidant compounds it was found that bl has a powerful free radical scavenging power bl belongs to thiol proteases in which the catalytic nucleophile is sulfhydryl groups of cysteine residues which in turn accounts for its antioxidant activity45the involvement of ros mda and pon1 are important mechanisms that play a vital role during radiation toxicity the use of antioxidants is an important preventive to decrease the toxic and pathological effects associated with oxidative stress caused by radiation46 the attained results show a hepatic impairment on the third day from exposure to Îradiation elevation of lpo and ros levels and inhibition of pon1 activity compared to normal mice however treatment with bl revealed an amelioration in hepatic damage caused by irradiation these results were in accordance with liu 47 who described the effect of radiation induced ros generation which in turn might attack cell membrane phospholipids and circulating lipids and thus increases production of mda48 lpo acts as a sensitive biomarker for oxidative stress that occurs as part of the pathogenesis of irradiation49 bl has sulfhydryl groups consequently accounting for its antioxidant activity45 thus it could act as ros scavengermeasurement of pon1 postradiotherapy could be an effective clinical biomarker of hepatic and systemic oxidative stress and may be used as an index of the usefulness of radiotherapy50 it has been demonstrated to catalyze hydrolysis of lipid hydroperoxides and lactones51 pon1 protects serum hdl and ldl ps against lipid peroxidation52 in the present study the decreased activity of pon1 upon radiation exposure might be due to its super saturation of lipid hydroperoxides and lactones upon treatment with bl the activity of pon1 was restored near to the normal level hence the pon1 | Colon_Cancer |
"adipogenesis is the process through which mesenchymalstem cells mscs commit to the adipose lineage and diï¬erentiate into adipocytes during this process preadipocytescease to proliferate begin to accumulate lipid droplets anddevelop morphologic and biochemical characteristics ofmature adipocytes such as hormoneresponsive lipogenesisand lipolytic programs currently there are mainly twomodels of benign adipocyte diï¬erentiation in vitro one isï¬broid pluripotent stem cells which can diï¬erentiate intonot only adipocytes but also muscle cartilage and othercells there are two kinds of ï¬broid pluripotent stem cellsbone marrow and adipose mesenchymal stem cells anothergroup is ï¬broblastic preadipocytes which have a single direction of diï¬erentiation namely lipid diï¬erentiation including3t3l1 and 3t3f422a cells cancer cells with tumorinitiation ability designated as cancer stem cells cscshave the characteristics of tumorigenesis and the expressionof speciï¬c stem cell markers as well as the longterm selfrenewal proliferation capacity and adipose diï¬erentiationpotential in addition to cscs cancer cells undergoing epithelialmesenchymaltransformation emt havebeen reported to be induced to diï¬erentiate into adipocytes[] lung cancer ncih446 cells can be induced to differentiate into neurons adipocytes and bone cells in vitro the adipogenesis diï¬erentiation treatment is promisingin the p53 gene deletion type of ï¬broblastderived cancer cancer cells with homologous recombination defectssuch as ovarian and breast cancer cells with breast cancersusceptibility genes brca mutations can be inducedto diï¬erentiate by poly adpribose polymerase parp 0cstem cells internationalinhibitors the nuclear receptor peroxisome proliferatoractivated receptor Î pparÎ agonist antidiabetic thiazolidinedione drug can induce growth arrest and adipogenicdiï¬erentiation in human mouse and dog osteosarcoma cells thyroid cancer cells expressing the pparÎ fusion proteinppfp can be induced to diï¬erentiate into adipocytes bypioglitazone adipogenesis can be induced in welldiï¬erentiated liposarcoma wdlps and dediï¬erentiatedliposarcoma ddlps cells by dexamethasone indomethacininsulin and 3isobutyl1methyl xanthine ibmx in this review we highlight some of the crucial transcription factors that induce adipogenesis both in mscs and inincluding the wellstudied pparÎ and ccaatcscsenhancerbinding proteins cebps as well as othercell factors that have been recently shown to have an important role in adipocyte diï¬erentiation we focus on understanding the complex regulatory mechanism of adipocytediï¬erentiation that can contribute to the clinical treatmentof human diseases including those caused by obesity andadipocytes dysfunction especially for the malignant tumorwhich can be transdiï¬erentiated into mature adipocytes adipocyte differentiationcell proliferation and diï¬erentiation are two opposingprocesses and there is a transition between these two processes in the early stages of adipocyte diï¬erentiation theinteraction of cell cycle regulators and diï¬erentiation factors produces a cascade of events which ultimately resultsin the expression of adipocyte phenotype adipogenesishas diï¬erent stages each stage has a speciï¬c gene expression pattern in general adipocyte diï¬erentiation ofpluripotent stem cells is divided into two phases the ï¬rstphase known as determination involves the commitment ofpluripotent stem cells to preadipocytes the preadipocytescannot be distinguished morphologically from their precursor cells but also have lost the potential to diï¬erentiate intoother cell types in the second phase which is known asterminal diï¬erentiation the preadipocytes gradually acquirethe characteristics of mature adipocytes and acquire physiological functions including lipid transport and synthesisinsulin sensitivity and the secretion of adipocytespeciï¬cproteins the diï¬erentiation of precursor adipocytes is also dividedinto four stages proliferation mitotic cloning early diï¬erentiation and terminal diï¬erentiation after the precursors are inoculated into the cell culture plates the cellsgrow exponentially until they converge after reaching contact inhibition the growth rate slows and gradually stagnatesand the proliferation of precursor adipocytes stops which isvery necessary for initiating the diï¬erentiation of precursoradipocytes adipocyte precursors exhibit transient mitosiscalled clonal expansion a process that relies on the actionof induced diï¬erentiation factors some preadipocyte cellsmouse cell lines 3t3l1 3t3f442a undergo one or tworounds of cell division prior to diï¬erentiation whereasother cell lines mouse c3h10t12 diï¬erentiating into adipocyte do not undergo mitosis clonal expansion whether mitotic clonal expansion is required for adiposediï¬erentiation remains controversial however it is certainthat some of the checkpoint proteins for mitosis regulateaspects of adipogenesis [ ] when cells enter the terminaldiï¬erentiation stage the de novo synthesis of fatty acidsincreases significantly the transcription factors and adipocyterelated genes work cooperatively to maintain precursor adipocyte diï¬erentiation into mature adipocytes containing largelipid droplets regulatory pathways inpreadipocytes commitmentadipocyte diï¬erentiation is a complex process in which geneexpression is ï¬nely regulated the most basic regulatory network of adipose diï¬erentiation has not been updated inrecent years but some factors and signaling pathways thatdo aï¬ect adipose diï¬erentiation have been continuouslyreported adipocyte diï¬erentiation is the result of the geneexpression that determines the phenotype of adipocyteswhich is a complex and delicate regulatory process figure wnt signal pathway in adipogenesis wnt signaling isimportant for adipocytes proliferation and diï¬erentiationboth in vitro and in vivo the wnt family of secretedglycoproteins functions through paracrine and autocrinemechanisms to uence cell fate and development wntprotein binding to frizzled receptors initiates signalingthrough catenindependent and independent pathways wnt signaling inhibits adipocyte diï¬erentiation in vitroby blocking the expression of pparÎ and cebpα constitutive wnt10b expression inhibits adipogenesis wnt10b isexpressed in preadipocytes and stromal vascular cells butnot in adipocytes in vivo transgenic expression of wnt10bin adipocytes results in a reduction in white adipose tissuemass and absent brown adipose tissue development wnt10a and wnt6 have also been identiï¬ed as determinantsof brown adipocyte development [ ] wnt5b is transiently induced during adipogenesis and promotes diï¬erentiation indicating that preadipocytes integrate inputs fromseveral competing wnt signals the hedgehog hh signaling pathway mechanismthree vertebrate hh ligands including sonic hedgehogshhindian hedgehog ihh and desert hedgehogdhh have been identiï¬ed and initiated a signaling cascademediated by patched ptch1 and ptch2 receptors [ ]hh signaling had an inhibitory eï¬ect on adipogenesis inmurine cells such as c3h10t12 ks483 calvaria mscslines and mouse adiposederived stromal cells thesecells were visualized by decreased cytoplasmic fat accumulation and the expression of adipocyte marker genes after hhsignaling was inhibited although it is generally agreedthat hh expression has an inhibitory eï¬ect on preadipocytediï¬erentiation the mechanisms linking hh signaling andadipogenesis remain poorly deï¬ned erkmapkppar signal pathway extracellularregulated protein kinase erk is required in the proliferativephase of diï¬erentiation erk activity blockade in 3t3l1 0cstem cells internationaldex insulin demxwnt 10band othersshhpbc smotgfð½p smad3 smad3testosteroneð½catentinarirspi3kaktcrebpkapcrebfoxo1a2tcflef gata23cebpð½mapkg3k3ð½p2cebpð½cebpαppará½»bmpssmad1srebpadipocytegenesfigure regulation pathways in preadipocytes commitment bmp and wnt families are mediators of mscs commitment to producepreadipocytes exposure of growtharrested preadipocytes to diï¬erentiation inducers igf1 glucocorticoid and camp triggers dnareplication leading to adipocyte gene expression due to a transcription factor cascade the dotted line indicates an uncertain molecularregulatory mechanismcells and embryonic stem cells can inhibit adipogenesis inthe terminal diï¬erentiation phase erk1 activity leads topparÎ phosphorylation which inhibits adipocyte diï¬erentiation this implies that erk1 activity must be reduced afteradipocyte proliferation so that diï¬erentiation can proceedthis reduction is mediated in part by mitogenactivatedprotein kinase mapk phosphatase1 mkp1 [ ]these extracellular and intracellular regulation factors causeadipocytespeciï¬c gene expression and eventually lead toadipocyte formation adipocyte differentiationregulatory proteins pparÎ and adipocyte diï¬erentiation pparÎ is a member of the nuclearreceptor superfamily and is both necessaryand suï¬cient for adipogenesis forced expression ofpparÎ is suï¬cient to induce adipocyte diï¬erentiation broblasts indeedthe proadipogenic cebps andkrüppellike factors klfs have all been shown to induceat least one of the two pparÎ promoters in contrast antiadipogenic transcription factor gata functioned in part byrepressing pparÎ expression pparÎ itself has twoisomers the relative roles of pparÎ1 and pparÎ2 in adipogenesis remain an open question pparÎ2 is mainlyexpressed in adipose tissue while pparÎ1 is expressed inmany other tissues although both can promote adipocytediï¬erentiation pparÎ2 could do so eï¬ectively at very lowligand concentration compared with pparÎ1 the twoprotein isoforms are generated by alternative splicing andpromoter usage and both are induced during adipogenesispparÎ1 can also be expressed in cell types other than adipocytes ren et al used engineered zincï¬nger proteins tothe expression ofthe endogenous pparÎ1 andinhibitpparÎ2 promoters in 3t3l1 cells ectopic expression ofpparÎ2 promotes adipogenesis whereas that of pparÎ1does not zhang et al reported that pparÎ2 deï¬ciencyimpairs the development of adipose tissue and insulin sensitivity there are transcriptional cascades between adipocytesgenes including pparÎ and cebpα which are the coreadipocyte diï¬erentiation regulators in the early stage of adipocyte diï¬erentiation the expression of cebp and cebpδincrease which upregulates cebpα expressionfurtheractivate pparÎ pparÎ activating cebpα in turn resultsin a positive feedback pparÎ binding with retinoic acid xreceptor rxr forms diï¬erent heterodimers the variousdimmers can combine with the pparÎ response elementppre and initiate the transcription of downstream genesfor diï¬erentiation into adipocytes cebps participate in adipogenesis and several cebpfamily members are expressed in adipocytesincludingcebpα cebp cebpÎ cebpδ and cebphomologous protein chop the temporal expression of thesefactors during adipocyte diï¬erentiation triggers a cascadewhereby early induction of cebp and cebpδ leads tocebpα expression this notion is further supported by thesequential binding of these transcription factors to severaladipocyte promoters duringadipocyte diï¬erentiationcebp is crucial for adipogenesis in immortalized preadipocyte lines cebp and cebpδ promote adipogenesis atleast in part by inducing cebpα and pparÎ cebpαinduces many adipocyte genes directly and plays an important role in adipose tissue development once cebpα isexpressed its expression is maintained through autoactivation despite the importance of cebps in adipogenesis 0cstem cells internationalthese transcription factors clearly cannot function eï¬cientlyin the absence of pparÎ cebp cannot induce cebpαexpression in the absence of pparÎ which is required torelease histone deacetylase1 hdac1 from the cebpαpromoter furthermore ectopic cebpα expressioncannot induce adipogenesis in pparÎï¬broblasts however cebpα also plays an important role in diï¬erentiated adipocytes overexpression of exogenous pparÎ incebpαdeï¬cient cells showed that although cebpα isnot required for lipid accumulation and the expression ofmany adipocyte genes it is necessary for the acquisition ofinsulin sensitivity [ ] figure human ï¬broblasts withthe ability to diï¬erentiate into adipocytes also do not undergomitotic cloning ampliï¬cation however pparÎ exogenousligands need to be added to promote adipocyte diï¬erentiation therefore it can be inferred that mitotic cloning expansion can produce endogenous ligands of pparÎ bmp and transforming growth factor tgf inadipocyte diï¬erentiation a variety of extracellular factorsaï¬ect the preadipocyte commitment of stem cells includingbone morphogenetic protein bmp transforminggrowth factor tgf insulininsulinlike growthfactor igf1 tumor necrosis factor α and interleukin matrix metalloproteinase ï¬broblast growthfactor fgf and fgf2 bmp and tgf have variedeï¬ects on the diï¬erentiation fate of mesenchymal cells the tgf superfamily members bmps and myostatinregulate the diï¬erentiation of many cell types includingadipocytes tgf inhibitor can promote adipose diï¬erentiation of cancer cells with a mesenchymal phenotypein vitro and transgenic overexpression of tgf impairsadipocyte development inhibition of adipogenesis couldbe obtained through blocking of endogenous tgf with adominantnegative tgf receptor or drosophila mothersagainst decapentaplegic protein smad inhibitionsmad3 binds to cebps and inhibits their transcriptionalactivity including their ability to transactivate the pparÎ2promoter [ ] exposure of multipotent mesenchymalcells to bmp4 commits these cells to the adipocyte lineageallowing them to undergo adipose conversion theeï¬ects of bmp2 are more complex and depend on the presence of other signaling molecules bmp2 alone has little eï¬ecton adipogenesis and it interacts with other factors such astgf and insulin to stimulate adipogenesis of embryonicstem cells bmp2 stimulates adipogenesis of multipotentc3h10t12 cells at low concentrations and can contribute tochondrocyte and osteoblast development at higher concentrations klfs in adipocyte diï¬erentiation during adipocyte differentiation some klf family members are overexpressedsuch as klf4 klf5 klf9 and klf15 while klf16 expression is reduced [ ] klf15 is the ï¬rst klf family members which were identiï¬ed to be involved in adipocytediï¬erentiation its expression increased significantly on thesixth day of 3t3l1 adipocyte diï¬erentiation and peakedon the second day of adipocyte induction in mscs andmouse embryonic ï¬broblasts inhibition of klf15 by sirnaor mutation led to a decrease in pparÎ cebpα fatty acidbinding protein fabp4 and glucose transporter glut4 however overexpression of klf15 in nih3t3cells was found to be associated with lipid accumulation aswell as increases in pparÎ and fabp4 mice with complete absence of klf5 showed embryonal lethality and micewith singlechromosome klf5 knockout showed a significant reduction in white fat in adulthood suggesting thatklf5 plays an important role in adipocyte diï¬erentiationklf5 can be activated by cebp or cebpδ which isinvolved in early adipocyte diï¬erentiation klf5 can beactivated by cebp or cebpδ which is involved in earlyadipocyte diï¬erentiation direct binding of klf5 to thepparÎ2 promoter in combination with cebps inducespparÎ2 expression transfection of klf5 dominantnegative mutants in 3t3l1 cells reduced lipid droplet accumulation and inhibited pparÎ and cebpα expressionwhereas overexpression of wild klf5 significantly promotedadipocyte diï¬erentiation even without exogenous hormonestimulation similar to klf5 klf9 knockdown can inhibitthe expression of a series of adipocyte diï¬erentiation genessuch as pparÎ cebpα and fabp4 hence inhibitingadipocyte diï¬erentiation however klf9 overexpressiondid not upregulate the expression of pparÎ and cebpα in addition klf4 can transactivate cebp by bindingto the region of kb upstream of the cebp promoter and promote lipid diï¬erentiation klf6 can forma complex with histone deacetylase3 hdac3 inhibitingpreadipocyte factor1 pref1 expression and promotinglipid diï¬erentiation klf2 is highly expressed in adiposeprogenitors and its expression decreases during the processof lipid diï¬erentiation overexpressed klf2 can bind to thecaccc region of pparÎ2 proximal promoter and inhibitlipid diï¬erentiation as well as the expression of pparÎcebpα and sterolregulated elementbinding proteinssrebp by inhibiting the promoter activity rnasequence analysisshowed that klfl6 expression wasdecreased on the ï¬rst day of adipocyte diï¬erentiation of3t3l1 cells adipocyte diï¬erentiation was promoted byklf16 knockdown but was inhibited by klf16 overexpression via inhibition of pparÎ promoter activity in addition klf3 and klf7 were also found to play a negativeregulatory role in adipocyte diï¬erentiation [ ] signal transducers and activators of transcriptionstats and adipocyte diï¬erentiation the activated statprotein enters the nucleus as a dimer and binds to the targetgene to regulate gene transcription in the adipocyte diï¬erentiation of mouse 3t3l1 cells the expression of stat1 andstat5 was significantly increased while that of stat3and stat6 was not significantly changed in the adipocyte diï¬erentiation of human subcutaneous adipose precursor cells stat1 expression was significantly decreased while the expression of stat3 and stat5 wasincreased and stat6 expression was unchanged therole of stat1 in adipocyte diï¬erentiation is not clearbecause its expression trend in humans and mice diï¬ersduring the adipocyte diï¬erentiation process early adipocytediï¬erentiation of 3t3l1 cells was inhibited by stat1 0cstem cells internationalklf5srebp1cklf15klf2chopcebpá½»krox20ligandcebpð½cebpð¿gata23ppará½»cebpð¼proadipogenicantiadipogenicgenes of terminaladipocytedifferentiationfigure a cascade of transcription factors that regulate adipogenesis pparÎ is one of the key transcription factors in adipogenesis and thecore of the transcriptional cascade that regulates adipogenesis pparÎ expression is regulated by several proadipogenic blue andantiadipogenic red factors cebpα is regulated through a series of inhibitory proteinprotein interactions some transcription factorfamilies include several members that participate in adipogenesis such as the klfs black lines indicate eï¬ects on gene expression violetlines represent eï¬ects on protein activityagonist interferon Î loss of stat1 in 3t3l1 cells can rescue the inhibition of adipocyte diï¬erentiation caused byprostaglandin factor 2α other studies have found thatstat1 is required for adipose diï¬erentiation and stat1overexpression in c3h10t12 cells can prevent the inhibition of lipid diï¬erentiation caused by bcell lymphoma6knockdown there was no abnormal adipose tissuein stat1 knockout mice stat3 not only aï¬ectsthe proliferation of 3t3l1 cells but also coregulates theiradipocyte diï¬erentiation with high mobility group protein the fabp4 promoter was used to speciï¬callyknock out stat3 in the adipose tissue of mice and theresults showed that mice weight significantly increasedand the adipocyte quantity increased compared with thewildtype mice stat5a and stat5b have diï¬erenteï¬ects on adipocyte diï¬erentiation abnormal adipose tissuewas found in the mice with stat5a or stat5b knockout ordouble knockout and the amount of adipose tissue was onlyoneï¬fth of the original adipose tissue in mice withoutknockdown histone modiï¬cation in adipocyte diï¬erentiation histone deacetylase sirtuin sirt plays an important rolein biological processes such as stress tolerance energymetabolism and cell diï¬erentiation during the adipocyte diï¬erentiation of c3h1012 cells sirt1 expressiondecreased overexpression of sirt1 activated thewnt signal which caused the deacetylation of cateninthe accumulation of catenin in the nucleus could inhibitadipocyte diï¬erentiation sirt1 knockdown resulted inincreased acetylation of the histones h3k9 and h4k16 inthe secreted frizzledrelated protein sfrp and sfrp2 promoters thereby promoting transcription of these genes andpromoting lipid diï¬erentiation forkhead box proteino foxo is a member of the transcription factor foxofamily it can recruit cyclic amp response elementbindingprotein cbphistone acetyltransferase p300 to initiate anacetylation the acetylated foxo1 can be phosphorylatedby phosphorylated protein kinase b pkbakt the phosphorylation of foxo1 by akt inhibits the transcriptionalactivation of foxo1 the acetylation of foxo1 lost the ability of dnabinding aï¬nity and promoted its shuttling fromnuclei to cytoplasm sirt1 and sirt2 can deacetylateand active foxo1 activated foxo1 nonphosphorylatednuclear foxo1 in the nucleus binds to the promoters of target genes encoding p21 p27 and pparÎ and initiates subsequent transcriptions sirt2 inhibits the acetylation andphosphorylation of foxo1 thereby induces the accumulation of activated foxo1 in the nucleus activated foxo1could inhibit adipogenesis via pparÎ [] lysinespeciï¬c histone demethylase lsd1 expression increasedduring the adipocyte diï¬erentiation of 3t3l1 cells lsd1could reduce the dimethylation levels of histone h3k9 andh3k4 in the cebpα promoter region thereby promotingadipocyte diï¬erentiation set domaincontaining setd8 catalyzed the monomethylation of h4k20 andpromoted pparÎ expression the activation of pparÎ transcriptional activity leads to the induction of monomethylatedh4k20 and modiï¬cation of pparÎ and its targets therebypromoting adipogenesis enhancer of zeste homolog ezh2 is a methyltransferase and can bind methyl groupsto histone h3k27 which is also necessary for lipid diï¬erentiation the absence of ezh2 in brown fat precursors results inreduced levels of the wnt promoter histone h3k27me3which is also saved by the ectopic ezh2 expression or theuse of a wntcatenin signal inhibitor in addition histone demethylases such as lysinespeciï¬c histone demethylase lsdkdm kdm6 and histone lysine demethylasephf2 are also involved in adipose diï¬erentiation andkdm2b inhibits transcription factor activator protein 2αpromoter via h3k4me3 and h3k36me2 role of microrna and long noncodingrna in adipogenesismicrorna mir can bind and cut target genes or inhibittarget gene translation endogenous sirna can be producedby the action of dicer enzyme and bind to a speciï¬c proteinto change its cellular location many kinds of mirsare involved in regulating adipocyte diï¬erentiation the 0cstem cells internationalexpression of mir143 increased during the diï¬erentiationof adipose progenitor cells overexpression of mir143promoted gene expression involved in adipose diï¬erentiationand triglyceride accumulation inhibition of mir143 prevented the adipose diï¬erentiation of human fat progenitorcells [ ] additionally mir8 promotes adipocyte diï¬erentiation by inhibiting wnt signaling moreover mir mir103 mir21 mir519d mir210 mir30mir204211 and mir375 also play a certain role in promoting adipocyte diï¬erentiation while mir130 mir448and let7y inhibit lipid diï¬erentiation [ ] in additionto mirs long noncoding rna lncrna is a type of noncoding rna and is important during epigenetic regulationand can form a doublestranded rna complex with mrnacauses protein transcription lncu90926 inhibits adipocytediï¬erentiation by inhibiting the transactivation of pparÎ2 as a novel lncrna hoxaas3 expression increasedduring the adipose diï¬erentiation of mscs and hoxaas3 silencing reduced the marker gene of adipose diï¬erentiation and inhibited the adipose diï¬erentiation zhu et al reported that hoxaas3 interacted with ezh2 toregulate lineage commitment of mscs hoxa as3 canregulate the trimethylation level of h3k27 in the runx2promoter region by binding to ezh2 therefore hoxaas3 is considered to be an epigenetic switch regulating mscslineage speciï¬city adipocyte diï¬erentiationassociatedlncrna can act as a competitive endogenous rna of mir in the process of lipid diï¬erentiation thereby promotingthe expression of sirt1 the target gene of mir204 and thusinhibiting lipid diï¬erentiation the lncrna neat1can also regulate adipocyte diï¬erentiation under the uence of mirna140 other lncrna including lncrnablnc1 and plnc are also involved in regulating adipocytediï¬erentiation [ ] other biochemical response involved inadipocyte differentiation unfolded protein responses in adipocyte diï¬erentiationin the endoplasmic reticulum of eukaryotes unfolded protein response involves three proteinsinositolrequiringenzyme 1α doublestranded rnadependent proteinkinaselike er kinase and activating transcription factoratf 6α knockdown of atf6α aï¬ects the expressionof adipocytes genes and inhibits c3h10t12 adipocyte differentiation the inhibitory eï¬ect of berberine on adipocyte diï¬erentiation of 3t3l1 cells is also due to inducedchop and decorin expressions and this inhibitory eï¬ectis ameliorated by chop knockout in the adipocytediï¬erentiation process of 3t3l1 cells increases in pparÎand cebpα as markers of adipocyte diï¬erentiation wereaccompanied by an increase in the corresponding proteinexpressions of phosphorylated eukaryotic translation initiaeif 2α phosphorylated endoribonucleasetion factorire1α atf4 chop and other unfolded protein responsesendoplasmic reticulum stress inducer or hypoxic endoplasmic reticulum stress can inhibit adipocyte diï¬erentiationadditionally eif2α mutation results in continuous activation or overexpression of chop which also inhibits adipocyte diï¬erentiation after the initiation of adiposediï¬erentiation numerous diï¬erentiationassociated proteinsare synthesized exogenous endoplasmic reticulum stressinducers can lead to excessive endoplasmic reticulumresponse which in turn aï¬ects the synthesis of proteinsrelated to diï¬erentiation and inhibits adipocyte formationfigure role of oxidative stress in adipogenesis during thedirectional diï¬erentiation of mscs mitochondrial complexi and iii and nadph oxidase nox4 are the main sourcesof oxygen species ros production currently it is believedthat ros aï¬ects not only the cell cycle and apoptosis but alsodiï¬erentiation through uencing the signaling pathwaysincluding the wnt hh and foxo signaling cascade duringmscs diï¬erentiation the diï¬erentiation ability ofstem cells is determined by the arrangement of perinuclearmitochondria which speciï¬cally manifests as low atpcellcontents and a high rate of oxygen consumption the lackof these characteristics indicates stem cell diï¬erentiation adipocyte diï¬erentiation is a highly dependent rosactivation factor related to mitosis and cell maturation schroder et al found that exogenous h2o2 could stimulate adipocyte diï¬erentiation of mouse 3t3l1 cells andhuman adipocyte progenitor cells in the absence of insulinh2o2 regulates adipocyte diï¬erentiation of 3t3l1 cells ina dosedependent manner high doses of h2o2 and μm promote adipocyte diï¬erentiation [ ] tormos et al found that ros synthesis increased in humanmscs at the early stage of adipose diï¬erentiation and targeted antioxidants could inhibit lipid diï¬erentiation byknocking down rieske ironsulfur protein and ubiquinonebinding protein ros produced by mitochondrial complexiii was found to be necessary in initiating adipose diï¬erentiation however other studies have shown that theexpression levels of adiponectin and pparÎ were decreasedby using h2o2 mm in 3t3l1 cells free radical nitric oxide no also promotes lipid diï¬erentiationbecause treatment with no inducer hydroxylamine or nosynthase nos substrate arginine can significantly induceadipose diï¬erentiation of rat adipose progenitor cells nosinduced adipose diï¬erentiation mainly via enos rather thaninos ros can induce adipose diï¬erentiation primarily by inhibiting wnt foxo and hh signaling pathwaysthat inhibit lipid diï¬erentiation autophagy in adipocyte diï¬erentiation the increase inautophagosomes during lipid diï¬erentiation indicates thatautophagy may play an important role in lipid diï¬erentiation baerga et al conï¬rmed that the adipocyte diï¬erentiation eï¬ciency was significantly inhibited in mouse embryonic ï¬broblasts lacking autophagyrelated gene atg agene encoding an essential protein required for autophagy knockdown of atg5 in 3t3l1 cells promotesproteasomedependent degradation of pparÎ2therebyinhibiting adipocyte diï¬erentiation zhang reportedthat autophagyrelated gene 7atg7 is also crucial for adipose development atg7deï¬cient mice were slim and onlyhad of white fat compared to wildtype mice and the 0cstem cells internationalcebpð½ geneebf1 geneklf4egr2cebpð½cytosolcebpð¿ genecebpð¿klf5geneppará½» geneklf5nr2f2nfkb11433relasrebf1a2rxrappará½»ppará½»rxra heterodimerppará½»rxracorepressor complexfabp4ligands of ppará½»fam120bthrap3ep300ncoa2ncoa3helz2ncoa1crebbpebf1adipoq geneaidrfcebpð¼ geneznf638znf467cebpð¼ncor1hdac3ncor2 slc2a4 geneglut4 genelep genefabp4 genecdk4ccnd3plin1 genepck1 genefabp4cd36 geneppararxracoactivator complexppará½»fatty acidrxramediatorcoactivator complexangptlgeneppargc1amediator complex consensuslpl genenucleoplasmproteins bind to gene promoterstranscription of genes into proteinsacting on proteins compoundingtgfð½1wnt1wnt10btnf77233adipoqglut4slc2a4 tetramerlepfabp4lipid dropletplin1pck1papa pa4xpalmccd36paangptl4lplfigure regulation of adipocyte diï¬erentiation a regulatory loop exists between pparÎ and cebp activation transcription factor coeebf activates cebpα cebpα activates ebf1 and ebf1 activates pparÎ cebp and cebpδ act directly on the pparÎ gene bybinding its promoter and activating transcription cebpα cebp and cebpδ can activate the ebf1 gene and klf5 the ebf1 and klf5proteins in turn bind the promoter of pparÎ which becomes activated other hormones such as insulin can aï¬ect the expression ofpparÎ and other transcription factors such as srebp1c pparÎ can form a heterodimer with the rxrα in the absence of activatingligands the pparÎrxrα complex recruits transcription repressors such as nuclear receptor corepressor ncor ncor1 andhdac3 upon binding with activating ligands pparÎ causes a rearrangement of adjacent factors corepressors such as ncor2 are lostand coactivators such as transcription intermediary factor tif2 cbp and p300 are recruited which can result in the expression of cyclicampresponsive elementbinding protein creb followed by pparÎ pparÎ expression initiates the expression of downstream genesincluding angiopoietinrelated protein pgar perilipin fabp4 cebpα fatty acid transportrelated proteins carbohydrate metabolismrelated proteins and energy homeostasisrelated proteinslipid metabolism and hormoneinduced lipolysis in the adipocytes were altered autophagy related gene atg4b isactivated by cebp in the process of lipid diï¬erentiationand autophagy activation is necessary for the degradationof klf2 and klf3 two negative regulators of lipid diï¬erentiation these results showed that adipose diï¬erentiation andautophagy are mutually complementary in 3t3l1cells autophagy was inhibited by aspartate ammonia or 0cstem cells internationalmethyladenine at diï¬erent lipid induction periods and days and only autophagy inhibition at days hindered the formation of lipid droplets and the expression of lipid marker genes indicating that autophagy wasvery important in the early stage of lipid diï¬erentiation recent studies showed that lc3 is overexpressed in3t3l1 cells further demonstrating the important role ofautophagy in lipid diï¬erentiation role of alternative splicing in adipogenesis selectivesplicing is uenced by splicing regulators which regulateadipocyte diï¬erentiation by regulating the selective splicingof genes speciï¬c to this process lipin1 is an important regulator in the process of adipocyte diï¬erentiation and includestwo isomers lipin1α and lipin1 which have diï¬erenteï¬ects high expression of lipin1α promotes adipocyte differentiation while that of lipin1 promotes lipid droplet formation in sam68deï¬cient mice the ï¬fth intron ofserinethreonineprotein kinase mtor was retained resulting in unstable and rapid mtor degradation and inhibitionof adipocyte diï¬erentiation furthermore there arefour isomers of pref1 pref1a and pr | Colon_Cancer |
n6methyladenosine m6a regulators are involved in the progression of various cancers via regulating m6amodiï¬cation however the potential role and mechanism of the m6a modiï¬cation in osteosarcoma remains obscurein this study wtap was found to be highly expressed in osteosarcoma tissue and it was an independent prognosticfactor for overall survival in osteosarcoma functionally wtap as an oncogene was involved in the proliferation andmetastasis of osteosarcoma in vitro and vivo mechanistically m6a dot blot rnaseq and meripseq meripqrtpcrand luciferase reporter assays showed that hmbox1 was identiï¬ed as the target gene of wtap which regulatedhmbox1 stability depending on m6a modiï¬cation at the ²utr of hmbox1 mrna in addition hmbox1 expressionwas downregulated in osteosarcoma and was an independent prognostic factor for overall survival in osteosarcomapatients silenced hmbox1 evidently attenuated shwtapmediated suppression on osteosarcoma growth andmetastasis in vivo and vitro finally wtaphmbox1 regulated osteosarcoma growth and metastasis via pi3kaktpathway in this study demonstrated the critical role of the wtapmediated m6a modiï¬cation in theprogression of osteosarcoma which could provide novel insights into osteosarcoma treatmentintroductionosteosarcoma is a primary malignant bone tumor thatis common among childhood and adolescents worldwide1despite the improvements including multiagent chemotherapy with surgery in recent years the 5year survival rate is for localized osteosarcoma and is for recurrent and metastatic osteosarcoma23 thereforea better understanding of molecular mechanism isurgent for developing novel therapeutic strategies forosteosarcomacorrespondence jinglei miao miaojinglei126com orjinsong li jinsongli_csu163com1department of orthopaedics the third xiangya hospital of central southuniversity tongzipo rd changsha hunan china2shanghai key laboratory of regulatory biology institute of biomedicalsciences and school of life sciences east china normal university shanghai chinafull list of author information is available at the end of the edited by a stephanouitn6methyladenosine m6a is the prominent dynamicmrna modiï¬cation which is involved in various biological process by regulating mrna translocation translation and stability4is catalyzed by m6a writermethyltransferase removed by erasers rna demethylases and recognized by m6a readers involving in variousbiological progression5 the formation of m6a is catalyzed by a methyltransferase mettl3 and mettl14form a core catalytic complex of methyltransferases that isstabilized by wtap8 recently mettl16910 mettl5zcchc411 and zc3h1312 were showed to play a criticalrole in compositing methyltransferase complex andfacilitating m6a methylation13 the eraser alkbh5 andfto has m6a demethylation activity to remove the m6amodiï¬cation the reader proteins are from yth familyheterogeneous nuclear ribonucleoprotein hnrp familyand insulinlike growth factor mrnabinding protein the authors open access this is licensed under a creative commons attribution international license which permits use sharing adaptation distribution and reproductionin any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the creative commons license and indicate ifchanges were made the images or other third party material in this are included in the s creative commons license unless indicated otherwise in a credit line to the material ifmaterial is not included in the s creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this license visit httpcreativecommonslicensesby40ofï¬cial of the cell death differentiation association 0cchen cell death and disease page of isfamily they recognize the m6a modiï¬cation to adjustrna metabolisms14dynamic and reversible m6a regulation was reported tobe involved in various physiological and pathologicalprocesses including stem cell differentiation cardiachomeostasis adipogenesis neuronal disorders and spermatogenesis15 recent years compelling evidence hasrevealed that m6a modiï¬cation plays an important role inthe tumorigenesis of various cancers20 for examplemettl3 was reported to play key role in the progressionof colorectal carcinoma25 bladder cancer2627 gastriccancer28 and pancreatic cancer29 ythdf2 was involvedthe progression of acute myeloid leukemia aml viaregulating hematopoietic stem cells hscs activity4 ftowas also reported to play a crucial role in the progressionof melanoma30 breast cancer31 gastric cancer32 andintrahepatic cholangiocarcinoma nevertheless the role ofm6a modiï¬cation in osteosarcomastill poorlycharacterizedwtap a wilms tumor wt1 associated protein33has been reported to be critical in the biological processesincluding g2m transition and premrna splicing34in addition accumulated studies identiï¬ed the importantrole of wtap in the progression of various cancers37 forexample wtap function as oncogene in cholangiocarcinoma38 acute myeloid leukemia39 colon cancer40 ovariancancer41 and diffuse large bcelllymphoma42 recentstudies reported that wtap is strictly connected to afunctional m6a methylation complex43 here we revealedthe increased expression of wtap in osteosarcoma tissuewhich was associated with clinicopathological features andpoor prognosis in osteosarcoma patients wtap functionas an oncogenic gene and it promotes the m6a modiï¬cation and progression of osteosarcoma mechanistically wtap induced the growth and metastasis ofosteosarcoma via downregulating hmbox1 expression inm6adependent manner further investigations demonstrated that wtaphmbox1 regulated osteosarcomagrowth and metastasis via pi3kakt pathway overallthese results imply that wtaphmbox1 may be animportant mechanism of osteosarcoma progression andserve as a novel therapeutically target of osteosarcomamaterials and methodsclinical samples and ethicsonehundredandfour paired fresh osteosarcoma metastatic os sample and nonmetastatic os sampleand normal tissues were obtained from patients withoutradiotherapy andor chemotherapy at the third xiangyahospital of central south university and then immediately frozen in °c for rna and protein extraction orformalxed and parafï¬nembedded for further usedinformed consent was obtained from each patient or theirguardians and the study were approved by the ethicsofï¬cial of the cell death differentiation associationcommittee of the third xiangya hospital of centralsouth universitycell culture transfection and infectionthe human hfob119 cells and osteosarcoma cell linessjsa1 mg63 hos u2os and 143b were obtainedfrom the cell bank of the chinese academy of sciencesshanghai china the osteosarcoma cells were culturedin dmem with fbs gibco grand island ny usathe hfob119 cells were cultured in dmemf12 df with fbsthe sequences of shrna cloned into plko1 vectorand then shwtapplko1 or shhmbox1plko1 werecotransfected with packing and pax2 plasmids into293tf cells fortyeight hours after transfectionthelentivirus was collected and infected hos and u2os cellsfor h the μgml puromycin was used for screeninginfected cells the primers were listed in table s1western blotproteintechantiythdf2antipi3k ab191606 abcamthe proteins were obtained from cells and tissues beinglysed with ripa buffer and then separated by sdspage the antiwtap ab195380 abcam antihmbox1161231apab220163abcamantippi3kab182651 abcam akt 9272s cell signaling paktser473and antigapdhab181602 abcam were used for pvdf membrane incubating overnight at °c the protein expression was detected by incubating with antirabbit secondary antibodies4051s cellsignalingrnaseq analysis and qrtpcrtotal rnas were extracted from osteosarcoma tissueand cells using trizol thermofisher usa for rnaseqanalysis the library construction and illumina sequencingusing the illumina truseq rna sample prep kit sandiego ca usa for qpcr validation the cdna wasobtained using first strand cdna synthesis kittoyobo finally the mrna expression was detectedusing sybr green biorad california usa all primers were in table s1rna m6a dot blot assay and rna m6a quantiï¬cationtotal rnas were extracted from osteosarcoma cells bytrizol thermofisher usa and then nanodrop3000was used for detecting rna quality the m6a content wasdetected by epiquik m6a rna methylation quantiï¬cation kit colorimetric epigentek usathe polya rnas and ng were spottedonto a nylon membrane ge healthcare for rna m6adot blot assay and then incubated with m6a antibody noabe572 merck millipore usa at °c overnight them6a dots was analyzed using an imaging system biorad usa 0cchen cell death and disease page of meripseq and meripqrtpcrtotal rnas were extracted from osteosarcoma cells bytrizol the dnafree fragmented rnas were incubatedwith magnetic dynabeads bounded antim6a antibodyabcam usa to enrichment the mrna with m6a andthen beads were treated with proteinase k and rna wasextracted for meripseq or validation by qrtpcr theprimers are in table s1cell proliferation assaysthe cck8 cell counting kit8 c0038 beyotimebiotechnology china was used for cell proliferationability44colony formation assaysthe cells were seeded into 6well plates with a density à cells per wells and cultured in dmem medium for weeks and then paraformaldehyde pfa was usedto ï¬x the colonies and crystal violet was used to stain asprevious described45woundhealing assayosteosarcoma cells were cultured for h to reach conï¬uency and then a straight artiï¬cial wound wasscraped with a μl pipette tip the cell migration abilitywas measured by photographing the distance at and h45transwell invasion assaysthe transwell chamber bd science bedford mausa was used for invasion assays as previously described1 in brief à osteosarcoma cells were seeded intothe upper chambers and incubated for h the invasivecells were counted and quantiï¬ed for cellinvasion asprevious described45luciferase reporter assaysthe ²utr of hmbox1 was cloned into pmiglovector promega usa the putative m6a sites bases awere mutated into bases c in ²utr using sitedirectedmutagenesis kit thermo usa the wt and mutplasmids were transfected in osteosarcoma cells and thenthe dualluciferase assay kit promega was used fordetecting the luciferase activity44animal experimentsnude mice weeks male were used for tumor modelall animal care and handling procedures were approvedby the institutional animal care and use committee ofthe third xiangya hospital of central south universitychangsha china for the subcutaneous tumor model Ãshwtapshhmbox1 u2os cells seeded into mice via subcutaneous injection tumor volume and tumor weightshhmbox1 orshncshwtap orofï¬cial of the cell death differentiation associationwere measured to analyze tumor growth as previousdescribed46 for orthotopic xenografttumor modelshnc shwtap shhmbox1 or shwtapshhmbox1u2os cells were labeled with a luminescent dye and gfpand injected into the cavity of the tibia of mice thirtydays after injection tumor growth was detected for themetastasis model the cells were injected into the tail veinand the lung metastasis were detected days afterinjection using in vivo bioluminescence imaging systemimmunohistochemistryimmunohistochemistry ihc analysis was performedusing antiwtap ab195380 abcam antihmbox1161231ap proteintech as pervious described26bioinformatics analysisthe geo dataset gse87624 and gse46705 weredownloaded and analyzed by r version httpswwwrproject the different expressed gene wascalculated with edger package and identiï¬ed by thethreshold criteria of log2 foldchange fc ¥ and adjp go and kegg analysis was performed toinvestigate the potential role of genes proteinproteininteraction ppi network was analyzed by the stringdatabasestatistical analysisthe spss spss inc chicago il usa was usedfor the statistical analyses using anova or students ttest in this study kaplanmeier analysis was used forsurvival the correlation between wtap and hmbox1were analyzed using pearson analysis statistical signiï¬cance was deï¬ned p resultselevated wtap is associated with poor prognosis ofosteosarcoma patientsto reveal the important role of m6a modiï¬cation inosteosarcoma we explored the expression levels of m6arelative genes in osteosarcoma tissue and normalbone tissue gse87624 normal and osteosarcomaas shown in figs 1a and s1a we examined the expressionof m6arelated genes in geo database and found thatwtap rbm15 ythdf1 and ythdf2 were differentlyexpressed in tumor tissue compared with control tissuethe m6a writers were reported to play key role on theprogression of various cancers so wtap was selected forfurther analysis next we detected the expression ofwtap in pairs of osteosarcoma tissue and the corresponding paratumor tissues from the third xiangyahospital of central south university txhcsu consistent with the geo results the signiï¬cant higher mrnaand protein levels of wtap in osteosarcoma was detectedusing the qpcr and wb analysis fig 1b c moreover 0cchen cell death and disease page of fig the expression levels of wtap in osteosarcoma tissue and cell lines a wtap expression in osteosarcoma tissue compared with normalbone tissue from gse87624 b qpcr assay and c western blot assay revealed the wtap expression in osteosarcoma tissue and the correspondingparatumor tissues from txhcsu d the kaplanmeier survival analysis e univariate and multivariate survival analysis f establishment of the overallsurvival nomogram for osteosarcoma patients g timedependent roc analysis h the mrna expression levels of wtap in osteosarcoma cell linesand hfob119 cell line using qpcrthe osteosarcoma patients with high wtap showed pooroverall survival in txhcsu fig 1d and the univariateand multivariate cox analysis showed that wtap andmetastasis were the independent prognostic factor foroverall survival in osteosarcoma patients fig 1e theprognostic nomogram wasthepredictusedtoprobabilities of overall survival rates of osteosarcomapatients and the calibration curves showed has a goodconsistency between the prediction and the actual observation fig 1f in addition the years roc curve aucof wtap in osteosarcoma patients from txhcsu was fig 1g besides the overexpression of wtap wasofï¬cial of the cell death differentiation association 0cchen cell death and disease page of table the association of wtap expression andclinicopathologic characteristics of osteosarcoma patientswtap acts as an oncogene that promoted cell proliferation migration and invasion in osteosarcomacharacteristics case number wtap expressionp valuehmbox1 is potential target of wtap in osteosarcomagendermalefemaleage¤tumor size¤ cm cmmetastasisyesnotnmiiiiiiivhighn lown p p p p p associated with tumor size metastasis and tnm stagetable and fig s1b finally the higher expression ofwtap was also detected in osteosarcoma cell lines sjsa mg63 hos u2os and 143b compared with that inthe human osteoblast hfob119 cell fig 1h collectively these results demonstrated that wtap is highlyexpressed in osteosarcoma and is correlated with its poorprognosissilenced wtap signiï¬cantly represses osteosarcomaprogression in vitroto generateto further clarify the role of wtap in osteosarcomashwtap1 andwe next used shrna lentivirusshwtap2stable wtapknockdownosteosarcoma cells and analyzed the role of wtap oncells migrationinvasion and proliferation of osteosarcoma the shwtap1 and shwtap2 signiï¬cantlyknockdown the expression levels of wtap in osteosarcoma cells fig 2a the cck8 assay and colonyformation assay showed that wtap deï¬ciency signiï¬cantly reduced proliferative capacity of osteosarcomacells fig 2b c the transwellinvasion and woundhealing assays showed that the invasion and migrationability of the osteosarcoma were signiï¬cant reduced bysilenced wtap fig 2d e these results determine thatofï¬cial of the cell death differentiation associationto reveal the potential mechanism by which wtapregulates the progression of osteosarcoma we performedrnaseq m6aseq in shwtapshnc u2os cell andclipseq to revealthe potential genes regulated bywtapmediated m6a modiï¬cation the rnaseq resultsrevealed downregulated degs and upregulateddegs with logfc and adjp in shwtapgroup compared with shnc group fig s2a geneontology go showed that degs were enriched in neutrophil activation cell cycle and so on fig s2b kegganalysis showed that degs were enriched in metabolismrelated signal pathway fig s2c the m6aseq analysisidentiï¬ed differentially m6a modiï¬cation genes inwtapsilenced u2os cell compared with normal u2oscells table s2 the clipseq from gse46705 revealed wtaptargeted rna in wtap overexpressed cellusing parclip technology and then we obtained theoverlapped genes in rnaseq m6aseq and clipseq asshown in fig 3a genes were overlapped in three groupsincluding two upregulated gene slc3a2 casp7 andfour downregulated gene abr hs6st1 cd59 andhmbox1 consistent with these results slc3a2 andcasp7 were signiï¬cantly upregulated and abr hs6st1cd59 and hmbox1 were signiï¬cantly downregulated inosteosarcoma tissues from geo gse87624 databasefig s3a these results were also conï¬rmed in theosteosarcoma tissues from our txhcsu data fig s3band then we evaluated the regulation of wtap on thesix candidates in osteosarcoma cells using qpcr amongwhich hmbox1 was the most signiï¬cantly upregulatedgene in shwtap osteosarcoma cells fig 3b and wasselected for further analysis consistent with the mrnaexpressionthe protein level of hmbox1 was alsoremarkably increased by silenced wtap fig 3c wenext evaluated the relationship between wtap andhmbox1 expression in gse87624 and txhcsu as ourspeculation hmbox1 expression was negatively correlated with wtap expression r in gse87624and r in txhcsu fig 3d moreover weanalyzed the relationship between hmbox1 expressionclinicopathological features in osteosarcoma patientsfrom txhcsu hmbox1 expression was signiï¬cantlyreduced with tumor size and metastasis table and figs3c and the survival analysis results showed that thepatients with low level hmbox1 had poor overall survival in osteosarcoma fig 3e moreover the univariateand multivariate analysis demonstrated hmbox1 as anindependent prognosticsurvivalp in osteosarcoma patients fig 3f we alsorevealedinof hmbox1expressionfactorfor overallthelower 0cchen cell death and disease page of fig silenced wtap signiï¬cantly repressed osteosarcoma progression in vitro a the shwtap1 and shwtap2 downregulated wtapexpression b cck8 assay revealed that silenced wtap reduced the cell proliferation ability in osteosarcoma c colony formation assay showed thatsilenced wtap decreased the cloning number of osteosarcoma cells d transwell invasion and e woundhealing assay revealed the inhibition ofsilenced wtap on osteosarcoma cell invasion and migrationosteosarcoma cell lines sjsa1 mg63 hos u2os and143b than that in the human osteoblast hfob119 cellline fig 3g in these results indicated thathmbox1 is a potential target of wtap and is related topoor prognosis of osteosarcoma patientswtap regulates hmbox1 expression via m6a modiï¬cationin osteosarcomaas meripseq data revealed different m6a modiï¬cationof hmbox1 in nc and wtapsilenced cells we nextanalyzed whether wtap regulated the hmbox1ofï¬cial of the cell death differentiation association 0cchen cell death and disease page of fig hmbox1 is a potential target of wtap a the venn diagram was generated from differentially expressed genes in rnaseq m6aseq andclipseq in gse46705 the expression of the overlapped genes in hos and u2os cells with silenced wtap using b qpcr assay and c western blotassay d correlation analysis of wtap and hmbox1 in osteosarcoma from gse87624 and txhcsu e the kaplanmeier survival analysis f univariateand multivariate survival analyses g wtap hmbox1 expression in osteosarcoma cell lines and human osteoblast hfob119 cell lineexpression in an m6adependent manner using m6a dotblot and rna methylation quantiï¬cation assay asexpected m6a levels were substantially decreased inwtapknockdown osteosarcoma cells compared controlosteosarcoma cells fig 4a moreover meripqpcrassay showed that hmbox1 was effectively enriched bym6aspeciï¬c antibody and enriched hmbox1 wasremarkably decreased by in wtapknockdown cells fig4b therefore we supposed that wtap could regulatem6a levels of hmbox1 according to the m6a rnaseqthe ²utr ofresulthmbox1 and the sramp httpwwwcuilabcnsramppredicted three very high conï¬dence m6a sites at the ²utr of hmbox1 fig s4 to further prove the directedthe m6a modiï¬cation was attarget role of wtap on hmbox1 with m6a modiï¬cation we cloned the hmbox1 ²utr containing these m6a sites into pmirglo vectors and then we mutant thebases a into bases c in the predicted m6a sites fig3f in fig 3g the luciferase activity of hmbox1 wassigniï¬cantly increased by silenced wtap however theluciferase activity of mut hmbox1 did not affected bysilenced wtap in both hos and u2os cells fig 4cythdf2 is a well know m6a reader and plays animportant role in the progression of several cancers viaregulating mrna degradation figure 1s showed thatythdf2 was upregulated in osteosarcoma tissues ingse87624 in fig 4e ythdf2 evidently upregulated inosteosarcoma tissue and cells we next shed light on theofï¬cial of the cell death differentiation association 0cchen cell death and disease page of table the association of hmbox1 expression andclinicopathologic characteristics of osteosarcoma patientscharacteristics case number hmbox1 expressionp valuehighn lown gendermalefemaleage¤tumor size¤ cm cmmetastasisyesnotnmiiiiiiivp p p p p expression of ythdf2 in osteosarcoma and the role ofythdf2 on hmbox1 expression in osteosarcoma disappointedly silenced ythdf2 showed no effects onhmbox1 expression in osteosarcoma cells fig 4f suggesting that wtap regulated m6amediated hmbox1expression in ythdf2independent mannertogether these data suggested that wtaprepressedhmbox1 expression via regulating m6a modiï¬cation ofhmbox1 at its ²utrhmbox1 is involved in wtapmediated osteosarcomaproliferation and metastasis in vitrowe next explored whether wtap promoted osteosarcoma progression by regulating hmbox1 expressionas shown in fig 5a hmbox1 expression was evidentlyincreased by wtap knockdown and was decreased byhmbox1 knockdown in osteosarcoma cells the cck8results showed that the proliferation levels were increasedby shhmbox1 in wtapsilenced hos and u2os cellsfig 5b consistent with the cck8 results silencedhmbox1 also alleviated shwtapmediated repressionof cell proliferation in colony formation assay fig 5cthe similar effects of hmbox1 were also observed inwtapsilenced osteosarcoma cell using woundhealingand transwell invasion assays fig 5d and e in additionofï¬cial of the cell death differentiation associationwestern blot results showed that silenced wtap evidently repressed the expression of mesenchymal markerscadherin and vimentin and induced the expression ofepithelial marker ecadherin which was attenuated byshhmbox1 in osteosarcoma cells fig 5f these datasuggested wtap promotes osteosarcoma cells proliferation and metastasis via repressing hmbox1 expressionhmbox1 inhibits osteosarcoma growth and metastasisin vivowe next veriï¬ed the role of hmbox1 in vivo byinjecting shnc shwtap shhmbox1 and shwtapshhmbox1 u2os cells to induce subcutaneous osteosarcoma mice model orthotopic xenograft tumor modeland tail vein metastasis model the expression levels ofhmbox1 was signiï¬cantly upregulated by silencedwtap in subcutaneous osteosarcoma tissue figs 6a ands5 and s6 moreover silenced wtap repressed thetumor size and tumor weight in subcutaneous osteosarcoma mice which was rescued by silenced hmbox1fig 6b we next used a luminescent dye and gfplabeled u2os cells to build an orthotopic xenografttumor model the bioluminescence imaging showed thatthe wtap knockdown reduced the proliferation ofu2os cells in situ while silenced hmbox1 alleviatedthis repression fig 6c to further detect the role ofwtap and hmbox1 on the metastatic ability ofosteosarcoma in vivo u2os cells were injected into nudemice via the tail vein the bioluminescence imagingshowed that silenced hmbox1 alleviated the repressionof silenced wtap on osteosarcoma metastasis fig 6dtaken together these results suggest that hmbox1 isinvolved in wtapmediated tumor growth and metastasis of osteosarcomawtaphmbox1 regulates the proliferation and metastasisof osteosarcoma partly by pi3kakt pathwaythe kegg results identiï¬ed that pi3kakt pathwayscould be regulated by wtap fig s2b pi3kakt signaling pathway promotes the growth migration andinvasion of various cancers including osteosarcoma4748we hypothesized that wtaphmbox1 was involved inthe progression of osteosarcoma via regulating pi3kaktsignaling pathway as shown in fig 7a phosphopi3k andphosphoakt were evidently repressed by shwtap andinduced by shhmbox1 and shhmbox1 signiï¬cantlyattenuatedandphosphoakt in both hos and u2os cells ly294002 api3kakt pathwaysreducedshhmbox1induced cell proliferation migration andinvasion in both hos and u2os cells fig 7b cthereforethese results imply that wtaphmbox1regulates the proliferation and metastasis of osteosarcomapartly via pi3kakt pathway fig shwtaprepressedphosphopi3kinhibitorremarkably 0cchen cell death and disease page of fig hmbox1 is negative correlation wtap expression and is associated to the poor prognosis of osteosarcoma a the m6a level in hosand u2os cells with silenced wtap b meripqpcr assay followed by qrtpcr revealed the hmbox1 m6a modiï¬cation c wildtype or m6a sitemutant hmbox1 were cloned in pmirglo d luciferase reporter assays revealed the target role of wtap on the ²utr of hmbox1 e ythdf2expression in osteosarcoma tissue and cells f the effects of silenced ythdf2 on hmbox1 expression in osteosarcoma cellsdiscussionin the past several years m6a modiï¬cation is considered as a pervasive internal modiï¬cation of mrna andplays critical roles in the progression of a variety of humandiseases including cancers20 however the underlyinginvolvement of m6a regulators in osteosarcoma progression is still unclear in the present study we focused onthe role and underlying mechanism of wtap and itmediated m6a modiï¬cation in the progression andmetastasis of osteosarcoma in this study wtap wasï¬rstly identiï¬ed to upregulated which was associated withpoor prognosis of osteosarcoma functionally wtappromoted the growth and metastasis of osteosarcomain vitro and vivo mechanistically hmbox1 was identiï¬ed as the target gene of wtap and it was regulated bywtap with m6a modiï¬cation at the ²utr finallywtaphmbox1 regulated osteosarcoma growth andmetastasis in a pi3kaktdependent patternactually wtap was reported as an oncogene in various cancers37 recent studies reported that wtapis strictly connected to a functional m6a methylationcomplex43 howeverfew study demonstrated theimportant role of wtap as a m6a regulator here weconcluded that wtap is not only upregulated but alsoplays key role on the m6a modiï¬cation in osteosarcomanotably the aberrant of m6a modiï¬cation is related toofï¬cial of the cell death differentiation association 0cchen cell death and disease page of fig hmbox1 as a tumor suppressor was involved in wtapmediated progression a hmbox1 expression in osteosarcoma cell with shwtapand shhmbox1 knockdown of hmbox1 effectively alleviated wtapdependent b cell proliferation c colony formation d transwell invasion ande woundhealing assay f the protein expression level of emt transition related protein p various biological processes via regulating mrna stability splicing and translation next we shed light on thedownstream mrna that modiï¬ed by wtapmediatedm6a modiï¬cation by combining the data from rnaseqm6aseqshowed thathmbox1 is a potential target gene of wtap subsequently meripqpcr western blotand luciferasereporter assay results revealed that wtap repressedand clipseq theresultshmbox1 expressed with wtapdependent m6a modthe ²utr of hmbox1 thus wtapiï¬cation atinvolved in tumorigenesis depending on its role of m6amodiï¬cation although ythdf2 is a wellknown m6areader in several cancers we found that ythdf2 showedno effects on hmbox1 expression suggesting thatwtap regulated m6amediated hmbox1 expression inythdf2independent manner and the m6a readerofï¬cial of the cell death differentiation association 0cchen cell death and disease page of fig silenced hmbox1 attenuated shwtaprepressed osteosarcoma growth and metastasis in vivo a the expression levels of hmbox1 inosteosarcoma mice models b knockdown of hmbox1 effectively alleviated shwtaprepressed osteosarcoma growth in mice c the orthotopicxenograft tumor and d lung metastasis were detected using a vivo bioluminescence imaging system representative images and a histogram areshown n each groupwhich was involved in the m6a modiï¬cation of hmbox1need be further investigatedhmbox1 a homeobox containing protein49 wasreported to be a transcriptional repressor involving in thebiological processes in bone marrowderived stromacells50 nk cells5152 and vascular endothelial cells53recent studies demonstrated the dysregulated hmbox1in various cancers for example high expression ofhmbox1 was observed in gastric cancer and it wasassociated with the poor prognosis of gastric cancer54moreover hmbox1 also observed as tumor suppressorin ovarian cancer55 and cervical cancer56 hmbox1repressed the progression of ovarian cancer via regulatingcell proliferation and apoptosis55 hmbox1 repressedliver cancer progression via regulating autophagy as wellas and immune escape57 consistently hmbox1 isofï¬cial of the cell death differentiation association 0cchen cell death and disease page of fig wtap hmbox1 was involved the progression of osteosarcoma via pi3kakt pathway a western blot analysis for the expression ofppi3k pi3k pakt and akt in osteosarcoma cells b cell proliferation c colony formation transwell invasion and woundhealing assay ofosteosarcoma cell treated with μm akt inhibitor ly294002 abcam ab120243 for hunclear how hmbox1 as a transcriptional repressorregulates pi3kakt pathway nonethelesssilencedhmbox1 only partly alleviated wtapmediated osprogressionthere are other potentialmolecular mechanisms regulated by wtapmediatedepigenetic modulation in osit means thatin summary we identiï¬ed the elevated wtap inosteosarcoma and which is associated with poor clinicaloutcome and serves as an independent prognostic factorfor osteosarcoma patients wtap dramatically promotedosteosarcoma proliferation and metastasis via regulatinghmbox1 mrna stability in a m6adependent mannerwtaphmbox1 regulated osteosarcoma growth andmetastasis via pi3kakt pathway altogether our resultsdetermined wtap hmbox1 as a potential therapeutictarget for osteosarcomaacknowledgementsthis study was supported by national natural science foundation of chinagrant nos national natural science foundation of china grantnos natural science foundation of hunan province grant nos2018jj2617 natural science foundation of hunan province grant nos2016jj3176 national key research and development program of china no2016yfc1201800 natural science foundation of hunan province no2018sk2090author details1department of orthopaedics the third xiangya hospital of central southuniversity tongzipo rd changsha hunan china 2shanghai keylaboratory of regulatory biology institute of biomedical sciences and schoolof life sciences east china normal university shanghai china 3fourgynecological wards ningbo women and childrens hospital ningbozhejiang china 4the second xiangya hospital of central southuniversity changsha china 5school of basic medical science central southuniversity changsha china 6department of anatomy histology andembryology changsha medical university changsha chinafig a schematic model illustrating wtapmediated m6a regulationin osteosarcomaevidentlydownregulated and closely related to the poor prognosisof osteosarcoma in the present study in addition silencedhmbox1alleviated wtapknockdownmediated repression of osteosarcoma progression whichimplied the import roles of hmbox1 in wtapdrivenosteosarcoma development finally we analyzed thedownstream pathway of wtaphmbox1 in osteosarcoma pi3kakt pathway was reported to an important role in the progression of various cancers includingosteosarcoma58 we found that pi3kakt pathway we | Colon_Cancer |
purpose to explore a new therapeutic option for patients with hepatocellular carcinoma hcc the efficacy and safety of a group of traditional chinese medicines banxia xiexin recipe as monotherapy for patients with advanced hcc was studied materials and methods the study included patients with advanced hcc from august to august for analysis these eligible patients received treatment with banxia xiexin recipe for at least month the primary endpoints were progressionfree survival pfs and overall survival os the secondary efficacy endpoints included objective response rate orr and disease control rate dcr in addition safety was also assessed results the median treatment duration of these patients was months range months and followup is still ongoing the median pfs was months confidence interval [ci] months and the median os was months ci months the orr was and the dcr was in the subgroup analysis the median os in the transcatheter arterial chemoembolization tace group was not reached and the median os in the no tace group was months ci months in addition no drugrelated serious adverse events were observed during the study this is the first clinical analysis of traditional chinese medicine as a single treatment for advanced hcc the obtained results are encouraging as they suggest that this panel of chinese herbs is safe and it may be effective for patients with advanced hcc in a realworld clinical settingkeywordshepatocellular carcinoma herbs tace banxia xiexin recipe clinical researchsubmitted december revised june accepted june introductionliver cancer is one of the most frequent malignant tumors and the third leading cause of cancerrelated deaths worldwide both the incidence and mortality rates for liver cancer are increasing while the overall incidence and mortality for all cancers combined tend to decline12 hepatocellular carcinoma hcc is the most common and deadly form of liver cancer in china chronic hepatitis b virus hbv infection and subsequent liver fibrosis and cirrhosis are the 1affiliated hospital of chengdu university of traditional chinese medicine chengdu sichuan province chinathese authors contributed to the work equally and should be regarded as cofirst authorscorresponding authorshaoquan xiong department of medical oncology affiliated hospital of chengdu university of traditional chinese medicine chengdu sichuan province china email xsquan106163comcreative commons non commercial cc bync this is distributed under the terms of the creative commons attributionnoncommercial license creativecommonslicensesbync40 which permits noncommercial use reproduction and distribution of the work without further permission provided the original work is attributed as specified on the sage and open access pages ussagepubcomenusnamopenaccessatsage 0c integrative cancer therapies leading causes of liver cancer due to the lack of effective treatment options as well as the high recurrence and metastasis rates hcc is associated with an extremely poor prognosis at present surgical resection and liver transplantation are effective treatments for liver cancer but only a few patients are surgical candidates due to tumor extension poor hepatic functional reserve or underlying liver cirrhosis34 even for the few lucky patients who have completed the surgery the recurrence rates at years following liver resection have been reported to exceed for patients with advanced liver cancer it is essential to improve general wellbeing promote quality of life and prolong survival time it is well known that chemotherapy effects in patients with advanced liver cancer are not outstanding67 some patients can consider local treatment such as transarterial chemoembolization tace the progressionfree survival pfs of patients receiving tace treatment is reported to be months8 molecular targeted drugs are also one of the options for patients with advanced liver cancer such as sorafenib and lenvatinib however according to reports on sorafenib the only approved molecular targeted therapy for advanced hcc the median pfs of patients treated with sorafenib was only months in the asiapacific region9 in recent years immunotherapy which has been attracting worldwide attention is also becoming an important treatment method for example nivolumab and pabolizumab which have been approved by the food and drug administration for secondline treatment of advanced hcc can bring certain benefits to patients with advanced liver cancer but are expensive and difficult to afford for many patientsin china traditional chinese medicine tcm therapies which can date back more than years are widely used in cancer treatment10 banxia xiexin decoction was created by the ancient doctor zhang zhongjing it is mainly used to treat digestive diseases such as bloating and abdominal pain it has a history of more than years in a previous retrospective study it was found that banxia xiexin decoction can bring survival benefits to patients with advanced liver cancer and one of them achieved complete response cr11 as a tcm hospital many patients with advanced liver cancer come to our hospital to undergo chinese medicine for treatment therefore we designed a prospective study of banxia xiexin decoction as monotherapy for advanced liver cancer and analyzed its treatment effect and safetymethodsstudy design and patient enrollmentin this prospective openlabel study patients from the oncology department of the affiliated hospital of the chengdu university of traditional chinese medicine chengdu china were recruitedaccording to the american association for the study of liver diseases criteria eligible patients were adults with advanced hcc confirmed by pathological assessment or noninvasive assessment12 these patients had not received any cancerrelated treatment within weeks before the start of study treatment and the disease had progressed after the prior treatment in addition these patients had to have at least one measurable lesion defined by modified response evaluation criteria in solid tumors for hcc mrecist and recist version inclusion criteria also included eastern cooperative oncology group ecog performance status of to an estimated life expectancy of months childpugh liver function class a or b adequate an function such as white blood cell count ¥ cellsl neutrophils ¥ cellsl and platelets ¥ cellsl and the absence of any serious comorbidities such as significant cardiac cerebrovascular hepatic or nephritic abnormities or other important an dysfunctionskey exclusion criteria included patients with low compliance or use of other antitumor treatments at the same time diagnosis of other malignant tumors and hcc with brain metastasisthe protocol was approved by the ethics committee of the affiliated hospital of chengdu university of traditional chinese medicine no 2015bl003 all patients provided written informed consent before study entrystudy treatment and proceduresthe enrolled patients received treatment with banxia xiexin recipe in the form of decoction the herbal components and their doses in the banxia xiexin recipe decoction are listed in the appendix with the use of an automatic herbboiling machine the decoction volume of each prescription was about ml patients were instructed to take ml each time times dailytreatment was continued until disease progression death development of unacceptable toxicity patients refusal to continue or at the discretion of the investigator when the disease progressed treatment could also be continued for month to assess tumor response as long as the patient agreed to do sofollowing an initial baseline assessment within days of the start of study treatment the investigator assessed tumor response by using computed tomography ct scan after to months of treatment subsequently followup assessments were done every months until the patient died adverse events were recorded continuously from enrollment to the end of the final study visitoutcomesthe primary endpoints were pfs time from enrollment to radiological disease progression or death and overall survival os time from enrollment to death due to any cause the secondary efficacy endpoints included the proportion 0cwang et al of patients who had an objective response complete or partial response [pr] according to recist version namely objective response rate orr and disease control rate dcr the proportion of patients who had a cr pr or stable disease besides safety was also assessed by the incidence and nature of adverse eventsstatistical analysisthe pfs and os were estimated using the kaplanmeier method all statistical analyses were performed using graphpad prism software version graphpad software inc and logrank testresultsfrom august to august a total of patients with advanced hcc were enrolled from the oncology department of the affiliated hospital of chengdu university of traditional chinese medicine however only the results of the mediumterm analysis are presented here since patients were still on treatment and had not yet reached the assessment time point therefore patients were finally included in the current analysis meaningful results were found to report in this midterm analysis of course we will update the results according to the latest followup data in the future the baseline characteristics of the included patients are shown in table notably patients were without any treatment in this study all patients who were previously treated with surgery or tace had progressive disease which was similar to the clinical trials of sorafenib9 the number of patients receiving chemotherapy and molecular targeted therapy is and respectively patients who had received previous systemic therapy were excluded in the clinical trials of sorafenib but these patients had progressive disease after the prior treatment according to recist version in this studythe data cutoff date for the current analysis was august the median treatment duration of these patients was months range months and followup is still ongoing since if the disease progressed treatment could also be continued for month to assess tumor response as long as the patient agreed to do so or if thepatient would like to use it as an adjuvant treatment to other treatment such as tace moleculartargeted therapy and so onin the primary analysis the median pfs was months confidence interval [ci] figure and the median os was months ci figure thirtysix patients died all due to disease progression the 1year survival rate was notably most of these patients received prior treatments such as tace and targeted molecular therapy before enrollment in order to explore the potential effect of previous treatment we performed a subgroup analysis the median pfs months ci was longer in the subgroup of patients who had previously received tace compared with the median pfs months ci who had not previously received tace thus the treatment effect in this type of patient seems to be better p figure the median os of advanced hcc patients who had previously received tace was not reached and the median os of subgroups who had not received tace was months ci as shown in figure in terms of tumor response after treatment with banxia xiexin recipe patients achieved a pr had stable disease the orr was and the dcr was table particularly the imaging data of typical cases were presented as follows the first patient was a 58yearold male who was diagnosed with advanced hcc with metastasis to the right kidney on august at baseline the ct scan showed that the size of the target lesion in the liver was cm cm cm figure 5a blood tests revealed elevated αfetoprotein µgml and the ecog performance status score was this patient had not received any anticancer treatment previously after months treatment with banxia xiexin recipe the ct scan showed that the target lesion in the liver shrank to cm cm figure 5b and reached a pr according to recist version the ecog performance status score improved from to the patient is still currently in treatment and has not yet had progressive diseaseone 54yearold male patient was first diagnosed to have hcc in december he was treated by surgical resection on january no antineoplastic treatment was done thereafter when a regular review was conducted on september it showed intrahepatic recurrence and right lung metastasis and then he received treatment with chinese herbs at baseline the ct scan showed that the size of the target lesion in the liver was cm cm cm figure 6a blood tests revealed elevated αfetoprotein µgml and the ecog performance status score was after a 32month treatment with modified xiexin recipe the ct scan showed that the target lesion in the liver shrank to cm figure 6b and reached a pr according to recist version the duration of the pr was months he continued to insist on treatment after the disease progressed and he died in october concerning safety no adverse events were observed except for the occurrence of mild nausea three patients reported mild nausea after taking pills but the symptom was relieved spontaneously after week without any 0c integrative cancer therapies table the clinical characteristics of the patients with advanced hepatocellular carcinomavariablesnumber of patientspercentageage years ¥sex male femaleecogps childspugh class a bhepatitis b virus infection yes noliver cirrhosis yes noafp µgl ¥ tumor size cm ¥pvtt yes noextrahepatic spread yes noinvolved disease sites per patient ¥previous treatment no sur tace rf che mttajcc stage iiibivaivbabbreviations ecogps eastern cooperative oncology groupperformance status pvtt portal vein tumor thrombus sur surgical resection tace transcatheter arterial chemoembolization rf radiofrequency che chemotherapy mtt molecular targeted therapy ajcc american joint committee on cancerfigure kaplanmeier analysis of progressionfree survivalfigure kaplanmeier analysis of overall survivalfigure kaplanmeier subgroup analysis of progressionfree survival stratified by prior tace treatmenttreatment in addition no serious adverse events such as liver or kidney damage were detected during the followup period thus the tcm treatment was safe and welltolerated in these patients with advanced hccaccording to the international tumor chemotherapy adverse drug evaluation systemctcae v4014 patients included in this study were evaluated for safety during the followup the adverse reactions were nausea 0cwang et al control effect of tcm as monotherapy for patients with liver cancer banxia xiexin decoction has been used for treating digestive diseases for more than years in our previous retrospective analysis it was found that banxia xiexin decoction can relieve the symptoms of patients with advanced liver cancer inhibit tumor growth and prolong survival one of the patients tumors bulk was significantly reduced to reach cr status this state has been maintained for up to months11 therefore it is necessary to carry out further scientific researchto the authors best knowledge this is the first prospective study of chinese herbs alone for the treatment of advanced hcc that used the widely accepted endpoints of pfs os orr and dcr according to recist version in the present study the median pfs was months in clinical studies of sorafenib an oral multikinase inhibitor which is the first molecularly targeted drug approved by multiple countries around the world for firstline treatment of advanced hcc the median pfs was months in western countries19 and only months in the asiapacific region9 in current research work all patients were from china and in particular of patients had been diagnosed with liver cirrhosis when enrolled table the manifestations of liver decompensation such as splenomegaly and portal hypertension had appeared however the childpugh class was a or b it had been reported that hcc patients with liver cirrhosis showed significantly worse os than noncirrhotic hcc patients months vs months p even so the median pfs reached months and the median os reached months in a realworld clinical setting these findings suggest that the efficacy of the banxia xiexin recipe may be superior to that of sorafenib in patients with advanced hcc although this was not a headtohead comparison a further study directly comparing chinese herbs versus sorafenib for advanced hcc will be conducted in the futurein the reflect trial21 a subgroup analysis of the chinese population found that the os of the lenvatinib group was significantly longer than that of the sorafenib group by months with statistical differences in addition when combined with hbv infection the survival benefit was significantly different between the lenvatinib group and the sorafenib group os months vs months therefore it was approved by the china food and drug administration on september and approved for domestic listing it is important to note that the experimental conditions for entering the reflect trial were strict excluding ecog performance status patients with a score of or and a large liver mass however in patients enrolled in the present study had liver masses cm and had ecog scores of to nevertheless the median os of this study was figure kaplanmeier subgroup analysis of overall survival stratified by prior tace treatmenttable tumor response according to recist version variablesnumber of patientspercentagecrprsdpdorrdcrabbreviations cr complete response pr partial response sd stable disease pd progressive disease orr objective response rate dcr disease control ratevomiting diarrhea abdominal distension oral ulcers the painless mass of the scalp and so on the overall adverse reaction rate was all of which were grade i to ii adverse reactions and responded to symptomatic treatment there was no adverse reaction of grade iii or above and no drugrelated lethal events occurred specific adverse reaction events and their incidence rates are shown in table discussionit is wellknown that hcc is one of the leading causes of cancerrelated mortality worldwide the longterm survival rate remains unsatisfactory due to the high recurrence and metastasis rates after conventional treatment to improve the outcome of patients with hcc there is an urgent need for more effective therapies at present tcm is often used as an adjuvant therapy to conventional treatments such as tace in treating patients with liver cancer1518 to reduce the toxicities of other treatments relieving symptoms and improving quality of life tcm has only been considered as a complementary or alternative treatment for cancer patients and there is a paucity of data regarding the tumor 0c integrative cancer therapies figure a b the imaging data of one partial response patient diagnosed as advanced hepatocellular carcinoma with metastasis to the right kidneyfigure a b the imaging data of another partial response patient diagnosed as advanced hepatocellular carcinoma with intrahepatic recurrence and right lung metastasismonths therefore for patients with worse economic and physical conditions banxia xiexin decoction may be a better choice for many patients with advanced liver cancer in addition some other new drugs have been tried for the treatment of hcc unfortunately all phase trials assessing novel systemic drugs2224 have failed to improve outcomes over sorafenibin immunotherapy immune checkpoint inhibitors represented by pd1pdl1 monoclonal antibodies have made rapid progress the results of the checkmate040 study25 showed that nivolumab achieved an os of months in newly diagnosed patients and a 150month survival benefit in patients who had undergone sorafenib treatment pabrizumab which is also a pd1 immunosuppressant used in the keynote224 study26 for patients with advanced hcc who had previously progressed with sorafenib achieved a pfs of months median os of months compared with the enrollment conditions for keynote224 the condition of the patients enrolled in the present study are more complicated the largest diameter of the mass in of patients is ¥ cm of patients are in stage iv of patients had hbv infection and of patients had hepatic cirrhosis even so this study still achieved a pfs of months higher than the above chemotherapy and immune drugs it can be seen that the efficacy of banxia xiexin decoction for the treatment of advanced liver cancer may be better than the abovementioned immune drugs and it has the potential to become one of the important methods for liver cancer treatmentfurther subgroup analysis revealed that previous treatment might affect the efficacy of chinese herbs in the current analysis patients had been treated with tace previously whose outcome was slightly better compared with those who had not been treated with tace previously pfs vs months p in terms of os the difference is more obvious os undefined vs months p subsequent treatment with banxia xiexin decoction seems to have a greater benefit for patients with advanced liver cancer who have progressed with interventional therapyoverall the orr was and the dcr was in this study in comparison with the data reported in 0cwang et al table adverse reactionsgrade igrade iigrade iiigrade ivany grade grade iii nauseavomitingdiarrheabloatingulcersuperficial painless mass related literature the orr and dcr were respectively and in the orient trial of sorafenib919 it should be noted that the ecogps score of the included patients was to in our study but to in the orient trial indicating that tcm treatment may have a wider scope of application and may benefit more patients compared with targeted therapyin terms of safety no serious adverse events such as liver or kidney damage were observed during the followup period thus banxia xiexin recipe was generally safe and welltolerated in patients with advanced hccas for the antitumor mechanism of tcm it has been reported that tcm or its extracts have direct antitumor effects such as pinellia27 ginseng2829 coptis30 glycyrrhiza31 atractylodes macrocephala32 tangerine peel33 and scutellaria34 the effect of inducing apoptosis is also noted such as with berberine contained in coptis3537 the effect of regulating immunity is noted such as with pinellia27 ginseng3839 atractylodes macrocephala40 and milkvetch root41 antitumor vascular growth such as that of scutellarin4243 can inhibit protein kinase b akt phosphorylation downregulate vegf and inhibit tumor angiogenesis thus playing a role in inhibiting tumor growth and metastasis also it has been reported that the imbalance of intestinal flora not only affects the occurrence and development of intestinal cancer and irritable bowel syndrome but also is closely related to the occurrence of breast cancer lung cancer liver cancer and other malignant tumors4446 however atractylodes macrocephala scutellaria baicalensis and coptis4047 can indirectly inhibit tumors by regulating intestinal flora it is worth mentioning that much basic research has found that pinellia48 coptis4950 ginseng51 scutellaria52 atractylodes macrocephala53 and astragalus54 have antihepatoma effects for example berberine4950 a constituent of the extract of coptis in banxia xiexin decoction can inhibit the pi3kakt pathway of liver cancer downregulate mhcc97h and hepg2 cells phosphorylate the expression of akt and pi3k and inhibit the growth of liver cancer cells in a dosedependent manner besides it can induce cell cycle arrest and promote apoptosis to treat hccthere are only a few patients with tumor shrinkage in the present work it is believed that the direct antitumor effect of banxia xiexin decoction is not outstanding and there may be other mechanisms after analysis it was observed that the more prominent effect of banxia xiexin decoction on liver cancer is reflected in os which is similar to the efficacy characteristics of pd1 immune checkpoint inhibitors banxia xiexin decoction contains pinellia27 ginseng3839 atractylodes40 and astragalus41 medicinal herbs with immunomodulatory effects for example ginseng38 could relieve immunosuppression by increased viability of natural killer cells enhanced immune an index improved cellmediated immune response increased content of cd4 and ratio of cd4cd8 and recovery of macrophage function therefore it was speculated that liver cancer patients in this study may benefit from indirect antitumor mechanisms such as antivascular immunomodulation and others in addition some studies5556 confirmed that some ingredients of banxia xiexin decoction could improve liver function and regulate gastrointestinal function these comprehensive effects can also partially explain why liver cancer patients in this study seem to obtain longer pfs and osin china most patients with hcc have liver diseases such as hepatitis and cirrhosis among the patients enrolled in this study patients had a history of hepatitis b and patients had liver cirrhosis hcc and basal liver disease often affect each other and form a vicious circle the scutellaria baicalensis ginseng licorice tangerine peel and astragalus in banxia xiexin decoction all have liverprotective effects and licorice has a direct antihbv and hepatitis c virus effects5758 from this point of view this is also one of the reasons why banxia xiexin decoction may be effective in treating liver cancer although much basic research has confirmed the antitumor effects of single herbal extracts in banxia xiexin decoction a single herb may not have a good antitumor effect in clinical use even though the decoction affects liver cancer after being prescribed still each herb may play its respective role whether it causes a new antitumor effect or through compatibility with the effects of other herbs the antitumor mechanism is unclear and needs further studylimitationsthe report is limited by its singlearm research design and cannot be directly compared with standard chemotherapy 0c integrative cancer therapies targeted therapy or immunotherapy at the same time the small number of cases in this study is also one of the disadvantages but this study is still continuing and there will be more data analysis in the future due to the willingness of patients to choose tcm for treatment in tcm hospitals it is difficult to avoid certain limitations when we adopt the single treatment of tcm although oral chinese medicine decoction is difficult to take according to scientific equal dosage and concentration it is in line with the use habits of tcmsin this is the first prospective study of chinese herbs as monotherapy for the treatment of advanced hcc that used the internationally accepted endpoints of pfs os orr and dcr the findings are encouraging as they suggest that this panel of chinese herbs is safe and may be effective for patients with advanced hcc in a realworld clinical setting further studies are required to assess the comparative efficacy of tcm treatment versus other antitumor therapies in this patient populationappendixcomponents and their doses in the modified xiexin recipe decoction chinese latin and english nameschinese namelatin nameenglish nameplace of origin production batch dose gbanxiahuangqinganjiangrenshenhuangliandazaogancaobaizhuchenpihuangqirhizoma pinelliaeradix scutellariaerhizoma zingiberisradix ginsengrhizoma coptidisfructus jujubaeradix glycyrrhizaerhizoma atractylodis macrocephalaepericarpium citri reticulataeradix astragalipinellia tuberbaikal skullcap rootdried gingerginsengcoptisjujubae chinese datelicorice rootlargehead atractylodes rhizometangerine peelmilkvetch rootsichuanshanxisichuanjilinsichuanxinjiangsichuanzhejiangsichuangansuacknowledgmentsthe authors acknowledge the contributions of the other investigators in this trialauthor contributionslijuan wang jianlong ke and shaoquan xiong conceived and designed the study lijuan wang jianlong ke cui wang yaling li qiuyue luo rui cai qian ding panpan lv and tingting song collected the data lijuan wang jianlong ke and cui wang processed the data lijuan wang and jianlong ke wrote the manuscript all authors have participated in the drafting review and approval of the report and the decision to submit for publicationdeclaration of conflicting intereststhe authors declared no potential conflicts of interest with respect to the research authorship andor publication of this fundingthe authors disclosed receipt of the following financial support for the research authorship andor publication of this this research was supported by the national natural science foundation of china no and sichuan provincial science and technology department project no2017jy0327orcid idslijuan wang jianlong ke panpan lv orcid0000000317048847 orcid0000000150587581 orcid0000000271727659references torre la bray f siegel rl ferlay j lortettieulent j jemal a global cancer statistics ca cancer j clin doi103322caac21262 chen w zheng r baade pd et al cancer statistics in china ca cancer j clin doi103322caac21338 hung h treatment modalities for hepatocellular carcinoma curr cancer drug targets doi102174 vitale a trevisani f farinati f cillo u treatment of hepatocellular carcinoma in the precision medicine era from treatment stage migration to therapeutic hierarchy hepatology published online february doi101002hep31187 bruix j sherman m practice guidelines committee american association for the study of liver diseases management of hepatocellular carcinoma hepatology doi101002hep20933 lee dw lee kh kim hj et al a phase ii trial of s1 and oxaliplatin in patients with advanced hepatocellular carcinoma bmc cancer doi101186s1288501840399 0cwang et al zaanan a williet n hebbar m et al gemcitabine plus oxaliplatin in advanced hepatocellular carcinoma a large multicenter ageo study j hepatol doi101016jjhep201209006 wang y cui w raltitrexed based transcatheter arterial chemoembolization tace for unresectable hepatocellular carcinoma a singlecenter randomized controlled study chemotherapy doi104172216777001000192 cheng al kang yk chen z et al efficacy and safety of sorafenib in patients in the asiapacific region with advanced hepatocellular carcinoma a phase iii randomised doubleblind placebocontrolled trial lancet oncol doi101016s1470204508702857 anonymous miraculous pivot ling shu jing originally published during warring states period to bc peoples health press shaoquan x lijian w qiuyue l et al retrospective analysis of modified xiexin recipe combined retrospective analysis of modified xiexin recipe combined chin j integr tradit western med bruix j sherman m american association for the study of liver diseases management of hepatocellular carcinoma an update hepatology doi101002hep24199 lencioni r llovet jm modified recist mrecist assessment for hepatocellular carcinoma semin liver dis doi101055s00301247132 gao wj liu yy yuan cr international evaluation system for adverse events of chemotherapeutic drugs in cancer treatment ctcae v40 tumor meng mb cui yl guan ys et al traditional chinese medicine plus transcatheter arterial chemoembolization for unresectable hepatocellular carcinoma j altern complement med doi101089acm20080060 xu h deng y zhou z huang y chinese herbal medicine chaihuhuaji decoction alleviates postem | Colon_Cancer |
biochemically interleukin6 belongs to the class of fourhelical cytokines the cytokine can be synthesised and secreted by many cells it acts via a cell surfaceexpressed interleukin6 receptor which is not signalling competent this receptor when complexed with interleukin6 associates with the signalling receptor glycoprotein kda gp130 which becomes dimerised and initiates intracellular signalling via the janus kinasesignal transducer and activator of transcription and rat sarcoma proto oncogenemitogenactivated protein kinasephosphoinositide3 kinase pathways physiologically interleukin6 is involved in the regulation of haematopoiesis and the coordination of the innate and acquired immune systems additionally interleukin6 plays an important role in the regulation of metabolism in neural development and survival and in the development and maintenance of various cancers although interleukin6 is mostly regarded as a proinflammatory cytokine there are numerous examples of protective and regenerative functions of this cytokine this review will explain the molecular mechanisms of the in part opposing activities of the cytokine interleukin6keywords gp130 sgp130fc il6 il6r sil6r transsignalling adam17invited reviewersversion aug faculty reviews are review s written by the prestigious members of faculty opinions the s are commissioned and peer reviewed before publication to ensure that the final published version is comprehensive and accessible the reviewers who approved the final version are listed with their names and affiliations elke roeb justus liebig university giessen germany hana alg¼l technical university of munich munich germany jacqueline bromberg department of medicine memorial sloan kettering cancer center new york usaany comments on the can be found at the end of the a0page of 0cf1000research 9faculty rev1013 last updated aug corresponding author stefan rosejohn rosejohnbiochemunikieldeauthor roles rosejohn s conceptualization data curation formal analysis funding acquisition investigation methodology project administration resources supervision writing original draft preparation writing review editingcompeting interests stefan rosejohn has acted as a consultant and speaker for abbvie amgen janssen chugai roche genentech roche pfizer eli lilly and sanofi he also declares that he is an inventor on patents owned by conaris research institute which develops the sgp130fc protein olamkicept and he has stock ownership in conarisgrant information the work of stefan rosejohn has been supported by grants of the deutsche forschungsgemeinschaft bonn germany under the grant numbers crc841 project c1 and crc877 project a1 and by the german excellence cluster inflammation at interfaces the funders had no role in study design data collection and analysis decision to publish or preparation of the manuscriptcopyright rosejohn s this is an open access distributed under the terms of the creative commons attribution license which permits unrestricted use distribution and reproduction in any medium provided the original work is properly citedhow to cite this rosejohn s interleukin6 signalling in health and disease [version peer review approved] f1000research 9faculty rev1013 1012688f1000research260581first published aug 9faculty rev1013 1012688f1000research260581 a0page of 0cintroductioninterleukin6 il6 is considered one of the most prominent proinflammatory cytokines1 blockade of il6 by the neutralising monoclonal antibody tocilizumab has been approved in more than countries for the treatment of patients with autoimmune disorders such as rheumatoid arthritis2 additionally the cytokine storm sometimes encountered when cancer patients are treated with chimeric antigen receptor car tcells3 could be effectively treated with the antibody tocilizumab leading to us food and drug administration fda approval of the drug for this condition even more recently it has been recognised that many patients experience a similar cytokine storm upon infection with sarscov2 covid19 virus4 and that these patients could also be treated with tocilizumab5 these new data led to a rekindled general interest in the cytokine il6il6 was initially discovered and cloned in the kishimoto laboratory as a bcell stimulatory factor6 immediately after the molecular cloning it was evident that il6 was identical to hepatocyte stimulating factor7 hybridomaplasmacytoma growth factor8 interferon 29 and kda protein10 this already indicated the pleiotropic nature of the cytokine later on it was also recognised that il6 shows profound activities in the brain1112 in the regulation of metabolism1314 in the response of the body to exercise15 and in the development and maintenance of various cancers16this review gives a short overview of the complex biology of il6 and explains how one cytokine can have extremely different biologic effects on different cells and in different physiologic states of the human body17the interleukin6 receptor complexthe fourhelical cytokine il6 figure on cells binds to a membranebound il6 receptor il6r and the complex of il6 and il6r associates with a second receptor protein glycoprotein kda gp130 which dimerises and initiates intracellular signalling via the janus kinase jaksignal transducer and activator of transcription stat and rat sarcoma proto oncogene rasmitogenactivated protein kinase and phosphoinositide3 kinase pathways figure importantly il6 exhibits only a measurable affinity to the il6r but not to gp130 and the il6r does not bind on its own to gp130 it is only the complex of il6 and il6r that binds to gp130 and induces its dimerisation figure all cells in the body express gp130 but only a few cells such as hepatocytes and some leukocytes express il6r it follows that cells that express only gp130 but not il6r cannot be stimulated by il61noteworthy gp130 is a component of the receptor complexes of the socalled gp130 cytokine family which besides il6 comprises il11 ciliary neurotrophic factor cntf cardiotrophin1 ct1 cardiotrophinlike cytokine clc leukaemia inhibitory factor lif oncostatin m osm and il27 for details please refer to recent reviews1920it has however been noticed that the membranebound il6r can be cleaved by the membranebound metalloprotease a f1000research 9faculty rev1013 last updated aug figure fourhelical topology of the interleukin6 il6 protein il6 belongs to the family of fourhelical cytokines the figure shows the four helices with the connecting loops the ab and the cd loops are long enough to reach the length of a helix whereas the bc loop is short consequently il6 has an upupdowndown topology meaning that helices a and b point upwards whereas helices c and d point downwards this topology is common to most cytokines such as il2 il4 il7 il11 il15 leukaemia inhibitory factor oncostatin m growth hormone leptin and many othersdisintegrin and metalloprotease adam17 to generate a soluble il6r sil6r21 to a minor extent the humanbut not the murinesil6r can be generated by translation from a differentially spliced mrna22 intriguingly the sil6r can still bind il6 and the complex of il6 and sil6r can associate with gp130 and induce signalling even on cells that lack the membranebound il6r23 this process has been named il6 transsignalling figure strikingly following this paradigm il6 can in the presence of sil6r stimulate any cell in the body since all cells express gp13017interestingly most il6rexpressing cells including hepatocytes express far more gp130 than il6r molecules therefore stimulation of such cells with il6 alone will only lead to engagement of few gp130 molecules whereas stimulation with the complex of il6 and sil6r will stimulate all cellular gp130 proteins a threshold for a given response might not be reached with il6 stimulation but only with stimulation of all gp130 molecules via il6 transsignalling this might be an explanation for the observed differences in signalling between transsignalling and classical signalling that lead to different phenotypes25molecular tools to elucidate the functions of interleukin6the concept of transsignalling has been corroborated by the use of two designer proteins the first such protein consists of il6 covalently fused to the sil6r via a flexible peptide linker which allowed the placement of il6 il6 page of 0cf1000research 9faculty rev1013 last updated aug figure stimulation of target cells by interleukin6 il6 il6 orange first binds to the il6 receptor il6r red the complex of il6 and il6r associates with glycoprotein kda gp130 blue which dimerises and leads to intracellular signalling it is important to note that il6 and il6r alone exhibit no measurable affinity to gp130 only the complex of il6 and il6r binds to and activates gp130 therefore il6 cannot stimulate cells that do not express il6r signalling occurs via the signal transducer and activator of transcription stat 1stat3 yamaguchi sarcoma viral oncogene homolog yesyesassociated protein yap phosphoinositide3 kinase pi3kakt and rat sarcoma proto oncogene rasmitogenactivated protein kinase mapk pathways jak janus kinaseat the correct distance to reach the il6 binding site of the sil6r this protein was called hyperil6 figure 3a26 this protein was shown to stimulate gp130expressing cells in vitro and in vivo and it was shown that liver regeneration27 stimulation of neural cells28 and expansion of hematopoietic cells29 was far more efficient in the presence of hyperil6 as compared to il6 alone30turned out while hyperil6 demonstrated only the biologic potential of il6 transsignalling these experiments did not prove that this process occurred in vivo a second soluble protein was designed which consisted of the entire extracellular portion of gp130 covalently fused to the fc region of human igg1 figure 3b the resulting protein named soluble gp130fc sgp130fc to exhibit similar properties as membranebound gp130 it did not bind il6 or il6r alone but it bound with high affinity the complex of il6 and sil6r3132 consequently the sgp130 protein in vitro and in vivo specifically inhibited il6 transsignalling without compromising il6 signalling via the membranebound il6r ie classic signalling32 the sgp130fc protein could be used to define il6mediated biologic responses which were dependent on classic or transsignalling this was accomplished by comparing the treatment of animals with sgp130fc or with neutralising antibodies against il6 or il6r which blocked all il6 signalling figure 3c d using animal models of human inflammatory diseases or inflammationassociated cancer it turned out inflammationassociated cancers were mainly driven by il6 transsignalling whereas regenerative and protective activities of il6 were mediated by classic il6 signalling via the membranebound il6r figure that autoimmune disorders and levels in physiologic and pathophysiologic functions of interleukin6under homeostatic conditions il6 the circulation are as low as pgml but during inflammatory states these levels can rise more than 1000fold and under extreme conditions leading to sepsis il6 levels in the µgml range have been reported33 il6 is produced by myeloid cells upon tolllike receptor stimulation together with the cytokines il1 and tumor necrosis factor α tnfα which via a feedforward loop lead to an immense amplification of il6 production during inflammatory conditions34 there is perhaps no other protein in the human body whose level can go up by six orders of magnitude this lets us conclude that il6 is the major alarm signal to infection inflammation and possibly cancer35the human body in response in however under normal conditions il6 plays an important role in ancellular homeostasis mice in which the il6 gene has been ablated il6 knockout mice become obese late in life13 cannot regenerate their liver upon hepatectomy36 and show no signs of osteoporosis upon ovariectomy37 indicating roles for il6 in body weight regulation liver physiology and bone metabolism in pathophysiologic states however there are marked differences between il6 knockout mice and wildtype mice il6 knockout mice are completely protected in animal models of rheumatoid arthritis38 and multiple sclerosis39 indicating a key role for il6 in these autoimmune disorderswith the help of the sgp130fc protein and of neutralising monoclonal antibodies it was possible to selectively block il6 transsignalling or to block all il6 signalling respectively page of 0cf1000research 9faculty rev1013 last updated aug tumour progression was induced by tumourinfiltrating myeloid cells which stimulated the neoplastic cells via il6 transsignalling47 selective blockade of this pathway by the sgp130fc protein blocked progression of pancreatic intraepithelial neoplasias to pancreatic ductal adenocarcinomas47 indicating a prominent role for il6 transsignalling in the development of pancreatic cancer in the murine apcmin model of colon cancer it was established that the genetic deletion of adam17 which is responsible for generating not only sil6r but also soluble tnfα and soluble ligands of the epidermal growth factor receptor egfr resulted in completely abrogated tumour development16 moreover the formation of neoplasias stimulated adam17 on macrophages leading to egfr ligand cleavage and subsequent egfr stimulation these macrophages now produced il6 and sil6r which led to the outgrowth of the tumours again selective blockade of the il6 transsignalling pathway by the sgp130fc protein blocked tumour development in the apcmin model and an additional mouse model of colon cancer16 this was highly reminiscent of a study in liver cancer in which it was shown that the egfr expressed in macrophages but not egfr in hepatocytes was involved in the development of hepatocellular carcinoma48 apparently macrophage activation may be an important step in the initiation and progression of tumours via the il6 transsignalling pathway20 figure therapeutic targeting of interleukin6 activitytherapeutic targeting of the proinflammatory cytokine tnfα was introduced as an efficient strategy to treat patients with autoimmune disorders such as rheumatoid arthritis and inflammatory bowel disease49 subsequently blockade of the biologic activity of the cytokine il6 was shown to be an efficient treatment for patients with rheumatoid arthritis and other autoimmune diseases2 and it was shown that blocking il6 activity was more efficient than blocking tnfα in a monotherapy trial50 blockade of il6 activity with the il6r neutralising monoclonal antibody tocilizumab was also highly effective in the treatment of patients with car t cellinduced severe cytokine release syndrome51 in patients with severe covid19 disease the administration of tocilizumab resulted in a marked improvement of the condition in the majority of patients the fever subsided creactive protein decreased and oxygen intake could be lowered no obvious adverse reactions were observed these preliminary data indicated that tocilizumab is a candidate for effective treatment of covid19 patients552 interestingly treatment of covid19 patients with the il6r neutralising monoclonal antibody sarilumab resulted in no significant difference in clinical improvement and mortality53summarythe discovery that the proinflammatory activities of il6 are mediated by il6 transsignalling whereas the protective and regenerative activities of il6 rely on classic signalling via the membranebound il6r suggested that the sgp130fc protein might be an ideal candidate for a more specific mode of cytokine blockade as opposed to global cytokine inhibition20 it was shown in appropriate animal models that blockade of il6 transsignalling was indeed superior to global il6 blockade in a bone healing model4445 in a sepsis model42 in abdominal page of figure designer proteins to probe for modes of interleukin il6 signalling a hyperil6 is a fusion protein between il6 and soluble il6 receptor sil6r b sgp130fc is a fusion protein of the extracellular portion of glycoprotein kda gp130 and the constant part of a human immunoglobulin g1 igg1 antibody c il6 can signal via the membranebound il6r classical signalling and via the sil6r transsignalling hyperil6 can be used to mimic il6 transsignalling b the sg130fc protein does not interfere with classical il6 signalling but it specifically blocks il6 transsignallingusing this approach it was shown that classic il6 signalling via the membranebound il6r was responsible for the defence of the body against bacteria4041 intestinal regeneration upon polymicrobial sepsis42 prevention of aortic rupture in animal models of abdominal aortic aneurysm43 and healing of bone fractures4445 important processes are severely compromised under blockade of global il6 activity46 it has been hypothesised that the same might apply for the treatment of covid19 patients46 figure indicating that these besides being the major alarm signal in the human body il6 plays a dominant role in various types of cancer one important reason could be that il6 via stimulation of the stat3 pathway is a prominent growth factor of many cancer cells the following scenario has been worked out in pancreatic cancer47 it was noted that in the krasg12d model the massive activation of the stat3 pathway which led to 0cf1000research 9faculty rev1013 last updated aug figure pro and antiinflammatory activities of interleukin6 il6 left antiinflammatory and protective activities of the cytokine il6 are associated with signalling via the membranebound il6 receptor il6r right proinflammatory activities of the cytokine il6 are associated with signalling via the soluble il6r sil6r the membranebound metalloprotease a disintegrin and metalloprotease adam17 orchestrates the pro and antiinflammatory activities of il6 treg regulatory t cellin patients with is presently ongoing aortic aneurysm models43 and in bacterial infection models4041 the sgp130fc protein was expressed and purified according to gmp regulations phase i clinical trials were successfully performed with healthy individuals and a phase ii clinical trial inflammatory bowel disease54 the future will tell whether this elegant therapeutic approach which was successfully tested in many animal models leads to a novel paradigm in cytokineblocking therapies in patients with autoimmune disorders46 similarly blockade of transsignalling while leaving classical signalling intact may prove to be beneficial for patients experiencing cytokine storms from covid19 or car tcell therapies finally we suggest that malignancies promoted by high levels of transsignalling could be contained by this therapeutic modalityinterleukin6 il6r abbreviationsadam17 a disintegrin and metalloprotease egfr epidermal growth factor receptor gp130 glycoprotein kda il6 interleukin6 receptor ras rat sarcoma proto oncogene sgp130fc soluble gp130fc fusion protein which under the name of olamkicept is in phase ii clinical trials sil6r soluble il6r stat signal transducer and activator of transcription tnfα tumor necrosis factor α yap yesassociated protein yes yamaguchi sarcoma viral oncogene homologacknowledgementsi thank all past and current colleagues of our laboratory for many helpful discussionsreferencesfaculty opinions recommended kishimoto t interleukin6 from basic science to medicine40 years in immunology annu rev immunol pubmed abstract publisher full text tanaka t narazaki m ogata a et al a new era for the treatment of inflammatory autoimmune diseases by interleukin6 blockade strategy semin immunol pubmed abstract publisher full text faculty opinions recommendation teachey dt lacey sf shaw pa et al identification of predictive biomarkers for cytokine release syndrome after chimeric antigen receptor tcell therapy for acute lymphoblastic leukemia cancer discov pubmed abstract publisher full text free full text page of 0c moore jb june ch cytokine release syndrome in severe covid19 science pubmed abstract publisher full text faculty opinions recommendation xu x han m li t et al effective treatment of severe covid19 patients with tocilizumab proc natl acad sci u s a pubmed abstract publisher full text free full text faculty opinions recommendation hirano t taga t yamasaki k et al molecular cloning of the cdnas for interleukin6b cell stimulatory factor and its receptor ann n y acad sci discussion pubmed abstract publisher full text gauldie j richards c harnish d et al interferon beta 2bcell stimulatory factor type shares identity with monocytederived hepatocytestimulating factor and regulates the major acute phase protein response in liver cells proc natl acad sci u s a pubmed abstract publisher full text free full text brakenhoff jp de groot er evers rf et al molecular cloning and expression of hybridoma growth factor in escherichia coli j immunol pubmed abstract zilberstein a ruggieri r korn jh et al structure and expression of cdna and genes for human interferonbeta2 a distinct species inducible by growthstimulatory cytokines embo j pubmed abstract free full text haegeman g content j volckaert g et al structural analysis of the sequence coding for an inducible 26kda protein in human fibroblasts eur j biochem pubmed abstract publisher full text rothaug m beckerpauly c rosejohn s the role of interleukin6 signaling in nervous tissue biochim biophys acta pt a pubmed abstract publisher full text willis ef macdonald kpa nguyen qh et al repopulating microglia promote brain repair in an il6dependent manner cell 833846e16 pubmed abstract publisher full text wallenius v wallenius k ahr©n b et al interleukin6deficient mice develop matureonset obesity nat med pubmed abstract publisher full text findeisen m allen tl henstridge dc et al treatment of type diabetes with the designer cytokine ic7fc nature pubmed abstract publisher full text faculty opinions recommendation pedersen bk febbraio ma muscles exercise and obesity skeletal muscle as a secretory an nat rev endocrinol pubmed abstract publisher full text faculty opinions recommendation schmidt s schumacher n schwarz j et al adam17 is required for egfrinduced intestinal tumors via il6 transsignaling j exp med pubmed abstract publisher full text free full text rosejohn s the biology of interleukin6 in the 21st century semin immunol pubmed abstract publisher full text schaper f rosejohn s interleukin6 biology signaling and strategies of blockade cytokine growth factor rev pubmed abstract publisher full text jones sa jenkins bj recent insights into targeting the il6 cytokine family in inflammatory diseases and cancer nat rev immunol pubmed abstract publisher full text faculty opinions recommendation garbers c heink s korn t et al interleukin6 designing specific therapeutics for a complex cytokine nat rev drug discov pubmed abstract publisher full text m¼llberg j schooltink h stoyan t et al the soluble interleukin6 receptor is generated by shedding eur j immunol pubmed abstract publisher full text lust ja donovan ka kline mp et al isolation of an mrna encoding a soluble form of the human interleukin6 receptor cytokine pubmed abstract publisher full text mackiewicz a schooltink h heinrich pc et al complex of soluble human il6receptoril6 upregulates expression of acutephase proteins j immunol pubmed abstract rosejohn s heinrich pc soluble receptors for cytokines and growth factors generation and biological function biochem j 300pt pubmed abstract publisher full text free full text rosejohn s the soluble interleukin receptor advanced therapeutic options in inflammation clin pharmacol ther pubmed abstract publisher full text fischer m goldschmitt j peschel c et al i a bioactive designer cytokine for human hematopoietic progenitor cell expansion nat biotechnol f1000research 9faculty rev1013 last updated aug pubmed abstract publisher full text galun e zeira e pappo o et al liver regeneration induced by a designer human il6sil6r fusion protein reverses severe hepatocellular injury faseb j pubmed abstract publisher full text m¤rz p otten u rosejohn s neural activities of il6type cytokines often depend on soluble cytokine receptors eur j neurosci pubmed abstract publisher full text audet j miller cl rosejohn s et al distinct role of gp130 activation in promoting selfrenewal divisions by mitogenically stimulated murine hematopoietic stem cells proc natl acad sci u s a pubmed abstract publisher full text free full text rosejohn s interleukin6 family cytokines cold spring harb perspect biol a028415 pubmed abstract publisher full text free full text horsten u schmitzvan de leur h m¼llberg j et al the membrane distal half of gp130 is responsible for the formation of a ternary complex with il6 and the il6 receptor febs lett pubmed abstract publisher full text jostock t m¼llberg j ozbek s et al soluble gp130 is the natural inhibitor of soluble interleukin6 receptor transsignaling responses eur j biochem pubmed abstract publisher full text waage a brandtzaeg p halstensen a et al the complex pattern of cytokines in serum from patients with meningococcal septic shock association between interleukin interleukin and fatal outcome j exp med pubmed abstract publisher full text free full text tanaka t narazaki m kishimoto t il6 in inflammation immunity and disease cold spring harb perspect biol a016295 pubmed abstract publisher full text free full text faculty opinions recommendation rosejohn s il6 transsignaling via the soluble il6 receptor importance for the proinflammatory activities of il6 int j biol sci pubmed abstract publisher full text free full text cressman de greenbaum le deangelis ra et al liver failure and defective hepatocyte regeneration in interleukin6deficient mice science pubmed abstract publisher full text poli v balena r fattori e et al interleukin6 deficient mice are protected from bone loss caused by estrogen depletion embo j pubmed abstract publisher full text free full text alonzi t fattori e lazzaro d et al interleukin is required for the development of collageninduced arthritis j exp med pubmed abstract publisher full text free full text okuda y sakoda s bernard cc et al il6deficient mice are resistant to the induction of experimental autoimmune encephalomyelitis provoked by myelin oligodendrocyte glycoprotein int immunol pubmed abstract publisher full text hoge j yan i j¤nner n et al il6 controls the innate immune response against listeria monocytogenes via classical il6 signaling j immunol pubmed abstract publisher full text sodenkamp j waetzig gh scheller j et al therapeutic targeting of interleukin6 transsignaling does not affect the outcome of experimental tuberculosis immunobiology pubmed abstract publisher full text barkhausen t tschernig t rosenstiel p et al selective blockade of interleukin6 transsignaling improves survival in a murine polymicrobial sepsis model crit care med pubmed abstract publisher full text paige e cl©ment m lareyre f et al interleukin6 receptor signaling and abdominal aortic aneurysm growth rates circ genom precis med e002413 pubmed abstract publisher full text free full text kaiser k prystaz k vikman a et al pharmacological inhibition of il6 transsignaling improves compromised fracture healing after severe trauma naunyn schmiedebergs arch pharmacol pubmed abstract publisher full text free full text prystaz k kaiser k kovtun a et al distinct effects of il6 classic and transsignaling in bone fracture healing am j pathol pubmed abstract publisher full text magro g sarscov2 and covid19 is interleukin6 il6 the culprit lesion of ards onset what is there besides tocilizumab sgp130fc cytokine x pubmed abstract publisher full text free full text faculty opinions recommendation lesina m kurkowski mu ludes k et al stat3socs3 activation by il6 transsignaling promotes progression of pancreatic intraepithelial page of 0cf1000research 9faculty rev1013 last updated aug neoplasia and development of pancreatic cancer cancer cell pubmed abstract publisher full text faculty opinions recommendation lanaya h natarajan a komposch k et al egfr has a tumourpromoting role in liver macrophages during hepatocellular carcinoma formation nat cell biol pubmed abstract publisher full text free full text faculty opinions recommendation sacre sm andreakos e taylor p et al molecular therapeutic targets in rheumatoid arthritis expert rev mol med pubmed abstract publisher full text gabay c emery p van vollenhoven r et al tocilizumab monotherapy versus adalimumab monotherapy for treatment of rheumatoid arthritis adacta a randomised doubleblind controlled phase trial lancet pubmed abstract publisher full text faculty opinions recommendation le rq li l yuan w et al fda approval summary tocilizumab for treatment of chimeric antigen receptor t cellinduced severe or lifethreatening cytokine release syndrome oncologist pubmed abstract publisher full text free full text faculty opinions recommendation campochiaro c dellatorre e cavalli g et al efficacy and safety of tocilizumab in severe covid19 patients a singlecentre retrospective cohort study eur j intern med pubmed abstract publisher full text free full text faculty opinions recommendation dellatorre e campochiaro c cavalli g et al interleukin6 blockade with sarilumab in severe covid19 pneumonia with systemic hyperinflammation an openlabel cohort study ann rheum dis pubmed abstract publisher full text faculty opinions recommendation safety and efficacy of tj301 iv in participants with active ulcerative colitis clinicaltrials reference sourcepage of 0cf1000research 9faculty rev1013 last updated aug open peer reviewcurrent peer review status editorial note on the review processfaculty reviews are review s written by the prestigious members of faculty opinions the s are commissioned and peer reviewed before publication to ensure that the final published version is comprehensive and accessible the reviewers who approved the final version are listed with their names and affiliationsthe reviewers who approved this areversion jacqueline bromberg department of medicine memorial sloan kettering cancer center new york ny usa competing interests no competing interests were disclosedhana alg¼l comprehensive cancer center munich university hospital klinikum rechts der isar mildredscheelchair of tumor metabolism technical university of munich munich | Colon_Cancer |
" the choice of treatment in patients with metastatic colorectal cancer mcrc is generally influenced by tumour and patient characteristics treatment efficacy and tolerability and quality of life better patient selection might lead to improved outcomesmethods this post hoc exploratory analysis examined the effect of prognostic factors on outcomes in the randomized double blind phase study of trifluridine tipiracil ftdtpi plus best supportive care bsc versus placebo plus bsc in patients with mcrc refractory to standard chemotherapies recourse trial patients were redivided by prognosis into two subgroups those with metastatic sites at randomisation low tumour burden and ¥ months from diagnosis of metastatic disease to randomisation indolent disease were included in the good prognostic characteristics gpc subgroup the remaining patients were considered to have poor prognostic characteristics ppcresults gpc patients n386 had improved outcome versus ppc patients n414 in both the trifluridinetipiracil and placebo arms gpc patients receiving trifluridinetipiracil n261 had an improved median overall survival vs months hr ci to p00001 and progression free survival vs months hr ci to p00001 than ppc patients receiving trifluridinetipiracil n273 improvements in survival were irrespective of age eastern cooperative oncology group performance status ecog ps kras mutational status and site of metastases at randomisation in the trifluridinetipiracil arm time to deterioration of ecog ps to ¥ and proportion of patients with ps0 discontinuing treatment were longer for gpc than for ppc patients vs months and vs respectively low tumour burden and indolent disease were factors of good prognosis in late line mcrc with patients experiencing longer progression free survival and greater overall survivalintroductioninclusion of new therapeutic options into the current treatment landscape in metastatic colorectal cancer mcrc has led to an increased survival in the last couple of key questionswhat is already known about this subject º the choice of treatment in patients with metastatic colorectal cancer mcrc is generally influenced by tumour characteristics and patient factors as well as treatment characteristics such as tolerability efficacy and quality of life effects trifluridinetipiracil is indicated in pretreated patients with mcrc based on results of the pivotal randomised double blind phase study of trifluridine tipiracil ftdtpi plus best supportive care bsc versus placebo plus bsc in patients with metastatic colorectal cancer refractory to standard chemotherapies recourse trial which demonstrated significantly improved overall survival os compared with placebo with a manageable safety profilewhat does this study add º in recourse classification of patients as having good prognostic characteristics gpc defined as those with low tumour burden metastatic sites at randomisation and less aggressive disease ¥ months from diagnosis of first metastasis at randomisation identified a subgroup of patients with improved os and progression free survival with trifluridinetipiracil compared with patients with poor prognostic characteristics treated with trifluridinetipiracil and gpc patients treated with placebohow might this impact on clinical practice º low tumour burden and indolent disease were shown to be factors of good prognosis in late line mcrc with these patients experiencing longer time on treatment and greater os this suggests that these patients could be candidates to receive further lines of therapy post trifluridinetipiracildecades1 first line treatment of patients typically involves the use of vascular endothelial growth factor vegf or epidermal growth factor receptor egfr targeted agents eg bevacizumab cetuximab panitumumab to fluoropyrimidine based fluorouracil or tabernero a0j et a0al esmo open 20205e000752 101136esmoopen2020000752 0copen accesscapecitabine chemotherapy regimens depending on the presence or absence of ras mutation positive disease2 in the usa immunotherapies nivolumab±ipilimumab or pembrolizumab are also recommended for the treatment of patients with mismatch repair deficient or microsatellite instability high disease4 in the second line setting vegf targeted treatments eg aflibercept ramucirumab can also be used in combination with chemotherapy2 the optimal chemotherapeutic regimen for use beyond third line remains unclear where resistantrefractory disease and residual toxicity potentially limit the treatment options with only two possible candidates at present5the general condition and performance status of a patient are strong prognostic and predictive factors for mcrc treatment2 fitter patients are typically assigned to a more intensive treatment approach ie a combination of cytotoxic agents with a biological agent than less fit patients2 the choice of treatment in the metastatic setting is generally influenced by tumour characteristics tumour burden localisation and biology patient characteristics age eastern cooperative oncology group performance status ecog ps an function and comorbidities and treatment characteristics efficacy toxicity profile administration and quality of life qol effects2the proportion of patients with mcrc receiving active treatment decreases from line to line leaving more than half of patients who received an active treatment in the first line without treatment in the third line setting even in randomised clinical trials in folfiri plus cetuximab versus folfiri plus bevacizumab as first line treatment for patients with metastatic colorectal cancer only of patients reached third line6 data from the usa indicate that only of patients receiving a first line of treatment move into the second line move to the third line and only will receive a fourth line of treatment7 being unable to receive a subsequent line of treatment therefore appears to have a negative impact on the patients survivalis trifluridinetipiracil ftdtpi lonsurf indicated for the treatment of adult patients with mcrc who have been previously treated with or are not considered candidates for available therapies including fluoropyrimidine based oxaliplatin based and irinotecan based chemotherapies anti vegf agents and anti egfr agents for eligible patient ras wild type combination of tipiracil hydrochloride with the nucleoside metabolic inhibitor trifluridine improves its bioavailability by inhibiting its catabolism by thymidine phosphorylase8 the relatively limited non haematological toxicity of trifluridinetipiracil makes it a good option in the third line and refractory settings2 in the pivotal phase iii randomised double blind phase study of trifluridine tipiracil ftdtpi plus best supportive care bsc versus placebo plus bsc in patients with metastatic colorectal cancer refractory to standard chemotherapies recourse trial conducted in patients with mcrc eligible for treatment in the third line and beyond treatment with trifluridinetipiracil versus placebo extended overall survival median os vs months hr p0001 and progression free survival median pfs vs months hr p000110 this effect was shown in all subgroups regardless of age ecog ps geographical region race and kras mutational status10 furthermore trifluridinetipiracil was well tolerated with few serious adverse events aes reported haematological toxicities were the most frequently observed aes10 also time to deterioration of ecog ps to ¥ was significantly improved median vs months hr p000110 with of patients treated with trifluridinetipiracil remaining at ps at discontinuation11 remaining at ecog ps is important as it could allow patients to further benefit from subsequent therapy and potentially extend their survival in recourse and of patients treated with trifluridinetipiracil remained alive at and months respectively in the refractory setting in the post hoc analysis described here we set out to explore other factors that could extend survival in the recourse population for the purposes of our exploratory analysis we defined the characteristics of good prognosis as low tumour burden metastatic sites by response evaluation criteria in solid tumors recist evaluation at randomisation and less aggressiveindolent disease ¥ months from diagnosis of first metastasis to randomisation which are known to be strong prognostic factors in patients with mcrc with good ecog ps12 our ultimate aim is to explore how clinicians can better predict individual treatment outcomes and support treatment selection through the continuum of carematerials and methodsstudy design and patientsthe study design and methodology of the recourse trial clinicaltrials gov number nct01607957 have been previously published10 in brief recourse was a phase iii randomised double blind placebo controlled study comparing the efficacy and safety of trifluridinetipiracil plus best supportive care with those of placebo plus best supportive care10 this study included patients with metastatic biopsy provendocumented adenocarcinoma of the colon or rectum who were previously treated with ¥ standard chemotherapy regimens or who had tumour progression within months of their most recent chemotherapy or who had clinically significant aes precluding readministration of standard chemotherapies patients were randomised to trifluridinetipiracil mgm2 two times a day on days and every weeks or matching placebo10 randomisation was stratified according to kras mutation status wild type vs mutant time from diagnosis of first metastasis to randomisation vs ¥ months and geographical region japan vs usa european union and australia10 all patients had adequate an function and were ecog ps tabernero a0j et a0al esmo open 20205e000752 101136esmoopen2020000752 0cof at inclusion10 the primary endpoint of the study was os and secondary endpoints included pfs objective response rate clinical benefit rate and safety10patient subgroupsin examining the effects of prognostic factors on treatment outcomes in the current analysis several subgroups of recourse patients were considered patients from recourse n800 were divided according to good prognostic characteristics gpc and poor prognostic characteristics ppc good prognosis was considered to be defined by low tumour burden metastatic sites by recist tumour evaluation at randomisation and less aggressiveindolent disease ¥ months from diagnosis of first metastasis to randomisation12 of the gpc subgroup n386 patients received trifluridinetipiracil and received placebo the remaining patients were included in the complementary ppc subgroup n414 of these received trifluridinetipiracil and received placeboanalysis outcomesos and pfs in the gpc subgroup were compared with those in the ppc subgroup these subgroups were then analysed according to other tumour and patient characteristics that is metastatic site at randomisation for those sites present in of the population liver lung lymph or peritoneum ecog ps vs kras mutation status wild type vs mutant and age vs ¥ years os and pfs with trifluridinetipiracil were compared with placebo and were analysed according to prognostic subgroups within each of the two arms finally the effect of prognostic classification of patients on ecog ps deterioration was analysed for all patients and subgroupsstatistical methodsdemographic and baseline characteristics of patients were summarised by treatment arm and subgroups using descriptive statistics n mean sd median minimum and maximum andor frequency distributions as appropriatethe differences in os pfs and time to ecog ps deterioration between trifluridinetipiracil and placebo patients or between subgroups of patients in a specific arm of treatment were assessed using the stratified log rank test stratification factors used for the randomisation from a cox proportional hazards model for each arm or each subgroup survival was summarised using kaplan meier curves and was further characterised in terms of the median with the corresponding two sided cisresultspatientsbaseline patient demographics and clinical characteristics were generally similar between gpc and ppc patients table in the trifluridinetipiracil arm slight imbalances were seen in ecog ps more gpc than ppc open accesspatients had an ecog ps of and kras status more gpc than ppc patients were kras wild type also more gpc than ppc patients had received ¥ prior regimens among the ppc group treated with trifluridinetipiracil of patients had ¥ months from diagnosis of first metastasis to randomisation but had ¥ metastatic sites and of patients had metastatic sites but months from diagnosis of first metastasis similar differences were observed in the placebo arm with the exception of kras status which was comparable in the gpc and ppc subgroupstreatmentamong trifluridinetipiracil treated patients those in the gpc group received more treatment cycles mean sd compared with patients in the ppc group mean sd online supplementary table s1 a higher proportion of gpc patients than ppc patients receiving trifluridinetipiracil had a dose delay vs respectively or dose reduction vs respectively which is consistent with a longer duration of treatment online supplementary table s1 however median dose intensity in the first four cycles was high ¥ and did not differ markedly between the groups cycle in the gpc group and the ppc group cycle and respectively cycle and respectively cycle and respectivelythe effect of good versus poor prognosis classifications on survivalsurvival curves for the gpc versus ppc subgroups are shown in figure median os was longer in the gpc subgroup than the ppc subgroup for both trifluridinetipiracil vs months hr ci to p00001 figure 1a and placebo vs months hr ci to p00001 figure 1b rates of month os and in gpc and and in ppc for trifluridinetipiracil and placebo respectively and month os and in gpc and and in ppc for trifluridinetipiracil and placebo respectively were also higher in gpc subgroups compared with ppc subgroups median pfs with trifluridinetipiracil was also longer in the gpc subgroup versus the ppc subgroup vs months hr ci to p00001 respective values for gpc versus ppc in the placebo arm were versus months hr p00699 pfs at and months in the ppc subgroup was and for trifluridinetipiracil and and for placebo respectively in the gpc subgroup these were and with trifluridinetipiracil and and with placebo respectivelyeffects of good prognostic factors on the relative efficacy of trifluridinetipiracilmedian os was prolonged with trifluridinetipiracil versus placebo in both subgroups but to a greater tabernero a0j et a0al esmo open 20205e000752 101136esmoopen2020000752 0copen accesstable baseline patient demographics and clinical characteristics according to prognosistrifluridinetipiracilplacebogpc subgroup n261 ppc subgroup n273 gpc subgroup n125ppc subgroup n141 females male asian other years to years ¥ yearsmedian age yearspatient age n gender n ethnicity n ecog ps n kras status n time since diagnosis of metastasis n number of prior regimens n number of metastatic sites n site of metastatic lesion n primary site of disease n liver lung lymph peritoneum ¥ ¥ mutant wild type months ¥ months colon rectum defined as metastatic sites and ¥ months since first metastasis only those in more than of the intent to treat population are included liver lung lymph and peritoneumecog ps eastern cooperative oncology group performance status gpc good prognostic characteristics ppc poor prognostic characteristicsextent in the gpc subgroup than in the ppc subgroup figure 2a similarly median pfs was prolonged with trifluridinetipiracil versus placebo in both subgroups with the greatest magnitude of benefit observed in the gpc patients figure 2banalysis of prognostic factorsthe effect of various prognostic factors on median os and pfs is shown in table their effect on month and month os and month month and month pfs is shown in online supplementary tables and for both tabernero a0j et a0al esmo open 20205e000752 101136esmoopen2020000752 0copen accessfigure overall survival os for the good prognostic characteristics gpc and poor prognostic characteristics ppc subgroups in patients receiving a trifluridinetipiracil or b placebo ap0001 one sided bp0001 two sided ftdtpi trifluridinetipiracil mos median overall survival nr not reachedtrifluridinetipiracil and placebo the gpc subgroup had better median os and pfs than the ppc subgroup irrespective of patient age ¥ vs years ecog ps vs kras mutation status mutant vs wild type and liver metastases yes vs nowhen analysing the gpc subgroup the absence of liver metastasis at randomisation n153 representing of the gpc and of the intent to treat population was found to be the best factor of prognosis further information on this group of patients is available in online tabernero a0j et a0al esmo open 20205e000752 101136esmoopen2020000752 0copen accessa overall survival os b progression free survival pfs and c time to eastern cooperative oncology group figure performance status ecog ps ¥ with trifluridinetipiracil versus placebo in the good prognostic characteristics gpc n386 and poor prognostic characteristics ppc n414 subgroups ftdtpi trifluridinetipiracil mos median overall survivalsupplementary table s4 and online supplementary figures s1 s3 among gpc patients treated with trifluridinetipiracil median os was months longer in patients with no liver metastases compared with those with liver metastases vs months table the month os rate in gpc patients treated with trifluridinetipiracil was in those without liver metastases and in those with liver metastases corresponding month os rates in these groups were and respectively online supplementary table s2 median os was also longer in patients with no liver metastases compared with those with liver metastases in the trifluridinetipiracil ppc subgroup vs months and both the gpc and ppc subgroups of the placebo arm vs months and vs months respectively table in the group of ppc patients treated with trifluridinetipiracil the month and month os rates were and respectively in those without liver metastases compared with and respectively in those with liver metastases online supplementary table s2 for the trifluridinetipiracil and placebo arms patients with baseline ecog ps had higher median os compared with ecog ps patients in both the gpc and ppc subgroups table in the trifluridinetipiracil arm age or ¥ years and kras status did not seem to affect the treatment outcome table similar results were found for pfs with an effect for all trifluridinetipiracil gpc and ppc subgroups with median pfs values ranging from to months table among gpc patients treated with trifluridinetipiracil the month pfs rate was in those with no liver metastases compared with in those with liver metastases corresponding month pfs rates in the ppc group of patients treated with trifluridinetipiracil were and respectively online supplementary table s3 no such effect was observed in the placebo arm with values ranging months whatever the prognosis at the outset for almost all subgroups median tabernero a0j et a0al esmo open 20205e000752 101136esmoopen2020000752 0ctable the effect of various prognostic factors on median overall survival os and progression free survival pfsnumber of patientsftdtpi placebomedian survival monthshr cinumber of patientsftdtpiplacebomedian survival monthshr ciopen accessosgpc subgroupppc subgroupgpc subgroupppc subgroupgpc subgroupppc subgroupgpc subgroupppc subgroupgpc subgroupppc subgroupgpc subgroupppc subgroupgpc subgroupppc subgrouppfsgpc subgroupppc subgroupgpc subgroupppc subgroupgpc subgroupppc subgroupgpc subgroupppc subgroupgpc subgroupppc subgroupgpc subgroupppc subgroupgpc subgroupppc subgroup vs vs no liver metastasesn97n56n35n21no lung metastasesn89n25n54n24no lymph metastasesn208n93n116n53no peritoneal metastasesn242n119n203n100ecog ps0n158n77n143n70age yearsn137n72n163n76kras wild typen142n61n120n70 vs vs vs vs vs vs vs vs vs vs vs vs no liver metastasesn97n56n35n21no lung metastasesn89n25n54n24no lymph metastasesn208n93n116n53no peritoneal metastasesn242n119n203n100ecog ps0n158n77n143n70age yearsn137n72n163n76kras wild typen142n61n120n70 vs vs vs vs vs vs vs vs vs vs vs vs vs vs to to to to to to to to to to to to to to to to to to to to to to to to to to to to liver metastasesn164n69n238n120lung metastasesn172n100n219n117lymph metastasesn53n32n157n88peritoneal metastasesn19n6n70n41ecog ps1n103n48n130n71age¥ yearsn124n53n110n65kras mutantn119n64n153n71liver metastasesn164n69n238n120lung metastasesn172n100n219n117lymph metastasesn53n32n157n88peritoneal metastasesn19n6n70n41ecog ps1n103n48n130n71age ¥ yearsn124n53n110n65kras mutantn119n64n153n71 vs vs to to vs vs to to vs vs to to vs vs to to vs vs to to vs vs to to vs vs to to vs vs to to vs vs to to vs vs to to vs vs to to vs vs to to vs vs to to vs vs to to good prognostic characteristics gpc were defined as metastatic sites at randomisation and ¥ months from first metastasis to randomisationftdtpi trifluridinetipiracil ppc poor prognostic characteristics ecog ps eastern cooperative oncology group performance statustabernero a0j et a0al esmo open 20205e000752 101136esmoopen2020000752 0copen accesstable effects of prognostic classification of patients on eastern cooperative oncology group performance status ecog psmedian time to deterioration to ecog ps ¥ monthsftdtpiplaceboitt population n80011good prognosis patients n386poor prognosis patients n414ftdtpi trifluridinetipiracil itt intent to treatpfs was longer and all hrs favoured treatment with trifluridinetipiracil table effects of prognostic classification of patients on ecog psdata relative to the effect of ecog ps are presented in table the proportion of gpc patients treated with trifluridinetipiracil with an ecog ps of at treatment discontinuation was among gpc patients with an ecog ps of at baseline had not deteriorated beyond a ps of at treatment discontinuation similarly among gpc patients with an ecog ps of at baseline had not deteriorated beyond a ps of at treatment discontinuation the median time to deterioration of ecog ps to ¥ in patients receiving trifluridinetipiracil was months in the gpc subgroup and months in the ppc subgroup figure 2ctolerability and safetythe most common aes in patients receiving trifluridinetipiracil were nausea anaemia neutropenianeutrophil count decrease diarrhoea fatigue and reduced appetite online supplementary table s5 the most common grade ¥ aes experienced by patients receiving trifluridinetipiracil were haematological anaemia neutropenianeutrophil count decrease white blood cell count decrease there was no evidence of a higher incidence of aes in patients with ppc versus gpc in the group receiving trifluridinetipiracil but there was a trend towards a higher incidence of aes in placebo recipients with ppc compared with gpc online supplementary table s5discussionthe results of our analysis show that patients in the gpc subgroup consistently performed better than those in the ppc subgroup in both the trifluridinetipiracil and placebo arms within the same subgroups patients treated with trifluridinetipiracil performed better than placebo trifluridinetipiracil has consistently been shown to provide a significant survival benefit to patients with mcrc refractory to standard therapy with a well tolerated safety profile in three large scale randomised clinical trials10 a previous subanalysis of recourse showed that trifluridinetipiracil was more effective than placebo in patients irrespective of region age racialhr ci to to to p valueecog ps at treatment discontinuation ftdtpiplaceboethnic differences or kras mutation status17 in the current analysis further categorisation of patients as having good prognosis using the criteria of metastatic sites by recist tumour evaluation at randomisation and ¥ months from diagnosis of first metastasis to randomisation12 identified a subgroup of patients with improved os and pfs with trifluridinetipiracil compared with poorer prognosis patients ie those with ¥ metastatic sites and months from first metastasis pfs and os were also improved in gpc patients treated with trifluridinetipiracil compared with gpc patients who received placebopatients with gpc received more cycles of treatment than patients with ppc because progression was delayed in this group which may have contributed to the better survival outcomes the difference cannot be explained by a difference in dose intensity since this was high and similar in both the ppc and gpc subgroups of patients receiving trifluridinetipiracil in addition there was no evidence for higher toxicity in the ppc than the gpc group in fact the haematological aes occurred at a slightly higher rate in gpc patients than in ppc patients who received trifluridinetipiracil which probably reflects a longer exposure to treatment in the gpc group more patients in the gpc than in the ppc subgroup had dose delays which suggests that grade ¥ haematological aes were appropriately managed during treatmentit is thought that the availability of more treatment options for mcrc has contributed to an improvement in os over the last years3 indeed a retrospective study in elderly patients aged ¥ years a patient population more prone to comorbidities poor performance status and the development of treatment related toxicity reported a correlation between os and the number of treatment lines received18 thus maintaining the general condition and performance status of a patient throughout the continuum of care is of great importance especially beyond the second line to ensure patients remain fit with good qol5 our analysis showed that the majority of patients in the gpc subgroup discontinued treatment with an ecog ps of at the time of disease progression suggesting that these patients could be candidates to receive further lines of therapy post trifluridinetipiracil this is important when sequencing through the continuum of care this is in line with other tabernero a0j et a0al esmo open 20205e000752 101136esmoopen2020000752 0canalyses indicating preservation of health related qol on treatment of patients with mcrc with trifluridinetipiracil19 while the post hoc nature of this analysis limits it to an exploratory analysis the relatively large number of patients analysed make these data a good tool to estimate the expected outcomes when treating patients with refractory mcrc with trifluridinetipiracil the smaller size of some of the subgroups may limit the s that can be drawn thus preventing an evaluation of other parameters that might impact on outcomes such as lactate dehydrogenase levels the exact definition of good and poor prognostic factors12 may require further validation in a prospective cohortthe current analysis shows that compared with poor prognosis patients treated with either trifluridinetipiracil or placebo and good prognosis patients treated with placebo patients with gpcs treated with trifluridinetipiracil adequate an function ecog ps metastatic sites by recist tumour evaluation at randomisation and ¥ months from diagnosis of first metastasis have an increased survival in terms of median os and month and month survival rates treatment with trifluridinetipiracil is effective and provides the majority of patients the opportunity to maintain ecog ps and the possibility to receive further treatment options through the continuum of careauthor affiliations1vall dhebron institute of oncology uvic ucc medical oncology vall d'hebron hospital barcelona catalunya spain2vall dhebron institute of oncology uvic ucc iob quironmedical oncology vall d'hebron hospital barcelona catalunya spain3medical oncology ospedale policlinico san martino istituto di ricovero e cura a carattere scientifico per l'oncologia genova liguria italy4department of medical oncology university hospital centre besançon besancon bourgogne franche comté france5kashiwa national cancer center hospital east kashiwa chiba japan6department of medical oncology dana farber cancer institute boston massachusetts usa7centre of excellence methodology and valorization of data centex mvd institut de recherches internationales servier suresnes france8global medical affairs les laboratoires servier sas suresnes île de france france9digestive oncology ku leuven university hospitals leuven leuven flanders belgiumacknowledgements the authors would like to thank andrea bothwell who wrote the first draft of this manuscript on behalf of springer healthcare communications this medical writing assistance was funded by institut de recherches internationales servier suresnes francecontributors jt and srmv contributed to the conception and design of the study all authors were involved in the acquisition analysis and interpretation of data and in writing andor revising drafts of the manuscript all authors have read and approved the final draft of the manuscript and accept responsibility for the finished article and the decision to submit the manuscript for publicationfunding the recourse study was funded by taiho oncology and taiho pharmaceutical co this analysis was funded by servier in partnership with taihocompeting interests jt has received personal fees from array biopharma astrazeneca bayer ag beigene boehringer ingelheim chugai genentech open accessgenmab as halozyme imugene limited inflection biosciences limited ipsen kura oncology eli lilly and company merck menarini merck serono merrimack pharmaceuticals merus molecular partners novartis peptomyc pfizer pharmacyclics proteodesign sl rafael pharmaceuticals f hoffmann la roche sanofi seattle genetics servier symphogen taiho pharmaceutical vcn biosciences biocartis foundation medicine haliodx sas pharmaceuticals and roche diagnostics ga has had an advisory role or received honoraria or travel grants from hoffmann la roche merck serono amgen sanofi bayer servier and bristol myers squibb afs has had an advisory role for amgen bayer celgene roche merck serono sanofi and servier and has attended a speakers bureau for amgen astrazeneca bayer bristol myers squibb celgene lilly merck serono roche sanofi and takeda evc has received research funding from amgen bayer boehringer ingelheim celgene ipsen lilly merck merck kga novartis roche sanofi and servier and has attended advisory boards for astellas astrazeneca bayer bristol myers squibb celgene lilly merck sharp dohme merck kgaa novartis roche and servier cb has attended advisory boards for roche servier and sanofi and has received a research grant from roche ao has received honoraria from ono bms chugai taiho eisai and amgen and has received research funding from bristol myers squibb an immediate family member of ao has been employed by celgene rjm declares no conflicts of interest lv and srmv are employees of servierpatient consent for publication not requiredethics ap | Colon_Cancer |
high throughput methods in biological and biomedical fields acquire alarge number of molecular parameters or omics data by a single experimentcombining these omics data can significantly increase the capability for recoveringfinetuned structures or reducing the effects of experimental and biological noise indataresultsfhclust for identifying patient subgroups from different omics information eg geneexpression mirna expression methylation in particular hierarchical structures of patientdata are obtained in each omic or view and finally their topologies are merged byconsensus matrix one of the main aspects of this methodology is the use of ameasure of dissimilarity between sets of observations by using an appropriate metricfor each view a dendrogram is obtained by using a hierarchical clustering based on afuzzy equivalence relation with Åukasiewicz valued fuzzy similarity finally a consensusmatrix that is a representative information of all dendrograms is formed by combiningmultiple hierarchical agglomerations by an approach based on transitive consensusmatrix construction several experiments and comparisons are made on real data egglioblastoma prostate cancer to assess the proposed approachs fuzzy logic allows us to introduce more flexible data agglomerationtechniques from the analysis of scientific literature it appears to be the first time that amodel based on fuzzy logic is used for the agglomeration of multiomic data theresults suggest that fhclust provides better prognostic value and clinical significancecompared to the analysis of singleomic data alone and it is very competitive withrespect to other techniques from literaturekeywords multiomics data data integration hierarchical clustering fuzzy similarityfuzzy aggregation the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriatecredit to the original authors and the source provide a link to the creative commons licence and indicate if changes weremade the images or other third party material in this are included in the s creative commons licence unlessindicated otherwise in a credit line to the material if material is not included in the s creative commons licence and yourintended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtain permission directlyfrom the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40 the creativecommons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to the data madeavailable in this unless otherwise stated in a credit line to the data 0cciaramella bmc bioinformatics 21suppl page of nowadays high throughput methods in biological and biomedical fields acquire a largenumber of molecular parameters by a single experiment in particular such measuredparameters are collected in omics datasets eg genomics transcriptomics methylomics among multiple measured parameters dna genome sequence rna expressionand dna methylation are representative instances for individually analysing suchdata several methodologies have been introduced in literature even though recentlya number of studies pointed out the best performance coming from the integration ofmultiomics data for instance analysing each omic or view in the machine learningjargon set separately fundamental patterns can be detected from data however somefinetuned structures such as cancer subtypes can be highlighted by both gene expression and dna methylation information so that multiomics analysis can reduce theeffects of experimental and biological noise in data from literature three kinds ofintegration methodologies emerge¢ early integration builds a single featurebased matrix by concatenating each omic¢ intermediate integration builds a joint representation of data given the views¢ late integration each omic is analysed separately and the solutions are integratedin general late integration methods and in particular clustering are preferred whenthe analysis combines continuous and discrete data together for a review of integrationapproaches and their comparisons the reader may refer to in this work a multiviewclustering methodology named fhclust is introduced see fig for its general schemafor identifying patient subgroups from different omics information or datasets eg geneexpression mirna expression methylation specifically for each omic dataset a fuzzybased hierarchical clustering approach is adopted and finally the results are mergedby consensus matrix the idea behind the proposed approach comes from observingthat a hierarchical clustering dendrogram can be associated with a Åukasiewicz fuzzydataset ie view and applies a singleomic analysisfig proposed approach a data preparation b data normalization and feature selection c multiomicshierarchical agglomerations d data integration e clustering and visualization 0cciaramella bmc bioinformatics 21suppl page of similaritybased equivalence relation so that a consensus matrix that is the representative information of all dendrograms is derived by combining multiple hierarchicalagglomerations following an approach based on transitive consensus matrix constructionmethodscluster analysis or clustering is an unsupervised technique that aims at agglomerating aset of patterns in homogeneous groups or clusters [ ] hierarchical clustering hc isone of several different available techniques for clustering which seeks to build a hierarchyof clusters and it can be of two types namely agglomerative where each sample starts inits own cluster and pairs of clusters are merged as one moves up the hierarchy or divisivewhere all samples start in one cluster and splits are performed recursively as one movesdown the hierarchy thus hc aims at grouping similar objects into a cluster and werethe endpoint is a set of clusters where each cluster is distinct from each other and theobjects within each cluster are broadly similar to each other hc can be performed eitheron a distance matrix or raw data agglomerative hc starts by treating each observationas a separate cluster and it repeatedly executes the following two steps identifies thetwo clusters that are closest together and merges the two most similar clusters thisprocess continues until all the clusters are merged togetherthe main output of hc is a dendrogram which shows the hierarchical relationshipbetween the clusters distances many distance metrics have been developed and thechoice should be made based on theoretical concerns from the domain of studylater on it is necessary to determine how the distance is computed eg singlelinkagecompletelinkage averagelinkage as with distance metrics the choice of linkage criteria should be based on theoretical considerations from the application domainin nonfuzzy clustering or hard clustering data is divided into distinct clusters andeach data point can only belong to exactly one cluster in fuzzy clustering data pointscan potentially belong to multiple clusters for example in hard clustering given someparameters a symptom can be in a mutually exclusive way present or absent red orblue whereas in fuzzy clustering that symptom could simultaneously be of somegrade red and some other grade blue in fig a comparison between hard and fuzzycategorisation is shown the reader can refer to for a recent comparison betweenhard and fuzzy clustering in this work we introduce a data integration methodologybased on fuzzy concepts in particular we associate a dendrogram to a fuzzy equivalencerelation ie Åukasiewicz valued fuzzy similarity so that a consensus matrix in a multiview clustering that is the representative information of all dendrograms can be obtainedfrom multiple hierarchical agglomerations [ ] the main steps of fuzzy agglomerationcan be summarised as follows characterisation of membership functions computation of a fuzzy similarity matrix or dendrogram for all models at a giventime construction of a consensus matrix for all hierarchical agglomerationsmembership functionswhen dealing with clustering tasks fuzzy logic fl permits to obtain a soft clusteringinstead of an hard clustering of data specifically data points can belong to more 0cciaramella bmc bioinformatics 21suppl page of fig hard vs fuzzy in symptom risk example a hard categorization b fuzzy categorizationthan one cluster simultaneously the fundamental concept in fl upon which all thesubsequent theory is constructed is the notion of fuzzy set a generalisation of a crisp setfrom classical set theorya fuzzy set generalises a crisp set by allowing its characteristic function ie itsmembership function assuming values in the interval [ ] rather than in the set in this way a given item belongs to the fuzzy set with a degree of truthranging from do not belong at all ie its membership function assumes value to completely belong ie the membership function assumes value in fl applications fuzzy sets make it possible to represent qualitative nonnumeric values ielinguistic variables such as high medium low for approximate reasoninginference or fuzzy control systems linguistic variables can be represented by fuzzy setsthrough a transformation step called fuzzification and it is achieved by using different types of membership functions representing the degree of truth to whicha given input sample belongs to a fuzzy set see membership functions sectionin supplementary material 0cciaramella bmc bioinformatics 21suppl page of fuzzy similarity matrixa measure of similarity or dissimilarity defines the resemblance between two samples orobjects similarity measure is a significant means for measuring uncertain informationfuzzy similarity measure is a measure that depicts the closeness among fuzzy sets and hasbeen used for dealing issues of pattern recognition and clustering analysisa binary fuzzy relation that is reflexive symmetric and transitive is known as a similarity relation fuzzy similarity relations are the generalisation of equivalence relationsin binary crisp relations to binary fuzzy relations in details a fuzzy similarity relationcan be considered to effectively group elements into crisp sets whose members are similar to each other to some specified grade and it is a generalization of classical equivalencerelation as described in fuzzy similarity section in supplementary material in orderto introduce the fuzzy similarity in the following we focus on the properties of theÅukasiewicz tnorm tl and the biresiduum in this way we obtain a fuzzy equivalencerelation that can be used for building dendrogram for more details in the derivation ofthese results see fuzzy similarity section in supplementary materialdendrogram and consensus matrixif a similarity relation is mintransitive ie t min then it implies the existence ofthe dendrogram see dendrogram and consensus matrix section in supplementarymaterial for details the mintransitive closure of a relation matrix r can be easilycomputed and the overall process is described in algorithm the last ingredient to accomplish an agglomerative clustering is a dissimilarity relationhere we considered the following result lemma letting r be a similarity relation with the elements rcid2x ycid3 [ ] and lettingd be a dissimilarity relation which is obtained from r bydx y rcid2x ycid3then d is ultrametric iif r is mintransitivein other words we have a onetoone correspondence between mintransitive similaritymatrices and dendrogram and between ultrametric dissimilarity matrices and dendrograms finally after the dendrograms have been obtained each time a consensus matrixie the representative information of all dendrograms is obtained by combining thetransitive closures ie maxmin operation the overall approach is described inalgorithm the overall workflow of the proposed approach is summarised in fig in particular for each omic data set xi a fuzzification step is adopted for obtaining thenew data set yi see supplementary material successively adopting a fuzzy similaritymeasure the similarity matrix si is computed and to guarantee the transitive closure ofthe matrix a new matrix ci is computed see algorithm finally all the ci matricesare collected for obtaining the consensus matrix a and the overall final dendrogram seealgorithm in fig we show an example that summarize a realistic agglomeration result we plotin figs 4abc three input hierarchies obtained on datasets that should be combinedin this case four sequences of patients are considered namely s1 s2 s3 and s4 respectively in fig 4d we show the final result by agglomerating dendrograms we observe that 0cciaramella bmc bioinformatics 21suppl page of fig workflow of the fuzzy based hierarchical clusteringthe output hierarchy contains clusters s1 s2 s3 and s1 s2 s3 s4 at different levels andeach of these clusters eg s1 s2 s3 are repeated at least in two out of the three inputdendrograms moreover it is worth stressing that the proposed approach based on theagglomeration of dendrograms can also be applied with commonly used metrics egeuclidean distance in fig we show a comparison between the dendrograms obtainedby using an euclidean metric and a similarity based approach ie Åukasiewicz tnormrespectively in this realistic example we simulate three omic data sets with rows ienumber of patients and columns ie features we split the single datasets in twopartitions or clusters such that the first rows are random samples from a standard normal distribution with variance and the other rows have the same distribution withfig combination algorithm abc input dendrograms d combined hierarchy 0cciaramella bmc bioinformatics 21suppl page of fig crisp hierarchical clustering vs fuzzy based hierarchical clustering a dendrogram of euclidean basedhierchical clustering b dendrogram of similarity based hierachical clusteringalgorithm mintransitive closure input relation si output transitive relation ci sti elaborate compute s if sielse ci sti si ª si ¦ sicid8 si replace si with si si and go to step i and the algorithm terminatesvariance obtaining a sort of an overlap we observe that both methods find two separated clusters but the similarity based approach in fig 5b permits to obtain a perfectseparation of the source partitionsresults and discussionin the following we describe the behaviour of the proposed methodology on multiomicsbenchmark datasetsalgorithm combination of dendrograms input ci ¤ i ¤ l l input similarity matrices dendrograms output similarity matrix dendrogram a aggregate the similarity matrices to a final similarity matrixa aggregate c1 c2 cla let a be the identity matrixb for each ci calculate e a a ª a ¦ cic if a is not changed a a and goto step else goto step 1b create the final dendrogram from aomics datasetswe consider multiomics cancer datasets available from the cancer genome atlastcga tcga is a large multiomic repository of data on thousands of cancerpatients all datasets contain three omics gene expression mirna expression and 0cciaramella bmc bioinformatics 21suppl page of table datasets description three omics are provided for each dataset respectively dna geneexpression mirna and methylationcases dnaoridatasetamlbiccoadgbmkirclihcluscskcmovsarclnrfmirnaorilnmethyorilnmultiomicsorilnrfrfrfthe number of features at each variable selection method is shown ori original variable dimension ln logarithm andnormalisation and rf random forest based on mean decrease gini indexdna methylation1 in table are summarised the main properties of the datasetsnamely acute myeloid leukemia aml breast invasive carcinoma bic colon adenocarcinoma coad glioblastoma multiforme gbm kidney renal clear cell carcinoma kirc liver hepatocellular carcinoma lihc lung squamous cell carcinomalusc skim cutaneous melanoma skcm ovarian serous cystadenocarcinoma ovsarcoma sarc the number of patients ranges from for aml to for bicmultiview clustering algorithmsfor validating the effectiveness of our model we compared it against several categories ofmultiview clustering algorithms2¢ kmeans and spectral clustering techniques ¢ lracluster it is a lowrank approximation based integrative probabilistic model¢ pins perturbation clustering for data integration and disease subtyping pinsis able to address subtype discovery as well as integration of multiple data types thealgorithm is built upon the resilience of patient connectivity and cluster ensembles toensure robustness against noise and biasto fast find the shared principal subspace across multiple data types¢ snf similarity network fusion snf allows for discovery of disease subtypesthrough integration of several types of highthroughput data on a genomic scale snfcreates a fused network of patients using a metric fusion technique and thenpartitions the data using spectral clustering snf appears to be the state of the art inthis area and has proven to be very powerful however the unstable nature ofkernelbased clustering makes the algorithm sensitive to small changes in molecularmeasurements or in its parameter settings¢ mcca multi canonical correlation analysis mcca which extends theapplication of canonical correlation analysis cca to more than two views is oneof the most widely used dimension reduction method for finding linear relationsbetween two or more multidimensional random variables1row data are available at httpacgtcstauacilmulti_omic_benchmarkdownloadhtml2httpsgithubcomshamirlabmultiomicscancerbenchmark 0cciaramella bmc bioinformatics 21suppl page of evaluation metricsin order to assess the performance of each method we adopt three evaluation metricsthat are the logrank test the enrichment of clinical labels in the clusters and the methods runtime the logrank test assumes that if clusters of patients have significantlydifferent survival they are different in a biologically meaningful way for the enrichmentof clinical labels in clusters six clinical labels are considered gender age at diagnosispathologic tumor pathologic metastases pathologic lymph nodes and pathologic stagethe four latter parameters are discrete pathological parameters measuring the progression of the tumor metastases and cancer in lymph nodes and the total progressionpathologic stage enrichment for discrete parameters was calculated using the Ï2 testfor independence and for numeric parameters using kruskalwallis test not all clinicalparameters were available for all cancer types so a total of clinical parameters wereavailable for testing to derive a pvalue for the logrank test the Ï2 test for independence the kruskalwallis test and the statistic for these three tests is assumed to have Ï2distribution preprocessingtcga datasets were preprocessed as follows patients and features with more than missing values were removed and missing values were imputed using knearest neighborimputation the sequence features were logtransformed the features with highestvariance from geneexpression and methylation omics were selected in the mirna omicfeatures with zero variance were filtered all features were then normalized to have zeromean and standard deviation for methylation we selected the features with maximal variance in each dataset and also adopted the standard pipeline proposed in whose procedure filters out the probes from the x and y chromosomes or probes that areknown to have common snps at the cpg sitea further unsupervised variable selection step has been performed by using the meandecrease gini based on random forest the mean decrease in gini is the average of a variable total decrease in node impurity weighted by the proportion of samplesfig mean performance of the algorithms on ten multiomics cancer datasets the xaxis measures thedifferential survival between clusters mean log10 of logranks test pvalue and the yaxis is the meannumber of clinical parameters enriched in the clusters 0cciaramella bmc bioinformatics 21suppl page of fig performance of the algorithms on ten multiomics cancer datasets for each plot the xaxis measuresthe differential survival between clusterslog10 of logranks test pvalue and the yaxis is the number ofclinical parameters enriched in the clusters red vertical lines indicate the threshold for significantly differentsurvival pvalue cid2 reaching that node in each individual decision tree in the forest this is effectively a measure of how important a variable is for estimating the value of the target variable acrossall of the trees that make up the forest a higher mean decrease in gini indicates highervariable importance therefore the most important variables to the model is the highestin the plot with the largest mean decrease in gini values conversely the least importantvariable is the lowest in the plot with the smallest mean decrease in gini values by following this strategy we cutoff all those variables whose importance is zero the numberof variable cutoff at each step is summarised in table experimental resultsin the experiments for all methods the number of searched clusters is selected in therange [ ] to determine the number of clusters for a method we used the elbowmethod to automatically pick out the optimal elbow rather than choose it manually weused as approximation the second derivative of a vector vv [i ] v [i ] 2v[ i] in particular we consider the index i that brings this expression to a maximum or minimum depending on whether v increases or decreases for all methods we adhered tothe guidelines for usage and parameter selection given by the developers in some casestable performance on ten multiomics number of clinical parameters enriched in the clustersfhclustcrisp hclustkmeansspectrallraclusterpinssnfmccaamlbiccoadgbmkirclihcluscskcmovsarcmeans 0cciaramella bmc bioinformatics 21suppl page of table performance on ten multiomics differential survival between clusters log10 of logrankstest pvaluefhclustcrisp hclustkmeansspectrallraclusterpinssnfmccaamlbiccoadgbmkirclihcluscskcmovsarcmeanswhere no information was provided by the authors we devised parameter selection methods we performed the same process pipeline used in for evaluating the performanceof our method all methods were run on a multicore intelr xeonr cpu e52620v3 240ghz with gb ram in the following the obtained experimental results aredescribedfigure shows the average performance for multiomics data and for each singleomicseparately across all cancer types and fig shows the performance on the different cancer datasets all algorithms show quite similar performance in either differential survivalor enriched clinical parameters with respect to survival our fhclust method achievedthe overall best prognostic value sum of log10 pvalues while pins and mcca ranked second and third respectivelyin table the differential survival between clusters mean log10 of logranks testpvalue are reported spectral achieved the highest total number of significant clinical parameters with parameters fhclust along with lracluster and kmeansplaced themselves second with parameters snf achieved the third position with parameterswith respect to survival table fhclust outperformed its competitors achieving parameters mcca pins and snf have achieved good results with and enriched parameters respectivelywe also counted the number of datasets for which a method solution obtains significantly different survival these results are reported in table all methods that weredeveloped for multiomics data had at least four cancer types with significantly differentsurvival in this case fhclust and pins had different cancer subtypes for which itsclustering had significantly different prognosis fhclust spectral clustering and mccahad enrichment in cancer typeson average fhclust pins and mcca had better prognostic value but found lessenriched clinical labels as compared to spectral clustering methodtable for each benchmarked algorithm the number of cancer subtypes for which its clusteringhad significantly different prognosis first row and had at least one enriched clinical label secondrow are shownsignificant different survivalsignificant clinical enrichmentfhclustkmeansspectrallraclusterpinssnfmcca 0cciaramella bmc bioinformatics 21suppl page of table number of clusters chosen by the benchmarked algorithms on ten multiomics cancerdatasetsfhclustcrisp hclustkmeansspectrallraclusterpinssnfmccaamlbiccoadgbmkirclihcluscskcmovsarcmeansthe number of clusters found for each dataset are presented in table ranging from to because of the good methods performance in the previous analysis partitioning thedata into a relatively high number of clusters could indicate that clustering cancer patientsinto more clusters improves prognostic value and clinical significanceconcerning with methods computational burden their run times are reported intable fhclust takes on average seconds per dataset while spectral and snf gotlower timing the worst method takes roughly minutes per dataset see fig finally fig shows the benchmarked methods performance for singleomic datamoreover for each dataset and method the single omic that gave the best results forsurvival and clinical enrichment are also shown these results suggest that fhclust provides better prognostic value and clinical significance on multiomics data compared tothe analysis of singleomic data used separately nevertheless the interested reader mayrefer to the supplementary material for details on additional results concerning singleomics we also stress that the proposed method differently from other methods suchas snf does not need any hyperparameter tuning moreover clustering is embeddedin the data integration and vice versa and the use of fuzzy concepts ie tnormsfrom one hand permits to obtain a generalisation of the clustering approaches whereason the other hand gives the possibility to apply an inference system eg mamdanifor a quantitative and qualitative measure eg high medium low in cancer riskassessmentsin this work we proposed a multiview clustering methodology for identifying patientsubgroups from different omics data in biological and biomedical fields combining theseomics data can significantly increase data mining capabilities one of the main aspects oftable runtime in seconds of the algorithms on ten multiomics cancer datasetsovfhclustcrisp hclustkmeansspectrallraclusterpinssnfmccacoadskcmbicamlgbmkirclihcluscsarcmeans 0cciaramella bmc bioinformatics 21suppl page of fig computational time comparisonsthis methodology is the use of a measure of dissimilarity between sets of observations byusing an appropriate metric and a consensus matrix that is a representative agglomerateinformation of all the dendrograms as emerged from the analysis of the scientific literature to the best of our knowledge our work concerns for the first time a model based onfuzzy logic used for the agglomeration of multiomic data the use of fuzzy logic allowsus to introduce more flexible data mining features also related to approximate reasoningseveral experiments and comparisons have been made on real data eg glioblastomaprostate cancer to assess the proposed methodology the results suggest that fhclustprovides better prognostic value and clinical significance compared to analysis of singleomic data alone fuzzy logic concepts and in particular membership functions permitsfig summarized performance of the algorithms across ten cancer datasets for each plot the xaxismeasures the total differential prognosis between clusters sum across all datasets of log10 of logranks testpvalue and the yaxis is the total number of clinical parameters enriched in the clusters across all cancertypes ac results for singleomic datasets d results when each method uses the single omic that achievesthe highest significance in survival e same with respect to enrichment of clinical labels 0cciaramella bmc bioinformatics 21suppl page of to develop a fuzzy inference model ie mamdani fuzzy cognitive maps for easilyobtaining a model for a quantitative and qualitative risk assessment of the cancer themodel based on approximate reasoning can be particularly useful for embedded devicesin future work it could be possible to improve results for multiomics analysis ina number of ways for instance more accurate feature selection algorithms couldbe adopted for improving the overall performance on one hand the integration oflabelled data could improve the feature selection step on the other hand some specific feature extraction strategies could be adopted indeed approaches based on thesignal analysis of gene expression data eg nonlinear principal component analysis compressive sensing could possibly further improve the performance [ ]in future it is possible to foresee a different weight for each omic data in order toobtain a more robust similarity and parametric similarity measures can be adoptedeg uninorm for generalizing the concept of and and or connections betweenclusterssupplementary informationsupplementary information accompanies this paper at httpsdoi101186s12859020035676additional file supplementary materialabbreviationsfhclust fuzzyhierarchical clustering dna deoxyribonucleic acid rna ribonucleic acid hierarchical clustering hccrisp hc crisp hierarchical clustering fl fuzzy logic tcga the cancer genome atlas aml acute myeloid leukemia bicbreast invasive carcinoma coad colon adenocarcinoma gbm glioblastoma multiforme kirc kidney renal clear cellcarcinoma lihc liver hepatocellular carcinoma lusc lung squamous cell carcinoma skcm skim cutaneousmelanoma ov ovarian serous cystadenocarcinoma sarc sarcoma pins perturbation clustering for data integrationand disease subtyping lracluster low rank approximation based multiomics data clustering snf similarity networkfusion mcca multi canonical correlation analysisabout this supplementthis has been published as part of bmc bioinformatics volume supplement proceedings from the 13thbioinformatics and computational biology international conference bbcc2018 the full contents of the supplement areavailable online at httpsbmcbioinformaticsbiomedcentralcomssupplementsvolume21supplement10authors contributionsac originally designed the methodology ac and dn worked on the developing of the method and the design of theexperiments ac dn and as contributed for interpreting and for analysing the results all authors contributed forwriting the manuscript read and approved the final manuscriptfundingpublication costs are funded by a grant from the dipartimento di scienze e tecnologie università degli studi di napoliparthenope tecniche di machine learning e soft computing per lelaborazione di dati multivariati softmulan piciaramellaavailability of data and materialscode and data of the proposed approach are available on multiomicscancerbenchmark github repositoryethics approval and consent to participateno ethics approval was required for the studyconsent for publicationnot applicablecompeting intereststhe authors declare that they have no competing interestsauthor details1dipartimento di scienze e tecnologie università degli studi di napoli parthenope centro direzionale c4 island naples italy 2hitachi rail sts via argine naples italypublished august 0cciaramella bmc bioinformatics 21suppl page of referencescamastra f di taranto md staiano a statistical and computational methods for genetic diseases an overviewcomput math meth med 20152015 id serra a fratello m fortino v raiconi g tagliaferri r greco d mvda a multiview genomic data integrationmethodology bmc bioinformatics rappoport n shamir r multiomic and multiview clustering algorithms review and cancer benchmark nucleicacids res reddy ck aggarwal cc data clustering boca raton chapman and hallcrc camastra f ciaramella a son lh riccio a staiano a fuzzy similaritybased hierarchical clustering for atmosphericpollutants prediction lncs ciaramella a staiano a on the role of clustering and visualization techniques in gene microarray data algorithmsbora dj gupta ak int j emerg trends technol comput sci napolitano f pinelli m raiconi g tagliaferri r ciaramella a staiano a miele g clustering and visualizationapproaches for human cell cycle gene expression data analysis int j approx reason ciaramella a cocozza sand clustering of genomic data neural netw iorio f miele g napolitano f pinelli m raiconi g tagliafer | Colon_Cancer |
cancer is a complex and heterogeneous disease with many possible genetic and environmentalcauses the same treatment for patients of the same cancer type often results in different outcomes in terms ofefficacy and side effects of the treatment thus the molecular characterization of individual cancer patients isincreasingly important to find an effective treatment recently a few methods have been developed to constructcancer samplespecific gene networks based on the difference in the mrna expression levels between the cancersample and reference samplesmethods we constructed a patientspecific network with multiomics data based on the difference between areference network and a perturbed reference network by the patient a network specific to a group of patients wasobtained using the average change in correlation coefficients and node degree of patientspecific networks of thegroupresultsnetworks with multiomics data the main differences of our method from previous ones are as follows networksare constructed with multiomics mrna expression copy number variation dna methylation and micrornaexpression data rather than with mrna expression data alone networks are constructed with bothnormal samples and cancer samples of the specified type to extract cancerspecific gene correlations and bothpatient individualspecific networks and patient groupspecific networks can be constructed the results of evaluatingour method with several types of cancer show that it constructs more informative and accurate gene networks thanprevious methodss the results of evaluating our method with extensive data of seven cancer types show that thedifference of gene correlations between the reference samples and a patient sample is a more predictive feature thanmrna expression levels and that gene networks constructed with multiomics data show a better performance thanthose with single omics data in predicting cancer for most cancer types our approach will be useful for finding genesand gene pairs to tailor treatments to individual characteristicskeywordsindividualspecific gene network groupspecific gene network cancer multiomics datacorrespondence khaninhaackr1department of computer engineering inha university incheon southkoreafull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriatecredit to the original authors and the source provide a link to the creative commons licence and indicate if changes weremade the images or other third party material in this are included in the s creative commons licence unlessindicated otherwise in a credit line to the material if material is not included in the s creative commons licence and yourintended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtain permission directlyfrom the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40 the creativecommons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to the data madeavailable in this unless otherwise stated in a credit line to the data 0clee bmc medical genomics 13suppl page of for the past years we have witnessed the rapid development of targeted cancer therapy targeted therapies forcancer work by targeting specific genes proteins or tissues that contribute to cancer growth and survival manytargeted therapies are effective only for patients with specific genetic alterations known as driver mutations thathelp cancer cells form and grow [ ] thus identifying genetic mutations specific to individual patients is ofutmost importance to determine targeted therapies thatcan effectively cure cancer patients while minimizing sideeffects motivated by a massive amount of data generated byhighthroughput technologies several cancer studies usedgene networks to explore gene expression characteristics [] however constructing a patientspecific genenetwork with a single sample obtained from a patient isdifficult because a gene network requires many samples tocompute genegene relationsrecently a few methods have been proposed to construct cancer samplespecific gene networks based onthe difference in the mrna expression levels betweenthe cancer sample and reference samples for exampleliu proposed a method to construct a samplespecific network by computing the difference between areference network from multiple reference samples anda network perturbed by a new sample however a slightchange to the reference samples can result in a significantly different samplespecific network for the samesample due to the small number of reference samples furthermore their samplespecific networks cannot reflectposttranslational modification and epigenetics becausethe networks are built using mrna expression data onlythis paper presents a new method for constructingcancer patientspecific and groupspecific gene networkswith multiomics data using a samplespecific networkand network propagation method network propagation strategies are widely used in recent cancerrelatedresearch li presented a synergy prediction algorithm using network propagation and predicted the drugsynergy in various cancers zhang introduceda propagation algorithm which learns the mutated subnetworks underlying tumor subtypes using a supervisedapproach and classified tumors to known subtypes onbreast and glioblastoma tumors peng identifiedbladder cancerrelated genes by propagating informationfrom seed genes to candidate genes the primary focusof our method is to construct a gene correlation network specific to cancer with multiomics data thus it isdifferent from a typical gene coexpression network thatrepresents coexpression relations between genes frommrna expression data our gene network is not a generegulatory network because our network does not showregulatory relations between genesthe main differences of our method from previous onesare as follows networks are constructed with multiomics mrna expression copy number variation dnamethylation and microrna expression data rather thanwith mrna expression data alone networks are constructed with both normal samples andcancer samples of the specified type to extract cancerspecific gene correlations and both patient individualspecific networks and patient groupspecific networks canbe constructed as shown later in this paper the resultsof evaluating our method with several types of cancershow that it constructs more informative and accuratetargetspecific networks than previous methodsmethodsat the top level our method consists of the followingsteps data processing constructing individualspecific gene networks and constructing a groupspecific gene networks a highlevel description of themethod is given in fig data collection and preprocessingfrom the broad institute tcga gdac firehose we obtained multiomics data of cancer samples of seventypes breast invasive carcinoma brca colon adenocarcinoma coad head and neck squamous cell carcinomahnsc pankidney cohort kipan liver hepatocellular carcinoma lihc lung adenocarcinoma luad andlung squamous cell carcinoma luscthe multiomics data used in this study includemrna expression mrnaseq copy number variationcnv dna methylation and mature mirna expression mirseq data the mrnaseq data were processedusing quartile normalized rsem and then log2transformed the segmented cnv data were convertedto genelevel data using the ensembl api and thecntools package of bioconductor the methylationdata were filtered to select the probe with the mean signal values for each gene the mirseq data were processedby rpm and log2transformed mrnas and mirnas thatwere not expressed in more than of the total sampleswere excluded in further analysis missing expression values of mrnas and mirnas were replaced by the smallestpositive normalized floatingpoint number realmin ofmatlab the number of samples and genes used in thisstudy are available in additional file individualspecific gene networkin each group of tumor samples and normal samples wefirst computed genegene relations by the pearson correlation coefficient pcc selected highly correlated genepairs ie those with pcc and constructed twosample networks one for each group from the tumorsample network we removed edges common to both 0clee bmc medical genomics 13suppl page of fig overview of constructing an individualspecific network and a groupspecific network with multiomics datatumor and normal sample networks and obtained a template reference network for cancer fig 2a the templatereference network consists of highlycorrelated gene pairsthat are specific to cancerwith n reference samples which may be different fromtumor samples used in the template network we computed pcc for every pair of genes in the template reference network and constructed a reference network forthe reference samples for a patient of interest we constructed a network which is a perturbed network byadding a single sample of the patient to the n reference samples a patientspecific network was obtainedby subtracting the reference network from the perturbednetwork 01pcc pccn1 pccnwe computed the difference in the absolute value ofpcc between the perturbed reference network and reference network by eq we also carried out a ztest ofpccn1pccn by eq for a large n we can approximatethe mean μ and standard deviation Ï of pccn1 pccn as and pcc2nn respectively pccchange pccn1 pccnz score pccchange μpccchangeÏ pccchange pccchangepcc2nnthe edges of the patientspecific network were classified into four types correlationgained edges fene pairs whose pccs are increased from the referencenetwork to the patientspecific network correlationlost edges for gene pairs whose pccs are decreased fromthe reference network to the patientspecific network correlationreversed edges for gene pairs whose signs ofpccs are changed from positive to negative or negativeto positive and correlationinvariant edges for genepairs with little change in pccs between the reference andpatientspecific networks ie those with zscore fig 2bthe edges were classified in the following way we firstselected gene pairs with zscore as correlationinvariant type and then selected gene pairs which havedifferent signs of pccs between the reference networkand the patientspecific network as correlationreversedtype the remaining gene pairs were classified into eithercorrelationgained or correlationlost type depending onwhether their pccs are increased correlationgained ordecreased correlationlost from the reference network tothe patientspecific network thus zscore ¥ in bothcorrelationgained and correlationlist gene pairsgroupspecific gene networka groupspecific gene network is useful when analyzinga large number of patientspecific gene networks afterconstructing patientspecific gene networks we obtained 0clee bmc medical genomics 13suppl page of fig process of constructing a patientspecific gene network a template of the cancer reference network obtained by removing edges common toboth networks b patientspecific gene network and four types of edges in the network edges with a zscore represent correlationinvariantgene pairs and edges with zscore ¥ and different signs of pccn and pccn1 represent correlationreversed gene pairs edges with zscore ¥ and 01pcc are correlatedgained gene pairs and those with zscore ¥ and 01pcc are correlationlost gene pairsa gene network specific to a group of patients based onthe average 01pcc and node degree of the patientspecificnetworks fig if the dominant type for a particular edge is correlationgained positive 01pcc in thepatientspecific networks the edge is represented in redin the groupspecific network in contrast if the dominant type for a particular edge is correlationlost negative 01pcc in the patientspecific networks the edgeis represented in blue in the groupspecific network inthe groupspecific network only the dominant type isshown for each edge if nondominant types are shownin addition to the dominant type for each edge the network becomes cluttered and unreadable the node sizeof a groupspecific gene network is proportional to theaverage degree of the nodeintegration of multiomics datato integrate multiomics data we first computed intergene correlations by pcc with four different types ofsingle omics data mrna expression cnv dna methylation and mirna expression separately and selectedsignificant intergene correlations only in mrna expression cnv and dna methylation data we select thetop pcc with pvalue in mirna expression data we selected the top pcc with pvalue due to a smaller number of mirnas inthe data the intergene correlations selected in eachsingle omics data are represented in four correlationmatrices mexpr mcnv mmethyl and mmirna andnormalizedusing the proteinprotein interactions ppis of thestring database we constructed separate weightednetworks from each omics data by eq in eq wexprwcnv and wmethyl denote the weighted networks andppiexpr ppicnv and ppimethyl are subnetworks of a ppinetwork consisting of genes present in each omics datasince the ppi network does not contain information onmirna a weighted network for mirna was not constructedcid3wexpr cid2 ppiexprwcnv mcnv à ppicnv wmethyl cid2 ppimethylwe then integrated the multiomics data by linear xmethylregression using eq in eq yi xcnvand xmirnadenote gene is expression level cnv levelmethylation level and mirna regulator expression levelrespectively βcnvdenote the regression coefficients of gene is expression level on cnv and methylation respectively βmirnais the regression coefficientof gene is expression level on its mirna regulator jsexpression leveland βmethyl mexpr mmethylcid3 à cid2cid3 à cid2cid3iiijiiijyi βcnvii βmethylxcnvixmethyli ncid4j1βmirnaijxmirnaij 01 0clee bmc medical genomics 13suppl page of fig process of constructing a groupspecific gene network from multiple patientspecific gene networks in the groupspecific gene network thenode size and node color are proportional to the average degree and average 01pcc of the node respectively if the dominant type for a particularedge is correlationgained positive 01pcc in the patientspecific networks the edge is represented in red in the groupspecific network if thedominant type for a particular edge is correlationlost negative 01pcc in the patientspecific networks the edge is represented in blue in thegroupspecific networkfrom the regression coefficients and the weighted networks a weight matrix w was derived and normalizedinto w eqs and the weight matrix w is symmetricso wij wji w11 w22 w33 and w44 represent wexprwcnv wmethyl and mmirna respectively the submatrices w21 and w31 contain regression coefficients βcnvand βmethylfor every gene i respectively w41 representsiβmirnaij the submatrices w32 w42 and w43 are emptyi¡¢¢£w w11 w12 w13 w14w21 w22 w23 w24w31 w32 w33 w34w41 w42 w43 w44¤¥¥¦eq and updated iteratively by eq in this iterative process the influence of the seed is propagated tothe neighbors until a mean squared error of st and st¤ à §ªªªª¨ªªªª©if v is a nonseed nv ¥ αif v is a nonseed nv αif v is a seednvnvenvα à nvnvdv st λ à st à w λ à d wheres1 d where nv is the number of neighbors of node v and nv isthe number of seeds in the neighbors the parameter αwhich is a threshold for nv was set to and λ was set to genes with the top st were used in findingcancerrelated genes and in classifying tumor samples andnormal samplesw i j w i jcid11cid12cid12cid13mcid4k1w i k à mcid4k1w k jin network propagation seed genes have greater impactthan nonseed genes on their neighbors thus only thegenes with a high average 01pcc were selected as seedgenes for a groupspecific network and their mirnasregulators extracted from mirtarbase were used asseed mirnas we calculated the cancerrelevance st ofeach gene to reflect the effect of the seed genes and mirnas on neighbors the initial score d was calculated byresultspatientspecific and groupspecific gene networksin this study we constructed patientspecific genenetworks for seven cancer types additional file foreach cancer type we also constructed groupspecific genenetworks as an example fig shows a groupspecificgene network derived from lung squamous cell carcinoma lusc patients 0clee bmc medical genomics 13suppl page of there are three distinct subnetworks in the networkfor the lusc group the subnetwork enclosed in box aof fig contains many hub genes large green nodesthe subnetwork in box b consists of correlationgainededges dark red edges whereas the subnetwork in box ccontains many correlationlost edges dark blue edgescomparison of multiomics data and singleomics datawe performed leaveoneout cross validation loocvto evaluate cancerrelevance score st of a gene and thecontribution of multiomics data to finding cancerrelatedgenes for comparison the cancerrelevance scores werecomputed with multiomics data and single omics dataseparately each seed gene was regarded as a nonseed anda new cancerrelevance score was calculated for the geneseed genes and nonseed genes were considered as positive and negative respectively seed genes included in thetop n genes were considered as true positives and seedgenes not included in the top n genes were consideredas false negatives similarly nonseed genes included infig groupspecific gene network for lung squamous cell carcinoma lusc patients a subnetwork of multiple hub genes large greennodes b subnetwork of correlationgained edges dark red edges c subnetwork with many correlationlost edges dark blue edges 0clee bmc medical genomics 13suppl page of the top n genes and nonseed genes not included in thetop n genes were considered as false positives and truenegatives respectivelywe carried out loocv with different ratios of seedgenes to nonseed genes figure shows the receiver operating characteristic roc curve and the area under thecurve auc of loocv of the cancer relevance of geneson data of breast cancer samples with various seedratios ranging from to enlarged plots of fig are available in additional file for comparative purposes we also computed the cancer relevance of geneswith single omics data as shown in fig multiomicsdata consistently exhibited better performance than singleomics data with any seed ratio between to forlater analysis the seed ratio was set to by default theaverage 01pcc and class label of each gene are available inadditional file indeed the superiority of multiomics data over singleomics data in determining the cancer relevance scoreof genes was observed in all seven types of canceradditional file in seven types of cancer the cancerrelevance score of genes computed with multiomics dataexhibited a good performance auc ¼ thecancer relevance score of genes computed with mrnaexpression data showed the second best performanceauc ¼ in particular the cancer relevancescore computed with mrna expression data showed avery similar performance to that with multiomics inbreast cancer brca the performance of the cancer relevance score computed with cnv auc ¼and dna methylation data auc ¼ alonewas lower than that with mrna expression data auc ¼fig roc curves of the leaveoneout cross validation of the cancer relevance score st of genes with different ratios of seed genes breastcancer samples were used in the leaveoneout cross validation the performance of multiomics data is always better than that of single omics data 0clee bmc medical genomics 13suppl page of evaluation of gene correlations and networksmany networkbased approaches to cancer research havefocused on finding genes that show differential expressions between tumor samples and normal samples genegene correlations ie intergene correlations may bemore helpful than individual genes because intergenecorrelations depend on the expression of neighbor genesin a gene regulatory network to compare the effect ofusing individual genes to that of intergene correlationsie 01pcc we constructed a support vector machinesvm model for classifying cancer samples and normalsamples the svm model was implemented using csvcand rbf kernel and the parameter values of the modelwere determined by the grid search algorithm mrnaexpression levels and 01pccs were used as features ofthe svm models for rigorous validation the test dataused in testing the models were not used in training themadditional file as shown in fig 6a 01pcc showed a better performance than mrna expression levels for six cancertypes except lusc the classification model with 01pccshowed mcc above in six cancer types except hnscwe also examined the effect of different networks on individualspecific networks in the workby liu ppi data with high confidence scoresin the string database were used to construct a network however the ppi data of stringdoes not reflect cancer typespecific characteristicsfigure 6b shows the performance of the classificationmodel with two different networks network from ppi data of string the approachby liu and cancer network ourapproach 01pcc was used as a feature of the classification model except for coad the performance ofthe classification model with the cancer network was better than the model with the string reference network in particular the classification model showed a significant difference for breastcancer brca mcc of vs mcc of detailed results of the classification model are available inadditional file discussionin the analysis of finding cancerrelated genes and genepairs we focused on a subnetwork of genes with a 01pcctable shows the top genes with a high average 01pcc in each groupspecific network of seven cancertypes in breast invasive carcinoma brca fam171a1showed the highest average 01pcc in the groupspecificnetwork fam171a1 is known as a potential biomarkerin triplenegative breast cancer foxc1 is involvedin tumor development and metastasis and associated withpoor prognosis in basallike breast cancer il33 isoverexpressed in various cancers and the serum concentration of il33 is a valuable indicator of poor prognosis inbreast cancer mamdc2 is significantly correlatedwith diseasefree survival of breast cancer patients mterfd1 is closely related to breast cancer recurrencefig results of evaluating features and networks by a validation set a comparison of mrna expressions of genes and 01pcc of genepairs b comparison of the cancer network with the network from ppi data 0clee bmc medical genomics 13suppl page of table top genes with a high average 01pcc in a groupspecific network for seven cancer typesbrcakipanlihccoadhnscfam171a1foxc1il33mamdc2mterfd1hoxa7cttnbp2znf204pjam3frem1gfra2scnn1bddx27rnps1ube2imdficctnnbl1cdk5rap1esf1trmt6cyp2j2barx2znf135ppfia1parlfaddcst6cobloraov1xpo7tfcp2l1kcnq1arl15kctd1oaz2tmem45bhpcal1tmem91sema5begln3slc19a3ecm1cyp2b6fbp1gpaa1f9pahagxt2hsd17b6rnase4luadcldn18adamts8pecam1sftpa1gimap6akr1c1mmeatad2cyp3a5chaf1bluscnsun2cct5fbxo45gpr116slc39a8fxr1inmtveph1crtamwdr53 and hoxa7 plays a critical role in regulating theproliferation of erpositive cancer cells in colon adenocarcinoma coad gfra2 showed thehighest average 01pcc in the groupspecific network itis known to be crucial for enteric neuron survival scnn1b and ddx27 are significantly related to colorectal cancer [ ] no direct relation of rnps1 withcolorectal cancer is known but rnps1 is essential tononsensemediated mrna decay that plays complexfunctions in cancer knockdown of sumo conjugating enzyme ube2i also known ubc9 or e2 inhibitsmaintenance and selfrenewal of colorectal cancer stemcell while overexpression of ube2i increases colorectalcancer cell stemness among the top genes with a high average 01pccin lung adenocarcinoma luad several genes such ascldn18 adamts8 pecam1 and sftpa1 have beenknown to be associated with luad in previous studies[] no direct relation of nsun2 and slc39a8 withlung squamous cell carcinoma lusc has been known sofar however recent studies [ ] reported that nsun2is correlated with survival in other types of squamous cellcarcinomas gao also showed that the epigeneticsilencing of slc39a8 expression by dna methylation isinvolved in the acquisition of resistance against cadmiumin lung cells and the relation between cadmium andlung cancer has received much attention many othergenes in table found in the groupspecific networksfor head and neck squamous cell carcinoma hnscpankidney cohort kipan and liver hepatocellular carcinoma lihc are also directly or indirectly related tocancerin addition to individual genes we identified genepairs of the same type ie either correlationgained orcorrelationlost in most patientspecific networks of thesame type table shows the most frequent gene pairs in breast cancer samples the most frequent gene pairsin other types of cancer are listed in additional file it isinteresting to note that all the gene pairs shown in table include at least one gene in the gene pair mamdc2hoxa7 and that they are correlationgained edges in thegroupspecific network for breast cancer figure showsa subnetwork containing mamdc2 and hoxa7 in thegroupspecific network of breast cancer the subnetworkwas obtained by selecting the edges for which the proportion of the same edge type ie correlationgained or lostis above in the total individualspecific networks ofbreast cancer patients it is interesting to note that all thegene pairs in table are included in the subnetworkto date the actual role of the mamdc2 gene in cancer is not clear but meng reported mamdc2as one of three genes mamdc2 tshz2 and cldn11that are significantly correlated with diseasefree survival of breast cancer patients mamdc2 is known as atarget of mir196a in head and neck squamous cell carcinoma as a member of the family of homeoboxgenes hoxa7 is associated with cell proliferation nerveinvasion distant metastasis and degree of tumor differentiation in several cancers [ ] hoxa7 is regulatedtable the most frequent gene pairs in breast cancersamples all the gene pairs are of a correlationgained type thegenes of table are shown in bold the proportion represents theratio of the gene pairs of the same type ie correlationgained orlost to the total number of patientspecific networksgene pairgene pairsproportion of the gene pairsin total cancer samplesmamdc2hoxa7mamdc2ccl14mamdc2znf204pmamdc2klmamdc2svep1mamdc2coro2bhoxa7meox2hoxa7hoxa9mamdc2sobpmamdc2hoxa9 0clee bmc medical genomics 13suppl page of fig a subnetwork of the groupspecific network of brca which contains mamdc2 and hoxa7 genes in the most frequent gene pairs shown intable are enclosed by a circleby several mirnas including mir196 [] thusboth mamdc2 and hoxa7 are related with mir196but a clear relation among them is to be uncoveredso far most approaches to constructing individualspecific gene networks have been constructed based onthe differential expressions between a small number ofreference samples and a sample of interest howeversuch networks cannot reflect posttranslational modification and epigenetics and are not reliable because a slightchange to the reference samples can result in a significantly different samplespecific network for the samesamplein this paper we presented a new approach to constructing cancer patientspecific and groupspecific networks with multiomics data the main differences ofour method from previous ones are as follows gene networks are constructed with multiomics mrnaexpression copy number variation dna methylationand microrna expression data rather than with mrnaexpression data alone networks can beconstructed with cancer samples of the specified type and both patient individualspecific networks and patientgroupspecific networks can be constructed the resultsof testing our method with several cancer types showedthat it constructs more informative and accurate genenetworks than existing methodsevaluation of our method with extensive data of sevencancer types showed that changes in gene correlations 01pcc between the reference samples and a patient sample is a more predictive feature than mrna expressionlevels and that gene networks constructed with multiomics data are more powerful than those with singleomics data in predicting cancer for most cancer typesmore work is required to validate the genes and genepairs identified in the cancer patientspecific and groupspecific networks however the method for constructingnetworks specific to individual patients or patient groupswith multiomics data should be useful aids in determining effective treatments to individual characteristicssupplementary informationsupplementary information accompanies this paper athttpsdoi101186s12920020007367additional file number of samples and genes in types of canceradditional file roc curve and auc of the cancerrelevance score ofbrca by various seed ratiosadditional file average 01pcc and class label of each gene in typesof canceradditional file roc curve of the cancerrelevance score of each cancertype with the seed ratio of additional file performance of classification of tumor samples andnormal samplesadditional file top gene pairs for each cancer type 0clee bmc medical genomics 13suppl page of abbreviationsauc area under the curve brca breast invasive carcinoma cnv copynumber variation coad colon adenocarcinoma gdac genome dataanalysis center hnsc head and neck squamous cell carcinoma kipan pankidney cohort lihc liver hepatocellular carcinoma loocv leaveoneoutcross validation luad lung adenocarcinoma lusc lung squamous cellcarcinoma mcc matthews correlation coefficient pcc pearson correlationcoefficient ppi proteinprotein interactions roc receiver operatingcharacteristic svm support vector machine tcga the cancer genome atlasacknowledgementsthe authors thank the anonymous reviewers for the constructive commentsthat contributed to improving the final version of the paperabout this supplementthis has been published as part of bmc medical genomics volume supplement selected s from the 15th international symposiumon bioinformatics research and applications isbra19 medical genomicsthe full contents of the supplement are available online at httpsbmcmedgenomicsbiomedcentralcomssupplementsvolume13supplement6authors contributionswl designed and performed all about this study and prepared the initialmanuscript dh helped integrating multiomics data of breast cancer khsupervised the work and wrote the manuscript all authors read and approvedthe final manuscriptauthors informationwook lee is currently working toward his phd degree at the department ofcomputer engineering inha university korea deshuang huang is a directorof the institute of machines learning and systems biology tongji universitychina kyungsook han is a professor at the department of computerengineering inha university koreafundingthis work was supported by the national research foundation of korea nrfgrant funded by the ministry of science and ict nrf2017r1e1a1a03069921and inha university research grant publication of this was funded bythe nrf grant nrf2017r1e1a1a03069921 the funders had no role in thedesign of the study data collection and analysis or writing the manuscriptavailability of data and materialsadditional files are available at httpbclabinhaackrcancernetworkethics approval and consent to participatenot applicableconsent for publicationnot applicablecompeting intereststhe authors declare that they have no competing interestsauthor details1department of computer engineering inha university incheon southkorea 2institute of machine learning and systems biology school ofelectronics and information engineering tongji university shanghaichinareceived may accepted june published august references widakowich c de castro g de azambuja e dinh p awada a reviewside effects of approved molecular targeted therapies in solid cancersoncologist liu s kurzrock r toxicity of targeted therapy implications for responseand impact of genetic polymorphisms cancer treat rev verma m personalized medicine and cancer j personalized medbarabasi al gulbahce n loscalzo j network medicine a networkbasedapproach to hum | Colon_Cancer |
" camp responsive element binding protein creb5 is a transcriptional activator in eukaryotic cells that canregulate gene expression previously we found that creb5 was involved in the occurrence and development of colorectalcancer crc using bioinformatics analysis however the biological roles and underlying regulatory mechanism of creb5 incrc remain unclearmethods realtime pcr western blotting and immunohistochemistry were used to examine creb5 expression in vitroexperiments including migration assay woundhealing assay chicken chorioallantoic membrane assay and human umbilicalvein endothelial cells tube formation assay were used to investigate the effects of creb5 on crc cell migration and tumorangiogenesis ability additionally an orthotopic implantation assay was performed in nude mice to confirm the effects ofcreb5 in vivo furthermore gene set enrichment analysis was performed to explore the potential mechanism of creb5 incrcresults we found that creb5 expression was highly upregulated in crc creb5 overexpression was positively correlatedwith advanced who stages and tnm stages and shorter survival in crc patients moreover creb5 overexpressionpromoted while creb5 silencing reduced the invasiveness and metastatic capacity of crc cells both in vitro and in vivofurthermore creb5 directly interacted with the met promoter and activated the hepatocyte growth factormet signallingpathway importantly inhibition of met reduced the invasion and metastasis of creb5overexpressing crc cells suggestingthat creb5 promotes metastasis mainly through activation of met signalling our study demonstrates a crucial role for creb5 in crc metastasis by directly upregulating met expressioncreb5 may be both a potential prognostic marker and a therapeutic target to effectively overcome metastasis in crckeywords colorectal cancer creb5 invasion metastasis met correspondence llifimmucom liaowt2002gmailcom shuyang wang junfeng qiu and lei liu contributed equally to this work1department of pathology nanfang hospital southern medical universityguangzhou guangdong chinafull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cwang of experimental clinical cancer research page of colorectal cancer crc is one of the most commoncancers ranking third in morbidity and tumorrelatedmortality among both men and women worldwidemoreover approximately of crc patients have metastases at the time of diagnosis metastasis is theleading cause of death among crc patients although systemic treatment of metastatic crc hasimproved the 5year survival rate is only andbecause ofthis poor prognosis understanding theunderlying mechanism of the metastatic process in crcis criticalfor both early detection of metastases andmore effective treatment the gene camp responsive element binding protein creb5 which is located on chromosome 7p151encodes a transcription activator in eukaryotic cells creb5 belongs to the atfcreb family the membersof which are characterized by a high affinity for campresponse elements cres the targets of the atfcreb family include transcriptional regulators including chromatinmodifying enzymes coactivators and corepressors genes involved in mitochondrial homeostasisand protein import and genes associated with proliferation and cell cycle entry metabolism proteases transporters and chaperones as an atfcreb familymember the creb5 protein contains several importantfunctional domainsincluding nterminal zinc fingerand cterminal bzip domains the latter of which includes a dna binding region and leucine zipper creb5 is a transcription factor that specifically binds tocre as a homodimer or a heterodimer with cjun orcrebp1 and functions as a credependenttransactivator creb5 is physiologically required for embryonic development in mice recent studies revealedthe roles of creb5 in the development and progressionof cancers examination of tcga pan cancer datasetsrevealed frequent creb5 amplification and overexpression in kidney cancers sarcomas lymphomas and lungadenocarcinomas as well as glioblastomas and gliomas experimentalinvestigations showed that creb5was upregulated in ovarian cancers hepatocellularcarcinoma and prostate cancer high creb5expression correlated with a poor prognosis in epithelialovarian cancer and hepatocellular carcinoma creb5 overexpression increased the proliferation of hepatocellular carcinoma moreover overexpression oramplification of creb5 promoted proliferation and mediated resistance to ar inhibition in metastatic castrationresistant prostate cancers in silico analysis showedthat the creb5 regulatory network was involved in crcmetastasis in addition qrtpcr assay revealed thatcreb5 mrna was upregulated in crc tissues and cells in vitro assays revealed that overexpression of creb5resulted in enhanced proliferation and migration andapoptosis inhibition in crc cells these findings suggest that creb5 may play an essential role in the progression of crc howeverthe specific function andmolecular mechanism of creb5 in crc metastasis remain largely unclearactivation of the hgfmet signalling pathway hasbeen reported to lead to the occurrence and metastasisof a variety of tumorsincluding crc breast cancerovarian cancer lung cancer and liver cancer as atyrosine kinase receptor met can be activated bydimerization multimerization and phosphorylation afterbinding to its ligand hepatocyte growth factor hgf activation of hgfmet can initiate downstreamsignalling pathways that drive malignant progression inmany types of tumors met is considered an essentialfactor for early invasion and metastasis of crc and canbe regarded as an important prognostic indicator in the present study we found that creb5 promotes crc invasion and metastasis by increasing metexpression to activate hgfmet signalling these results uncover a new molecular mechanism for cancermetastasis and suggest that creb5 may be a promisingtarget for crc treatmentmaterials and methodspatients and specimensa total of pathological specimens were obtainedfrom colon cancer patients between and atthe department of pathology nanfang hospital southern medical university the medical records of these patients provided information on sex age and thefollowing essential factors tumor pathological characteristics pathologic stage t stage dukes stage lymph nodemetastasis and distant metastasis ten pairs of fresh biopsies collected from crc patients and matched noncancerous mucosaltissue were obtained from theoperating room of nanfang hospital the fresh biopsieswere stored in liquid nitrogen before usein addition a tissue microarray no co802 containing colon cancer tissue specimens and adjacentnoncancerous tissue specimens was purchased fromailina biotechnology company approval for the use ofclinical materials for research purposes was obtainedfrom the southern medical university institutionalboard guangzhou china all samples were collectedand analysed with the prior written informed consent ofthe patientscell culturesthe human crc celllines sw480 ht29 hct15hct116 sw620 ls174t sw837 lovo dld1 andrko were purchased from the american type culturecollection sw620 ht29 and lovo cells were culturedin dmem medium gibco supplemented with 0cwang of experimental clinical cancer research page of foetal bovine serum fbs gibco sw480 hct116hct15 ls174t sw837 and dld1 cells were culturedin rpmi medium gibco with fbs gibcoplasmidscreb5 constructs were generated by cloning pcramplifiedfulllength human creb5 cdna into psinef2puro thefollowing primers were used for cloning including enzymesforward primer ² cgcgaattcatgatttatgaggaatccaa3² ecor i reverse primer ²ccggctagcttaaagaatcggattcaggt3² nhe i for deletion ofcreb5 short hairpin rna shrna sequences creb5²aacaagtcatccagcataa3² creb5shrna1shrna2 ²ggaatatctcgatgcataa3² were separately cloned into a gv248 vector psinef2puro and gv248were purchased from addgene incthe intensity of staining was graded according to thefollowing criteria no staining weak staining lightyellow moderate staining yellow brown and strong staining brown the staining index was calculated as the staining intensity score à the proportion ofpositive tumor cells using this method of assessmentwe evaluated the expression of creb5 in benign breastepithelium and malignant lesions by determining thestaining index with scores of and cutoff values for creb5 were selected on the basis of ameasure of heterogeneity with the logrank statisticaltest with respectto overall survival optimal cutoffvalues were identified a staining index ¥ was used todefine tumors with high creb5 expression and anindex ¤ was used to define tumors with low creb5expressionrna isolation reverse transcription rt and realtimepcrtotal rna samples from cultured cells and primarytumor tissues were extracted using trizol reagent invitrogen usa according to the manufacturers instruction realtime rtpcr was performed at least threetimes in triplicate using sybr green mix toyobojapan and the abi prism sequence detectionsystem applied biosystems usa the data were normalized to the geometric mean of the housekeeping genegapdh and calculated using the 2δδct method primerexpress was used to design the realtime pcr primersand primer sequences for amplification are shown insupplementary table s2luciferase reporter assaygenomic dna extracted from sw480 cells was used as atemplate to amplify met promoter fragments met promoter fragments were obtained by pcr and constructedinto pgl3basic plasmid using a fast ligation kit followingmanufacturers instructions sangon b620511 thesequences of the pcr primers are listed in supplementarytable s4 cells at confluence in a 24well plate weretransfected using lipofectamine fortyeight hoursafter transfection luciferase activity was measured usingthe dualluciferase reporter assay system promega corpmadison wi usa and normalized to renilla luciferasegene expression all the experiments were performed intriplicatewestern blotting analysiswe carried out western blotting as previously described using anticreb5 abcam ab168928 antimetcell signaling technology antipmetcellsignaling technology antiakt cell signalingtechnology antipaktcell signaling technology antierkcell signaling technology antiperk cell signaling technology and antisnail cell signaling technology antiantiαtubulin antibodybodies mouse monoclonalsigma was used as the internal controlimmunohistochemistryimmunohistochemistry ihc staining was performed aspreviously described using creb5 antibody abcamusa ab168928 the degree of ihc staining wasreviewed and scored independently by two observersbased on both the proportion of positively stained tumorcells and the intensity of staining [ ] the proportion of tumor cells was scored as follows no positivetumor cells positive tumor cells positive tumor cells and positive tumor cellschromatin immunoprecipitation chip assaychip assays were carried out as previously described precleared lysates were incubated with creb5 antibody abcam ab168928 or normal mouse immunoglobulin g cst as a negative control overnightat °c with rotation the human met promoter wasamplified by pcr all chip assays were performed threetimes and the sequences of the pcr primers are listedin supplementary table s3migration assaya boyden chamber with an 8μmpore filter membranewas used for the in vitro migration and invasion assaybriefly cells à in culture medium containing fbs were seeded in the upper chamber and culturemedium with fbs was added in the lower chamberas a chemoattractant the upper side of the filter wasfirst coated with matrigel bd biosciences sanjose ca usa after incubation for h cells on theupper side of the filter were removed with cotton swabscells that migrated to the lower surface of the filter werefixed in paraformaldehyde and stained with giemsa 0cwang of experimental clinical cancer research page of the migratory cells were counted random Ãfields per well three independent experiments wereperformed and the data are presented as the mean ±semwoundhealing assaycells were seeded in 6well plates and incubated underpermissive conditions until confluence after serumstarvation for h wounds were created in the confluent cells using a pipette tip wound healing within thescrape line was then observed and photographed at indicated time points each experiment was repeated at leastthree timeschicken chorioallantoic membrane assaya chicken chorioallantoic membrane cam assay wasperformed on the sixth day of development of fertilizedchicken eggs as previously described human umbilical vein endothelial cell tube formationassayfirst μl of matrigel was pipetted into each well of24well plates and polymerized for min at °c human umbilical vein endothelial cells huvecs à in μl of conditional medium were added to eachwell and incubated at °c in co2 for h imageswere obtained under a brightfield microscope and thecapillary tubes were quantified by the counting lengthorthotopic mouse metastatic model to 6weekold balbc athymic nude mice nunuwere obtained from the animal center of southernmedical university guangzhou china all mice werehoused in a sterile environment cells à permouse were orthotopically inoculated into the caecumof anaesthetized nude mice the mice were sacrificedwithin weeks after surgery individual ans were excised and metastases were observed by histological analysis tissues were then fixed with formaldehyde andparaffinembedded and then 5mm sections were cutand stained with haematoxylin and eosin he thenumbers of gross metastatic foci were determined usinga dissection microscope all the mice used in this studywere maintained under specific pathogenfree conditions and all animal experiments were conducted in accordance with standard procedures and approved by theinstitutional use committee for animal carestatistical analysisall statistical analyses were carried out using spss version pearson correlation analysis was used for expression correlation analysis the survival curves of crcpatients in low and highcreb5 expression groups wereanalysed by the kaplanmeier method and the logranktest was used to compare differences p was considered significantresultscreb5 is upregulated in crc and associated with a poorprognosisthe expression of creb5 was analysed in differenttypes of malignant tumors in the public database oncomine wwwconominecom revealing that creb5was upregulated in crc tissues in of crc datasetssupplementary fig s1a additionally a gene set enrichment analysis gsea plot showed significant enrichment of tumorrelated genes and crcrelated genesets in the highcreb5 expression group supplementary fig s1b realtime pcr and western blotting analyses showed that creb5 was differentially expressed incrc cell lines supplementary fig s1cd in additioncreb5 was significantly upregulated in ten crc tissuescompared with adjacent normal intestinal epithelial tissues fig 1a and b ihc showed that creb5 proteinwas weakly expressed in normal tissue but markedly increased in adenocarcinoma cells and was mainly localized in the nucleifig 1c kaplanmeier survivalanalysis showed that crc patients with higher creb5protein expression levels had a poorer prognosis fig1d in addition creb5 expression levels were significantly correlated with the t classification lymph nodemetastasis and distant metastasis p supplementary table s1 creb5 expression was substantiallyhigher in tumors from patients with distant metastasismoreover high creb5 expression was also positivelycorrelated with who stages p supplementarytable s1 these data suggested that creb5 expressionis significantly correlated with advanced stages of crccreb5 activates met signallinggseas of creb5regulated gene signatures revealed thathigher creb5 expression was positively correlated withenrichment of an met signalling pathway signaturegse17538 fig 2a to validate this result we establishedstable creb5overexpressing and creb5knockdowncrc cell lines fig 2b upregulation of creb5 significantly increased while knockdown of creb5 decreasedthe expression of total met at both translational andtranscriptional levels fig 2c and d in addition creb5overexpression markedly increased but creb5 downregulation significantly attenuated the expression of phosphorylated met perk pakt and snail fig 2c moreovermet expression was increased in a dosedependent manner at both translational and transcriptional levels by transiently transfecting sw480 cells with a creb5 expressionvector fig 2e 0cwang of experimental clinical cancer research page of fig creb5 is upregulated in crc and associated with a poor prognosis a and b realtime pcr and western blotting analysis of creb5 expression inpaired human colon cancer tissues and adjacent noncancerous tissues p quantity one software was used to quantify the protein expressionlevels c ihc representative images of creb5 expression in normal intestinal epithelium and crc tissues scale bar μm d the paraffin samples of crc patients were divided into a lowcreb5 expression group n and a highcreb5 expression group n based on ihc results thekaplanmeier method was used to analyse survival curves and the logrank test was used to compare differences p fig creb5 activates the met signalling pathway a gsea of gse17538 in met signalling pathways es p b stable overexpressionand interference cell lines were detected by western blotting and realtime pcr c the expression of met and downstream signalling moleculesin creb5knockdown or creb5overexpressing cells was observed by western blotting d creb5 had an effect on met by realtime pcr in theindicated cells e after transient transfection of different amounts of the creb5overexpression plasmid in sw480 cells the protein and mrnalevels of met were detected by western blotting and realtime pcr respectively p 0cwang of experimental clinical cancer research page of creb5 associates with the met promotergiven that creb5 is a transcriptional factor and upregulatesmet at the transcriptional level we performed a luciferasereporter assay to investigate whether creb5 can increasemet promoter activity a 27kb fragment of the fulllengthmet promoter region was subcloned into a luciferase vector met promoter activity wasincreased by cotransfection with a creb5 expression vector in sw480 cellsbut decreased in hct116 cells expressing creb5 shrna ina dosedependent manner compared with empty vectorsfig 3a to determine the effective regions of the met promoter that creb5 may affect we transfected met promotertruncations fig 3b into hct116 cells expressing eithercreb5 shrna or a scramble control sequence as shown infig 3c luciferase activity was increased in cells carrying thefulllength met promoter and truncations and bp upstream of the transcription start site but not incells carrying truncations to bp or to bp knockdown of creb5 expression bycotransfection of creb5 shrna significantly decreased metpromoter activity fig 3c furthermore we performed chromatin immunoprecipitation chip assays and identified thatthe to 223bp region of the met promoter whichcontains an ap1 motif was a creb5 protein binding sitefig 3d these data identify met as a direct transcriptionaltarget of creb5downregulation of creb5 represses invasiveness andreduces the metastatic potential of crc cellsnext we investigated the role of creb5 in invasivenessand metastasis in crc cells silencing of creb5 significantly compromised the migratory and invasive abilitiesof crc cells fig 4a and b supplementary fig s2a andb the tubule formation and chicken cam assays revealed that knockdown of creb5 strongly inhibited theformation of tubules by huvecs and inhibited angiogenesis in cams fig 4c and d supplementary figs2c orthotopic inoculation assay showed that knockdown of creb5 inhibited liver metastases fig 4eknockdown of creb5 also obviously extended the overall survivaltime of nude mice inoculated with thecrc cell lines fig 4f these results indicate that silencing creb5 inhibits the invasiveness and metastasis of crc cellsinhibition of met attenuates the invasion and metastasisof crc cells by creb5 in vivo and in vitroto determine the functional relationship between creb5and met in the invasion and metastasis of crc weknocked down met using two met shrnas or suppressed met activation using the met inhibitor crizotinib emd silencing or inhibition of met insignificantlysw480creb5or ht29creb5cellsfig creb5 regulates met and binds directly to the met promoter a the met promoter sequence was cloned into pgl3basic vector containing theluciferase reporter gene and then transfected into crc cells with the indicated treatments b schematic diagram of the full and truncated met promoterc the fulllength met promoter or its truncations were cloned into pgl3basic vector containing the luciferase reporter gene and then transfected intohct116 cells with creb5 shrna or empty vector d chip analysis of creb5 binding to the met promoter in sw480 cells p 0cwang of experimental clinical cancer research page of fig downregulation of creb5 inhibits the invasion and metastasis of crc cells in vivo and in vitro a and b woundhealing assay and transwellmigration assay were performed to evaluate the invasive and migratory abilities of crc cells with different treatments in vitro c huvec tubeformation after stimulation with the indicated conditioned medium d representative images of the cam assay histograms show the formationof secondary and tertiary blood vessels after stimulation with the indicated conditional medium scale bar mm e orthotopic transplantationwith the indicated hct116 cells in nude mice n in each group was performed and representative gross images of the livers and intestinesare shown the arrows indicated the tumors liver sections were stained with haematoxylin and eosin he scale bar μm f the kaplanmeiermethod was used to analyse survival curves in the specified treatment groups and the logrank test was used to compare differences p decreased the expression of phosphorylated met perkand pakt as well as snail supplementary fig s3a inaddition the invasive and migratory abilities of sw480creb5 or ht29creb5 cells were partially diminished byinhibition of met fig 5a and b supplementary fig s3band c moreover upregulation of creb5 expression enhanced the capacity of crc cells to induce tube formationand angiogenesis in cams in contrast angiogenesis ability was partially diminished by met inhibition fig 5cand d supplementary fig s3d orthotopic inoculationassay showed that creb5 significantly promoted liver metastases and decreased the overall survival of mice fig 5eand f conversely inhibition of met significantly attenuated the formation of metastatic foci by sw480creb5cells and extended the survival time of mice inoculatedwith sw480creb5 cells fig 5e and fcreb5 expression positively correlates with metexpression in crcto assess a potential link between creb5 and met expression in human crc we analysed tcga crc dataand identified a strong positive correlation between highexpression levels of creb5 and met p r fig 6a in addition analyses of fresh crc tissuesshowed that creb5 expression was positively correlatedwith met expression at both mrna p r and protein levels p r fig 6b and c 0cwang of experimental clinical cancer research page of fig overexpression of creb5 promotes the invasion and metastasis of crc cells but inhibition of met weakens these effects a and b theinvasive and migratory abilities of crc cells in vitro with different treatments were evaluated by woundhealing assay and transwell migrationassay c huvec tube formation after stimulation with the indicated conditional medium d representative images of the cam assay histogramsshow the formation of secondary and tertiary blood vessels after stimulation with the indicated conditional medium scale bar mm eorthotopic transplantation with the indicated sw480 cells in nude mice n in each group was conducted and representative gross images ofthe livers and intestines are shown the arrows indicate the tumors liver sections were stained by he scale bar μm f the kaplanmeiermethod was used to analyse the survival curves of different treatment groups and the logrank test was used to compare differences p p p furthermore ihc revealed that creb5 expression waspositively correlated with met fig 6d p discussionmetastasis of crc is a multistep process requiring the accumulation of genetic andor epigenetic alterations andabnormal expression of genes involved in signal transduction pathwaysincluding oncogenic mutation of krasand activation of the erkmapk pathway wntβcatenin signalling and tgfβ signalling ap1 dnabinding sequences act as crucial response elements fortranscriptional activation by the raserk pathway creb5 is a credependent transactivator downstream ofthe raserk signalling pathway since it interacts with cjun which is one of the ap1 subunits to form a homodimer or a heterodimer along with foxd1 and atf3creb5 formed a transcription factor regulatory networkthat negatively regulates mapk signalling which is suppressed by fzd3 in melanoma previous studies haverevealed that creb5 mrna was upregulated in crc asdemonstrated by bioinformatics analysis or qrtpcrexamination in human cancer tissues [ ] in thecurrent study we demonstrated that creb5 was highlyupregulated in crc at both the mrna and protein levelsoverexpression of creb5 was significantly associatedwith aggressive cellular characteristics of crc eg an 0cwang of experimental clinical cancer research page of fig creb5 positively correlated with met expression in crc a correlation analysis of creb5 and met in tcga crc data r p band c correlation analysis of creb5 and met at the mrna r p and protein levels r p in fresh crc tissues dthe expression of creb5 and met protein in specimens including colon cancer tissue specimens and adjacent normal tissue specimens wasdetected by ihc representative ihc images left and correlation analysis right of creb5 and met expression scale bar μm high expressionof creb5 n high expression of met n low expression of creb5 n low expression of met n p advanced who stage and an advanced tnm stage andpoorer patient outcomes suggesting that creb5 might bean oncogene and a prognostic marker of crc progressionconsistent with our results upregulation of creb5 hasbeen reported to be responsible for poorer outcomes inpatients with epithelial ovarian cancer and hepatocellular carcinoma in prostate cancers creb5 overexpression occursthrough both copy number gain and increased gene expression however examination of tcga colorectalcancer datasets via cbioportal revealed rare amplificationof creb5 suggesting that overexpression of creb5 iscontrolled attranscriptional and posttranscriptionallevels creb5 has been shown to be a downstream target of lncrna snhg5mir1323p and circularrna circvapamir125a in addition fzd3 inhibits transcriptional networks controlled by creb5 however the alternative mechanisms involved inupregulation ofthe creb5 gene and activation ofcreb5mediated signalling require further investigationthe effects of creb5 overexpression on promotingcell proliferation and migration have been demonstratedusing in vitro assays in human hepatocellular carcinomacells and crc cell lines [ ] in the current studywe showed that creb5 overexpression promoted whilecreb5 silencing reduced the invasiveness and metastaticcapacity of crc cells both in vitro and in vivo mechanistically creb5 directly interacted with the met promoter and activated the hgfmet signalling pathwayimportantlyinhibition of met reduced the invasion 0cwang of experimental clinical cancer research page of and metastasis of creb5overexpressing crc cells suggesting that creb5 promotes metastasis mainly throughactivation of met signalling our data provide solid evidence that upregulation of creb5 plays an essential rolein crc metastasis recently overexpression or amplification of creb5 was reported to promote proliferationand mediate resistance to ar inhibition in metastaticcastrationresistant prostate cancers these datasuggest that creb5 may function as a multitaskingregulator in cancer progression and clinical outcomessignallingtransportimmunegrowth factorscreb family members can be phosphorylated via various intracellular signal transduction pathways such asprotein kinase a pka calmodulindependent proteinkinasecamk mitogenactivated protein kinasesmapks and other kinases upon phosphorylationcreb recruits crebbinding protein cbp and binds tothe cres of the promoters of its target genes target genes containing consensus sites for creb bindinginclude those related to metabolism transcription neuropeptidesneurotransmitters cell cyclecellsurvivalregulationdna repairreproductiondevelopmentandstructure specifically knockdown of creb1creb5increased tumor necrosis factor alpha tnfα levelsenhanced the expression of phosphonfκb p65 andnfκb p65 and induced immunosuppression in monocytes in prostate cancer creb5 could improve resistance to enzalutamide with the help of foxa1 andselectively enhance the interaction of ar with targetgenes critical for survival however little is knownabout the downstream targets of creb5 involved in theprogression of crc our results showed that creb5 directly interacted with the met promoter and activatedthe hgfmet signalling pathway which in turn increased the expression of downstream erk and pi3ksignalling cascades meanwhile the expression of snailan essential emt transcription factor was also upregulated via the creb5hgfmet axistranscriptional factor that interacts with the met promoter at the ap1 motif and activates met expressionin our data suggest that creb5 has an essential role in crc metastasis by regulating the protooncogene met interfering with creb5 may representan alternative therapeutic target to prevent or reducemetastasis in crcsupplementary informationsupplementary information accompanies this paper at httpsdoi101186s13046020016730additional file table s1 the relationship between creb5 expressionand clinicopathological parameters table s2 primer sequences used forrealtime pcr ² to ² table s3 sequence of primers for chip assaytable s4 sequence of primers for luciferase reporter assayadditional file figure s1 bioinformatics analysis of creb5expression the expression of creb5 in crc and other malignant tumorswas analyzed by oncomine database the inclusion criteria were that thedifference of creb5 expression between tumor tissue and normal tissuewas more than times and the arrangement of gene position was lessthan with p the outliers in the red and blue boxes representthe number of data sets with high and low expression of creb5respectively the right table of a represents the copa score of creb5 in crc data sets b the two crc chips gse17538 n andgse35896 n from the public database of geo was analyzed bygsea the plot showed significant enrichment of tumorrelated gene setkegg_pathway_in_cancer and colorectal cancerrelated gene setkegg_colorectal_cancer in the creb5 high expression group cand d realtime pcr and western blotting analysis of creb5 endogenous expression in indicated crc cellsadditional file figure s2 representative images of woundhealingassay a transwell migration assay b and huvec tube formation assayc with i | Colon_Cancer |
microbiota involves communities ofhepatitis is generally known as an ammation of the liver that can be caused by hepaticand nonhepatic viruses can be caused by alcohol can be drug induced and can be caused byautoimmunity gut microbiota composition is known to be associated with disease pathogenesishowever dynamic alteration of the gut microbiota in disease pathogenesis is not wellunderstoodsymbiotic as well as pathogenicmicroanisms found in anisms ie plants and animals microbiota of a healthy individualshows more of commensalism or symbiosis without causing any disease these microbes mainlycolonize humans during birth or shortly thereafter and remain throughout the course of life thesecan be found in many areas like skin respiratory tract urinary tract and digestive tract whilebrain lungs and the circulatory system are free of microbes approximately microbes arepresent in a healthy individual gut minemura and shimizu therefore gut microbiota hasan important role to modulate the immune system in disease progression or recoverycommensaltranslocation of microbes or their metabolic products cause intestinal ammation leadingto impairment of the primary barrier hill there is limited available informationregarding the role of gut microbiota in hepatitis which makes it important to majorly focus onclinical data of gut microbiota linked with hepatitis b and c virusgut microbiotagut or gastrointestinal tract starts from the mouth and ends at the back passage anus gut helpsin the digestion of food by absorbing energy and nutrients majority of gut microbiota to contains good bacteria and only to are harmful bacteria in diï¬erent parts of the intestineedited bymilan surjittranslational health science andtechnology institute thsti indiareviewed byjawed iqbaljamia millia islamia indiabinod kumarloyola university chicagounited statescorrespondencenirupma trehanpatitrehanpatigmailcomspecialty sectionthis was submitted tovirus and hosta section of the frontiers in cellular and infectionmicrobiologyreceived march accepted june published august citationsehgal r bedi o and trehanpati n role of microbiota inpathogenesis and management ofviral hepatitisfront cell infect microbiol 103389fcimb202000341frontiers in cellular and infection microbiology wwwfrontiersinaugust volume 0csehgal microbiota and liver diseasesbajaj in mouth and upper respiratory tract normalï¬ora is more of the commensal bacteria like streptococcusmoraxella neisseria and haemophilus very few species ofbacteria are present in the stomach and small intestine whilethe large intestine and colon contain dense population ofmicrobes ie up to cellsg along with bacteria many othermicroanisms like fungi protists archaea and viruses alsosymbiotically harbor in the gutthere are four dominant phyla of bacteria present in thegut and they are firmicutes bacteroidetes actinobacteriaand proteobacteria khanna and tosh most importantgenera in which bacteria belong are bacteroides clostridiumfaecalibacterium eubacterium ruminococcus peptococcuspeptostreptococcus and biï¬dobacterium fern¡ndez some of the fungal species that also coexist in the gut arecandida saccharomyces aspergillus penicillium rhodotorulatrametes pleospora sclerotinia bullera and galactomycesamong others raimondi functions of gut microbiotagut microbiota plays an important but diverse role such asbarrier eï¬ect vitamin synthesis and fermentation residentbacteria of the gut acts as a barrier and protect the intestinalmucosa from invasion of the other potential pathogens hooper many factors including diet age medicationillness stress and lifestyle uence the gut microbiota whichhave a great impact on disease pathogenesis in fact manybacteria ie bacteroides eubacterium propionibacterium andfusobacterium are instrumental in the synthesis of vitamins kand b ie folate b12 and biotin canny and mccormick they are also involved in the fermentation of nondigestible carbohydrates for the production of shortchain fattyacids scfas which are helpfulin maintaining metabolichomeostasis in addition to the production of scfa glycolysisand pentose phosphate pathway also produce butyrate whichpromotes the growth of lactobacilli and biï¬dobacteria bacteriain the colon venegas various studies supported thefact that nutrients derived from microbiota play a pivotal rolein the normal functioning of the hepatic system li zheng moratalla jiminez cremer wang 2017agut microbiota in liver diseasescommensal bacteria play a decisive role in maintaining immunehomeostasis figure and also guard immune reactions atmucosal surfaces ichinohe intestinal microï¬ora isa dynamic and complex ecosystem which helps in proliferationgrowth and diï¬erentiation of epithelial cells to ï¬ght infectionsand improve immunity despite its crucial role in the synthesisfolate scfa and peroxides gut microbiotaof vitamin kacts as a chief environmental as well as etiologicalfactorfor the progression of many liver diseasesohara andshanahan particularly gut microbiota has a largeruence on alcoholic liver disease nonalcoholic fatty liverdisease viral hepatitishepatitis b and c autoimmunehepatitis aih primary sclerosing cholangitis psc andprimary biliary cholangitismohamadkhani pbclactobacillus biï¬dobacterium saccharomyces boulardii andlactobacillus plantarum play a bigger role in the managementof various metabolic disorders and hepatitis mohamadkhaniincludingvirusesandseveralpathogensintestinalmicroanisms use mucous membranes as a doorwaykarst hepatic viruses breach the intestinal permeabilityleading to gut dysbiosis and release proammatory cytokinesinstrumental in developing liver cirrhosis and hcc it is alsoobserved that the use of probiotics reduces the tolerogenicresponse and enhances the mucosal defense against viralpathogens rigoadrover m del lactobacillusalone can uence the production of interferon by modulatingthe antiviral eï¬ects of vitamin a lee and ko themixture of various probiotics and biï¬dobacterium with galactooligosaccharides and fructooligosaccharides has a defensiveeï¬ect against rotavirus infection by aggregating the productionof tnfα il4 ifnÎ and tlr2 expression rigoadrover mdel in most of the liver disease especially cirrhosisdysbiosis of the gut increases proteobacteria enterobacteriaceaeand veillonellaceae while it decreases bacteroidetes andlachnospiraceae sanduzzi zamparelli recentlythe cirrhosis dysbiosis ratio cdr is coined for deï¬ningthe changes in gut microbiome in cirrhosis patients withbeneï¬cial lachnospiraceae and ruminococcaceae and harmfulenterobacteriaceae bacteria bajaj other groupshave also associated patients with severe cirrhosis and hepaticencephalopathy with overgrowth of enterobacteriaceae bacteriachen role of gut microbiota in hepaticviral infectionsacute viral hepatitis due to hepatitis a and e viral infectionsis a major community health problem especially in developingcountries hepatitis a and e cause acute infection whichcould be shortlived and selfclearing unless the subjects areimmunocompromised or in transplant settings acute hepatitise infection also becomes detrimental and lifethreatening duringpregnancy aï¬ecting both the mother and the childboth hepatitis a and e are rna viruses thattransmitthrough oral fecal routes lemon and may havedevastating eï¬ects on intestinal microï¬ora it was observedthat administration of the healthy probiotic bacterium likeenterococcus faecium ncimb aï¬ects the reduction as wellas the removal of enteric hev viruses in pigs kreuzer however there is lack of relevant data in humansas per the world health anization who hepatitisb virus hbv infection caused deaths in and million diagnosed with chronic infection in similarly hepatitis c virus hcv caused deaths with anestimated million diagnosed with chronic infection in both these viruses cause chronic infections at in hbv andmore than in hcv leading to cirrhosis and hepatocellularcarcinoma hepatic viruses have evolved mechanisms to avoidtheir detection from the host innate and adaptive immunityfrontiers in cellular and infection microbiology wwwfrontiersinaugust volume 0csehgal microbiota and liver diseasesfigure protective role of fecal microbiota transplantation and use of probiotics in immune restorationand characterized as viral escape visvanathan itis observed that chronic hepatitis patients have largertranslocation of the intestinal microbiota lu li bacterialtranslocation cause intestinalammation viadysregulation of immune cell overgrowth of pathogenic bacteriaas well as dysfunction of the primary barrier hill xu also supported the fact that intestinal ï¬oraloses homeostasis during dysbiosis which in fact helps theadvancement of hepatitis viral infection xu itthereforeis now understood that during chronicitycommensal microbiota have greater impact not only on viral hostcell interaction but also on viral replicationin viral hepatitis few harmful bacteria like escherichia colienterobacteriaceae enterococcus faecalis and faecalibacteriumprausnitzii directly alter the proï¬le of good intestinal microbiotawith a lower number of intestinallactic acid species suchas lactobacillus pediococcus weissella and leuconostoc bajaj chen some of the bacterial species ieneisseria e coli enterobacteriaceae e faecalis f prausnitziiand gemella are also found responsible for the progression ofhepatitis b and c virusrelated cirrhosis and primary biliarycirrhosis chen mohamadkhani candidais also frequently found in patients with hepatitis brelatedcirrhosis cui role of gut microbiota in hepatitis b viralinfectiondysbiosis of gut microbiota in chronic hepatitis b infectionaï¬ects disease pathogenesis and causes liver failure in alarge proportion lps lipopolysaccharides from the outermembrane of gramnegative bacteria help in the activationof innate immune response by recognizing tlrs especiallytlr2 and hbv infection leads to progressive declinein butyrateproducing bacteria however lpsproducinggenera is enriched in hbv infection in hbv infection abeneï¬cial bacterium lachnospiraceae plays a role in themanagement of hbv infection via reduction in lps sectionand bacterialtranslocation chen ren studies have shown the role of faecalibacteriumpseudobutyrivibrioruminoclostridiumprevotella alloprevotella and phascolarctobacterium in potentialantiammatory scfa activity which increases the abundanceof butyrate compared to normal subjects liu lu have demonstrated that copy numbers of fprausnitzii e faecalis enterobacteriaceae biï¬dobacteria andlactic acid bacteria lactobacillus pediococcus leuconostocand weissella have marked variation in the intestine of hbvcirrhotic patients during hbv infection dysbiosis in theoral microbiota was observed and yellow tongue coating issuggestive of a reduction in bacteroidetes but an increaselachnoclostridiumfrontiers in cellular and infection microbiology wwwfrontiersinaugust volume 0csehgal microbiota and liver diseasesin proteobacteria zhao also suggested positivecorrelation of neisseriaceae with the serum hbvdnacirrhotic patients with hbv infection showed a signiï¬cantdecrease in the biï¬dobacteriaceaeenterobacteriaceae be ratiolu while yun observed no diï¬erence in thebe ratio in hbsag with normal or high alt and in noncirrhotic hbv carriers yun it means the be ratiois disturbed only in cirrhosis however other study observedthat the megasphaera genus from the firmicutes phylum wasabundant in the hbsag high alt group than the normal altin patients with normal alt butyrateproducing bacteria likeanaerostipes are more in feces compared to hbsagve yun it is interesting to note that both megasphaeraand anaerostipes produce scfa as a byproduct of lactatefermentation and butyrate however butyrate is known asanticarcinogenic and antiammatory and plays a role inoxidative stress hamer another study suggests thatchronic hepatitis b infected cirrhotic patients exhibit a decreasein biï¬dobacteria and lactobacillus levels while signiï¬cantlyincreasing enterococcus and enterobacteriaceae levels comparedto healthy individualsbacterial translocation is also observed in the developmentof hepatocellular carcinoma hcc recently wang havedeï¬ned the serum zonulin as an intestinal permeability markerand showed its association with afp levels in hbvassociatedliver cirrhosis and hcc they are helpful in correlating it withadvanced stages of the diseases fasano the use of probiotic in hbvinfected patients showed beneï¬tand suggested that probiotic vsl3 plays an important role in themanagement of hbv viral infection dhiman role of gut microbiota in hepatitis c viralinfectionchronic hepatitis c infection is another leading cause ofcirrhosis hcc and in some casesliver failure and deathin majority enterobacteriaceae and bacterioidetes increased inchronic hcv patients but firmicutes found to be decreasedhcv infection cause marked elevation in lps which issuggestive of microbial translocation and ammation duringdisease progression dolganiuc inoue on the other hand it was observed that antiviral treatment ofhcv with ribavirin rbv and immune modulator pegylatedinterferon pegifn has no direct impact on gut dysbiosisin factit increases the production of bile acids which isimportant for gut microbiota ponziani somepathogenic bacteria such as enterobacteriaceae staphylococcusand enterococcus decreased the bile acid in hcvinfectedcirrhotic patients which normalized after a directacting antiviraltreatment oral directacting antivirals daas were also foundto be helpful in improving gut especially lachnospira and doreagenera and restored tnfα levels p©rezmatute but after daa treatment expression of calprotectin zo1and lps was found more in hcv patients with cirrhosis itwas also suggested that during hcv infection l acidophilusand biï¬dobacterium spp can act as a supportive supplementwith antiviral and antibacterial activities dore immune response in hcv patients can be stimulated by usefulmicrobiota via activation of cd3 cells and cd56 nk cellcounts which were explained by doskali and furthersuggested that good ï¬ora increases the cytotoxic eï¬ects of nkcells against viral infected cells inhibiting the replication of hcvuse of probiotics in hcvinfected patients with cirrhosis wassigniï¬cantly beneï¬cial preveden another hepatic virus hepatitis d virus is a new playerand not much is known about it yet it was also suggestedthat endotoxemia in hcv and hdv patientstobe multifactoriallikely depending on impaired phagocyticfunctions and reduced tcellmediated antibacterial activitykefalakes and rehermann seemsmicrobiota modulates molecularsignaling in hepatitisactivereceptorthe keycomponent of gramnegative bacterialps isie enterobacteriaceae thefor lps iscd14tlr4md2 receptor complex on induction whichsecretes many proammatory cytokines including tumornecrosis factorα il1 il6 and chemokines through the nfκbsignaling fooladi seki and schnabl bryant to cause liver injury in the intestinal tract lpsdownregulates the expression of various tight junction proteinszo1 and closed protein by increasing the permeability ofthe intestinal mucosa and enters the blood ï¬ow through theportal venous system park in liver kupï¬er cells asspecialized macrophages are induced by the lpstlr4 pathwayfor the release of immunosuppressive mediators such as il10which in turn suppress the release of ammatory mediatorsby kupï¬er cells dixon in this way during viralhepatitis virus speciï¬c immune responses are suppressed andultimately inhibit eï¬cient clearing of bacteria as well as virusesin addition to lps unmethylated cpg dna bacterialdnarna bacterial cell wall also contains teichoic acidpeptidoglycan and specialized proteins ï¬agellin bacterialdnarna is recognized by tlrs as well as all components ofcellwalllike teichoic acid and peptidoglycan also recognized bytlr2 while tlr5 got activated by ï¬agellin dsrna bacteriaare recognized by tlr3 ssrna activates receptors of bothtlr7 and tlr8 all these tlrs ultimately stimulate the jakstat pathway hepatitis viruses are also recognized by tlrs inthe liver or in the intestine and activate downstream signalingpathways mencin unmethylated cpg dnas are found abundantly in thelactobacillus family ie l casei l plantarum l rhamnosus andothers like biï¬dobacteria proteobacteria and bacteroidetes inthe intestinal ï¬ora of animals unmethylated cpg dna is sensedby tlr9 expressed on various mononuclear cells and stimulatesboth innate immune response as well as adaptive immuneresponse krieg kauppila activation the ofcpgtlr9 pathway stimulates downstream molecules of myd88such as irak4 traf6 and irak1 ultimately triggering nfκb and mapk signaling pathways these downstream pathwayshelp in the activation of dcs for the secretion of cytokines andfrontiers in cellular and infection microbiology wwwfrontiersinaugust volume 0cfrontiersincellluarandifnectionmcroboogyiilwwwfrontiersniaugustlvoumearticeltable randomized fmt clinical trials for the treatment of chronic hepatitis b infectionsnostudy titlestudy typeno ofsubjectsinterventiontreatmentstatusphaseprimary outcomemeasuresrandomized controlled trialcomparing the efï¬cacy andsafety of fmt in hepatitis breactivation leads to acuteon chronic liver failurelocationinstitute of liver and biliarysciencesnew delhi delhi indiastudy on effect of intestinalmicrobiota transplantationin chronic hepatitis blocation zhongshanhospital afï¬liated to xiamenuniversityxiamen fujian chinainterventionalclinical trialdrug tenofovirdrug fecal microbiotatransplantation fmtcompletedcompletedtransplant free survival[time frame months]interventionalclinical trialother intestinalmicrobiota transplantdrug antiviral agentsrecruitingnachange of serum hepatitis bvirus e antigenhbeag level[time frame months]serum hepatitis b virus eantigenhbeag levels ismeasured in scosecondary outcome measuresclinicaltrialsgovsehgaletalidentiï¬ernct02689245nct03429439reduction in hepatitis b virus dnalevel ¥ log [time frame weeks]improvement in meld model forend stage liver disease score[time frame weeks]change of serum hepatitis b virussurface antigenhbsag level [timeframe months] serumhepatitis b virus surfaceantigenhbsag levels is measuredin iumlchange of serum antihepatitis bvirus e antigenantihbe [timeframe months] appearanceof serum antihepatitis b virus eantigenantihbe suggest the abilityof body to resistant hbvchange of serum antihepatitis bvirus surface antigenantihbs[time frame months]appearance of serum antihepatitisb virus surface antigenantihbssuggest the ability of body toresistant hbvchanges of gut microbiota [timeframe months] alpha andbeta diversity of gi microbiota byhighthroughput sequencing 16srrna on baseline line and months after treatmentrelief of constipation [time frame months] relief of diarrhea[time frame months] reliefof abdominal pain [time frame months] the onset andduration of constipation will beassessed by evaluation scoretable of gastrointestinalsymptomsimcrobotaiandlveriidseases 0csehgal microbiota and liver diseaseschemokines krieg kauppila chronic hbvpatients have reduced lactobacillus and biï¬dobacteria both arerich in unmethylated cpg dna levels ultimately aï¬ecting thecpg dnatlr9 pathway and immune response on hbv linand zhang role of fecal microbialtransplantation fmt in viralhepatitisfmt mainly involves the insertion of healthy microbiota in thediseased gut in brief fecal matter derived from a healthy familymember of the patient receiving the same diet as the patient isprocessed and introduced in the intestinal tract of the patientthese have minimal side eï¬ects and proved helpful in reinstatinghealthy gut ï¬ora in the patient fmt administration can bedone using several routes such as oral nasogastric nasoduodenalnasojejunal endoscopic rectal and colonoscopic or midguttransendoscopic enteral tubing cui tang for cirrhotic patients with dysbiosis small bowel route ismost aï¬ected while mostly used route is oral delivery in severealcoholic hepatitis sah in comparison to steroids fmt isassociated with decreased disease severity and improved survivalearlier wang 2017b have observed that fmt restored thecognitive function liver function indexes and tlr response incarbon tetrachloride ccl4induced acute hepatitis in ratswoodhouse have observed in a profit clinical trialthe beneï¬ts of fecal microbiota transplantation in the smallbowel of cirrhotic patients woodhouse meiglani also observed that cirrhotic patients with antibioticresistant clostridioides diï¬cile infection cdi responded wellafter fmt treatment in factfecal microbiota of alcoholresistant mice when given to alcoholsensitive mice has reducedbacteroidetes and increased actinobacteria as well as firmicutesand protected steatosis development ferrere limited studies are published yet on fmt administration inalcoholrelated liver disease however all these studies showedimmense beneï¬t of fmt bajaj observed the recoveryof cognitive function and hepatic encephalopathy in patientsunder clinical trial after administration of fmt studies recentlypublished from our center have found better eï¬ciency of fmtreferencesbajajj s heuman d m hylemon p b sanyal a the cirrhosis dysbiosisb monteith p changescomplicationsin the gut microbiomej hepatolassociated with cirrhosisj white mratio deï¬nesand its101016jjhep2013bajaj j s kassam z fagan a gavis e a liu e cox i j fecal microbiota transplant from a rational stool donor improveshepatic encephalopathy a randomized clinical trial hepatology 101002hep29306bryant c e symmons m and gay n j tolllike receptor signallingthrough macromolecular protein complexes mol immunol 101016jmolimm201406033in severe alcoholic patients than standard medical treatmentsarin there are only a couple of randomizedfmt clinical trials for chronic hepatitis b infected patientstable recently groups have addressed how fmt is modulatingimmunity in gut and liver mucosaassociated invariant tmait cells are found abundant in liver to ofintrahepatic t cells gut peripheral blood as well as lungs gao have observed that functional mait cells were altered insah resulting in more bacterial infection in patients alterationin circulating mait cells is observed with defective antibacterialcytokinecytotoxic response against the infection gao they believe that fmt administration has a profoundeï¬ect on the expression of mait cells in alcoholrelated diseasessummary and conclusionlikeruminoclostridiumgut microbiota has an important role in viral alcoholicand metabolic liver diseases gut microbiota plays a crucialrole in modulating the tolllike receptors nfκb signalingjanus kinasesignal transducer and transcription jakstatpathway and cd4t cell activation numerous usefulmicrobiotasfaecalibacteriumlachnoclostridium prevotella alloprevotella pseudobutyrivibrioand phascolarctobacterium play an important role in potentiatingantiammatory short chain fatty acid scfa activity andincreased the butyrate abundance which play a crucial role inthe management of various hepatitisrelated viral infectionsfecal microbiota transplantation became an attractive andsafest mode of treatment for the management of various liverdiseases especially in severe alcoholic hepatitis despite recentpublications there are still gaps in understanding the role ofmicrobiota in viral hepatitis especially in acute hav and hevviral infections therefore there is a need to explore more inthese infectionsauthor contributionsrs and ob written the review nt provide valuablesuggestions corrected and revised all authors contributed to the and approved the submitted versioncanny g o and mccormick b a bacteria in the intestine helpfulresidents or enemies from within infection and immunity am soc microbiol 101128iai0018708chen y ji f guo j shi d fang d and li l dysbiosis of smallintestinal microbiota in liver cirrhosis and its association with etiology sci rep 101038srep34055chen y yang f lu h wang b chen y lei d characterization of fecal microbial communities in patients with liver cirrhosishepatology 101002hep24423cremer j arnoldini m and hwa t eï¬ect of water ï¬ow and chemicalenvironment on microbiota growth and composition in the human colon procnatl acad sci usa 101073pnas1619598114cui b feng q wang h wang m peng z li p fecalmicrobiota transplantation through midgut for refractory c rohns diseasefrontiers in cellular and infection microbiology wwwfrontiersinaugust volume 0csehgal microbiota and liver diseasessafety feasibility and eï¬cacy trial results j gastroenterol hepatol 101111jgh12727cui l morris a and ghedin e the human mycobiome in health anddisease genome med 101186gm467dhiman r k rana b agrawal s garg a chopra m thumburu k k probiotic vsl\\ reduces liver disease severity and hospitalization inpatients with cirrhosis a randomized controlled trial gastroenterology 101053jgastro201408031dixon l j barnes m tang h pritchard m t and nagy l e kupï¬ercells in the liver compr physiol 101002cphyc120026dolganiuc a norkina o kodys k catalano d bakis g marshall c viral and host factors induce macrophage activation and loss of tolllikereceptor tolerance in chronic hcv infection gastroenterology 101053jgastro200708003dore g ward j and thursz m hepatitis c disease burden andstrategies to manage the burden guest editors mark thursz gregory doreand john ward j viral hepat 21suppl 101111jvh12253doskali m tanaka y ohira m ishiyama k tashiro h chayama k possibility of adoptive immunotherapy with peripheral bloodderived cd3cd56 and cd3cd56 cells for inducing antihepatocellularcarcinoma and antihepatitis c virus activity j immunother 101097cji0b013e3182048c4efasano a intestinal permeability and its regulation by zonulin diagnosticand therapeutic implications clin gastroenterol hepatol 101016jcgh201208012fern¡ndez m f reinap©rez i astaj m rodriguezcarrillo aplazadiaz j and fontana l breast cancer and its relationshipwith the microbiotaj environ res public health int 103390ijerph15081747ferrere g wrzosek l cailleux f turpin w puchois v spatz m fecal microbiota manipulation prevents dysbiosis and alcoholinduced liver injury in mice j hepatol 101016jjhep2016fooladi a i tavakoli h and naderi a detection of enterotoxigenicjin domestic dairy productsisolatesiranstaphylococcus aureusmicrobiol gao b ma j and xiang x mait cells a novel therapeutic target foralcoholic liver disease gut 101136gutjnl2017315284hamer h m jonkers d venema k vanhoutvin s troost f and brummerr j the role of butyrate on colonic function aliment pharmacol ther 101111j13652036200703562xhill d a hoï¬mann c abt m c du y kobuley d kirn t j metagenomic analyses reveal antibioticinduced temporal and spatial changesin intestinal microbiota with associated alterations in immune cell homeostasismucosal immunol 101038mi2009132hooper l v xu j falk p g midtvedt t and gordon j i amolecular sensor that allows a gut commensal to control its nutrient foundationin a competitive ecosystem proc natl acad sci usa 101073pnas96179833ichinohe t pang i k kumamoto y peaper d r ho j h murray ts microbiota regulates immune defense against respiratorytract uenza a virus infection proc natl acad sci usa 101073pnas1019378108inoue t nakayama j moriya k kawaratani h momoda r ito k gut dysbiosis associated with hepatitis c virus infection clin infectdis 101093cidciy205jiminez j a uwiera t c abbott d w uwiera r r and inglis gd impacts of resistant starch and wheat bran consumption onenteric ammation in relation to colonic bacterial community structuresand shortchain fatty acid concentrations in mice gut pathog 101186s1309901601496karst s m the uence of commensal bacteria on infection with entericviruses nat rev microbiol 101038nrmicro201525kauppila j h karttunen t j saarnio j nyberg p salo t gravesd e short dna sequences and bacterial dna induceesophageal gastric and colorectal cancer cell invasion apmis 101111apm12016kefalakes h and rehermann b ammation drives an alteredphenotype of mucosalassociated invariant t cells in chronic hepatitis d virusinfection j hepatol 101016jjhep201905024khanna s and tosh p k a clinicians primer on the role of themicrobiome in human health and disease mayo clin proc 101016jmayocp201310011kreuzer s machnowska p amus j sieber m pieper r schmidt m f feeding of the probiotic bacterium enterococcus faecium ncimb diï¬erentially aï¬ects shedding of enteric viruses in pigs vet res krieg a m therapeutic potential of tolllike receptor activation natrev drug discov 101038nrd2059lee h and ko g antiviral eï¬ect of vitamin a on norovirus infection viamodulation of the gut microbiome sci rep 101038srep25835lemon s m ott j j van damme p and shouval d typea viral hepatitis a summary and update on the molecular virologyepidemiology pathogenesis and preventionj hepatol 101016jjhep201708034li d yan p abousamra a b chung r and butt a proton pumpinhibitors are associated with accelerated development of cirrhosis hepaticdecompensation and hepatocellular carcinoma in noncirrhotic patients withchronic hepatitis c infection results from erchives aliment pharmacolther 101111apt14391li h gao z zhang j ye x xu a ye j sodium butyratestimulates expression of ï¬broblast growth factor in liver by inhibition ofhistone deacetylase diabetes 102337db110846lin l and zhang j role of intestinal microbiota and metabolitesand human diseases bmc immunolon gut homeostasis 101186s1286501601873liu q li f zhuang y xu j wang j mao x alterationin gut microbiota associated with hepatitis b and nonhepatitis virus relatedhepatocellular carcinoma gut pathog 101186s1309901802816lu h wu z xu w yang j chen y and li l intestinal microbiotawas assessed in cirrhotic patients with hepatitis b virus infection microb ecol 101007s0024801098018meiglani a alimirah m ramesh m and salgia r fecal microbiotatransplantation for clostriodioides diï¬cile infection in patients with chronicliver disease int j hepatol mencin a kluwe j and schwabe r f tolllike receptors as targets inchronic liver diseases gut 101136gut2008156307minemura m and shimizu y gut microbiota and liver diseases worldj gastroenterol 103748wjgv21i61691mohamadkhani aintestinal microbialcommunity in hepatocarcinogenesis in chronic hepatitis b cancer med 101002cam41550 on the potential role ofmoratalla a gmezhurtado i santacruz a moya ¡ peir g zapater p protective eï¬ect of biï¬dobacterium pseudocatenulatum cect against induced bacterial antigen translocation in experimental cirrhosisliver int 101111liv12380ohara a mand shanahan ftherapeutic potential clin gastroenterol hepatolfor 101016jcgh200612009 gut microbiota miningpark e j thomson a b and clandinin m t protection of intestinaloccludin tight junction protein by dietary gangliosides in lipopolysaccharideinduced acute ammation j pediatr gastroenterol nutr 101097mpg0b013e3181ae2ba0p©rezmatute p ±iguez m villanuevamill¡n m j reciofern¡ndez e andv¡zquez a m s¡nchez s c shortterm eï¬ects of directactingantiviral agents on ammation and gut microbiota in hepatitis cinfectedpatients eur j inter med 101016jejim201906005ponziani f r putignani l paroni sterbini f petito v picca a del chiericof uence of hepatitis c virus eradication with directactingantivirals on the gut microbiota in patients with cirrhosis aliment pharmacolther 101111apt15004preveden t scarpellini e mili´c n luzza f and abenavoli l gut microbiota changes and chronic hepatitis c virus infection expert revgastroenterol hepatol frontiers in cellular and infection microbiology wwwfrontiersinaugust volume 0csehgal microbiota and liver diseasesraimondi s amaretti a gozzoli c simone m righini l candelieref in the human gutits proï¬ling phenotyping and colonization front microbiol 103389fmicb201901575 longitudinalsurvey offungiren z li a jiang j zhou l yu z lu h gut microbiomeanalysis as a tooltowards targeted noninvasive bioma | Colon_Cancer |
" the empirical dietary index for hyperinsulinemia edih score is a validated foodbased dietary scorethat assesses the ability of wholefood diets to predict plasma cpeptide concentrations although the edih hasbeen extensively applied and found to be predictive of risk of developing major chronic diseases its influence oncancer survival has not been evaluated we applied the edih score in a large cohort of colorectal cancer patients toassess the insulinemic potential of their dietary patterns after diagnosis and determine its influence on survivaloutcomesmethods we calculated edih scores to assess the insulinemic potential of postdiagnosis dietary patterns andexamined survival outcomes in a sample of stage iiii colorectal cancer patients in the nurses health studyand health professionals followup study cohorts multivariableadjusted cox regression was applied to computehazard ratios hr and confidence intervals ci for colorectal cancerspecific mortality and allcause mortalitywe also examined the influence of change in diet from pre to postdiagnosis period on mortalityresults during a median followup of years there were deaths which included colorectal cancerspecific deaths in the multivariableadjusted analyses colorectal cancer patients in the highest compared tolowest edih quintile had a greater risk of dying from colorectal cancer hr ci and a greater risk of allcause death hr 95ci compared to patients who consumed low insulinemicdiets from pre to postdiagnosis period patients who persistently consumed hyperinsulinemic diets were at higherrisk of colorectal cancer death hr151 95ci and allcause death hr 95ci our findings suggest that a hyperinsulinemic dietary pattern after diagnosis of colorectal cancer isassociated with poorer survival interventions with dietary patterns to reduce insulinemic activity and impactsurvivorship are warrantedkeywords colorectal cancer survival insulinemic dietary patterns insulin cpeptide correspondence fredtabungosumcedu1division of medical oncology department of internal medicine the ohiostate university college of medicine west 12th avenue 302b wisemanhallccc columbus oh usa2the ohio state university comprehensive cancer center arthur g jamescancer hospital and richard j solove research institute columbus oh usafull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0ctabung bmc cancer page of canceristhefourth most colorectalcommonlydiagnosed cancer in the united states while there ishigh potential for dietary patterns as a modifiable riskfactor for colorectal cancer development [ ] verylimited evidence exists among colorectal cancer survivors for example in a recent review we identified s published up to that reported on theassociation between dietary patterns and colorectalcancer development but only about five s onthe association between dietary patterns and outcomesamong colorectal cancer survivors [] the evidenceshowed that the western dietary pattern often characterized by high intakes ofred andprocessed meats desserts and potatoesis associatedwith higher risk of allcause mortality but generally notwith colorectal cancerspecific mortality in patients withcolorectal cancer the prudent dietary pattern oftencharacterized by high intakes of fruits vegetables wholegrains and poultry showed similar results with inverseassociations for allcause mortality but no consistent association with colorectal cancerspecific mortality []higher adherence to other dietary patterns including themediterranean diet score dietary approaches to stophypertension meal plan american cancer society cancerprevention guidelines score healthy eating index scorewere generally associated with lower risk of allcausemortality but the associations were inconsistent acrossstudies [ ]refined grainschronic diseasesfurther research is therefore needed to clarify ifdietary patterns are importantfor colorectal cancerprognosis and if dietary changes can maximally impactoverall and cancerspecific survival biomarkerbaseddietary patterns may be helpfulin this regard forexample hyperinsulinemia and insulin resistance areconsidered important underlying mechanisms linkingpoor dietary patterns and lifestyle behaviors to the development of multipleincludingcolorectal cancer [] studies have shown positiveassociations between circulating cpeptide concentrations a marker of beta cell secretory activity and colorectal cancer risk and prognosis [] therefore adietary pattern associated with hyperinsulinemia may bemore predictive of outcomes following colorectal cancerdiagnosis than a dietary pattern not associated with thispathway we previously derived the empirical dietaryindex for hyperinsulinemia edih score to assess thepotential of dietary patterns to influence insulinemia which has been extensively applied in large cohortstudies and found to be predictive of nonfasting cpeptide concentrations [ ] longterm weight gain risk of developing colorectal cancer other digestive system cancers and other cancers however the influence of dietary insulinemic potentialon cancer survival outcomes has not yet been evaluatedthe objective of the current study is to apply the edihscore in a large cohort of colorectal cancer patients toassess the insulinemic potential of their dietary patternsafter diagnosis and determine its influence on survivaloutcomesmethodsstudy populationwe used data from the nurses health study nhs andthe health professionals followup study hpfs twoongoing cohorts in the united states hpfs was initiatedin and enrolled male health professionalsbetween the ages of and years nhs initiatedin enrolled registered female nurses aged to years fig data on medical lifestyleand other healthrelated factors was collected at baselineand have been updated every years thereafter ethicalapproval for our study was provided by the harvardth chan school of public health and those of participating registries as required and the institutional reviewboards of the brigham and womens hospital studyparticipants provided consent by completing and submitting study questionnaires participants were free toterminate participation in the study at any timeassessment of diet and the empirical dietary index forhyperinsulinemia edih scorein both cohorts diet was assessed using a validated selfadministered food frequency questionnaire ffq thatassessed how often on average participants consumed astandard portion size of various foods in the past yearin the nhs diet was assessed in andevery years thereafter whereas in the hpfs diet wasassessed in and every years thereafter theedih score developed to empirically measure the insulinemic potential of whole diets using food groups hasbeen described in detail briefly thirtynine foodgroups were entered into stepwise linear regressionmodels to identify a dietary pattern most predictive ofplasma cpeptide levels the edih score represents aweighted sum of food groups with higher scores indicating hyperinsulinemic diets hyperinsulinemia andlower scores indicating low insulinemic diets the foodgroups contributing to lower edih scores are winecoffee fullfat dairy products whole fruit and green leafyvegetables whereas the food groups contributing to highedih scores arelowfat dairy products french frieslowenergy beverages cream soups processed meat redmeat margarine poultry nondark fish high tomatoesenergy beverage and eggs in the current study we calculated edih scores foreach participant based on the selfadministered ffqspostdiagnosis edih score was calculated based on the 0ctabung bmc cancer page of fig flow chart describing the flow of participants from the full cohorts to the final analytic sample in the nurses health study nhs andhealth professionals followup study hpfsfirst ffq returned at least months but not more than years after colorectal cancer diagnosis thus avoidingdiet assessment during active cancertherapy themedian time from diagnosisto postdiagnosis dietassessment was years prediagnosis edih score wascalculated based on the cumulative average of edihscores up to the last diet assessment before colorectalcancer diagnosis the median time from prediagnosisdiet assessment to diagnosis was yearspatients with colorectal cancer and mortality assessmentwhen a colorectal cancer diagnosis was reported duringthe previous years on the followup biennial questionnaires we requested permission to obtain hospital records and pathology reports blinded study physiciansthen reviewed these records and recorded data on tumorcharacteristics for nonrespondents the national deathindex was used to identify deaths and ascertain anydiagnosis of colorectal cancer that contributed to deathafter years of followup for disease diagnoses in nhs and to in hpfs we identified patients with pathologically confirmed colorectalcancer we excluded participants who died before in nhs or in hpfs had reported any cancerexcept nonmelanoma skin cancer before colorectalcancer diagnosis who died at diagnosis who did nothave prediagnosis diet or postdiagnosis diet patientswho did not complete a diet assessment between months and years after diagnosis or had diet assessedoutside of this period who had diabetes at colorectalcancer diagnosis and patients with stage iv or unknownstage at diagnosis therefore the current analysis included patients with stage i ii or iii colorectal cancerincluding participants from hpfs and from nhs fig deaths were ascertained throughreporting by family for persistent nonresponders wequeried the national death index with their names up 0ctabung bmc cancer page of to june for nhs and january for hpfs cause of death was assigned by blinded physicianscovariate assessmentboth cohorts assessed covariate data eg medical historylifestyle and health factorsthrough selfadministeredquestionnaires every years these factors included physical activity smoking habits alcohol intake multivitaminuse endoscopy status regular use of aspirin and othernonsteroidal antiinflammatory drugs nsaids familyhistory of colorectal cancer weight height menopausalstatus and postmenopausal hormone use only forwomenin both cohorts as previously described dietassessment was conducted every years [ ]statistical analysiswe categorized the edih score into quintiles withcohortspecific cutoffs then pooled the data for analysispersontime of followup was calculated from the dateof postdiagnosis diet assessment to death or to lastfollowup date january in hpfs or june innhs whichever was first we used the kaplanmeiermethod to generate survival curves by quintiles of edihscore and tested group differences highest vs lowestquintile using the logrank test for this test the edihscore was adjusted for total energy intake and bmi usingthe residual methodcox proportional hazards regression was used to calculate hazard ratios hrs of colorectal cancerspecificdeath or allcause death in edih quintiles quintile cutpoints were created separately by sex and applied in thepooled sample given that participants must survivefrom diagnosis until postdiagnosis diet assessment weused time since diagnosis as the underlying time scale toaccount for left truncation due to staggered entry thecox models were tested for the assumption of proportionality using timecovariate interaction terms andstratified by age sex and stage we fitted two models tothe data as follows model minimally adjusted modelincluded bmi demographic factors sex age at diagnosis and tumor characteristics stage subsite within thecolon grade of tumor differentiation model fullyadjusted model included all the covariates in model and postdiagnosisfactors packyears ofsmoking physical activity regular aspirin use pre topostdiagnosis weight change total alcohol intake andprediagnosis dietary pattern edih score testforlinear trend of risk across edih quintiles was performedusing the median postdiagnosis edih score in eachedih quintile as a continuous variable in the cox regression models and interpreting the pvalue of thisvariable as the pvalue for linear trendlifestyleto determine how changes in the insulinemic potential of diet before and after diagnosis influence survivalwe dichotomized pre and postdiagnosis edih scores atthe median and used to create a change variable withlow indicating a score below the median and high ascore above the median lowlow consistently lowdietary insulinemic potential before and after diagnosisie both scores below the median lowhigh patientsconsuming low insulinemic diets before diagnosis andmore hyperinsulinemic diets after diagnosis highlowpatients consuming hyperinsulinemic diets before diagnosis and then changed towards low insulinemic dietsafter diagnosis and highhigh patients who consistentlyconsumed hyperinsulinemic dietary patterns before andafter diagnosis we then applied these dietary patternchanges in multivariableadjusted cox models to examine risk of death from colorectal cancer and from othercauseschange preand postdiagnosisfrom postdiagnosis weightwe conducted exploratory subgroup analysesincategories of the following potential effect modifiers sexweightand prediagnosis edih score we categorized prediagnosisedih at the median median and ¥ median weightchange was calculated by subtracting prediagnosisweightthe continuousweight change variable was categorized as follows thosewho gained more than kg had a stable weight kg to kg or lost more than kg we also conducted subgroup analyses by time since diagnosis years ¥ years and age at diagnosis years ¥years tests of interaction between postdiagnosis edihscore and the potential effect modifiers were assessed byentering in the modelthe cross product of postdiagnosis edih score and the stratification variable andevaluated by the wald test all analyses were performedusing sas for unix all pvalues were two sidedresultscharacteristics of patients women with colorectal cancer after diagnosis are shown in table meanage at diagnosis was years and mean postdiagnosisbmi was kgm2 with of patients classified asoverweight or obese regarding disease stage hadstage i or ii and had stage iii disease during amedian followup of years there were allcausedeaths which included colorectal cancerspecificdeaths median overall survival by cancer stage was years for those with stage i disease years for thosewith stage ii and years for those with stage iiidisease fortyone percent of patients maintained astable weight between kg and kg between theprediagnosis and postdiagnosis period while lostmore than kg body weight and gained kgbody weight in the same period colorectal cancer patients with the most hyperinsulinemic dietary patterns 0ctabung bmc cancer page of table postdiagnosis characteristics of colorectal cancer patients by quintiles of postdiagnosis empirical dietary index forhyperinsulinemia edih score n characteristictotal population n quintiles of the empirical dietary index for hyperinsulinemia edih scoreabquintile quintile to to n n quintile to n quintile to n quintile to n female age at diagnosisdage at diagnosis by sexdfemalemalecurrent smoker packyears of smokingbody mass index kgm2overweight bmi ¥ obese bmi ¥ physical activity methweekcphysical activity methweekcd by sex femalemale nonalcohol drinkers regular aspirin use location of cancer in the colon proximal colondistal colonrectumunspecifiedstage at diagnosis stage istage iistage iiimedian survival time yearsmedian survival time by staged years stage istage iistage iiiweight change categoriesd stable weight to kggained more than kglost more than kg avalues are means sd for continuous variables and percentages for categorical variables and are standardized to the age distribution of the study populationbedih scores were adjusted for total energy intakecmetabolic equivalents from recreational and leisuretime activitiesdvalue is not age adjustedafter diagnosis quintile tended to have higher bodyweight and lower physical activity for example theaverage bmi among those classified in quintile was kgm2 and the average physical activity was methourweek compared with kgm2 and methourweek among those in quintile alsopatients consuming the most hyperinsulinemic dietarypatterns were less likely to have stage i disease and theyexperienced shorter survival times table patients consuming low insulinemic dietary patternshad higher intakes of wholegrains nuts vegetables wholefruits and coffee and lower intakes of refined grainscream soup eggs french fries butter margarine sugarsweetened beverages red meat and processed meat in 0ctabung bmc cancer page of table median 5th 95th percentile food and nutrient intake profiles of colorectal cancer patients by quintiles of postdiagnosisempirical dietary index for hyperinsulinemia edih scoretotal population n quintiles of the empirical dietary index for hyperinsulinemia edih scoreabquintile n quintile n quintile n quintile n quintile n foods servingsweekprocessed meatred meat highenergy sugary beverages lowenergy sugary beverages margarinebutterfrench friesnondark fisheggslowfat dairycream souprefined grainstomatopoultrydark fishfullfat dairycoffeeteawhole fruitfruit juicepotatoesgreenleafy vegetablesdarkyellow vegetablesother vegetablesnuts total alcohol intake drinksweek whole grainsnutrient profile total carbohydrates gdtotal protein gd branchedchain amino acids gd total fat gdtotal fiber gd avalues are means sd for continuous variables and percentages for categorical variables and are standardized to the age distribution of the study populationbedih scores were adjusted for total energy intaketerms of the nutrient profile resulting from this postdiagnosis dietary pattern patients consuming a lowinsulinemic dietary pattern had higher intakes of totalcarbohydrates and total fiber and lower intakes of total fattotal protein and branchedchain amino acids table kaplanmeier curves by quintiles of edih score areshown in fig with patients consuming a lowinsulinemic diet quintile experiencing better survivalfor colorectal cancerspecific and overall mortalitycompared to those consuming hyperinsulinemic dietsquintile in the multivariableadjusted analyses wefound that a hyperinsulinemic postdiagnosis dietarypattern was associated with higher risk of colorectalcancerspecific mortality and allcause mortality table 0ctabung bmc cancer page of fig kaplanmeier curves of a colorectal cancerspecific and b overall survival among patients with colorectal cancer by quintile of postdiagnosis empirical dietary index for hyperinsulinemia edih score logrank pvalues were calculated to test group differences quintile vs and adjusted for postdiagnosis total energy intake and postdiagnosis body mass indexcomparing colorectal cancer patients classified in the highestedih quintile to those in the lowest quintile there was a higher risk of colorectal cancerspecific death hr 95ci ptrend and a higher risk of allcause death hr 95ci ptrend afteraccounting for prediagnosis dietary insulinemic potentialamong other confounding variables table in relation to changes in the insulinemic potential ofthe diet before and after diagnosis patients whoconsumed a more hyperinsulinemic dietary patternconsistently before and after diagnosis were at higherrisk of dying from colorectal cancer hr ci and from other causes hr ci compared to patients who consistentlytable hazard ratios ci for colorectal cancerspecific and allcause mortality among patients with colorectal cancer byquintile of postdiagnosis edih scorestatistical modelcolorectal cancerspecific mortalityquintiles of the empirical dietary index for hyperinsulinemia edih scorequintile quintile quintile quintile ptrendquintile deathspatients aliveminimallyadjusted model reference fully adjusted model reference allcause mortalitydeathspatients aliveminimallyadjusted model reference fully adjusted model reference the minimallyadjusted models was adjusted for age at diagnosis postdiagnosis body mass index total energy intake sex race year of diagnosis cancer stagegrade of tumor differentiation and location of primary tumor within the colon the fullyadjusted model was additionally adjusted for postdiagnosis physicalactivity postdiagnosis pack years of smoking postdiagnosis regular aspirin use weight change pre to postdiagnosis postdiagnosis total alcohol intake andprediagnosis edih score 0ctabung bmc cancer page of fig hazard ratios for the association of change in dietary insulinemic potential between prediagnosis diet and postdiagnosis diet and risk ofdying form colorectal cancer crcsurvival and from all causes combined overall survival edih scores were dichotomized at the median lowlow the reference category represents participants who persistently consumed low insulinemic diets below the median edih from pre to postdiagnosis period lowhigh are those who changed from low insulinemic diets towards more hyperinsulinemic diets highlow represents thosewho changed from consuming hyperinsulinemic diets prior to diagnosis towards consuming low insulinemic diets after diagnosis whereas highhigh represents those who persistently consumed hyperinsulinemic diets prior to diagnosis and after diagnosis the number of deaths patientsalive in the four categories were as follows crcsurvival lowlow lowhigh highlow highhigh overallsurvival lowlow lowhigh highlow highhigh models were adjusted for age at diagnosis postdiagnosisbody mass index total energy intake sex race year of diagnosis cancer stage grade of tumor differentiation location of primary tumor withinthe colon postdiagnosis physical activity postdiagnosis pack years of smoking postdiagnosis regular aspirin use weight change pre to postdiagnosis postdiagnosis total alcohol intake and prediagnosis edih scoreconsumed a low insulinemic dietary pattern before andafter diagnosis fig in subgroups of potential effect modifiers risk of colorectal cancerspecific mortality was significantly elevatedamong women and among those who lost body weightthose who were consuming a hyperinsulinemic dietarypattern before diagnosis and those younger than years for these subgroup analysesinteractions werestatistically significant only for sex in allcause mortalitytable discussionin the current study we showed that habitual consumption of hyperinsulinemic dietary patterns after colorectalcancer diagnosis or consumption of a hyperinsulinemicdietary pattern consistently before and after diagnosiswas associated with higher risk of dying from colorectalcancer and from all causes combinedthe insulinemic potential of diet was first estimatedby the insulin index which is based on a conceptsimilar to the more widely used glycemic index thatcharacterizes carbohydratecontaining foods accordingto their ability to raise blood glucose concentrationspostprandially compared with a reference food glucoseor white bread though carbohydrate content isone important factor influencing insulin response foodscan also stimulate insulin secretion in a carbohydrateindependent manner the insulin index directly quantifies the postprandial insulinemic potential of a food andtakes into account foods with a low or no carbohydratecontent it is important to understand that theinsulin index which was used in most previous studiesof dietary insulinemic potential and colorectal cancersurvival is conceptually and technically different fromthe edih and essentially uncorrelated spearman r the principle ofthe insulin index is how aparticularfood item stimulated insulin secretionindependent of underlying insulin resistance whereasthe edih is primarily driven by insulin resistance forcolorectal cancer the only other paper using the edihwas on cancer incidence both the insulin index and glycemic index assess thepostprandial shortterm effects of the diet unlike theedih score which predicts integrated insulin exposureie both fasting and nonfasting based on habituallongterm dietary intake postdiagnosis insulinindex and insulin load have been linked to higher risk ofdying from colorectal cancer [ ] higher dietary insulin load and insulin index after diagnosis of colorectalcancer were associated with increased risk of colorectalcancerspecific and overall mortality the association of postdiagnosis glycemic indices with colorectal 0ctabung bmc cancer page of table subgroup analyses of the association between dietary insulinemic potential and colorectal cancerspecific and allcausemortalitysubgroupptrendpinteractionquartile quartile quartile deathspatientsaliveedih quintilesquartile colorectal cancerspecific mortalitysexmenwomenweight change post minus prediagnosis weightstable weight to kgweight gain kgweight loss kgprediagnosis edih score median¥ medianage group at diagnosis years¥ yearstime since diagnosis years¥ yearsallcause mortalitysexmenwomenweight change post minus prediagnosis weightstable weight to kgweight gain kgweight loss kgprediagnosis edih score median¥ medianage group at diagnosis years¥ yearstime since diagnosis years¥ years ref ref ref ref ref ref ref ref ref ref ref ref ref ref ref ref ref ref ref ref ref ref models were adjusted for age at diagnosis postdiagnosis body mass index total energy intake sex race year of diagnosis cancer stage grade of tumordifferentiation and location of primary tumor within the colon postdiagnosis physical activity postdiagnosis pack years of smoking postdiagnosis regular aspirinuse weight change pre to postdiagnosis and postdiagnosis total alcohol intake and prediagnosis edih scorecancer prognosis has been inconsistent whereas onestudy found higher risk of colorectal cancer recurrenceand death associated with higher glycemic load but nothigher glycemic index another found no associationbetween glycemic load or glycemic index and colorectalcancer survival glycemic scores are primarilyreflective ofthe postprandial glucose responses ofcarbohydratecontaining foods whereas the edih scoredirectly reflects insulin increases induced by componentsof the dietary pattern that may or may not be contributing to calories eg coffee current study findingstherefore suggest that the direct effect of the diet on 0ctabung bmc cancer page of insulin may be more important than the effect of diet onglucose for colorectal cancer prognosis though theglycemic index is a measure of the shortterm postprandial effect of the diet on glucose concentrations it ispossible that such a habitual dietary pattern could overtime lead to sustained hyperinsulinemia and insulin resistance which could then mediate colorectal cancerprognosis however a previous study in these cohortsdid not observe an association between an overall lowcarbohydrate diet score and colorectal cancer or overallmortality although those who consumed a plantrichlowcarbohydrate diet which emphasized plant sourcesof fat and protein with moderate consumption of animalproducts had lower risk of colorectal cancerspecificmortality insulin is a growth factor and major regulator of cellmetabolism and its effects in target cells are mediatedby the insulin receptor a transmembrane protein withenzymatic activity evidence suggest that insulinstimulates growth mainly via its own receptor and notthe igf1 receptor and that in many cancer cells theinsulin receptor is overexpressed and the a isoformwhich has a predominant mitogenic effectis morerepresented than the b isoform the metabolicpathway stimulated by the activated insulin receptor toregulate glucose protein and lipid metabolism involvesthe pi3kakt pathwaycharacteristicsprovide a selective growth advantage to cancer cellswhen exposed to insulin therefore all conditions ofhyperinsulinemia both endogenousdiabetes metabolic syndrome obesity and exogenouseg hyperinsulinemic diets which also influence someof the endogenous conditions [ ] willincreasecancer risk and mortality theseegtypefor most ofalthough interactionsthe subgroupanalyses were not statistically significant some of thefindings merit some discussion the associations werestronger among women than among men which may berelated to severalfactors the larger sample size andstatistical power in our evaluation of women potentialconfounding with age as women were younger on average than men and a true biological interaction basedupon endocrine and associated metabolic factors wealso observed that there were worse outcomes amongpatients who lost weight than among those who maintained a stable weightto postdiagnosisperiod which may be consistent with complications ofprogressing disease leading to poor diet intakefrom premajor strengths of our study include the use of afoodbased edih score that is correlated with circulating cpeptide concentrations [ ] we had access tocomprehensive pre and postdiagnosis data on diet andimportant covariates which reduces the potential for residual confounding and recall bias our findings alsoaccounted for potential bias from staggered entry due todifferences between participants in the time betweendiagnosis and postdiagnosis diet assessment limitations to be considered in interpreting our findings include potential measurement error in the selfreporteddietary and lifestyle data though prior studies in thehpfs and nhs that evaluated the relative validity offfq data have shown reasonably good correlations between ffq and diet records [ ] though we adjusted for several potential confounding variables ahyperinsulinemic dietary pattern may be associated withother factors not included in the current study therefore we cannot completely rule out confounding byunmeasured variables given that we did not have information on cancer treatment which could influence dietary choices of cancer patients or modify the diet andsurvival association we adjusted all analyses by cancerstage at diagnosis which is the principal determinant ofcolorectal cancer treatmentin this large prospective study a higher edih scorereflecting higher insulinemic potential of the diet wasassociated with higher risk of death from colorectalcancer and from all causes taken together our resultssuggest that this association may be mediated partlythrough mechanisms involving hyperinsulinemia interventions with dietary patterns to reduce insulinemia mayenhance survivorship among colorectal cancer patientsabbreviationsbmi body mass index ci confidence interval edih empirical dietary indexfor hyperinsulinemia score ffq food frequency questionnairehpfs health professionals followup study hr hazard ratio methourweek metabolic equivalent hours per week nhs nurses health study nsaids nonsteroidal antiinflammatory drugs pi3kakt phosphatidylinositol kinaseprotein kinase b sas® statistical analysis software®acknowledgementswe would like to thank the participants and staff of the nurses health studyand health professionals followup study for their valuable contributions asw | Colon_Cancer |
specialty sectionthis was submitted tomolecular and cellular oncologya section of the frontiers in cell and developmentalbiologyreceived april accepted july published august citationchen w yang j fang h li l andsun j relevance functionof lincror in the pathogenesisof cancerfront cell dev biol 103389fcell202000696cancer is a serious disease that aï¬ects human health being one of the main causes of death all overthe worldwide according to research in of new tumor cases and of the cancerassociated deaths occurred in lowincome and developing countries kumar and sharawat noorolyai owing to a shortage in eï¬ective screening methods and lack of identiï¬cationof early symptoms most patients were already in advanced stages when they were diagnosedwith cancer bray koo additionally some clinical studies have shownthat polarity and adhesion of cancer cells was decreased leading to heir increased mobility andinvasion which is a key step in the development of cancer yan therefore studies haveshown that the high mobility of cancer cells is the main factor leading to high mortality rates inpatients with cancer currently there are many ways employed in the treatment of cancer includingsurgery radiotherapy chemotherapy biotherapy and targeted therapy nie howeverin the past years the survival rate of patients with cancers remains dismal nakashima abbreviations bc breast cancer cenas competing endogenous rna emt epithelialmesenchymal transition hcchepatocellular cancer ipscs induced pluripotent stem cells lincror long intergenic nonprotein coding rna regulatorof reprogramming lncrnas long noncoding rnas pc pancreatic cancerfrontiers in cell and developmental biology wwwfrontiersinaugust volume 0cchen relevance function of lincrorin the process of developing human antitumorthereforestrategiesto ï¬nd new earlyitbiomarkers and thus identify potential regulatory mechanisms toimprove the survival rate of patients with cancersis particularly importantover the past decades ncrnas constitute more than of the rnas made from the human genome but mostof the known noncoding rnas ncrnas havebeen discovered and remain largely unstudied bhan slack and chinnaiyan transfer rna trna and ribosomal rna rrna constitute themajority of ncrnasfollowed in abundance by messengerrnas mrnas thus the remaining ncrnas includingcircular rna circrna small nuclear rna snrna smallnucleolar rna snorna microrna mirna and long nonfor ¼ ofcoding rna lncrna together accounttotalncrna despite their low abundancethese ncrnas havebeen reported to play critical roles in transcription posttranscriptional processing and translation such as epigeneticsposttranscriptional regulation chromatin modiï¬cation andregulation of the cell cycle huarte kondo peng 2017b additionally because ncrnas can be packagedinto extracellular vesicles ev including exosomes meldolesi they have been shown to provide a mechanism forintercellular communication through the transfer of mirnaand lncrna to recipient cells both locally and systemicallysun it is important to note that the expressionof ncrnas their posttranscriptional modiï¬cation particularlylncrnas and their subcellular distribution have been shownto be important to when assigning their potential functionpalazzo and lee recently nextgeneration sequencingand bioinformatics technology have revealed that circrnasplay crucial role in diagnosis and prognosis of various diseasespamudurti brieï¬y circrnas are singlestrandedtranscripts generated by backsplicing jeck and sharpless with covalently linked headtotail closed loop structures withneither 5cid483cid48 polarity nor a polyadenylated tail memczak that range in length from a few hundred to thousandsof nucleotides and are widely expressed in mammals therebyshowing higher stability compared to that in linear rnas chenj and exhibiting a celltype or developmentalstagespeciï¬c expression pattern barrett and salzman wang j j many functions of circrnas have alsobeen identiï¬ed including their role as mirna sponges bindingto rnabinding proteins and protein decoys and functioningas regulators of transcription hansen du yang y interestingly many circrnas havebeen shown to be dysregulated in pathophysiological processesand circrnas are known to regulate the expression of geneby acting as mirna sponges in a mechanism that is termedas competitive endogenous rna cerna mechanism zheng wang j j for example circmto1have been demonstrated to harbor conventional mirna bindingsites and has been identiï¬ed as an inhibitor of mirna9 inhepatocellular carcinoma hcc han additionallyour previous study has demonstrated that mirna plays arole in limiting the development of liver ï¬brosis by markedlyblocking the activation and proliferation of hepatic stellatecells hscs suggesting that mirnas might be involved inthe development and progression of several forms of cancersyang j yang of notelncrnaswhich are mainly transcribed by rna polymerase ii are anew kind of ncrna that are longer than nucleotidesma owing to the lack of open reading frameslncrnas have extremely limited or no protein coding capacityruan li j these new regulatorswere initially regarded as transcriptional noise with no speciï¬cbiologicalfunctions kim and sung recently ourlaboratory found that epigenetic silencing of lncrna anrilpromoted the progression of liver ï¬brosis thereby indicating thatlncrnas were associated with the progression of cancers yang interestingly increasing evidence have shown thatcellular events including diï¬erentiation proliferation invasionapoptosis and migration have all been associated with lncrnasguttman additionally there has been new evidencesuggesting that lncrnas may regulate a variety of biologicaland disease processes from gene transcription and translationto posttranslational modiï¬cations davalos and esteller pang more importantlylncrnas have beenreported to be used as tumor suppressor genes or oncogenesthus aï¬ecting the proliferation and metastasis of various typesof tumors during tumorigenesis chen q n lu subsequent studies have demonstrated thatlncrnas may serve as cernas for mirnas and in chromatinremodeling during the development of cancers huang wang c j figure illustrates the functionsof lncrnas at the molecular level regarding certain cancerassociated human lncrnasit was demonstrated that lincror was demonstrated to be predominantly upregulated intumors peng 2017a the abnormal expression of lincror in tumors has been suggested to be one of the mainleading factors driving the development the main ways torevert this eï¬ect would be to aï¬ect cell growth migrationand invasionthus leading to the inhibition of epithelialmesenchymal transition emt enhancement of the sensitivityto chemotherapy etc chen 2016a zhao forexample the expression level of lincror in hcc tissues wasinhibited compared with the adjacent tissues at the same timethe downregulation of lincror was linked to the aggressiveprocess of the disease in patients with hcc furthermore theability of migration and invasion of hcc cells may be delayedby the low expression level of lincror in this review weattempted to introduce the latest research on the biologicaleï¬ects potential clinical applications and molecular mechanismsof lincror in human tumors and discuss its prognostic andtherapeutic valuesoverview of lincrorlincror isamong lncrnasand importantcarcinogenic kb lncrna located in chromosome which was initially identiï¬ed as a highly expressed transcriptof pluripotent and embryonic stem cells chen 2016bstudies found that the octamerbinding transcription factor a novelfrontiers in cell and developmental biology wwwfrontiersinaugust volume 0cchen relevance function of lincrorfigure paradigms for the function of long ncrnas recent studies have identiï¬ed a variety of regulatory paradigms for the mechanism by which long ncrnasfunction many of which are highlighted here transcription from an upstream noncoding promoter pink can negatively or positively affect the expression ofthe downstream gene purple by inhibiting the recruitment of rna polymerase ii or inducing chromatin remodeling respectively an antisense transcript orangeis able to hybridize to the overlapping sense transcript purple and block the recognition of the splice sites by the spliceosome thus resulting in an alternativelyspliced transcript alternatively hybridization of the sense and antisense transcripts can allow dicer to generate endogenous sirnas by binding to speciï¬cprotein partners a noncoding transcript blue can modulate the activity of the protein serve as a structural component that allows the formation of a largerrnaprotein complex or alter where the protein localizes in the cell long ncrnas green can be processed to yield small rnas such as mirnaspirnas and other less wellcharacterized classes of small transcriptsoct4 srybox transcription factor sox2 and nanoghomeobox nanog key pluripotency factors could regulatelincror wang howeverlincror was alsofound to be expressed in several ans including lungliver breast and colon since its discovery research in thisï¬eld has been extensively expanded during the past yearsrevealing the important role of lincror in tumorigenesislincror has been suggestedadditionally upregulation ofto mediate the reexpression offetal and cardiomyocytehypertrophyrelated genes wang li inmany reportsrecent years have revealed thatlincror is positively correlated with the clinicopathologicalcharacteristics and poor prognosis of tumorsincluding thestages of advanced tumor node metastasis tnm positivelymph node metastasis lnm and lower survival rate buthigher recurrence rateregarding lincror and tumorigenesisthe upregulation ofcurrent evidence have strongly indicated thatlincrormay exert an impact on a variety of cancers pan furthermore both tumorigenesis and metastasis havebeen shown to be induced by lincror via activation of theemt in various cancers hou huang zhan for example lincror was demonstratedto be upregulated thereby promoting emt in hcc li j besides it was also reported that selfrenewal anddiï¬erentiation of glioma stem cells was signiï¬cantly aï¬ectedby lincror zhang feng moreimportantly the chemoresistance of pancreatic cancer pcand breast cancer bc li as well as radioresistance of colorectal cancer crc cells were observed to beelevated by lincror yang p moreover lincror has also been shown to exert a signiï¬cantly eï¬ect onthe stem celllike characteristics and tumorigenic potential ofpc recentlyit was also reported that lincror could beused as a biomarker in the ï¬eld of diagnosis and prognosisof bc and oral cancer arunkumar zhao notably increasing studies showed that lincror couldbe used as a cerna thus exerting its impact in the posttranscriptional network of tumor pathogenesis for examplein triplenegative bc lincror has been shown to serve asa cerna therefore promoting the migration and invasion ofbc cells signal overall lincror is a typicallncrna that plays important regulatory roles in interactionwith mirnas and maintenance of stem cell pluripotencytriggering the emt as well furthermore lincror has alsobeen involved in various key roles under hypoxia and in thepromotion of tumorigenesis figure therefore lincrormay be considered as an oncogene aï¬ecting the progressionof tumor and a promising predictor for the poor prognosis inpatients with cancer the transition of lincror from basicresearch to clinical application requires further investigations asearly as possiblefrontiers in cell and developmental biology wwwfrontiersinaugust volume 0cchen relevance function of lincrorfigure lincror is a typical lncrna that plays important regulatory roles in interacting with mirnas and maintaining stem cell pluripotency as well astriggering the emt as well lincror is also involved in various key roles under various stresses and in epigenetic regulationregulatory role of lincror invarious types of cancerincreasing evidence has shown that the lincror was abnormalexpression in many cancers and its dysregulation was associatedwith cellular functions fu spinelli additionally studies found that the expression level of lincror was substantially upregulated in samples of papillarythyroid carcinomas ptcs and ptcs cell lines as well as inmetastatic ptcs samples and ptcs cell lines zhang simultaneously cell migration and invasion could be regulatedby lincror via aï¬ecting emt pastushenko and blanpain more importantly studies demonstrated that lincrorwas abnormally expressed in several cancers and led to elevatedthe invasion and metastasis of cancer cells to promoting theprogression of tumors hashemian li 2020bcthis review summarizes the status of lincror research invarious human cancers and discusses its mechanism and clinicalsigniï¬cance in the development and progression of tumor theexpression pattern functional role and regulatory mechanism oflincror are summarized in table and depicted in figure breast cancerbc which accounts for a quarter of all female cancer casesis the most commonly diagnosed cancer and the leading causeof cancerassociated deaths among women worldwide li z in it was estimated that there would be million or so newly diagnosed cases of female bc bray hannafon the main risk factors forbc which is the diï¬cult to change due to prolonged exposureto endogenous hormones is diï¬cult to control rudel bray however comprehensive treatmentapproaches have resulted in relatively good clinical outcomesfor some patients with bc rudel goel kumler nevertheless it has been reported that aboutonethird of the patients with bc have the potential for cellmetastasis chemotherapy resistance and even recurrence goel kumler hence there is an urgent need todevelop new therapies targeting various molecular mechanismsof tumorigenesis for the treatment of bcthe level ofthe level ofhou the expression ofinvestigated the expression level of lincrorin patients hou their results revealedlincror was increased in bc tissuesthatmoreoverlincror in theperipheral blood ofthe patients with bc was shown tobe closely related to tnm phase and lnm in additionthe woundhealing assay showed that overexpression of lincror increased bc cells mcf10a mobility transwell assayrevealed that lincror overexpression remarkably increased themigration ability hou more importantly theyfound that ectopic expression of lincror induced an emtprogram in mcf10a cells fluorescence activated cell sorteranalysis demonstrated that the subpopulation of the stem cellphenotype cd44highcd24low was elevated in mcf10a cellstransfected with lincror plasmid mechanistically the resultsof bioinformatic analysis and rna immunoprecipitation analysisfrom hou demonstrated thatlincror functions asa cerna to regulate mir205 activity toward prevention ofthe degradation of transcripts of mir205 target genes suchas zeb1 and zeb2 from degradation additionallyit wasshown that the expression levels of mir205 members weredecreased upon lincror overexpression in mcf10a cellshou more importantly zhao recentlydemonstrated that crisprcas9generated brca1knockdownadiposederived stem cells stimulated a more aggressive behaviorfrontiers in cell and developmental biology wwwfrontiersinaugust volume 0cchen relevance function of lincrorlateillateoaglatenahzlatenehclatecillateihzlateilnusdnanaylatenapalategnahzlateuohsecnereferbezrmrorcniilmkppbtprmrorcniilgonanrmrorcniilnirehdacehzerorcnilsosfdmnlegatsmnttmeinosavnitmeinosavninoitargminoitarefilopicnegocnoldetaugerpuamoncraciraulllecotapehllseuceomyrotaugerllnoitaerroclacniilcleorlanoitcnufleorinosserpxesepytrecnacsrecnacnamuhnirorcnlielbatirmrorcnilevruccormnlegatsmntnoitargmiissotpopanoitarefilorpicnegocnoldetaugerpurecnactsaerbtmeinosavnipbezrorcnilliavvrusroopmnlegatsmntnoitargminoitarefilorphtwicnegocnoldetaugerpurecnaccitaercnapmxofprmrorcniilncsfrmrorcniprmrorcniililirmrorcnilecnatsserigurdtmeinosavnitmelecycllecinosavniliavvrusroopmnlegatsmntnoitargmiissotpopanoitarefilorpicnegocnoldetaugerpurecnacdoryhtisosfdmnlegatsmntinoitargmecyclllecissotpopaicnegocnoldetaugerpurecnacgnulecnatsseriyparehtodaritmeinosavnisosfdmnlegatsmntnoitargmiissotpopanoitarefilorpicnegocnoldetaugerpurecnaclatcerooclin bc cells than wildtype adiposederived stem cells zhao therefore we believe that crisprcas9 may beused to in the treatment of bc by inhibiting the expressionof lincror during the progression of bc conclusively thelincrormirnas axis has been reported to closely aï¬ect theoccurrence and development of bcpancreatic cancerpc is the fourth most common cause of cancerrelated mortalityworldwide leading to approximately deaths annuallysiegel sabater the year relativesurvival of patients with pc remained at approximately for siegel hence pc has been proposedto be one of the top two cancers in terms of fatalities in thenext decade rahib surgical resection remainsthe exclusive potential curative treatment xiong however approximately half ofthe patients present withmetastasis at the time of diagnosis missing the opportunityfor an eï¬ective treatment vincent xiong a growing body of literature has demonstrated that bothmetastasis and limited eï¬ective biomarker for the diagnosis andtreatment are the main obstacles for the eï¬cient medical therapyof pc vincent boj basuroy thus it is an absolute necessity to identify potential biomarkersand therapeutic targets in pczhan have highlighted the oncogenic eï¬ectsof lincror in the initiation and progression of pc theirstudy demonstrated that the level of lincror was signiï¬cantlyelevated in pc tissues zhan moreoverthewoundhealing assay and boydens chamber assay results showedthat lincror silencing reduced the migratory capability andmetastasis of pc cells zhan another study bychen showed that the proliferation rates of shrorcellsin which the level of lincror was suppressed were evidentlylower than those of shnccells this result was conï¬rmed bycolony formation assay suggesting that lincror accelerated thegrowth of pc cells chen 2016a interestingly silencingof lincror was shown to result in increased levels of theepithelial markers ecadherin and αcatenin and decreased levelsof mesenchymal markers ncadherin and vimentin indicatingthat lincror plays an important role in the regulation ofemt in pc cells zhan chen moreimportantly microarray analysis identiï¬ed zeb1 as potentialtarget gene of lincror further the expression of lincrorand zeb1 were observed to be negatively correlated with thatof p53 suggesting that lincror might mediate migration andmetastasis in pc cells may partly via activation of zeb1 throughthe inhibition of the expression of p53 zhan interestingly the ï¬uorescence in situ hybridization and luciferasereporter assay results showed that the expression of lincrorwas demonstrated to be negatively correlated to that of mir145mir145 can induce posttranscriptional silencing of its targetedgenes by binding to the mrna utr or lincror speciï¬csites indicating that lincror can act as a cerna to decreasemir145 in pc cells thereby activating expression of nanogthus leading to the proliferation of pancreatic cancer stem cellspcscs gao additionally li furtherfrontiers in cell and developmental biology wwwfrontiersinaugust volume 0cchen relevance function of lincrorfigure underlying molecular mechanisms of lincror in multiple cancers a lincror binds to mir205 to upregulate zeb2 while it also regulated theexpression of emt markers b lincror could interact with mir145 to inhibit fscn1 and upregulated emtassociated proteins while it decreases g0g1 arrestand facilitated drug resistance c lincror facilitated emt through upregulate ezhz and regulated zeb2 by competitively binding to mir145 and zeb2overexpression leads to increased emt in addition lincror binds to mir8765p to upregulate foxm1 d lincror downregulated through emt production andrepress the expression of mir145 e lincror binds to mir68333p while also regulating the process of emt f lincror upregulated zeb1 to attenuate theexpression of p53 while also decreasing the expression of mir145 to increase the level of nanog and reduce that of mir124 to suppress pkm2 detailedmechanisms of lincror in other cancers are provided in the reviewproved that the impact of lincror can be partly reversed byoverexpression of mir124 consistently lincror was observedto exhibited a negative correlation with the expression of mir li hence a lincrormir124ptbp1pkm2axis was identiï¬ed in pc shedding new light on the lncrnabased diagnosis and therapeutic approaches in pc li 2020b notably recent studies showed that pc cellderivedevs could be used as eï¬ective carriers of paclitaxel to theirparental cells thereby bringing the drug into cells through anendocytic pathway and increasing its cytotoxicity saari additionally it was demonstrated that vesiclecontainingncrnas could serve as evassociated pc detection markersworst thus the presence of lincrors in evs frompatients with pc could serve as a potential diagnostic biomarkerand a novel target for the therapy of patients with pc this isworthy of further and wider research attentionhepatocellular carcinomaas the sixth most international commonly occurring cancer in hcc has become the fourth cause of cancerassociateddeaths worldwide it has been estimated that new casesand deaths will occur each year bray brieï¬yhcc has been reported to account for of all the livercancer cases half of which have been detected in china omata bray as such hcc poses a huge threatto the worldwide health especially that of the chinese peopleomata about of the patients is expected torecrudescent within years after hepatectomy and of thepatients will die from this tumor vigano thereforeon the basis of studying the pathogenesis of hcc it is apparent tolook for more eï¬ective molecular markers and therapeutic targetsfor the management of hccli c and chen reported that theexpression level of lincror was obviously elevated in hcctissues and four cell lines compared to the corresponding nontumor tissues and normal liver cell lines respectively suggestingthat lincror might be critical regulator in the progression ofhcc furthermore biological function assay demonstrated thatlincror could play promoting role in regulating migration andinvasion of hcc chen moreover downregulationof lincror could result in a signiï¬cant increase in g1g0phase and an obvious decrease in s phase li c more importantly silencing of lincror could lead to theincreased expression of ecadherin and decreased expressionlevel of ncadherin in hcc cell lines li c furtherconï¬rmed that lincror could bind to the zeste homolog ezh2 thereby aï¬ecting the expression of ecadherin furtherindicating that lincror could regulate the progression of emtmoreover lincror was further determined to be associatedwith dna repair currently mounting studies have identiï¬edreliable indicators of dna damage such as phosphorylatedhistone h2ax γh2ax chen uncover thatfrontiers in cell and developmental biology wwwfrontiersinaugust volume 0cchen relevance function of lincrorlincror could obviously decrease theoverexpression ofexpression level of γh2ax illuminating the suppressive eï¬ectsof the overexpression of lincror on dna repair in hccfurther research demonstrated that lincror could interactwith mir and dramatically downregulate the expression ofmir in hcc cells li c the abovementionedresults revealed that lincror might play a promoting role inthe proliferation migration invasion and emt of the hcc cellwhich was contrary to the inï¬uence of mir enrichmentli c it suggested that overexpressed mircould eï¬ectively reverse the promotion of hcc tumorigenesisinduced by the overexpression of lincror li c liand his colleagues proposed a mechanistic model that lincrorpromotes hcc tumorigenesis and autophagy partly throughnegatively regulating the expression of mir aside fromthat it has been reported that mir145 represses emt tumormigration and invasion by directly targeting the utrs ofzeb2 in the tumor the decrease in mir and increase inzeb2 can obviously reversed the inhibition of cell migrationand invasion mediated by the lincror knockdown thereforeit was suggested that targeting the lincrormir145zeb2axis might represent a novel therapeutic application in hccli c similarly zhi similarly showedthat the migration and invasion of cells was reduced by theknockdown of lincror moreover they further conï¬rmedthat foxm1mediated activation of lincror contributes tothe poor sensitivity of hcc cells to sorafenib via partiallyregulating the mir8765pfoxm1 axis which forms a positivefeedback loop further evaluation of the regulatory mechanisminvolving this axis may provide new insights for exploringa potential therapeutic strategy for the management of hcczhi consequently these studies may oï¬er newinsights regarding the pathology of hcc and provide potentialstrategies for lncrnadirected treatment however both thein vivo inï¬uence and other underlying mechanisms of lincrorstill remain to be determined and clariï¬ed in the future researchthe prognosiscolorectal cancerthere are approximately million new crc cases and crcrelated deaths worldwide each year thus making crc thethird most common cancer in the world torre although the treatment of crc has signiï¬cantly improvedin recent decadesremains unsatisfactoryespecially in case of advanced tumors with distant metastasesbogousslavsky torre current studiesresults showed that approximately of cases with crchave synchronous liver metastases during the time of diagnosiskawaguchi these patients have inherently lowsurvival rates of less than within years with an even worseprognosis hu kawaguchi thus there isan urgent need to better understand the progression of crc andto identify novel and sensitive biomarkers for the diagnosis andtreatment of patients with crcyang detected the expression of lincror in crctissues compared to normal tissues by using qrtpcr theyfound that the expression of lincror was remarkably increasedin crc tissues compared with normaltissues similarlylincror was shown to be overexpressed in ï¬ve crc cell linesyan and sun li 2020a then they also performeda series of functional assays to clarify the biological eï¬ects ofthe aberrant expression of lincror on proliferation viabilityapoptosis migration and invasion of crc cells knockdownof lincror was shown to eï¬ectively inhibit the proliferationof crc cells whereas its overexpression obviously increasedthe proliferative capacity of crc cells accordingly silencing oflincror strongly inhibited the migratory and invasive abilitiesof crc cells compared with that in the control cells li 2020a in contrast the migratory and invasive ability of cells wasactivated following the overexpression of lincror the mts345dimethylthiazol2yl53carboxymethoxyphenyl24sulfophenyl2htetrazolium assay results showed thatthelincror could enhance the viability ofoverexpression ofcrc cells furthermore the ï¬ow cytometric analysis resultsrevealed that the percentage of apoptotic cells in lincroroverexpression group was reduced by ± indicatingthat the overexpression of lincror could inhibit apoptosisin the crc cell lines li 2020a more importantly arecent study revealed the role of lincror in the emt it wasrevealed that the upregulation of lincror could increase theexpression of ncadherin and vimentin as well as decrease thelevel of ecadherin leading to the promotion of the progressionof emt zhou yan and sun meanwhilethe high expression of lincror in crc was also conï¬rmedby hu mechanistically li 2020a provedthat that lincror could bind to mir68333p which wasdetermined to be signiï¬cantly downregulated in crc tissuesadditionally a negative correlation was exhibited between theexpression of lincror and mir68333p in bc tissues antiago2 rna immunoprecipitation assay further conï¬rmed theseresults li 2020a besides rescue assays demonstratedthat downregulation of mir68333p could partly reversed theinhibition of tumorigenesis induced by lincror knockdown inbc cells li and his colleagues uncovered that lincror exertedits oncogenic role through negatively regulating the expressionof mir68333p during the progression of crc which mightgive new insights into molecular diagnosis and treatment li 2020a in addition li 2020a further uncoveredthat lincror could mediate the expression level of smc bysponging mir68333p in crc cells thus promoting crcprogression as for the eï¬ects of lincror on radiotherapyresistance yan and sun showed that overexpression oflincror increased the ability of crc cells for radiotherapyresistance collectively these ï¬ndings indicated that lincrormight be engaged in the metastatic process of crc cells andcould promote the development of crc through a variety ofmolecular mechanismslung cancerlung cancer is the leading cause of cancerrelated deathsworldwide bray nonsmall celllung cancernsclc accounts for about of the lung cancer typesincluding squamous cell carcinomalung cancerand lung adenocarcinoma herbertz bray brainard and farver although there are variousapproaches for its diagnosis and treatments the 5year overallsurvival os rate for patients with advanced lung cancer is lesslarge cellfrontiers in cell and developmental biology wwwfrontiersinaugust volume 0cchen relevance function of lincrorthan zhou therefore in order to carry outan early diagnosis and treatment of lung cancer the search forvaluable and eï¬cient tumor markers is very urgentin recent yearslincror has appeared as an importantregulator oflung cancer research from qu demonstrated that the expression of lincror was increasedin nsclc tissues compared to that in the normal tissuesthe overexpression of lincror was shown to be closely relatedto the poor prognosis of lnm histological grade and stageof tnm pan qu another study bypan demonstrated that the decreased expressionof lincror could obviously impair the proliferative capacityof lung adenocarcinoma lad cells cause a g0g1 phasearrest and increase the ratio of apoptotic lad cells moreoverdownregulation of lincror substantially inhibited the invasiveand metastatic ability of lad cells meanwhile forced expressionof lincror was observed to reduce the expression of ecadherinand βcatenin which are the characteristic biomarkers ofepithelial cells whereas it increased the expression of ncadherinand vimentinthus displaying a mesenchymal phenotypeconversely downregulation of lincror was demonstrated toresult in increased the expression of epithelial markers anddecreased the expression of mesenchymal markers this resultuncovered the fact that the prometastatic eï¬ects of lincrorwere induced by the regulation of the expression of a number ofgenes involved in cell metastasis and emt progress meanwhileoverexpression of lincror was shown to enhance the resistanceof lad to docetaxel dtx pan suggestingthat lincrorinduced resistance of lad cells to dtx andemt through regulation the expression of mir145 whichwas predicted to interact with lincror more importantlyfurther research conï¬rmed that mir145 could bind to lincror and its downregulation could partly inhibit the resistanceof lad cells to dtx and emt aside from that pan andhis colleagues discovered that a decrease in the expression ofmir145 and increase in the expression fscn1 could obviouslyreverse the inhibition of cell proliferation chemoresistanceand emt mediated by lincror knockdown they identiï¬edthat dysregulation of the lincrormir145fscn1 axis wasassociated with the therapeutic resistance and emt transition inlad cells thereby providing potential therapeutic strategies formanaging drug resistance in patients with lad pan taken together these studies collectively suggested that lincrorcould activate the malignant phenotype of nsclc cells with theguidance of a mechanism involving mirnas hence more eï¬orts | Colon_Cancer |
" patients with pelvic malignancies often receive radiosensitising chemotherapy with radiotherapy toimprove survival however this is at the expense of increased normal tissue toxicity particularly in elderly patientshere we explore if an alternative lowcost and nontoxic approach can achieve radiosensitisation in mice transplantedwith human bladder cancer cells other investigators have shown slower growth of transplanted tumours in mice fedhighfibre diets we hypothesised that mice fed a highfibre diet would have improved tumour control followingionising radiation ir and that this would be mediated through the gut microbiotaresults we investigated the effects of four different diets lowfibre soluble highfibre insoluble highfibre and mixedsolubleinsoluble highfibre diets on tumour growth in immunodeficient mice implanted with human bladder cancerflank xenografts and treated with ionising radiation simultaneously investigating the composition of their gutmicrobiomes by 16s rrna sequencing a significantly higher relative abundance of bacteroides acidifaciens wasseen in the gut faecal microbiome of the soluble highfibre group and the soluble highfibre diet resulted indelayed tumour growth after irradiation compared to the other groups within the soluble highfibre groupresponders to irradiation had significantly higher abundance of b acidifaciens than nonresponders when allmice fed with different diets were pooled an association was found between the survival time of mice andrelative abundance of b acidifaciens the gut microbiome in responders was predicted to be enriched forcarbohydrate metabolism pathways and in vitro experiments on the transplanted human bladder cancer cellline suggested a role for microbialgenerated shortchain fatty acids andor other metabolites in the enhancedradiosensitivity of the tumour cellss soluble highfibre diets sensitised tumour xenografts to irradiation and this phenotype wasassociated with modification of the microbiome and positively correlated with b acidifaciens abundance ourfindings might be exploitable for improving radiotherapy response in human patientskeywords gut microbiome dietary fibre inulin cellulose radiotherapy radiosensitisation pelvic tumour bacidifaciens parabacteroides correspondence annekiltieoncologyoxacuk chee kin then and salome paillas contributed equally to this work1crukmrc oxford institute for radiation oncology department ofoncology university of oxford old road campus research building offroosevelt drive oxford ox3 7dq ukfull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cthen bmc biology page of patients with pelvic tumours including bladder cervixand rectal cancers who are receiving radiotherapy areoften given additional radiosensitising chemotherapy toimprove cure rates at the expense of increased toxicityin local ans and tissues [ ] with an ageing population new approaches to radiosensitisation are urgentlyrequired one such approach might be to modify the intake of dietary fibre by supplements before and duringradiotherapy or current standard chemoradiation schedules which would be a very costeffective strategy notexpected to add to normal tissue toxicity [ ]wei showed slower growth rates of subcutaneouslymphoma xenografts in mice fed a highfibre diet compared to mice on a lowfibre diet with similar findings in both immunedeficient and immunecompetentmodels this was associated with increased plasmaand tumour butyrate levels but the authors did not investigate the effects of the diet on the gut microbiomedietary fibre manipulation can very rapidly alter thehuman gut microbiome with changes in faecal shortchainfatty acid scfa levels seen only day after the dietreaches the distal gut dietary fibre can also mediate systemic immune effects as can the microbiotaderivedscfas [ ] furthermore dietary fibre structures alignwith phenotypes of specific microbes that differ in theirmetabolic pathways scfas are known to confer anticancer effects [ ] other metabolites including smallintermediate and end byproducts of endogenous metabolicpathways products of microbehost cometabolism andexogenous signals arising from diet drugs and other environmental stimuli might also be important we hypothesised that systemic effects of altered metabolites secreted by gut bacteria on tumours due to dietaryfibre modification could be exploited in conjunction withionising radiation ir to achieve radiosensitisationthe aims of this study were to examine the impact ofthe diet on the microbiome before and after irradiationand to correlate dietinduced microbiome changes withtumour growth and response to radiation treatmentresultsthe environmental microbiome had minimal impact ongut microbiome analysisfemale cd1 nude mice were injected subcutaneouslywith rt112 bladder carcinoma cells and at the sametime they commenced a modified diet namely one ofthe following low dietary fibre cellulose low fibrewith butyrate in drinking water high soluble fibre inulin high insoluble fibre cellulose and highmixed fibre cellulose inulin fig 1a and additional file table s1 we quantified bacterial loadsusing pcr and gel electrophoresis compared to knownnumbers of e coli colonyforming units cfus ourmouse samples contained more than bacterial cfuswhich appeared to override contaminating species in thesample microbial communities fig 1b the pbs negative control was processed from the start of the dna extraction identically to the luminal contents and tissuesamples the amount of nucleic acid detected in thepbs negative controls was extremely low compared tothat in the gut microbiota fig 1c furthermore thecommunity microbiome in this negative control differedmarkedly from the gut microbiome of the mice fig 1dtherefore the environmental microbiome had minimalimpact on the analysis of the gut microbiomes of interest in this studythe landscape diversity and enrichment of bacterial taxain the gut microbiome samples collected when thetumours reached mm3in samples collected when the tumours reached mm3and mm3 the faecal hereinafter referred to as gutmicrobiome and caecal microbiomes were found tohave similar bacterial components additional file figure s1tumoursreached mm3 butyrate levels in the faeces measured by highperformance liquid chromatographyhplc were found to be in the millimolar rangeand were generally higher in the low fibre with butyrate and high soluble fibre groups p ns additionalfile figure s2a the mean time for tumours toreach mm3 was ± days additionalfile figure s2bculled when theirin micein faeces collected from culled mice when the tumoursreached mm3 abundance analysis revealed the fivebacterial taxa with the highest abundance were bacteroides acidifaciens parabacteroides akkermansia muciniphila lachnospiraceae and s247 fig 2a in terms ofalpha diversity the soluble highfibre group had a lowershannons index p fig 2b this could be dueto the higher abundance of b acidifaciens which lowered the diversity within groups in terms of beta diversity principal coordinate analysis showed a notablecluster effect among different groups which indicatesthat samples within groups were more similar to eachother than to those from the other groups fig 2c thissuggested that the gut microbiome was indeed modifiedin this study which might be a dieteffect or a cageeffect see later regarding the abundance of specifictaxa in different diet groups fig 2d the high solublefibre diet significantly increased b acidifaciens abundance p the lowfibre diet increased parabacteroides abundance p the lowfibre diet withincreased akkermansia muciniphilaadded butyrateabundance p and the high mixed fibre diet increased lachnospiraceae abundance p 0cthen bmc biology page of fig the environmental microbiome had minimal impact on gut microbiome analysis a two microbiomes were analysed from the intestinaltract namely faecal and caecal content samples collected when tumours reached mm3 and mm3 respectively b quantification ofbacterial load from different tissue and luminal contents from mice with e coli à à à cfus as controls n mouse ccomparison of the total amount of nucleic acid quantified by picogreen assay in all samples collected when the tumours reached mm3 and mm3 d common bacterial taxa at the species level in samples of pbs as negative controls of dna extraction by 16s rrna sequencingthe landscape diversity and enrichment of bacterial taxain the gut microbiome samples collected when thetumours reached mm3when the tumours reached mm3 abundance analysisof the gut microbiome of both ir and nonir cohorts revealed that the top bacterial taxa with the highestabundance were s247 akkermansia muciniphila bacteroides lachnospiraceae clostridiales and b acidifaciensfig 3a in terms of alpha diversity the soluble hf grouphad a significantly lower shannons index p forthe kruskalwallis test fig 3b in terms of beta diversity principal coordinate analysis using the braycurtis 0cthen bmc biology page of fig dietary fibre shapes the baseline gut microbiome when tumours reached mm3 a stacked bar plot of phylogenetic composition ofcommon bacterial taxa at the species level when tumours reached mm3 faeces were collected from mice fed with lowfibre lowfibre with butyratehigh mixed fibre high insoluble fibre and high soluble fibre diets n for each group b shannons index of gut microbiomes by the kruskalwallis testerror bars represent the interquartile range of diversity scores c principal coordinate analysis of gut microbiomes using the braycurtis dissimilarity anotable clustering effect by diet was seen in the gut microbiome d differentially abundant taxa when the tumours reached mm3 all comparisonsamong different diet groups were performed by the kruskalwallis test and dunns multiple comparison tests all tests compared each median with thecontrol denoted the diet with the highest abundance of taxa was denoted as the control p p p dissimilarity showed a notable clustering effect among different groups which indicates that samples within groupswere more similar to each other than to those from theother groups fig 3c the composition of the gut microbiome continued to evolve on the diets to the time the tumours reached mm3 regardless of whether the micewere irradiated or not the taxonomic cladogram of lefselinear discriminant analysis effect size of the gut microbiome showed that the high soluble fibre diet increasedrelative abundance of s247 additional file figure s3b acidifaciens an acetateproducing bacterium [ ]was found to be of highest abundance in all but the lfdiet group at mm3 but by mm3 was evenly distributed across the different diet groups except the solublehf group treated with radiation p forthekruskalwallis test fig 3dbacteroidales s247 was the highest abundance bacterial taxa in the second cohort its relative abundance was 0cthen bmc biology page of fig composition of the gut microbiome when tumours reached mm3 a stacked bar plot of the phylogenetic composition of commonbacterial taxa at the species level when tumours reached mm3 samples were collected from mice fed with lowfibre high mixed fibre high insolublefibre and high soluble fibre diets n for each group be results for irradiated mice only b shannons index of gut microbiomes by the kruskalwallistest error bars represent the interquartile range of diversity scores c principal coordinate analysis of gut microbiomes using the braycurtis dissimilarityrelative abundance of d b acidifaciens and e bacteroidales s247 in mice with or without irradiation p p p significantly higher in the mixed hf and soluble hfgroups compared to the lf and insoluble hf groupsp for the kruskalwallis test fig 3esoluble high fibre causes increased growth delay inirradiated bladder cancer cell xenograftsto investigate the effect of different diets on the tumourresponse in mice irradiated when the tumour had grownto mm3 tumour growth was monitored to mm3 0cthen bmc biology page of fig soluble high fibre causes increased growth delay in irradiated bladder cancer cell xenografts and responses are influenced by gutmicrobiota composition a tumour growth in rt112 flank xenografts irradiated with gy ir in mice fed lowfibre high mixed fibre highinsoluble fibre and high soluble fibre diets n for each group tumour curve slopes were calculated by linear regression to represent tumourgrowth rates and compared by anova b the kaplanmeier survival curves for mice showing plots of time to treble tumour volume c mice inthe soluble hf group were stratified into responders and nonresponders based on tumour radiation response d shannons index of gutmicrobiota in responders and nonresponders by the kruskalwallis test error bars represent the interquartile range of diversity scores e principalcoordinate analysis of gut samples n in the soluble hf group by response using the braycurtis dissimilarityslopes of the tumour growth curves were obtained usinglinear regression to indicate the tumour progression ratesfig 4a the high soluble fibre diet group had the slowesttumour growth rate the slopes were ± for lf ± for mixed hf ± for insoluble hf and ± for soluble hf fig 4a and additional file figures4 for individual irradiated mouse tumour growth curvesthe kaplanmeier survival curves for time to treble tumourvolume showed that the soluble hf group had the longestmedian survival time days for lf days for mixed hf days for insoluble hf days for soluble hf p logrank test fig 4bamong the eight mice fed the soluble highfibre dietfour mice were classified as responders as using linearregression they had shallower slopes to the tumourgrowth curves namely ± ± ± and ± the other four mice were classified asnonresponders with steeperto the tumourgrowth curves namely ± ± ± and ± fig 4c in terms of alpha diversity therewas no significant difference in shannons index betweenslopes 0cthen bmc biology page of responders and nonresponders fig 4d in terms ofbeta diversity principal coordinate analysis of the braycurtis dissimilarity showed the gut microbiomes of responders and nonresponders were more similar withingroups than between groups fig 4edifferences in composition of the gut microbiomebetween responders and nonresponderslinear discriminant analysis showed that mice respondingto the soluble highfibre diet with a slower tumour growthrate were enriched with bacteroidaceae f flavobacterium g flavobacteriales o lactococcus g streptococcusg streptococcaceae f allobaculum g and erysipelotrichales o the nonresponding tumourbearing mice wereenriched with bifidobacterium g bidifobacteriaceae fbifidobacteriales o parabacteroides g porphyromonadaceae f lactobacillus g lactobacillaceae f and lactobacillales fig 5a in terms of effect size b acidifacienssp and bacteroidaceae f had the largest enrichment inresponders and parabacteroides g and porphyromonadaceae f had the largest enrichment in nonrespondersfig 5b to further explore these findings the discretefalsediscovery rates within all taxonomic levels were calculated fig 5c in responders b acidifaciens species andthe allobaculum genus and in nonresponders lactobacillus and parabacteroides genera had p values the bacidifaciens abundance was significantly higher in responders than that in nonresponders p fig 5dwhile the bacteroidales s247 abundance was similar between responders and nonresponders in the soluble hfgroup p fig 5emetagenomics functional prediction of the gutmicrobiome by response in the soluble hf group andin vitro functional effects of shortchain fatty acidsfunctional prediction at kegg kyoto encyclopedia ofgenes and genomes pathway level revealed that thegut microbiome in responders was enriched for carbohydrate metabolism pathways and in nonresponders formembrane transport pathways fig 5f for carbohydratemetabolism the pathway with the highest level of enrichment statistical analysis showed that responders had a significantly higher levelthan nonresponders fig 5gshortchain fatty acids scfas including acetate propionate and butyrate are major products of fibre fermentation by the gut microbiota we showed that all threescfas increased histone acetylation p for mmacetate p for mm propionate p for mm butyrate fig 5h and tend to increase radiosensitivityp ns for acetate p ns for propionate p forbutyrate in gy fig 5i of rt112 bladder cancer cellssingle scfas reduced cell proliferation fig 5j while aphysiologicalstrongerscfa mixtureconferred aphenotype fig 5j purple bar which was shown in atimedependent pattern as well additional file figures5ato validate the antitumoural effects of b acidifaciens wetreated the bladder tumour cells with bacterial supernatantsof b acidifaciens and its crossfeeding with f prausnitziiand compared their effects with bifidobacterium acetateproducer and f prausnitzii butyrateproducer bacterialsupernatants of b acidifaciens and its crossfeeding with fprausnitzii significantly increased cytotoxicity of bladdertumour cells compared to the other supernatants in day fig 5k and in day additional file figure s5bcorrelation between the abundance of b acidifaciens orparabacteroides genus and mouse survival time in ir andnonir cohortsas b acidifaciens was the top hit for responders andthe parabacteroides genus was one of the top two hitsfor nonresponders in the soluble hf group we exploredhow specific bacterial taxa affected mouse survival timethe correlation between b acidifaciens abundance andtime to culling was investigated across the diet groupssome mice in the nonir cohort lived as long as thosein the ir cohort ie days which may be a reflectionof gy being a relatively low dose of radiation in the ircohort the time of culling positively correlated with bacidifaciens abundance r2 p howevera similar correlation was not seen in the nonir cohortr2 p additional file figure s6ausing the time for tumours to treble in volume as theoutcome measure mice with high b acidifaciens abundance had a significantly prolonged median survival timelogrank test p fig 6a a similar finding wasseen in the ir cohort p but not in the nonircohort p in the ir cohort the time to cullingnegatively correlated with the abundance of parabacteroides genus additional file figure s6b r2 p however a similar correlation was not seenin the nonir cohort r2 p mice in thelow parabacteroides genus abundance group had no significant difference in median time to treble in volumecompared to the high abundance group logrank testp fig 6b b acidifaciens p for thekruskalwallis test and parabacteroides genus p for the kruskalwallis test abundance was evenlydistributed among all cages which suggested that the existence of these taxa in the gut microbiome was not acagespecific effect additionalfile figure s7 andadditional file table s2discussionto date the literature studying the effects of dietaryfibre intake or manipulation of the gut microbiota ontumour growth is limited wei showed that dietary 0cthen bmc biology page of fig differences in composition of the gut microbiome between responders and nonresponders a taxonomic cladogram from lefse showingdifferences among taxa between responders and nonresponders in the soluble hf group dot size is proportional to the abundance of thetaxon b linear discriminant analysis lda scores computed for differentially abundant taxa in the microbiomes of responders green and nonresponders red length indicates the effect size associated with a taxon p for the kruskalwallis test c discrete falsediscovery rate ofdifferent abundant taxa in responders and nonresponders in the soluble hf group differential abundance within all taxonomic levels inresponders versus nonresponders by mannwhitney u test dots are overlapping between bacteroides acidifaciens and allobaculum and betweenlactobacillus and parabacteroides relative abundance of d b acidifaciens and e bacteroidales s247 and in responders and nonresponders in thesoluble hf group f g metagenomic functional prediction by picrust of the gut microbiome in responders n and nonresponders n inthe soluble hf group with reference to the kegg database level columns represent mice responders orange nonresponders blue and rowsrepresent enrichment of predicted kegg pathways red low enrichment yellow medium enrichment blue high enrichment h western blotanalysis of histone acetylation levels of rt112 cells treated with scfas n i linear quadratic survival curves of ic10treated rt112 cells withreceiving irradiation of gy n j cell survival analysis of rt112 cells treated with single scfa and combined scfas mixture n combined purple bar denotes scfa mixture of mm acetate mm propionate and mm butyrate k reduced cell survival of rt112 cells bybacterial supernatants at day n bafp denotes the crossfeeding of b acidifaciens and f prausnitzii while biffp denotes the crossfeedingof bifidobacterium and f prausnitzii p p p 0cthen bmc biology page of fig abundance of otus within the gut microbiome is predictive of response to ionising irradiation the kaplanmeier km plots of time fortumours to treble in volume whole cohorts ir cohorts and nonir cohorts based on a b acidifaciens or b parabacteroides genus abundance inir and nonir cohorts from different diet groups combined comparison km plots by logrank test in mice with high abundance green relativeabundance intermediate abundance red relative abundance or low abundance blue relative abundance of bacidifaciens in all ir and nonir cohorts for parabacteroides genus relative abundance more than or equal to was classified as high bluewhile less than was classified as low greenfibre and the associated butyrate production reducedsubcutaneous lymphoma tumour growth with associatedupregulation of histone acetylation hardman found slower growth of breast cancer xenografts in micefed with fish oil concentrate in contrast cougnoux found that colibactinproducing e coli enhancedtumour growth of colon cancer xenografts in this study faecal and caecal microbiomes were investigated in mice fed the following lowfibre diet lowfibre diet with butyrate high mixed fibre diet high 0cthen bmc biology page of insoluble fibre cellulose diet and high soluble fibreinulin diet and profiles of both microbiomes were correlated the gut microbiomes were shaped by the different modified diets within weeks and homogeneousgut microbiomes were seen in samples within groups adistinct bacterial taxon was seen in each group enrichment of b acidifaciens in soluble hf parabacteroides inlf akkermansia muciniphila in lf with butyrate andlachnospiraceae in mixed hfthe tumours were irradiated when they reached a volume of mm3 with no effect on diet on the time toreach this point and monitored until they reached mm3 during the course of the study all mice developedan increased relative abundance of s247 family bacteriathus indicating that their gut microbiomes had alteredover the tumour growth period although the gut microbiomes became more heterogeneous a notable clustereffect still existed in samples within groups miceresponding to radiation in the soluble hf group wereenriched with b acidifaciens and nonresponding micewere enriched with the parabacteroides genusa predictive metagenomics study of the gut microbiome in responders was enriched for carbohydrate metabolism pathways this implies a higher level of fibrefermentation occurring in responders reflecting the selection of bacteria more able to ferment carbohydratesthis could result in more scfa production in the faeces both butyrate and propionate have been proposedto increase histone deacetylase inhibition which is aknown mechanism of cellular radiosensitisation [ ]in this study we demonstrated that these phenotypesexist in bladder cancer cells fig 5h i furthermorein vitro studies showed that scfa reduced cell proliferation in liver cancer induced apoptosis in lung cancer and did both in breast cancer vecchia found that acetate and propionate potentiated theanticancer effects of butyrate in leukaemic cervicaladenocarcinoma melanoma and breast cancer cells we demonstrated similarin bladdercancer cells in this study fig 5j and additional file figure s5a altogether these data are supportive ofcarbohydrate metabolism or scfa production being importantincludingradiosensitisationin enhancing anticancer effectsfindingswhen the mice from different diet groups werepooled b acidifaciens abundance was positively correlated with survival time and mice with high b acidifaciens had the longest median survival times using thekaplanmeier survival analysis bacteroidetes includingb acidifaciens have been proposed to produce themetabolic end products acetate succinate and possiblypropionate but not butyrate [ ] high acetate levelscould act as a substrate for butyrate production giventhat acetate is necessary for butyrate productionparticularly in the butyrylcoaacetate coatransferasepathway [ ] we speculate that the faecal butyratelevels could have been enhanced due to crossfeeding ofbutyrateproducing bacteria by b acidifaciens proofofconcept was previously demonstrated for this by crossfeeding butyrateproducing bacteria faecalibacteriumprausnitzii and acetateproducing bacteria bifidobacterium adolescentis of note ramirezfarias inulin increased both faecalibacteriumshowed thatprausnitzii and bifidobacterium adolescentis in a humanvolunteer study limited to chemotherapya modified gut microbiota can augment the efficacy ofantitumoural treatment however most studies to dateareand immunotherapyreviewed in [ ] recently herranz found thatdepletion of grampositive bacteria in the gut by vancomycin enhanced radiotherapyinduced antitumour immune response and further delayed tumour growthhowever the authors gave very large 21gy single fraction irradiation doses furthermore reductions in abundanceabsence of gramnegative bacteriaincludingbacteroides and s247 were also seen with increasedparabacteroides which could be of significance herewe only gave a 6gy single fraction of ir which is moreclinically relevant and our findings were not immunologically mediated via t cells as cd1 nude mice lack tcellsto our knowledge ours is the first study to provideevidence that a high soluble fibre diet and its subsequent modification of the gut microbiome can act toradiosensitise tumoursfurthermore b acidifaciens was identified as a potential radiosensitiser because its abundance was enhancedby a high soluble fibre diet and positively correlated withtumour response to radiation and survival time in the ircohort this bacterium was first isolated in andwas so named because it reduces the ph level ofpeptoneyeast broth with fildes digest consistentwith our findings marques demonstrated that ahighfibre diet markedly increased the prevalence of bacidifaciens another study showed b acidifaciensto be enriched in normal human subjects compared topatients with inflammatory bowel disease and bacidifaciens increased insulin sensitivity and preventedfurther obesity however the effect of these bacteriaon tumour growth is still controversial a study foundincreased b acidifaciens abundance associated with hepatocellular carcinoma induced by a streptozotocinhighfat diet in contrast b acidifaciens reduces the isoflavone genisten which is associated with increased riskof breast cancer and b acidifaciens was shown tocontribute to the antitumour effect of medicinal gynostemma saponins in this study we revealed that bacterial supernatant from b acidifaciens and its cross 0cthen bmc biology page of feeding with f prausnitzii caused significantly higherlevels of cytotoxicity compared to the other supernatantsfig 5k and additional file figure s5b this resultsupports our finding that b acidifaciens may drive theradiosensitising effect moreover b acidifaciens in vitrohas a greater effect on cell kill than f prausnitzii butyrateproducer p implying that metabolites otherthan butyrate may be involved in its effect studies suggest that a broad range of gut microbiotaderived metabolites can enhance antitumoural effects or tumourresponse to anticancer treatments [] so futurework should include undertaking a global metabolomicanalysis of b acidifaciensproduced metabolites to identify radiosensitisers otherthan scfas with similarprofileswe also found bacteroidales s247 or candidatushomeothermaceae or muribaculaceae an uncultured bacterium with limited characterisation to behighly abundant in our study especially in the mixed hfand soluble hf groups the prevalence of this bacteriumin humans is however increased prevalence ofbacteroidales s247 of up to has been shown in thehadza tribe of tanzania who consume tubers containinglarge amounts of soluble fibre this indicates thathigh abundance of bacteroidales s247 found in thisstudy when the tumours reached mm3 might be induced by soluble high fibreenvironmental contamination is an inevitable issue inmicrobiome studies to minimise the influence ofcontamination in this study bacterial loads of sampleswere quantified and appropriate negative controls wereincluded bacterialloads from luminal contents andtissue samples contained more than cfus whichoverrode the environmental bacteria communities furthermore the bacterial compositions of the pbs negativecontrols were very different from those of the studygroups so the environmental microbiome was considered not to be a major source of bias in this studyalthough a strong correlation between b acidifaciensabundance and tumour response to irradiation was seena limitation of this study is that only one cohort of micewas studied further studies are needed to determine thecausal relationship between b acidifaciens and radiosensitisation and its underlying aetiology this could beachieved by oral gavage of b acidifaciens with or without other bacteria in gnotobiotic mice and in vitro culture studies of b acidifaciens as both gut bacteria and irradiation directly interact with the immunesystem further in vivo studies need to be conducted inan immunecompetent model to reveal how immunomodulation might contribute to the radiosensitisationa high soluble fibre diet increased responses of rt112subcutaneous xenografts in cd1 nude mice to ionisingradiation and this phenotype was associated with higherrelative abundance of b acidifaciens possible mechanismsmediating this effect which require further investigationinclude the following increased concentrations of metabolitesincluding butyrate or other shortchain fattyacids in tumours acting via hdac inhibition or via otherpathways suppression of overgrowth of unfavourablebacteria such as parabacteroides genus andor enhancement of antitumoural immunity our findings suggest that dietary fibre modification and the resultantmodification of the gut microbiome might be exploited toimprove tumour responses to radiotherapy in humanpatientsmethodsmice and mouse dietsall animal work was done in accordance with uk homeoffice guidelines following the arrive animal research reporting of in vivo experiments guidelinesand approved by the university of oxford animalwelfare and ethical review body awerb underuniversity of oxford project licences p4b738a3b andp8484edae group sizes were chosen to detect large effect sizes by using a gpower analysis program all micewere purchased from charles rivers uk ltdcd1 nu | Colon_Cancer |
the periparturient period is one of the most challenging periods in dairy cows and encompasses the a0wk prior to and a0wk after parturition the nutrient requirements of dairy cows change greatly during this time largely due to the exponential growth of the gravid uterus and fetus followed by the demands of lactation nrc inflammation oxidative stress and adipose tissue mobilization lead to a reduction in dry matter intake dmi during the periparturient period this reduction in dmi leads to a negative nutrient balance nnb with a shortfall in the nutrient availability for the cow and fetus ingvartsen and andersen additionally this reduction in dmi also increases the risk of metabolic ketosis fatty liver milk fever and immunerelated disorders the risk of these diseases poor reproduction and low efficiency is greatly impacted by the degree and length of time during which these systems metabolism and immune response remain out of balance loor et a0al 2013a much of the research over the last decade have examined these biological interactions to identify the mechanisms behind the metabolic physiologic and immune adaptations that occur during the periparturient period loor et a0al 2013a 2013b roche et a0al bradford et a0al it is now known that nutrients such as amino acids aa serve functional roles outside of their use as building blocks for proteins and have immunomodulatory properties and interact through common biochemical pathways eg 1carbon metabolism figure a0 this concept has been well explored in nonruminant species li et a0 al sikalidis with nutritional management during the periparturient period continuing to be an active area of research it is important to develop a system understanding the potential immunometabolic role that dietary aa may play during this period thus the objective of this review is to provide an overview of physiological adaptations during the periparturient period the immune system and methods to assess immune function and oxidative stress this will be followed by a more specific discussion of the immunometabolic roles of specific aa and their potential effects in dairy cow during the periparturient period the potential effects of enhanced aa supply during the preweaning period will also be discussed brieflybiological adaptations in the transition cowa brief overviewduring the nnb associated with the periparturient period biological mechanisms coordinate the mobilization of body reserves in order to support fetal growth and milk production bauman and currie insulin concentrations are reduced and the response of hormonesensitive lipase in adipose tissue eg low insulin high growth hormone and catecholamines or high glucocorticoid concentrations is greater to facilitate lipid mobilization this periparturient period is also characterized by a state of inflammation encompassing an increase in hepatic production of positive acutephase proteins app such as haptoglobin and serum amyloid a a0saa and a decrease in the production ingvartsen the authors published by oxford university press on behalf of the american society of animal sciencethis is an open access distributed under the terms of the creative commons attribution noncommercial license httpcreativecommonslicensesbync40 which permits noncommercial reuse distribution and reproduction in any medium provided the original work is properly cited for commercial reuse please contact spermissionsoupcoms175 0cs176 of animal science vol no suppl abbreviationsaa akt app asct2 bcaa bcat bcka bhmt bmec bsa cbs csad cth dmi gator1 gcl gclc gpx gsh gsr gss il inos kegg klh lps mpo mtor mtorc1 mtr nfe2l2 nfκb nnb nos pc pmn pmnl pmtor rns rom ros rpm saa sahh sam samtor sell shmt slc1a1 slc1a5 sod stat tag th tlr tnfα vldl amino acidsprotein kinase bacutephase proteinsalanineserinecysteine transporter branchedchain amino acidsbranchedchain amino transferasesbranchedchain αketoacidsbetaine homocysteine methyltransferasebovine mammary epithelial cellsbovine serum albumincystathionine synthasecysteine sulfinic acid decarboxylasecystathionine gammalyasedry matter intakegtpaseactivating protein activity towards rags glutamate cysteine ligaseglutamate cysteine ligase catalyticglutathione peroxidaseglutathioneglutathione reductaseglutathione synthaseinterleukininducible noskyoto encyclopedia of genes and genomeskeyhole limpet hemocyaninlipopolysaccharidemyeloperoxidasemechanistic target of rapamycinmtor complex 5methyltetrahydrofolatehomocysteine methyltransferasenuclear factor erythroid 2like2nuclear factor kappa bnegative nutrient balancenitric oxide synthasephosphatidylcholinepolymorphonuclear neutrophilspolymorphonuclear leukocytesphosphorylated mtorreactive nitrogen speciesreactive oxygen metabolitesreactive oxygen speciesrumenprotected metserum amyloid asadenosyl homocysteine hydrolasesadenosyl methioninesadenosylmethionine sensor upstream of mtorlselectinserine hydoxymethyltransferasesolute carrier family member solute carrier family member superoxide dismutasesignal transducer and activator of transcriptiontriacyglycerolthelpertolllike receptorstumor necrosis factorαverylowdensity lipoproteinsof negative app such as albumin bertoni et a0 al it has been well established that these responses are mediated by the proinflammatory cytokines interleukin il6 il1 and tumor necrosis factorα tnfα kindt et a0al additionally oxidative stress also occurs during this period and is driven by the imbalance between the production of reactive oxygen metabolites rom reactive nitrogen species rns and the neutralizing capacity of antioxidant mechanisms in tissues and blood some of the wellestablished cellular antioxidants include glutathione gsh taurine superoxide dismutase sod and vitamins a a0and e bernabucci et a0al when oxidative stress overwhelms cellular antioxidant capacity rom induce an inflammatory response that is controlled via changes in mrna abundance of transcription regulators eg signal transducer and activator of transcription [stat3] nuclear factorkappa b [nfκb] the increase in oxidative stress and inflammation during this period is also negatively associated with a reduction in liver functionality and measurement of app can provide a useful tool to assess liver function as well as inflammation bertoni and trevisi during the periparturient period aa metabolism is also altered with moderate carcass protein losses reported even when animals are fed to their predicted metabolizable protein requirements bell et a0 al additionally circulating aa concentrations change dramatically with favorable circulating profiles of many aa not being restored until d postpartum zhou et a0al 2016b furthermore the total concentration of aa in plasma reaches a nadir around day postpartum zhou et a0al 2016b which corresponds to a peak in total disease incidence during early lactation ingvartsen this decrease in circulating aa is likely associated with the increased use of aa for gluconeogenesis as well as for hepatic production of app thus it is important to investigate how supplemental aa during the periparturient period may modulate metabolism and immune responses to promote production and reduce susceptibility to diseasegeneral overview of the immune a0systemthe immune system consists of both the adaptive and innate immune responses which are linked together through signaling molecules such as cytokines the innate immune system is the first line of defense and includes physical barriers such as epithelial cell layers that express tight cell junctions and the mucus layer of the respiratory gastrointestinal and genitourinary tracts chaplin cells involved in the innate immune response include macrophages polymorphonuclear neutrophils pmn dendritic cells mast cells eosinophils natural killer cells and natural killer t cells turvey and broide the focus of the present review is on the effects of aa on phagocytic cells that is cells that engulf and kill such as macrophages and pmn macrophages not only phagocytose invading pathogens but also produce cytokines such as ils and tnfα which initiate innate and adaptive immune responses and recruit pmn to the site of infection chaplin the adaptive immune response consists of t lymphocytes b lymphocytes and humoral factors marshall et a0 al there are two types of t lymphocytes cytotoxic t cells cd8 cells and thelper th cells cd4 cells marshall et a0al cytotoxic t cells detect and eliminate infected cells while th cells produce ils and interferonγ ingvartsen and moyes the b lymphocytes are cells that produce antibodies that bind to antigens on the surface of pathogens to mark them for destruction marshall et a0al 0ccoleman et a0al s177aa immune function and oxidative a0stressduring the periparturient period the metabolic status is associated with the inflammatory regulation of peripheral blood mononuclear cells with a more pronounced inflammatory response in those cells during the nnb immediately postpartum agrawal et a0 al mann et a0 al this period is also associated with altered signaling of nutrientsensing kinases in immune cells such as the protein kinase b akt and mechanistic target of rapamycin mtor pathway which may modulate cytokine production mann et a0al aa can directly and indirectly alter the immune system besides being used in energy metabolism reactions and the synthesis of proteins aa are critical for the synthesis of other functional molecules such as the antioxidants gsh and taurine no histamine and hydrogen peroxide li et a0 al thus this section of the review will focus on the role that aa play in modulating immune function and oxidative stress in dairy cows during the periparturient period a a0special focus will be placed on aa involved in 1carbon metabolism as this represents an interconnected route through which aa could impact molecular events such as epigenetic regulation protein synthesis via mtor energy metabolism and antioxidant synthesis a a0summary of studies investigating the effects of aa on immune function and oxidative stress in dairy cows is provided in table a0 a a0summary model of how aa might alter immune function and oxidant status is depicted in figure a0methioninemethionine not only is essential for protein synthesis but also serves as a functional nutrient that is needed for the production of the antioxidants gsh and taurine atmaca and provision of methyl groups finkelstein these features are exemplified by the central role of met in 1carbon metabolism figure a0 in these pathways met is used to synthesize sadenosyl methionine sam which can be used to methylate dna and to support phosphatidylcholine pc and carnitine synthesis for fatty acid metabolism vance et a0al during the periparturient period triacyglycerol tag accumulate in the liver leading to mitochondrial dysfunction inflammation and reduced liver function li et a0al du et a0al pc is the main phospholipid component of verylowdensity lipoproteins vldl vance thus enhancing pc synthesis through greater met supply may help improve vldl synthesis and reduce hepatic tag accumulationas part of 1carbon metabolism homocysteine can be remethylated to met using betaine or folate as methyl donors bhmt and via betaine homocysteine methyltransferase 5methyltetrahydrofolatehomocysteine methyltransferase mtr respectively homocysteine is also used to synthesize cystathionine via cystathionine synthase cbs in the first reaction of the transsulfuration pathway banerjee et a0 al cystathionine is used to make cys which is utilized to synthesize the antioxidants taurine or gsh cbs is allosterically figure interrelationships among components of the 1carbon metabolism pathway methionine cycle folate cycle and transsulfuration pathway 5mthf 5methyltetrahydrofolate amd1 adenosylmethionine decarboxylase arg arginase b12 cobalamin b2 riboflavin b6 pyridoxal ²phosphate cbs cystathionine betasynthase dcsam decarboxylated sadenosylmethionine dhfr dihydrofolate reductase dnmt dna methyltransferase dtmp thymidine monophosphate dump deoxyuridine monophosphate ftcd formimidoyltransferase cyclodeaminase gnmt glycine nmethyltransferase mat methionine adenosyltransferase mthfr methylenetetrahydrofolate reductase mtrr 5methyltetrahydrofolatehomocysteine methyltransferase reductase odc1 ornithine decarboxylase pemt phosphatidylethanolamine nmethyltransferase sahh sadenosylhomocysteine hydrolase shmt serine hydroxymethyltransferase sms spermine synthase srm spermidine synthase tdh threonine dehydrogenase thf tetrahydrofolate tyms thymidylate synthetase 0cs178 of animal science vol no suppl activated by sam banerjee et a0 al therefore enhanced sam production with increased met supply can help enhance the flux of the transsulfuration pathway increasing taurine and gsh production to help reduce oxidative a0stressin dairy cattle low levels of serum met postpartum are associated with severe hepatic lipidosis shibano and kawamura and other than his met is the only aa for which net uptake by the liver increased pre and postpartum larsen and kristensen therefore enhancing postruminal met supply during the periparturient period is of interest to increase circulating concentrations of met for its functional roles in the body the work from dalbach et a0 al demonstrated that rumenprotected met rpm can be used to increase serum concentrations of met in the first 2wk postpartum which will enhance the availability of met for protein synthesis and metabolism via the 1carbon pathways in terms of production supplementation of met during the peripartal period concomitantly increases milk yield milk protein and milk fat soon after calving ordway et a0 al osorio et a0 al these responses are in large part driven by enhancing met availability and by the additional flux of met through the met cycle in the liver which consequently increases the production of downstream compounds such as sam pc gsh and taurine additionally work feeding rpm during the periparturient period has detected positive responses in maintaining consistent rates of dmi prepartum last d and faster and greater rates of dmi during the first to d after calving osorio et a0al zhou et a0 al 2016c batistel et a0 al the same milk production response was also observed when met was supplemented postpartum as the isopropyl ester of 2hydroxy4methylthiobutanoic acid stpierre and sylvester as described earlier the transient inflammatorylike status around parturition appears to be a normal aspect of the adaptations to lactation as cows approach parturition those with greater but still subclinical concentrations of circulating cytokines have greater inflammation and oxidative stress and lower liver function along with lower milk yield and lower postpartum dmi bertoni et a0 al in addition to their function in the immune system cytokines interferons and tnfα also elicit pathophysiological effects leading to sickness table summary of studies in dairy cows investigating the effects of supplemental aa on immune function oxidative stress and inflammation tissuecellstreatmentmain outcomeplasmarpm for d prepartum and d postpartumimprovements in plasma biomarkers indicating reduced oxidative stress and inflammation and enhanced liver function increased neutrophil phagocytosis and oxidative burstreferencebatistel et a0al plasmaabomasal infusion of glu for first infusions of gln increased the abundance of cd4 tcells on day doepel et a0al d postpartumpostpartum and increased the abundance of monocytesmammary rpm for d prepartum and d methionine supply upregulated expression of genes involved in han et a0al glandplasmapostpartumantioxidant metabolism and increased activation of nfe2l2intravenous infusions of gln for glutamine infusion decreased plasma haptoglobin and increased jafari et a0al d postcalvinglpsbinding protein and saa subcutaneous rpm for d prepartum and d enhanced met supply increased mrna and protein abundance of liang et a0al 2019aadiposepmnlpostpartumenzymes related to gsh metabolismincubation with met andor supplemental met coupled with adequate choline enhanced gene lopreiato et a0al cholineexpression of pathogen recognition mechanisms methionine ameliorated the increased inflammation and oxidative stress observed when cells were incubated without choline plasma and protected gln for d postpartum increased total blood protein and albumin decreased plasma nemati et a0al milkwhole bloodrpm for d prepartum and d aspartate aminotransferase and milk somatic cell countincreased whole blood neutrophil phagocytosis on day osorio et a0al 2013apostpartumpostpartum with supplemental metliverrpm for d prepartum and d methionine supply altered flux through 1carbon metabolism via osorio et a0al 2014aliver and plasmaplasmapostpartumchanges in mrna to support antioxidant and met synthesisrpm for d prepartum and d methionine increased liver gsh and decreased concentrations of osorio et a0al 2014bpostpartum plasma biomarkers of inflammationrpm for d during midlactation increased proliferative ability of peripheral blood t lymphocytes soder and holden with supplemental metplasmarpm for d prepartum and increased antioxidant capacity of plasma and cd4cd8 t sun et a0al postpartumlymphocyte ratio with met supplywhole bloodrpm for d prepartum and d methionine damped the hyperresponse of il1 during an lps vailatiriboni et a0al plasmajugular infusion of arg and lps in arginine alleviated lpstriggered inflammation by decreasing il6 zhao et a0al 2018apostpartumchallenge through improvements in oxidative stressserumjugular infusion of arg and lps in infusion of arg promoted antioxidant mechanisms during lpszhao et a0al 2018bmidlactation cowsinducible nos and lpsbinding proteinmidlactation cowsrpm for d prepartum and d postpartumrpm for d prepartum and d triggered inflammation by increasing total antioxidant capacity and gsh peroxidase activity and decreasing malondialdehyde increased hepatic gsh and improved plasma biomarkers of liver function and inflammation with met neutrophil phagocytosis capacity and oxidative burst were also increased with metenhanced met supply increased mrna expression of genes zhou et a0al 2016azhou et a0al 2017bpostpartumassociated with pc and antioxidant synthesisrpm for d prepartum and d decreased expression of genes related to inflammation and zhou et a0al 2018bliver and plasmaliver pmnlpostpartumoxidative stress 0ccoleman et a0al s179figure the theoretical model of cellular aa utilization amp adenosine monophosphate atp adenosine triphosphate ctp cytidine triphosphate dttp deoxythymidine triphosphate gmp guanosine monophosphate imp inosine monophosphate no nitric oxide r5p ribose 5phosphate tca cycle tricarboxylic acid cycle ump uridine monophosphate utp uridine5²triphosphatebehaviors whose primary manifestation is satiety larson and dunn an example of this behavior in dairy cows is the reduction in dmi around calving in mice these cytokines have been shown to reduce meal size and duration as well as decrease meal frequency and prolong intermeal intervals platasalamán furthermore cytokines directly affect the hypothalamus il1 and ifn act directly and specifically on the glucosesensitive neurons in the brain satiety and hunger sites platasalamán in addition to increases in dmi and milk production rpm supplementation during the periparturient period has been associated with positive health responses across four studies osorio et a0al 2014b sun et a0al zhou et a0al 2016a batistel et a0 al there have been consistent improvements in the concentrations of plasma biomarkers of inflammation where il1 and haptoglobin have decreased and albumin has increased summarized in table a0 improvements in biomarkers of oxidative stress have also been observed with enhanced met supply during the periparturient period in the study by batistel et a0al plasma concentrations of ferricreducing antioxidant power carotene tocopherol and total and reduced gsh were increased with rpm while rom were lower sun et a0al also observed an improvement in blood antioxidant status with rpm increasing total antioxidant capacity glutathione peroxidase gpx activity and vitamin e a0a a0similar effect was observed in liver tissue by osorio et a0 al 2014b where cows fed rpm had greater hepatic concentrations of total and reduced a0gshfrom a mechanistic standpoint changes in the mrna abundance of sadenosyl homocysteine hydrolase sahh mtr sod1 glutamate cysteine ligase catalytic gclc subunit and dna methyltransferase 3a suggested alterations in flux through 1carbon metabolism osorio et a0al 2014a importantly sahh the enzyme that makes homocysteine from sadenosylhomocysteine was upregulated with met which would support a supply of homocysteine to be used for antioxidant and met synthesis furthermore in the study by zhou et a0al 2016a greater met supply compared with rumenprotected choline increased antioxidant concentration in liver tissue despite a lower concentration of pc those responses were due to the greater abundance of phosphatidylethanolamine methyltransferase the enzyme that utilizes sam and phosphatidylethanolamine to make pc and cbs zhou et a0al 2017b additionally enhanced met supply during the periparturient period was observed to increase mrna and protein abundance of enzymes related to gsh metabolism in subcutaneous adipose tissue suggesting greater activation of those pathways liang et a0al 2019a a a0greater dietary supply of choline did not change the mrna abundance of bhmt and mtr in cows zhou et a0al 2017ba positive effect of met supplementation on mammary gland antioxidant mechanisms was observed by han et a0 al mrna abundance of gpx1 gclc glutamate cysteine ligase gcl modifier subunit malic enzyme ferrochelatase and ferritin heavy chain and genes involved in gsh and iron metabolism were upregulated protein abundance of phosphorylated nuclear factor erythroid 2like2 nfe2l2total nfe2l2 was also increased han et a0al nfe2l2is a regulator of transcription of antioxidant genes hence an increase in phosphorylated nfe2l2 suggested greater activation of antioxidant systems and is likely one of the mechanisms behind the changes in mrna abundance lastly across studies there has also been a consistent improvement in the concentrations of plasma biomarkers of liver function such as increases in paraoxonase and cholesterol with rpm osorio et a0al 2014b zhou et a0al 2016a batistel et a0 al which is likely linked to the reduction in inflammation and oxidative stress thus the consistent changes across studies in metabolites and plasma biomarkers as well as mrna abundance across tissues indicate that enhanced met supply during the periparturient period reduces oxidative stress and inflammation however more work is needed to verify the exact mechanisms behind the observed changes 0cs180 of animal science vol no suppl enhanced postruminal supply in the form of rpm during the periparturient period has been associated with improvements in immune cell function when rpm was provided for d prepartum and d postpartum whole blood neutrophil phagocytosis was increased compared with control cows at d postpartum osorio et a0 al in the study by zhou et a0 al 2016c rpm supplementation from day prepartum to day postpartum increased neutrophil phagocytosis capacity and oxidative burst activity zhou et a0 al 2016a this same improvement in neutrophil immune function was observed when rpm was supplied from day prepartum to day postpartum batistel et a0 al furthermore an increase in the proliferative ability of peripheral blood t lymphocytes was observed when rpm was supplemented to midlactation cows soder and holden zhou et a0 al 2018b isolated polymorphonuclear leukocytes pmnl and observed lower abundance of genes related to inflammation il1b tlr2 nfκb and stat3 and oxidative stress cbs gpx1 glutathione synthase [gss] and sod2 as well as an increase in plasma taurine with supplemental met suggesting a better redox and inflammatory status of those cells additionally those same cows were used for an ex vivo whole blood challenge with lipopolysaccharide lps to further investigate immune cell responses during this challenge a table summary of additional beneficial effects of feeding rpm during the transition period and early lactationbiomarkerresponse12sitebiological functionmetabolism carnitine cholesterolonecarbon metabolism cystathionine betasynthase activity cystathionine cystine homocysteineinflammation il1beta haptoglobin albumin oxidative stress romliveroxidation of fatty acidsplasmalipoprotein metabolismliverplasmaplasmaplasmaantioxidant synthesish2s biosynthesis redox statusredox statusmethylation reactionsplasmaproinflammatory cytokineplasmainflammation signalplasmaacutephase responseplasmaperoxides gsh taurine antioxidant capacity paraoxonasesuperoxide ohradicalsliver blood antioxidantantioxidantplasmaplasmatotal antioxidants in bloodplasmaantioxidant enzyme beneficial increase beneficial decrease no change in concentration2relative to a control or rumenprotected choline supplemented diet osorio et a0al 2014b zhou et a0al 2016a sun et a0al batistel et a0al vailatiriboni et a0al hyperresponse in il1 was observed around parturition which likely arose from oxidative stress vailatiriboni et a0 al however rpm supplementation dampened this hyperresponse likely through improvements in oxidative stress vailatiriboni et a0al the recent work by lopreiato et a0 al investigated the effects of incubating bovine pmnl with met andor choline and observed that supplemental met coupled with adequate choline enhanced gene expression of tlr2 and lselectin sell which are pathogen recognition mechanisms in the same experiment cells incubated without choline had greater mrna abundance of il1b il6 il10 and myeloperoxidase mpo glutathione reductase gsr gss cystathionine gammalyase cth and cysteine sulfinic acid decarboxylase csad indicating greater inflammation and oxidative stress this effect however was ameliorated by supplementing additional met lopreiato et a0 al thus the increased dmi and milk production observed when feeding rpm can be partly explained by a reduction in inflammation as it directly at the hepatic level and by dampening the immune cell overresponse and indirectly reducing oxidative stress decreases circulating proinflammatory cytokinesenhanced met supply during the periparturient period has also been associated with changes in mtor signaling mtor is a serinethreonine kinase that plays a central role in integrating environmental cues from growth factors nutrients particularly aa and energy powell et a0al in dairy cattle mtor has traditionally been studied in the context of its role in regulating protein synthesis however work from humans and nonruminant species has indicated that mtor is an important regulator of immune responses powell et a0 al jones and pearce when activated by aa mtor directs an activation of anabolic metabolism which allows growth proliferation and development this makes the activation of mtor in immune cells particularly important for maintaining proliferation and without proper activation cells may enter periods of growth arrest jones and pearce methionine specifically may interact with mtor via the production of sam specifically sam can bind to sadenosylmethionine sensor upstream of mtor samtor a protein that inhibits mtor complex mtorc1 by interacting with gtpaseactivating protein activity towards rags gator1 gu et a0al when sam binds to samtor it inhibits the association of samtor and gator1 allowing mtorc1 to be activated gu et a0al to our knowledge there is only one study investigating the expression of mtor signaling proteins in immune cells in dairy cattle and work is also lacking in nonruminant species in periparturient cows the activation of aktmtor signaling in immune cells was reduced postpartum compared with prepartum mann et a0al importantly agrawal et a0al also identified the expression of aa transporters and the kyoto encyclopedia of genes and genomes kegg pathways related to 1carbon metabolism and mtor in pmnl from peripartal cows a a0 list of these transporters and kegg pathways related to aa and 1carbon metabolism are summarized in table a0 together these studies support the potential importance of aa for nutrient signaling in immune a0cellsrecent work indicating that enhanced met supply activates mtor signaling in the mammary gland supports its role in enhancing protein synthesis for example in vitro enhancing met supply to immortalized bovine mammary epithelial cells bmec by varying the ratio of lys to met increased the concentration and utilization of essential aa particularly branchedchain aa bcaa dong et a0 al this change 0cwas potentially mediated by alterations in aa transporters controlled by mtor dong et a0al other studies with bmec have also revealed a potential for greater mtor activation when met supply is enhanced nan et a0al sala ma et a0al in vivo the effects of supplemental met during the periparturient period on mtor signaling were explored using cows from the study by batistel et a0al in the mammary gland cows receiving rpm had lower protein abundance of total mtor and phosphorylated mtor pmtor compared with control cows on day postpartum but the ratio of pmtortotal mtor was not different suggesting that there was no difference in mtor activation between treatments ma et a0 al however changes in aa transporters and insulin signaling indicated that insulin sensitivity of the mammary gland was enhanced with supplemental met ma et a0al in subcutaneous adipose tissue the mrna and protein abundance of some aa transporters and pmtor were upregulated with enhanced met supply while changes in insulin signaling and plasma glucose also indicated that met helped improve insulin sensitivity liang et a0 al 2019b thus enhancing met supply during the periparturient period may lead to mtor activation in immune cells as well as improved nutrient uptake which could help to support proliferation and development additional work in ruminants and nonruminants is needed to understand whether met modulates mtor signaling in immune cellscysteinecysteine can be synthesized endogenously from homocysteine as described earlier and is needed to synthesize gsh and taurine gsh is synthesized via two enzymes gcl and gss lushchak to synthesize taurine cysteine sulfinic acid is first synthesized from cysteine by cysteine dioxygenase and can then be utilized by csad to produce taurine park et a0al dietary cys has been explored as a way to improve health in nonruminant species and humans under a variety of conditions including type2 diabetes cardiovascular disease and liver disease to name a few yin et a0 al across these studies increased dietary cys increased the concentrati | Colon_Cancer |
Subsets and Splits
No community queries yet
The top public SQL queries from the community will appear here once available.