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" 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 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 |
"annual meeting of the european associationfor the study of diabetes september sindex of oral presentationsop diabetes complications new insights from cutting edge epidemiologyop news on the insulin secretion frontop insulin sensitivity and biomarkersop central actions in diabetesop glucoselowering therapies and the liverop uncomplicating the pathogenesis of diabetes complications inhumansop smoke on the water is bat still hotop charting human beta cell failure in type diabetesop novel agents in type diabetesop developing better insulinsop from diagnostics to the endstage of diabetic kidney diseaseop nafld is it all about the liverop diabetic retinopathy see what's newop taking the long view of diabetesop pregnancy in diabetes prediction and outcomesop signals and networks in beta cell failureop broken heart in diabetesop unlocking the potential of digital healthop decoding the heritable basis of type diabetesop feeding the pipeline from drugs to surgeryop sglt2 inhibitors at the heart of the matterop new treatments for nafld hope or hypeop addressing potential new treatments of diabetic kidney diseaseop glucagon and hormones beyondop incretin based therapiesop unusual forms of diabetesop macrovascular complications and beyondop linking inflammation to metabolismop what's new in automated insulin deliveryop understanding the mechanisms of diabetic kidney diseaseop novel aspects of diabetic neuropathyop reducing the burden of hypoglycaemiaop what exercise doesop back to the future risk markers in diabetesop diet not only quantity mattersop on the road to human islet failure in type diabetesop a deep dive into the mechanisms of diabetesop triggers and drivers of beta cell failure in type diabetesop gastroentero pancreatic factors anoids mice and menop new aspects of novel therapiesop fatty mattersop diabetes care is expensiveop developing beta cellsop modelling metabolism lessons from animalsop diabetic foot new developments in wound healingop challenges in delivering diabetes care new solutionsop thinking about diabetes complications in the brainop insulin secretion in various subgroupsindex of poster sessionsps diabetes and early deathps living with chronic diabetes complicationsps micro and macrovascular complications of diabetesps global view on diabetes complicationsps type diabetes treatment irlps unusual forms of diabetesps molecular insights into glucose abnormalitiesps pathophysiology of glucose homeostasisps the inner workings of the pancreasps islets and antibodies in type diabetesps markers and phenotypes of glucose traitsps global aspects on the epidemiology of type diabetesps risk factors for type diabetesps prevalence of type diabetes around the worldps risk factors in type diabetesps islet transplants revisitedps islets in type diabetes new playersps beta cells under stressps to live and let die a beta cell perspectiveps job description insulin secretionps further down the road to human islet failure in type diabetesps sitting and exercising does it allps the ins and outs of carbohydrate metabolismps pregnancy in vitro and in vivo studiesps pregnancy epidemiologyps pregnancy who is at riskps incremental studies on gut hormonesps the fundamentals of insulin resistanceps studies on insulin resistanceps treatment of hyperglycaemia in pregnancyps pancreatic hormonesps insulin secretion in mice and menps something more about obesityps more about metabolismps inflammation in type diabetesps models of prediabetes and diabetesps models of obesity and insulin resistanceps lipid metabolismps adipokine signallingps drugs and environment in obesityps weight loss interventionsps brain mattersps sglt2 inhibitors clinical aspectsps different aspects of sglt2 inhibitorsps basic aspects of incretinbased therapiesps clinical outcome of incretinbased therapies 0cdiabetologiaps glycaemic control and incretinbased therapiesps various clinical aspects of incretinbased therapiesps various aspects of nutrition and dietps oral therapies metformin sensitizers and other nonsecretagoguesps novel agents to treat diabetes and its consequencesps novel glucoselowering agents in type diabetesps key issues in improving outcomes in people with diabeteseducation and costsps how to improve diabetes careps the impact of new basal insulinsps insulin therapy real world studiesps insulin therapy fast acting insulin analoguesps the challenges of insulin therapy in type diabetesps different aspects of insulin therapyps the continued advance of continuous glucose monitoringps insulin pump therapyps automated insulin deliveryps the varied use of technologies in type diabetesps novel applications of technology in diabetesps novel therapies to reduce hypoglycaemiaps mechanisms and clinical consequences of hypoglycaemiain diabetesps emerging topics in hypoglycaemiaps investigating diabetes distress and depressionps aspects of quality of life and well beingps digital health in type diabetesps is telehealth the answer to improving care in diabetesps predicting prognosis of diabetic kidney diseaseps clinical aspects of diabetic kidney diseaseps the rock and role of experimental kidney diseaseps new tools to view diabetic retinopathyps diabetic retinopathy screening and interventionps focus on diabetic foot ulcersps hypertension and vascular diseaseps cure the pain of diabetic neuropathyps understanding clinical neuropathyps from artificial intelligence to treatment of diabetic footps from biomarkers to genetics of diabetic kidney diseaseps treatment of nafld and diabetes from food to pharmacologyps mechanisms and prevalence of nafldps lipids everywhere lipid metabolism in the liver and the heartps all about coronary arteries and diabetesps lipids and glucose not so good for the heartps cardiac complications of mice rats and cellsps atherosclerotic complications stemming from cells to the kidneyps stiff arteries and how to avoid themps cardiac function and dysfunctionps cardiovascular complications in humans through and throughps diabetes and neoplasiaps contemplating cognitive dysfunction in diabetesps endothelial cell circulation and the heartps tradition no nontraditional complications of diabetes 0cdiabetologiaop diabetes complications new insightsfrom cutting edge epidemiologycirculating metabolites significantly improve the prediction of renaldysfunction in type diabetesm scarale1 s de cosmo1 c prehn2 f schena3 j adamski2 vtrischitta4 c menzaghi11fondazione irccs casa sollievo della sofferenza san giovannirotondo italy 2helmholtz zentrum m¼nchen germany 3universityof bari bari italy 4sapienza university roma italy and aims chronic kidney disease ckd mainly indicated by a reduced glomerular filtration rate gfr remains one of theleading causes of reduced lifespan in patients with type diabetest2d discovering novel biomarkers able to predict low gfr will helpidentify highrisk patients to be targeted to more aggressive and burdensome preventive and treatment strategiesmaterials and methods we measured serum metabolites byabsoluteidqtm p180 kit biocrates life sciences ag innsbruckaustria and investigated their association with egfr calculated with theckdepi formula in a discovery sample of patients with t2d cases and controls with egfr60 and ¥70mlmin173m2 respectively a threshold p value of 28x104 ie followingbonferroni's rule was used as statistical significance in a model comprising age sex smoking bmi hba1c diabetes duration albumintocreatinine ratio acr and ongoing treatments metabolites associatedin the discovery sample were validated threshold p value of 005numberof surviving validation metabolites in a second cohort comprising diabetic patients cases and controls for egfr60 or ¥70mlmin173m2 respectively standardized values of each validated metabolitesweighted for the effect size ie observed in the discovery samplewere then summed up in a metabolic score metscore to be used as agfr prediction tool to this purpose metscore was used on top of anestablished clinical model comprising sex age bmi hba1c and acrand then discrimination [δ area under the receiver operating characteristic roc curve auc and the relative integrated discriminationimprovement ridi] and reclassification [the categoryfree net reclassification index cnri] measures were evaluatedresults thirteen metabolites six acylcarnitines six biogenic amines andone amino acid were independently associated to low egfr [ors range for 1sd increase p range 13x107 20x104] in the discoverysample all of them but one a biogenic amine were validated in thereplication sample [ors range for 1sd increase p range32x1018 43x106 below the threshold of 0051242x103] theauc of the abovementioned clinical model was and in the discovery the replication and the pooled sample respectively the addition of metscore on top of the clinical model improvedboth discrimination and reclassification measures in the discovery δauc4 p14x103 ridi29 p20x1011 ½cnri54p15x1014 the replication δ auc39 p16x103 ridi28p38x108 ½cnri30 p22x1010 and the pooled samples δauc39 p40x106 ridi29 p22x1017 ½cnri35p19x108conclusion we have discovered and validated metabolites that arestrongly associated with low egfr in patients with t2d a metscorecomprising these metabolites improves an established clinical prediction model of low egfr in terms of both discrimination and reclassification encouraged by these findings we are now investigating the ability ofmetscore to improve prediction of gfr decline in prospective cohorts oft2d with the aim of improving risk stratification and therefore refiningprevention efforts of kidney dysfunction in diabetic patientssupported by italian ministry of health rf201302356459disclosure m scarale noneassociation between insulinlike growth factor binding protein2 andinsulin sensitivity metformin and mortality in patients with newlydiagnosed type diabetesmr kristiansen12 js nielsen12 i brandslund3 da olsen3 jvstidsen2 sk nicolaisen4 r hjortebjerg25 j frystyk561danish centre for strategic research in type diabetes dd2odense 2steno diabetes center odense odense 3irs lillebaelthospital biochemistry and immunology vejle 4department ofclinical epidemiology aarhus 5department of clinical researchuniversity of southern denmark odense 6department ofendocrinology odense university hospital odense denmark and aims insulinlike growth factor binding protein2igfbp2 is engaged in metabolism circulating concentrations ofigfbp2 are positively correlated to insulin sensitivity overexpressionof igfbp2 protects against obesity and diabetes in mice and metforminincreases igfbp2 gene expression indicating that igfbp2 is a target ofmetformin action interestingly igfbp2 appears to predict mortalityindependently of insulin sensitivity this study aimed to investigate theassociation between indices of insulin sensitivity metformin treatmentand mortality in patients with newly diagnosed type diabetes t2dmaterials and methods in this crosssectional study we included newlydiagnosed patients with t2d enrolled in the danish centre for strategicresearch in type diabetes dd2 cohort patients were continuouslyenrolled from to throughout denmark and followed usingdanish healthcare registries unbound fractions of igfbp2 were determinedin serum from fasting drug na¯ve n864 and metformin treated ¥ twoprescriptions months prior enrollment patients n558 using an inhouseassay developed on the simoa platform values are given as medians iqrassociation was analyzed using a pearsons regressioncox regression amultivariable model was used to adjust for age bmi and homasresults a total of patients with median age of medianbmi of and median diabetes duration of yearswere included igfbp2 level was positively correlated with homasr2026 and p0005 and inversely correlated with cpeptide r2018and p0005 both associations persisted following adjustments for ageand bmi the igfbp2 level in metformin treated patients was slightlylower ngml than in drug na¯ve patients ngml p0026 a total of patients suffered from one or morecomorbidities from charlson comorbidity index their igfbp2 levelswere higher than patients with no comorbidity vs ngml p0001 during a median of years offollowup a total of patients died igfbp2 level was significantly higher at baseline in patients that died vs not died vs ngml p0001 igfbp2 was associated withmortality with a hazard ratiohr ci per doubling in proteinconcentration of p0001 this association was notobserved when analyzing patients without comorbidities but was significant in patients with other comorbidities hr p0001conclusion this is the first larger study to confirm that igfbp2 isassociated with indices of insulin sensitivity but is not largely affectedby metformin treatment interestingly increased igfbp2 level is associated with high mortality rates but the association was mainly driven bythe presence of comorbidities at baselinesupported by university of southern denmark and region of southerndenmarkdisclosure mr kristiansen nonebuilding clinical risk score systems for predicting allcause andcardiovascularspecific mortality among type diabetes patientscs liu1 tc li2 cc lin1 ci li11china medical university hospital taichung 2china medicaluniversity taichung taiwan 0c and aims no prior prediction model for mortality considered the effect of glycemic variability and blood pressure variabilitywhich have been broadly reported as the important clinical predictors ofmortality especially in diabetes patients the aim of this study was todevelop and validate risk score systems with considering the effects ofglycemic and blood pressure variability on allcause and cardiovascularspecific mortality in persons with type dmmaterials and methods this is a retrospective cohort study consistingof type diabetic patients aged years during allparticipants were randomly allocated into two groups derivation andvalidation sets in ratio and were followed up until death or august cox proportional hazards regression were used to develop allcauseand cardiovascularspecific mortality prediction model prediction modelperformance was assessed by the area under the receiver operating characteristics curve aurocresults overall deaths were identified after a mean of years offollowup the prediction accuracy measured by auroc of and 15year allcause mortality based on a model containing the identifiedtraditional risk factor biomarkers and variability in fasting plasmaglucose and hba1c and diastolic blood pressure variability were and respectively in derivation set and the corresponding values forcardiovascularspecific mortality were and respectively the predictionaccuracy in the validation set for allcause mortality were and respectively and for cardiovascularspecific mortality were and respectivelyconclusion our prediction model considering glycemic and blood pressure variability had good accuracy of prediction of cardiovascularspecific and allcause mortality in patients with type diabetessupported by ministry of science and technology of taiwandisclosure c liu noneincident cardiovascular disease by clustering of favourable riskfactors in type diabetes the eurodiab prospectivecomplications studys soulimane1 yd vogtschmidt12 m toeller3 b balkau4 nchaturvedi5 jh fuller6 ss soedamahmuthu121department of medical and clinical psychology center of research onpsychological and somatic disorders corps tilburg universitynetherlands 2institute for food nutrition and health university ofreading reading uk 3heinrichheineuniversity d¼sseldorfd¼sseldorf germany 4clinical epidemiology universit© parissaclayuvsq inserm cesp villejuif france 5institute of cardiovascularscience university college of london london uk 6department ofepidemiology and public health eurodiab london uk and aims the incidence of cardiovascular diseases cvdis up to eight times higher in people with type diabetes t1d greaterclustering of adverse risk factors is thought to contribute to excess cvdrisks in type diabetes though not explored in t1d the aim of this studywas to examine a cvd risk reduction for those in the most favourablethird of individual risk factors compared to the least favourable two thirdsand b cvd risk reduction by clustering of favourable cvd risk factorsmaterials and methods we analysed data of participants from theeurodiab prospective complications study a european t1d cohortrecruited in countries between were men with a meanage of ± years we studied seven cvd risk factors namely hba1csmoking bmi combined systolic and diastolic bp ldl cholesterolphysical activity pa and diet table cox proportional hazards analyses were used to calculate hazard ratios hr [95ci] of incident cvdfor each cvd risk factor adjusted for age sex retinopathy comparingthose in the most favourable tertiles with the least favourable two tertilesdiabetologiawe then scored each individual by the number of risk factors for whichthey occupied the most favourable tertilesresults there were incident cvd cases after a mean followup of± years multivariable cox models showed that participants withthe most favourable hba1c57 [39mmolmol] had a significantlylower cvd risk hr [95ci] [] than the least favourabletwo tertiles nonsignificant inverse associations were found withfavourable bmi [] pa [] diet score[] and bp [] no associations were foundwith smoking or ldlcholesterol greater clustering of favourablecvd risk factors was associated with a lower risk of cvd in univariatemodels with a significant linear trend in multivariate models the resultswere partly attenuated with the lowest hr of [ ] in peoplewith clustering of favourable cvd risk factors tableconclusion greater clustering of favourable cvd risk factors was associated with a lower risk of incident cvd in people with t1d with a doseresponse relationship hba1c remained the most protective factor againstcvd in t1d targeting combined risk factors could be more effective inpreventing cvd risk than targeting single risk factorssupported by welcome trust the european community and diabetesukdisclosure s soulimane nonebidirectional association between type diabetes and obstructivesleep apnoea a metaepidemiological studyt karagiannis1 e athanasiadou1 a tsapas12 e bekiari11clinical research and evidencebased medicine unit aristotleuniversity of thessaloniki thessaloniki greece 2harris manchestercollege university of oxford oxford uk and aims individual epidemiological studies suggest acomplex relationship between type diabetes and obstructive sleepapnea we aimed to assess whether there is a bidirectional associationbetween the two conditions by conducting a metaanalysis of longitudinalcohort studiesmaterials and methods we included cohort studies that evaluated theassociation between type diabetes and obstructive sleep apnea in eitherdirection published until january we pooled cohortspecific estimates by means of random and fixed effects metaanalyses and calculatedodds ratios ors with confidence intervals cis to measure theassociation of prevalent obstructive sleep apnea with incident type diabetes and of prevalent type diabetes with incident obstructive sleepapnearesults out of records identified through the search cohortstudies were included in the metaepidemiological analysis ten studiesevaluated the association between prevalent obstructive sleep apnea andincident type diabetes one study assessed the association betweenprevalent type diabetes and incident obstructive sleep apnea while fourstudies evaluated a bidirectional association duration of study followupranged between and years median years the random effectsmetaanalysis for prevalent obstructive sleep apnea and incident type diabetes patients yielded an or of ci to 0cdiabetologiaresults were consistent in the fixed effects metaanalysis figureprevalent type diabetes increased the odds of incident obstructive sleepapnea patients with an or of ci to and ci to for the randomeffects and fixedeffects metaanalysis respectively metaanalyses of effect estimates adjusted forconfounding factors were similar to those of the main analysisconclusion pooled evidence from large cohort studies suggests thatpresence of obstructive sleep apnea at baseline is associated withincreased risk for developing type diabetes while presence of type diabetes is associated with increased risk for developing obstructive sleepapnea thus effective management of either condition could preventdevelopment of the otherfigure odds ratio for developing type diabetes in patients with obstructive sleep apnea versus those without obstructive sleep apneaalzheimer hr [ ic ] vascular dementia hr [ ic ] and nonvascular dementia hr [ ic ] when a 3years landmark analysis was conducted the associations remained similar for vascular and nonvascular dementia but disappeared for alzheimers diseasesconclusion the association of t2d with neurodegenerative diseasesdiffer by type of dementia the strongest detrimental association wasobserved for vascular dementia moreover t2d patients with polycaemic control have an increased risk of developing vascular andnonvascular dementiadisclosure c celismorales nonesupported by greece and the european social fund esfdisclosure t karagiannis noneglycated haemoglobin type diabetes and the links to dementia andits major sub types findings from the swedish national diabetesregisterc celismorales1 s franz©n2 am svensson3 n sattar1 sgudbjornsdottir21institute of cardiovascular and medical sciences university ofglasgow glasgow uk 2department of molecular and clinicalmedicine university of gothenburg gothenburg sweden 3swedishnational diabetes register gothenburg sweden and aims type diabetes t2d has been associated withhigh dementia risk however the links to different dementia subtypes isunclear we examined to what extent t2d associated with alzheimervascular and non vascular dementia incidence and whether such associations differed by glycaemic controlmaterials and methods in this swedish national diabetes registerstudy we included patients with t2d and matchedcontrols the outcomes were incidence of alzheimer vascular and nonvascular dementia the association of t2d with dementia was stratifiedby baseline glycated haemoglobin hba1c concentrations cox regression was used to study the excess risk of outcomesresults the followup median years t2d patientsand controls developed dementia the strongest association was observed for vascular dementia here patients with t2d had ahr of [ ci ] compared to controls the association oft2d with nonvascular dementia was more modest hr [ ci ] however risk of alzheimer was lower in t2d patientscompared to controls hr [ ci ] when the analyseswere stratified by circulating concentrations of hba1c a doseresponseassociation was observed compare to patients with t2d with hba1c mmolmol those with hba1c mmolmol had a higher risk of 0cop news on the insulin secretion frontwhat makes beta cells 1st responders and are they temporallyconsistentv kravets we schleicher jm dwulet am davis rkpbenningerbioengineering university of colorado aurora usa and aims calcium ca2 uptake drives glucosestimulated insulin secretion from the pancreatic cells functionalsubpopulations of cells disproportionally control the oscillatory phaseof ca2 uptake which is disrupted with ageing and in diabetes less isknown about cells which impact the 1st phase of ca2 uptake disruptedin early diabetes here we determine whether 1stresponder cells thatlead the 1st phase of ca2 uptake are the same as hub cells that coordinate oscillatory ca2 2nd phase we study what makes cell a1st responder and whether 1st responders are a transient state or a distincttemporally stable subpopulationmaterials and methods we used mipcreer gcamp6s mouse modelwhich expresses ca2sensitive gfp specifically in cells weperformed simultaneous recording of ca2 dynamics and gap junctionpermeability in individual islets we stimulated islets with glucosekatp channel blocker glibenclamide and kcl based on ca2 dynamicswe defined the of cells responding to the glucose stimulation soonerthan the rest of the islet as 1st responders and the of cellsresponding slower as last responders we tested their temporal consistency over and hours we used laser ablation to remove specificcells from the islet we performed computational modelling of the isletelectrophysiologyresults we found that ca2 wave coordination of the 1st responders wasnot greater than the isletaverage and hence they are not overlapping withhighlycoordinated hub cells in fact according to our gap junctionpermeability data 1st responders had lower than average electricalcoupling p00157 furthermore our computational model showedlower electrical coupling conductance in both 1st and last respondersp00447 p00279 this may be explained by our finding that1st responders are located at the islets periphery at ± of the isletsradius we found 1st responders to be consistent under glibenclamidestimulation cells which respond first to the glucose remained in the15th percentile of the time response distribution when stimulated withglibenclamide sem this is consistent with our computationalresults 1st responders had lower katp conductance hence highermembrane depolarization probability p00086 glucose elevationswith 1h period showed that 1st responders remained consistent withreaction time within the of the reaction time distribution for all cellswith an elevation period of hours their reaction time shifted to thesecond quartile of the distribution and with hours to the medianunlike 1st responders last responder cells were not consistent at any timeinterval ablation of the 1st responders discoordinated but did notdisrupt the ca2 response of the islet a different cell took over the roleof the 1st responder postablation this new 1st responder was a cell whichoriginally preablation was within a leading 7th percentile of the timeresponse distribution sem conclusion in conclusion 1st responders are distinct from hub cellsubpopulation have higher membrane depolarization probability and areless strongly coupled to other cells after the laser ablation of a1st responder new 1st responder taking on its role comes from a poolof original leading cells while initially consistent over a short 1h periodof time 1st responders may be losing temporal consistency over longertime periodssupported by nr01 dk102950 dk106412 jdrf 3pdf2019741andisclosure v kravets nonediabetologiabetaarrestin is absolutely required for the potentiation of insulinsecretion by gipma ravier1 j obeid1 m leduc1 s costes1 p gilon2 s dalle1 gbertrand11igf univ montpellier cnrs inserm montpellierfrance 2universit© catholique de louvain brussels belgium and aims the scaffold protein betaarrestin2 arrb2 isknown to uncouple g protein coupled receptors gpcrs from the gprotein and to recruit new signaling pathways such as the erk12pi3k fak¯ in non beta cells arrb2 interacts with a wide rangeof gpcrs but its interaction with the gip receptor gipr is still unclearour aim is to determine if arrb2 is involved in the signaling of thegipr in pancreatic beta cellsmaterials and methods the experiments were carried out in beta cellsfrom fivemonthold arrb2 and arrb2 male mice camp productioncampsepac endogenous pka akar3 and erk12 ekaractivations [ca2] in the cytosol [ca2]c fura2lr and in the endoplasmic reticulum [ca2]er d4er were assessed by live cell imagingin mouse pancreatic beta cells epac2 epac2gfp recruitmentbeneath the plasma membrane was monitored by total internal reflectionfluorescence microscopy factin depolymerisation was evaluated byphalloidin staining alexa fluor 488conjugated phalloidin and thep h o s p h o r y l a t i o n o f f o c a l a d h e s i o n k i n a s e f a k b yimmunofluorescenceresults insulin secretion from arrb2 islets was reduced by compared to arrb2 islets in response to gip 100pm10nm p001when arrb2 arrb2gfp was reexpressed in arrb2 beta cells insulin secretion in response to gip was restored to a similar level thanin arrb2 islets surprisingly upon gip stimulation 10pm10nm thecamp production pka activation and epac2 recruitment were similarin arrb2and arrb2 beta cells both [ca2]c and [ca2]er remainedcomparable finally the activation of erk12 was also similarin arrb2 and arrb2 beta cells by contrast the factin depolymerisationinduced by 10nm gip was significantly reduced p001 in arrb2 beta cells pi3kγ and fak have been reported to be involved in factindepolymerisation in response to gip and glucose respectively and to berequired for optimal insulin secretion as expected the pi3kγ inhibitoras604850 1μmoll reduced factin depolymerisation p001by gip stimulation in arrb2 beta cells but no additional effect wasobserved in arrb2 beta cells moreover gipinduced fak activationwas also reduced by in arrb2 beta cellsconclusion our study revealed that arrb2 is required for the potentiation of insulin secretion by gip through factin depolymerisation probably via fak activation and pi3kγ recruitment but independently fromthe canonical camp signalling pka and epac2 and the erk12 pathway therefore any variation in the expression of arrb2 as observed indiabetic states should functionally affect the incretin effect produced bygipsupported by soci©t© francophone du diabete sfddisclosure ma ravier nonepancreatic beta cellselective deletion of the mitofusins and mfn1and mfn2 impairs glucosestimulated insulin secretion in vitro andin vivoga rutter1 e geiadou1 t rodriguez2 c muralidharan3 mmartinez3 p chabosseau1 a tomas1 g carrat1 a di gregorio2 ileclerc1 ak linnemann31cell biology functional genomics faculty of medicine imperialcollege london london uk 2national heart and lung instituteimperial college london london uk 3center for diabetes andmetabolic diseases indiana university school of medicineindianapolis usa 0cdiabetologia and aims mitochondrial metabolism of glucose is essential for the initiation of insulin release from pancreatic beta cellsalthough altered in subjects with type diabetes whether mitochondrialultrastructure and the proteins controlling the fission and fusion of theseanelles are important for glucose recognition is unclear here wegenerated mice with beta cellselective adultrestricted deletionof mfn1 and mfn2 essential for mitochondrial fusion and studied theimpact on insulin secretion and glucose homeostasis in vivo and in vitromaterials and methods c57bl6 mice bearing mfn1 and mfn2 alleleswith floxp sites were crossed to transgenic animals carrying aninducible cre recombinase under pdx1 promoter control pdxcreertrecombination was achieved by daily tamoxifen injections for one weekislets were isolated and used for live beta cell fluorescence imaging ofcytosolic cal520 or mitochondrial rgeco free ca2 concentrationand membrane potential tetramethyl rhodamine methyl ester tmrmusing spinning disc confocal microscopy nikon ti2 mitochondrialnetwork characteristics were quantified using super resolution fluorescence zeiss lsm and transmission electron microscopy intravitalimaging was performed in mice injected with an adenoassociated virusto express the cytosolic ca2 sensor gcamp6s selectively in beta cellsunder the control of the rat insulin promoter using multiphoton microscopy leica tcs sp8 dive blood flow through the islet was visualisedsimultaneously after injection of fluorescent albumin647results mitochondrial length was sharply to ± of controlsp00001 reduced in the mfn12 ko mice and these animals displayedhigher fasting glycaemia than control littermates at weeks vs mmoll p005 in vivo an increase in circulating glucose levelswas also observed p005 at min and p001 at min and wasassociated with a substantial fivefold decrease in plasma insulin min p00001 postintraperitoneal glucose injection mitochondrialca2 accumulation and membrane potential were significantly reducedp001 in response to high glucose in the ko animals examined byintravital imaging of the exteriorised pancreas antiparallel changes incytosolic ca2 and mitochondrial membrane potential observed incontrol animals were largely suppressed after mfn12 deletionconclusion mitochondrial fusion and fission cycles are essential in thebeta cell to maintain normal mitochondrial bioenergetics and glucosesensing both in vitro and in the living mouse such cycles may bedisrupted in some forms of diabetes to impair mitochondrial functionand consequently insulin secretio | Colon_Cancer |
" tumor associated macrophages tam constitute the most abundant immune cells in the tumor stroma initiating pro inflammatory m1 or immunosuppressive m2 responses depending on their polarization status advances in tumor immunotherapy call for a detailed understanding of potential immunogenic mechanisms of irradiation routinely applied in rectal cancer patientsmethods to test the effects of radiotherapy on tam we ex vivo irradiated tissue samples of human rectal cancer and assessed the phenotype by flow cytometry we furthermore evaluated the distribution of leucocyte subsets in tissue sections of patients after short course radiotherapy and compared findings to non pretreated rectal cancer using an immunostaining approach anotypic assays ota consisting of macrophages cancer associated fibroblast and cancer cell lines were used to dissect the immunological consequences of irradiation in macrophagesresults we demonstrate that short course neoadjuvant radiotherapy in rectal cancer patients is associated with a shift in the polarization of tam towards an m1 like pro inflammatory phenotype in addition ex vivo irradiation caused an increase in the phagocytic activity and enhanced expression of markers associated with stimulatory signals necessary for t cell activation in ota we observed that this alteration in macrophage polarization could be mediated by extracellular vesicles ev derived from irradiated tumor cells we identified high mobility group box in ev from irradiated tumor cells as a potential effector signal in that crosstalks our findings highlight macrophages as potential effector cells upon irradiation in rectal cancer by diminishing their immunosuppressive phenotype and activate pro inflammation our data indicate that clinically applied short term radiotherapy for rectal cancer may be exploited to stimulate immunogenic macrophages and suggest to target the polarization status of macrophages to enhance future immunotherapeutic strategiesintroductionsince the introduction of immune checkpoint blockade immunotherapy has become an attractive therapeutic option in cancer1 irradiation used as standard therapy in a number of solid malignancies induces immunogenic cell death icd by the release of damage associated pattern damp4 studies based on murine models indicated that irradiation induced dna damage of tumor cells elicited an antitumor immune response5 it was shown that alterations of the infiltrating immune cells by irradiation might be augmented when combined with immune modulating drugs6 a detailed understanding of the impact of radiotherapy on the immune system in humans should allow application of radiotherapy as a part of novel immunotherapeutic concepts however little is known about the detailed regulation of the immunogenic effect of irradiation in clinical settingsmacrophages are one of the most abundant immune cell subsets in tumor tissue9 and play a key role in the cancer microenvironment11 tumor associated macrophages tam are functionally diverse13 and display high plasticity upon immunological stimuli14 the concept of m1 and m2 macrophages was introduced to describe the heterogeneity of this cell subset16 m1 like macrophages possess the capacity to clear infections in support of a t helper type th1 immune response17 whereas m2 like macrophages respond in general to th2 cytokines and are strongly enriched in the tumor microenvironment18 the concept of m1m2 has been challenged and is seen as an oversimplified approach to the phenotype of macrophages but can be viewed as a linear scale on which m1 and m2 present two extremes19 tam recognize damp and respond by producing a variety of cytokines and growth factors to promote innate and stary a0v et a0al j immunother cancer 20208e000667 101136jitc2020000667 0copen access adaptive immunity9 in response to these signals macrophages are able to undergo reprogramming with enhanced antitumor features making them an attractive target for anticancer therapies22 there are conflicting results with respect to the effect of irradiation on the macrophage phenotype klug et al demonstrated in a murine model of breast cancer and human pancreatic cancer that low dose irradiation gy induced repolarization of m2 like macrophages to m1 like macrophages via induction of nitric oxide synthase inos24 moreover expression of inos correlated with vessel normalization and an influx of cd8 t cells suggesting a tumor ablative as well as pro inflammatory effect of repolarized macrophages upregulation of inos was also observed in murine prostate cancer which has been irradiated with up to gy25 similarly agonists of the toll like receptor further stimulated the m1 phenotype of macrophages and enhanced the antitumor effects of irradiation in a murine model of breast cancer26 other studies suggested that irradiation of tumors was associated with a more immunosuppressive phenotype of tam27 jones found that the depletion of macrophages by an anti csf antibody greatly increased tumor ablation upon irradiation gy in murine tumors generated from a colorectal and a pancreatic cell line obviously the effect of irradiation appears to depend on the model irradiation dose tissue as well as on the investigated time point after irradiation however despite the controversial reports an important role for tam in response to radiotherapy seems evidentmore recently extracellular vesicles ev have attracted attention in mediating signals to immune cells ev are rich in molecular cargos and are emerging as critical messengers in the cell to cell crosstalk they contain a variety of small signaling molecules which can be transferred to other cell types to modulate cell functions30 thus we hypothesized that ev might be involved in macrophages regulation in the tumor microenvironment in irradiated cells using a clinically relevant approach we show that short course irradiation increased the proportion of m1 like tam in human rectal cancer tissue primary short term cultures and anotypic tumor assays ota consisting of tumor cell lines macrophages and cancer associated fibroblasts allowed to further dissect irradiation induced changes in colorectal tam using irradiated tumor cell lines we demonstrate that ev are able to mediate irradiation induced repolarization of macrophagesmethodspatients and tissue materialpatients with clinical t3 rectal cancer patients characteristics online supplementary table received neoadjuvant hyperfractionated radiotherapy over the course of days within the frame of a controlled clinical study34 as a control group we used historical surgical tumor specimen of non pretreated rectal cancer lesions of patients from the same institution with no history of irradiation therapy or cytoablative treatment for immunofluorescence and immunohistochemical stainings surgical paraffin embedded specimen of the cancer lesions were cut in µm sections and mounted on slides for ex vivo cultures and subsequent flow cytometric stainings rectal cancer tissue was obtained from patients with histologically verified rectal cancer with no history of irradiation therapy or cytoablative treatment studies involving patient material were performed according to the declaration of helsinki and approved by the local ethics committee of the medical university of vienna for ota hct116 ccl147 and dld1 ccl221 were purchased from atcc peripheral blood mononuclear cells for macrophage differentiation were isolated from healthy blood donorsprimary ex vivo cultures of leucocytes and flow cytometrytissue samples of non irradiated rectal cancer were minced resuspended in rpmi medium with fetal bovine serum and plated in a mm petri dish irradiation protocol was applied as indicated below after hour of incubation °c co2 a single cell suspension from cancer tissue was prepared briefly tissue was digested with collagenase iv unitsml and dnase i mgml for hour min in rpmi supplemented with fetal bovine serum and hepes buffer solution at °c afterwards cell suspension was rinsed through a µm mesh and leucocytes were isolated by density gradient centrifugation using ficoll gradients for flow cytometry analysis cells were stained with fluorescence antibodies listed in the online supplementary table livedead discrimination was performed with fixable viability dye biolegend samples were acquired on a facsaria iii bd and analyzed with flowjo software v1061irradiation protocola theratron mds nordion radiotherapy unit with a cobalt60 source was used for γirradiation of ota ex vivo cultures of rectal cancer tissue monocyte derived macrophages and cancer cell lines tissue samples of rectal cancer were minced and cultivated petri dishes with cancer tissue were irradiated with à gy after hours of incubation a single cell suspension was prepared ota and cancer cell lines hct116 and dld1 were irradiated with à gy or à gy and subsequently cultivated over the course of hours monocyte derived macrophages were irradiated with gy or gy a negative control gy was always transported to the radiotherapy unit but was not exposed to γirradiationex vivo phagocytosis assaymononuclear cells isolated of rectal cancer lesions were spun down at g for min at °c for ex vivo escherichia coli e coli phagocytosis assay irradiated and non irradiated leucocytes were resuspended in µl phrodo red e coli bioparticles thermo fisher scientific as per the manufacturers instruction a same set of cells were resuspended in phosphate buffered saline pbs as controls after hours incubation at °c stary a0v et a0al j immunother cancer 20208e000667 101136jitc2020000667 0copen accessleucocytes were washed and stained for flow cytometry tam cd11b cd14 hla dr viable cd45 cells that were pe high were considered to be phagocytosing for ex vivo tumor cell phagocytosis assay tam were isolated via fluorescence activated cell sorting facs cd11b cd14 hla dr viable cd45 cells from healthy colon mucosa or colorectal cancer lesions and incubated overnight with ev isolated from irradiated à gy and non irradiated gy dld1 cells evtam after washing dld1 were labeled with carboxyfluorescein diacetate succinimidyl ester cfse thermo fisher µm and cocultured with tam at an effector to target cell ratio of the proportion of cfse tam was assessed by flow cytometry after hours of incubation at °cat the air liquid interface in dmem supplemented with egm for seeding of tumor cells nylon discs with collagen cell cylinders were transferred into well plates five hundred microlitres of a tumor cell suspension with à cellsml hct116 or dld1 were added to each well after hours the tumor cells attached to the surface of the collagen gels and the nylon meshes with the tumor cell colonized collagen cell cylinders were put back onto the metal grids and incubated for days with media changes every days prior to further experiments for further assessment ota were embedded in optimal cutting temperature media snap frozen in liquid nitrogen and stored at °c until processing or fixed and embedded in paraffinimmunofluorescence and immunohistochemical stainingdirectly and indirectly labeled monoclonal antibodies as listed in online supplementary table and dapi as a nuclear marker were used an isotype was used as negative control in brief after incubation with the primary antibodies overnight at °c an appropriate secondary fluorescence labeled antibody was applied for min at room temperature followed by staining of dapi nuclear marker for immunohistochemical staining antibodies were mixed with bovine serum albumin in pbs and applied overnight to sections in a humid chamber at °c for visualization of the cells aec was used as chromogen sections were counter stained with hematoxylin for evaluation of staining results images of whole tissue sections one section per patient were acquired using a z1 axio observer microscope equipped with a ld plan neofluar à objective zeiss for leucocyte evaluation the tumor normal interface mm on tumor and normal zone was defined as regions of interests roi for γh2ax staining only tumor tissue was selected as roi roi were automatically quantified using tissuefaxstissuequest image analysis software tissuegnostics gmbhpreparation of otaota were set up as previously described35 briefly cancer associated fibroblasts caf were isolated and cultured from fresh samples of colon adenocarcinomas for the preparation of macrophages monocytes were isolated using the easysep direct human monocyte isolation kit stemcell technologies according to the manufacturers instructions à caf and monocytes per ota were mixed and pelleted by centrifugation at g for min for the collagen gel preparation all steps were performed on ice collagen solutions were prepared by mixing mgml of collagen i rat tail mgml becton dickinson and the fibroblastmonocyte suspension in dulbecco's modified eagle medium dmem supplemented with endothelial cell growth medium mv2 egm promocell a total of µl each of the collagen cell suspension were transferred into silicone gel casting devices after polymerization of the collagen solution the gels were lifted up on nylon mesh discs and transferred onto metal grids in well plates and cultured macrophage differentiationpbmc were obtained by ficoll plaque density gradient centrifugation pbmc were seeded at a concentration of Ã106well well plates in rpmi medium monocytes were isolated using the ability of monocytes to adhere to non tissue culture treated plastic culture dishes attached cells were cultivated in rpmi medium with glutamax thermo fisher scientific supplemented with ngml macrophage colony stimulating factor m csf thermo fisher scientific fetal bovine serum uml penicillin uml streptomycin and µgml fungizone in a humidified atmosphere at °c cells were cultivated for days with two medium changes to obtain m1 lpsifnγ polarized macrophages cells were stimulated with ngml lipopolysaccharide lps sigma aldrich and ngml interferonγ ifnγ thermo fisher scientific for hours m2 il4il13 polarized macrophages were generated using ngml interleukin il4 strathmann and ngml il13 biolegendev preparation and characterizationev were isolated by differential centrifugation hct116 and dld1 cells were cultured in mccoys 5a medium supplemented with exosome depleted fetal calf serum briefly cancer cell line culture medium was centrifuged at g for min to pellet cells supernatant was transferred to new falcon tubes and then centrifuged at g for min to pellet dead cells and apoptotic bodies rotanta 460rc hettich supernatant was transferred to the high speed centrifugation tubes and large ev were removed by ultracentrifugation at g for min sorvall evolution rc thermo fisher scientific after filtration of the supernatant through a µm syringe filter small ev were pelleted by ultracentrifugation at g for min rotor t1250 suspended in ice cold pbs and collected after another ultracentrifugation at g for min rotor type sorvall wx ultra thermo fisher scientific the ev pellet was resuspended in cold pbs for further use ev count was determined using a nanoparticle tracking analyzer zetaview particlemetrix results were analyzed with zetaview software ev were added to macrophages at a stary a0v et a0al j immunother cancer 20208e000667 101136jitc2020000667 0copen access concentration of Ãà cells we have submitted all relevant data of our experiments to the ev track knowledgebase ev track id ev20006537western blotcells were scraped from cell culture plates washed in ice cold pbs and lysed in à ripa buffer mm trishcl ph mm nacl np40 sodium deoxycholate sds containing à halt protease inhibitor cocktail thermo fisher scientific ev suspensions were mixed with à ripa buffer containing à halt protease inhibitor cocktail to obtain à concentration protein extracts were incubated at °c for min and centrifuged at g for min clear protein extract supernatants were quantified with the bca protein assay kit thermo fisher scientific one to five micrograms of protein were denatured with laemmli buffer loaded into each well of a acrylamide bisacrylamide gel containing sds and electrophoresed with magel for vhours proteins were transferred to a polyvinylidene fluoride membrane using the transblot turbo and the rta ready to assemble transfer kit bio rad after several hours of blocking pvdf membranes were incubated with primary antibodies online supplementary table at °c overnight after washing with tris buffered saline tween detergent membranes were incubated with the secondary antibody anti rabbit immunoglobulin g igg hrp linked antibody cell signaling technology or anti mouse igg hrp linked antibody cell signaling technology at room temperature for hour detection was done with clarity or clarity max western ecl substrate bio rad protein band intensities were quantified with imagequant tl ge healthcareelectron microscopyafter mounting cu mesh r22 holy carbon grids quantifoil with a tweezer into a leica gp leica microsystems grid plunger µl of ev sample solution were applied to grids front side and blotted for s grids were plunge frozen into liquid ethane for instant vitrification and transferred to a glacios cryo transmission microscope thermo fisher scientific equipped with a x feg images were recorded in low dose mode using the serialem software mastronarde with a falcon3 direct electron detector at magnifications of and with pixel sizes of and respectivelystatistical analysisstatistical analysis was performed using graphpad prism graphpad software statistical significance was determined by student's t test when comparing two groups the two way analysis of variance followed by tukey's multiple comparison test was used when comparing three or more groups significance was set at a p value of less than resultstam in rectal cancer polarize towards m1like phenotype upon irradiationwe first investigated the effect of irradiation on human tam in a primary ex vivo culture of rectal cancer specimen for this purpose minced tumor tissue was irradiated with gy and subsequently cultivated for hours before leucocyte isolation figure 1a the gating strategy for macrophages isolated from viable cd45 mononuclear cells of human rectal cancer is shown in figure 1b tam as defined by cd14cd11bcd68hla dr accounted for over of all viable cd45 leucocytes in rectal cancer figure 1c with over cells per gram of tissue figure 1d there was no significant difference in percentage or absolute numbers of tam in non irradiated versus corresponding ex vivo irradiated tissue samples figure 1cd we then assessed the phenotype of treatment naïve and ex vivo irradiated tam using flow cytometry in naïve rectal cancer lesions tam were characterized by high expression of cd206 cd163 and cd64 and low levels of the chemokine receptor ccr7 indicating that the m2 like macrophage phenotype was present in untreated rectal cancer figure 1e this was also reflected in their cytokine pattern as more tam in untreated rectal cancer samples produced il10 il13 and il4 while few interferonγ ifnγ and tumor necrosis factor alpha tnfα producing tam were found in untreated samples in contrast ex vivo application of gy γirradiation to primary rectal cancer leucocytes resulted in a reduced presence of cd163 tam this phenotype correlated with enhanced levels of tnfα and inos tam as well as diminished detection of il10 and il13 tam hence low dose irradiation of rectal cancer tissue can polarize tam towards a pro inflammatory m1 like phenotype and may therefore directly contribute to antitumor activity by tnfα and inos productionlowdose irradiation reduces pd1 expression and enhances phagocytosis in tam to e colito further assess the distinct functional phenotype of tam on irradiation we investigated the ability of tam to phagocyte as an aspect of direct effector function and activation to model the phagocytic behavior of irradiated tam we incubated leucocytes derived from irradiated ex vivo tumor culture with ph sensitive pe labeled e coli particles in the phagosome the fluorescence of e coli particles increases as demonstrated in a representative example figure 2a phagocytosis by tam was significantly elevated upon irradiation with gy figure 2b demonstrating that the acquired shift towards m1 polarization due to irradiation was also functionally relevant as pd1 was previously shown to inhibit phagocytosis38 we next determined pd1 expression on irradiated ex vivo tumor culture derived tam we found variable pd1 expression on tam in non irradiated rectal cancer and observed a significant decrease of pd1 on tam derived from irradiated cultures figure 2c these stary a0v et a0al j immunother cancer 20208e000667 101136jitc2020000667 0cchanges induced by irradiation can be instrumental for phagocytic activity of tamirradiation promotes antigen presentation and cytokines associated with th1 responseone of the hallmarks of m1 polarization of macrophages is the acquisition of antigen presenting features leading to efficient th1 response17 as a first step to determine whether irradiation may result in improved capability of tam to initiate immune responses we analyzed the expression of cd86 on tam derived from irradiated ex vivo tumor cultures irradiation correlated with a significant increase of cd86 on the cell surface of tam as compared with tam derived from non irradiated naïve cultures figure 2d we next investigated hla dr on non irradiated tam versus tam irradiated with gy we observed hla dr low expressing tam and high expressing tam clearly dividing them into two groups figure 2e in non irradiated samples more than of tam expressed high levels of hla dr while irradiation significantly increased the fraction of hla drhigh tam to over figure 2f we next investigated whether irradiation can induce the expression of il12 p70 and il23 p19 as markers for the initiation of adaptive immune responses whereas il23 p19 showed a tendency to be produced by tam on irradiation il12 p70 was significantly induced in irradiated tumor tissue compared with open accesstreatment naïve samples figure 2g together these results suggest that low dose irradiation influences tam polarization in rectal cancers by equipping the cells with an hla drhiil12hi il23hi cytokine profile this observation emphasizes that irradiated tam have a higher probability to participate in antitumor immune responses directly by phagocytosis and cytokine secretion as well as indirectly by exhibiting a broad armamentarium that potentially activates t cells as main players of antitumor adaptive immunityshortcourse irradiation of patients with rectal cancer increases the m1m2 ratio of tamto corroborate our ex vivo data with the in vivo situation on short course irradiation we examined tam polarization and function in rectal cancer patients treated by a routinely applied radiotherapy protocol we made use of a cohort of patients in clinical stage t3 rectal cancer who received neoadjuvant hyperfractionated short course radiotherapy two times gy per day over the course of days figure 3a surgery was performed days after radiotherapy in these patients as a control we used surgical specimen from treatment naïve clinical t3 rectal cancer patients importantly the indication for radiotherapy was not correlated with a more severe tumor progression compared with the treatment naïve cohort figure ex vivo γirradiation induces polarization of tumor associated macrophages a tissue samples of naïve rectal cancer were minced and irradiated after incubation a single cell suspension of cancer tissue was prepared b representative example of the gating strategy for macrophages isolated from viable cd45 mononuclear cells of human rectal cancer c percentage of macrophages compared to total of viable cd45 cells in non irradiated and ex vivo irradiated rectal cancer lesions assessed by flow cytometry and presented as mean±sd n10 d mean numbers of macrophages in rectal cancer per gram in non irradiated and ex vivo irradiated rectal cancer presented as mean±sd n10 e expression of intracellular and extracellular markers in macrophages assessed by flow cytometry bars presented as mean percentage of indicated markers±sd of non irradiated and ex vivo irradiated macrophages n10 p005 p0001 p00001 by two tailed paired students t test gy gray tam tumor associated macrophages ifnγ interferonγ tnfα tumor necrosis factorα il interleukin inos inducible nitric oxide synthasestary a0v et a0al j immunother cancer 20208e000667 101136jitc2020000667 0copen access irradiation increases phagocytosis and marker associated with antigen presentation and th1 response a figure representative histogram and flow cytometry plots indicating difference in pe fluorescence of non irradiated blue versus irradiated red tam in ex vivo phagocytosis assay total phagocytosis was analyzed by first gating on tam and then gating on pe cells b analysis of tam phagocytosis of phrodo e coli particles data presented as percentage of phagocytosis of non irradiated tam and corresponding irradiated tam gy n5 c percentage of tam positive for pd1 non irradiated versus ex vivo irradiated and representative histogram non irradiatedblue vs irradiatedred data presented as mean±sd n10 d percentage of expression of cd86 in non irradiated versus ex vivo irradiated tam and representative histogram non irradiatedblue vs irradiatedred data presented as mean±sd n10 e representative plots histogram and f expression of hla dr on tam of non irradiated and ex vivo irradiated rectal cancer data given as mean±sd n10 g expression of il12 p70 and il23 p19 data presented as mean percentage±sd of total tam n10 representative example of il12 p70 and il23 p19 expression on tam p005 p0001 by two tailed paired students t test th1 t helper type gy gray tam tumor associated macrophages il interleukinstary a0v et a0al j immunother cancer 20208e000667 101136jitc2020000667 0copen accessfigure short course irradiation in patients modulates the immune infiltrate with induction of macrophage polarization a irradiation protocol of patients with neoadjuvant hyperfractionated à gy per day over the course of five days radiotherapy for clinical t3 rectal cancer surgery was performed the following week b quantitative in situ assessment of infiltrating macrophages cd68 t cells cd3cd56 nk cells cd56cd3 and nkt cells cd56cd3 in non irradiated n25 and irradiated rectal cancer n45 assessed by multicolor immunofluorescence staining data are given as absolute numbers of positive cells per mm2±sd c analysis of immunohistochemical staining for γh2ax as irradiation response in non irradiated n25 and irradiated tissue sections data are given as mean±sd d representative examples of immunohistochemical staining for γh2ax images below are magnified à e representative immunohistochemical staining of cd68 cells in non irradiated n25 and irradiated tissue sections n45 images below are magnified à f representative example of immunofluorescence multicolor staining of cd68 macrophages pe m1inos fitc m2 cd163 apc g quantitative in situ analysis of immunofluorescence staining of ratio of inos m1 to cd163 m2 tams cd68 macrophages presented as m1m2 in non irradiated n25 compared to irradiated sections n45 data are given as mean±sd h percentage of il10 cells to total cd68 cells data are presented as mean±sd i representative image of multicolor immunofluorescence staining of il10 in macrophages cd68 peil10 fitc of irradiated rectal cancer p0001 p00001 by two tailed unpaired students t test gy gray inos inducible nitric oxide synthase tam tumor associated macrophages il interleukinstary a0v et a0al j immunother cancer 20208e000667 101136jitc2020000667 0copen access since the stage in the tnm classification of malignant tumors and cancer differentiation of the two cohorts were comparable online supplementary table to assess the composition of the leucocytic infiltrate and the impact of radiotherapy we evaluated the distribution of macrophages t cells nk cells and nkt cells via immunofluorescence staining and automated image analysis irradiated tumor sections showed significantly less infiltration of cd68 cells which correlated with a tendency for a decrease of cd3 t cells while the amount of nk cells cd56 cd3 or nkt cd56 cd3 cells did not show significant differences figure 3b immunostaining of phosphorylated γh2ax was used to confirm radiotherapy induced dna damage in analyzed tumor specimen irradiated tumors revealed a significantly higher amount of γh2ax positive cells than non irradiated tissue figure 3c γh2ax foci were primarily located in the nuclei of tumor cells rather than in the cells of the tumor infiltrating microenvironment figure 3d in both patient cohorts cd68 cells accumulated in regions surrounding tumor cell clusters figure 3eto evaluate the in vivo effect of irradiation on macrophage polarization and to correlate results with ex vivo irradiated tumor specimens we determined the ratio of m1 like tam to m2 like tam by staining for inos and cd163 in cd68 cells irradiation was associated with higher numbers of inos expressing tam while cd163 tam were drastically reduced resulting in a significant increase in the m1m2 ratio figure 3fg similar to the ex vivo cultures we observed a sixfold decrease of il10 tam in tumors from irradiated patients as compared with treatment naive tumors figure 3hi these data are corroborating hallmarks of our ex vivo irradiation protocols with macrophages becoming active players within the tumor microenvironment upon γirradiationirradiation of anotypic cultures promote macrophage activation towards an m1like phenotypeto be able to further dissect the irradiation induced findings in patients tissue sections and ex vivo cultures we used anotypic cultures which were established with colorectal cancer cell lines hct116 or dld1 primary caf and peripheral blood derived macrophages online supplementary figure a in collagen i gels in contrast to primary cultures ota allowed culture periods of up to days to mimic the clinical protocol of short course irradiation ota containing either hct116 or dld1 were exposed to irradiation with two times gy or two times gy over the course of hours compared with non irradiated controls figure 4ab frozen tissue sections of ota were assessed for macrophage marker using immunostaining and automated image analysis the number of macrophages in ota was comparable and not significantly different among the distinct conditions online supplementary figure b non irradiated gy ota harbored macrophages resembling an m2 like phenotype of cd11bcd68 macrophages as indicated by high expression of cd163 figure 4cd low expression of ccr7 figure 4e and modest expression of il10 figure 4fg upon irradiation of ota cd163 and il10 were reduced whereas the expression of the pro inflammatory marker ccr7 was elevated thus the marker profile of ota correlated with those of primary ex vivo tumor cultures and surgical specimen following neoadjuvant irradiation these observa | 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 |
" the development of a safe effective reversible nonhormonal contraceptive method for men hasbeen an ongoing effort for the past few decades however despite significant progress on elucidating the functionof key proteins involved in reproduction understanding male reproductive physiology is limited by incompleteinformation on the genes expressed in reproductive tissues and no contraceptive targets have so far reachedclinical trials to advance product development further identification of novel reproductive tractspecific genesleading to potentially druggable protein targets is imperativeresults in this study we expand on previous single tissue single species studies by integrating analysis of publiclyavailable human and mouse rnaseq datasets whose initial published purpose was not focused on identifyingmale reproductive tractspecific targets we also incorporate analysis of additional newly acquired human andmouse testis and epididymis samples to increase the number of targets identified we detected a combined totalof genes for which no previous evidence of male reproductive tractspecific expression was annotated manyof which are potentially druggable targets through rtpcr we confirmed the reproductive tractspecific expressionof novel orthologous human and mouse genes without a reported mouse model of these we ablated fourepididymisspecific genes spint3 spint4 spint5 and ces5a and two testisspecific genes pp2d1 and saxo1 inindividual or double knockout mice generated through the crisprcas9 system our results validate a functionalrequirement for spint45 and ces5a in male mouse fertility while demonstrating that spint3 pp2d1 and saxo1 areeach individually dispensable for male mouse fertilitys our work provides a plethora of novel testis and epididymisspecific genes and elucidates thefunctional requirement of several of these genes which is essential towards understanding the etiology of maleinfertility and the development of male contraceptiveskeywords contraception drug target male reproductive tract paralog sperm maturation spermatidspermatozoa correspondence coarfabcmedu thomasgarciabcmedu1dan l duncan comprehensive cancer center baylor college of medicine baylor plaza houston tx usa3department of pathology and immunology baylor college of medicine baylor plaza houston tx 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 0crobertson bmc biology page of the world human population reached nearly eight billion people in august this number continues torise and is predicted to reach nearly ten billion by theyear the increasing need to promote familyplanning through the development of reliable contraceptive options available to both men and women is widelyrecognized currently there are numerous contraceptiveoptions available to women however identification of asafe nonhormonal contraceptive option for men is stillan ongoing challenge although several different fertilitycontrol alternatives for men have been investigatednone are currently clinically approved for use our understanding of the mechanisms underlying male reproductive physiology is still at an early stage as theidentification and elucidation of the function of key reproductive proteins is still an ongoing effort identifyingdruggable protein targets expressed in the male reproductive tract has been the focus of numerous studiesdedicated to the development of male contraceptionessentialroletheitsthe mammalian epididymis is a segmented ancomprised of a single highly coiled tubule with functionally and morphologically distinct regions that can besubdivided most simplistically into a proximal centraland distal region conventionally named the caput corpus and cauda regions respectively as mammalianspermatozoa transit through the epididymis they acquire the ability to recognize and fertilize an egg properties that they did not possess upon exiting the testis epididymisinconsideringaddition to maturing germ cells of the testis and spermatozoais a prime target for the development of a malecontraceptive to advance progress towards the development of a nonhormonal male contraceptive several previous highthroughput studies have been published thatidentified a number of human mouse and rat genes astestisspecific or epididymisspecific [ ] in schultz conducted the first study to identify malereproductive tractspecific genes using microarraysthroughgeneexpression analysis of meiotic and postmeiotic spermatogenic cells together with parallel analysis of available data from the ncbi unigene database the authorsidentified mouse genes as testisspecific which included genes with both known and unknown function atthe time in the following years through two additional microarraybased studies of rat testes and purifiedrat testicular cells johnston identified and additional or overlapping genes astestisspecific in as part of the continued effort to identify novel contraceptive targets the newer rnaseqbased transcriptomics methodology was utilized identifying human genes as testisspecific together withantibodybased protein profiling many of these genesaffymetrixbasedgenomewidewere characterized in terms of the spermatogenic cellpopulations showing expression the first highthroughput transcriptomics study to identify epididymisspecific genes was a mouse epididymal transcriptome studyin which rna isolated fromeach of the epididymal segments was analyzed bymicroarray analysisidentifying epididymisspecificgenes with distinct patterns of segmental gene regulation later in additional transcriptome profiling utilizing whole genome microarrays resulted in identification of previously unreported epididymisspecific transcripts inthe mouse and epididymisspecific transcripts inthe rat a significant number of the identified mouseand rat genes in these studies were not known at the timeand only the probe identification numbers were presentedwhen evaluating potential druggability in a targetbased drug discovery process one must consider theprotein properties that are required for safe and effectiveinhibition among the most significant is tissue expression specificity to minimize potential adverse effectsprotein function and whether protein activity or interaction with other proteins is potentially druggable sequence similarity to closely related paralogs that may beubiquitously expressed and whether genetically manipulated animal models demonstrate a functional requirement for the target of interest several noteworthyreview publications have mentioned numerous geneswhose critical functions high expression and specificityto the testes or epididymides make them viable nonhormonal male contraceptive targets [] howeveramong the identified genes a significant number either are required for fertility but are expressed in nonreproductive tissues or are reproductive tractspecific but when disrupted lead to subfertility ineither case both are ineffective and highly undesirableoutcomes for a potential male contraceptive targettherefore the identification of additional novel male reproductive tractspecific genes would allow for furtheradvances to be made in the quest to develop an effectiveand safe nonhormonal male contraceptivein this study newly acquired and previously published human and mouse rnaseq datasets [ ]were processed in parallel through a custom bioinformatics pipeline designed to identify novel reproductive tractspecific and reproductive tractenriched transcripts additional databases obtained from illuminating the druggable genome mouse genome informatics andensembl biomart were utilized to stratify the resultsinto subgroups based on protein druggability and on theavailability of a mouse model numerous reproductivetractspecific and reproductive tractenriched potentiallydruggable targets for which no published mouse modelexists congruent in expression across both mouse and human datasets were identified through our analysis and 0crobertson bmc biology page of verified through conventional polymerase chain reactionpcr we present the data in a manner that should bemost relevant and of substantialinterest to the malecontraceptive development field since identification ofnew targets worthy of consideration for further functionalanalysis in a knockout animal model and potential drugtargeting continues to be of vast importanceresults wethrough ouridentified four novelepididymisspecific genes spint3 spint4 spint5 andces5a and two novel testisspecific genes pp2d1 andsaxo1 worthy offunctional validation in an animalmodel through the crisprcas9 system we generatedfour individual gene knockouts spint3 ces5a pp2d1and saxo1 and one double knockout mouse modelspint45 revealing an essential requirement for spint4and spint5 in male mouse fertility and the potential utility of pursuing spint4 in humans as a nonhormonalcontraceptive targetresultsstudy approaches and datadespite significant advances in our understanding of thehuman and rodent testis and epididymis transcriptomemostly through microarraybased studies no prior studies have utilized purified human testis cells for the identification of human testisspecific transcripts no priorstudies have utilized the more stateoftheart rnaseqbased transcriptomics methodology for analysis of human epididymisspecific transcripts and no prior studieshave utilized rnaseq analysis of rodent reproductivetissues or cells to identify rodent reproductive tractspecific transcripts to address these gaps in knowledgeand to increase the number of identified reproductivetractspecific genes in both species using the most relevant highthroughput transcriptomics methodology weanalyzed in parallel on a custom bioinformatics pipelinea large number of published and newly acquired humanand mouse rnaseq datasets one hundred and sixtytwo previously published human and previously published mouse rnaseq datasets were retrieved from thesequence read archive sra the sra value for eachsample is listed in additional file table s1 and additional file table s2 we also generated new humanand new mouse reproductive tissue rnaseq samplesgeo accession gse150854 the final dataset is comprised of new and previously published human testisdatasets previously published purified humangerm cell datasets [ ] previously published purified human sertoli cell datasets [ ] new and previously published human epididymis segment datasets previously published mouse testis datasets new mouse epididymis datasets previouslypublished purified mouse germ cell datasets [ ]and previously published purified mouse sertoli celldatasets an additional previously publisheddatasets contributed to the nonreproductive humantissues and previously published datasets contributed to the nonreproductive mouse tissues figure 1a b summarizes all the samples acquired for thestudywe performed a principal component analysis tovisualize the variation in the samples after correcting forbatch effects human reproductive and nonreproductivetissues grouped according to sample type the reproductive tissue samples clustered by tissue type whetheror not they were newly generated or acquired from thesra fig 1c mouse data showed a similar variation inthe samples based on the tissue type fig 1d for bothhuman and mouse reproductive tissues samples separated by whether or not the rnaseq was performed onisolated cells or the whole tissue epididymal tissue wasdistinct from testis tissue in both human and mousefig 1c didentified in the mouseto identify potential male reproductive tractspecificdrug candidates we analyzed the aggregated rnaseqdata to find genes that were statistically significant in expression when compared to the nonreproductive tissuethat had the maximum expression for that gene thisgene list was then further refined by filtering for genesthat were lowly expressed in the nonreproductive tissuethat had the maximum expression for that gene tpmless than or equal to for human tpm less than orequal to for mouse finally this tpm filtered listwas then filtered for the genes that had a reproductivetissue or cell expression value greater than or equal to tpm for human or tpm for mouse fig 2aacross all the reproductive tissues candidate geneswere identified in the human and candidate geneswerefig 2badditional file fig s1 additional file table s3 andadditional file table s4 summarize the differentialfold change identity of the nonreproductive tissue withmaximal gene expression based on the differential geneanalysis fdr average and standard deviation tpm expression values and log2 cpm gene expression value forthe human and mouse samples respectively the resultsfrom the fdr and tpm expression value filtering forthe human and mouse samples are summarized inadditional file table s5 and additional file tables6file table s5 andadditional file table s6 report the log2 fold changefor the reproductive tissue or cell of interest comparedto the tissue with maximal gene expression the genesidentifiedandadditional file table s6 pass the filters in at least oneofinadditional file table s5 and additional file tables6 a value of zero for a given gene and fold expressionthe reproductive tissues or cells ofrespectively additionalinterestsamplesin additionalfile tables5 0crobertson bmc biology page of fig see legend on next page 0crobertson bmc biology page of see figure on previous pagefig summary of the human and mouse rnaseq samples used in the identification of novel male reproductive tractspecific drug targets thernaseq samples used in the human a and mouse b analyses are schematically shown principal component analysis was performed on thehuman c and mouse d nonreproductive and reproductive samples separately the colors of the circles next to the tissues listed in a and bcorrespond to the colors used in the circles for the pca in c and d sample size n values in red andor black denote the number of new redand previously published black samples included in our analysiscomparison indicates that for that comparison the genedid not pass the filters the majority of genes weredownregulated in the reproductive tissue ofinterestcompared to the maximal geneexpressing nonreproductive tissue additional file fig s2 from theanalysis the majority of the candidate genes that passedthe fdr and tpm filters were identified in the testis orspermrelated cells in both human and mouse samplesadditional file fig s2the majority of candidate genes identified in ourscreen that were testisspecific were already identified bythe human protein atlas andor our reanalysis offig identification of candidate drug male reproductive gene targets a diagrammatic representation of overall methodology used to identifyreproductive tractspecific candidate genes in humans genes and in mice genes the maximum gene expression was determinedacross all the nonreproductive tissue samples for each gene for a reproductive tissue or cell sample of interest genes were then filtered forsignificance using a false discovery rate fdr of less than or equal to based on the differential gene expression analysis for the nonreproductive tissue with maximum gene expression and reproductive tissue or cell sample of interest genes that passed the fdr filter werefiltered such that the average tpm expression value of the maximum expressing nonreproductive tissue was less than or equal to tpm andthe average tpm expression value of the reproductive tissue or cell of interest was greater than or equal to tpm b diagrammaticrepresentation of the number of human and mouse candidate genes in terms of the number of orthologs in the opposite species thenumber of genes previously or not previously identified in a prior transcriptomicsbased drug target report the availability and phenotypicoutcome of any reported mouse models and the number of novel genes without a reported mouse model congruent across both speciesthe main value in each bubble represents the total number of candidate genes identified regardless of tissue or cell identified in the numbers inparentheses comprise the total number of candidate genes that are either epididymisspecific or specific to testis and epididymis but not testisandor testis cellspecific only 0crobertson bmc biology page of the hpa testis datasets additional file fig s3 andadditional file table s7 thirtysix out of the genes that were identified across all the human epididymis tissue were also identified by the human combinednewly acquired and previously published datasets testiscandidate gene list finally the majority of the candidategenes identified from the combined newly generated and previously published human testis datasetswere shared with genes identified from the various testiscell datasets we identified more candidate genes in thenewly generated human epididymis tissues compared topreviously published data out of genes wereunique to the newly generated caput samples comparedto only out of genes which was unique to the previously published samples out of genes were uniquein the newly generated corpus samples compared to out of genes which were unique to the previously published corpus samples and genes were unique to thenewly generated cauda samples compared to genes inthe previously published cauda data with no overlap between the two cauda gene lists additional file fig s3and additional file table s7 there were candidategenes that overlapped between the newly generated human testis samples and mouse testis sample gene listswhile there were candidate genes that overlapped between the previously published human testis sample andmouse testis sample gene lists additional file fig s3and additional file table s7 across all human epididymis tissue samplesincluding the newly generated andpreviously published samples there were genes in common with the combined list of candidate genes across allthe mouse epididymis tissue samples there was a smalloverlap between the human and mouse samples when thenewly generated human caput corpus and cauda tissueswere individually compared to the mouse caput corpusand cauda tissues there was an overlap of and forrespectivelyadditional file fig s3 and additional file table s7this trend was continued for the candidate gene lists derived from the previously published human caput corpusand cauda samples when compared to the candidate genelist from the mouse caput corpus and cauda with and genes in common for the caput corpus and caudacomparisons respectively additional file fig s3 andadditional file table s7 additional file table s7 details the genes that are unique and in common for each ofthe comparisonscorpuscaputtheandcaudato assess the potential usefulness of the candidategenes identified in each human reproductive tissue asdrug targets we assigned the genes to a protein familyie gpcr or ion channel the majority of identifiedgenes were not from a traditional drug target family likekinases or enzymes the testis and germ cell datasetsprovided the most potential targets while the epididymisdatasets provided the fewest additionalfile figs4a the protein family classification for each candidate gene identified in each reproductive tissue is detailed in additional file table s8 the majority of thecandidate genes do not have a reported mouse modeladditional file fig s4b additional file table s9summarizes mouse model availability for each candidategene identified from human reproductive tissues or cellsfigure shows the complete list of novel human geneswithout a reported mouse model as identified in each ofthe respective cell andor tissue datasets digital pcrsheatmap and conventional pcrs demonstrating expression of a subset of the novel human reproductivetractspecific genes without a reported mouse modelthat we identified are shown in figs and respectively additional file fig s5 shows the complete listof previously identified human genes that remain without a reported mouse model as identified in each of therespective cell andor tissue datasets additional file fig s6 shows the complete list of male reproductivetractspecific human genes for which a previously generated mouse model shows male infertility phenotype asidentified in each of the respective cell andor tissuedatasetsreproductive tractspecific genes identified throughhuman datasetsthrough our bioinformatics analysis of previously published and newly acquired rnaseq datasets we identified a total of genes as reproductive tractspecific inhumans fig of these genes genes do not have amouse gene ortholog while genes have a mousegene ortholog fig of those with a mouse geneortholog have a single gene ortholog have thesame symbolin mouse while have a differentsymbol in mouse while have two or more orthologous mouse genes seventysix human genes had orthologous mouse symbols genes had orthologous mouse symbols and genes fam205a krtap106 magea10 or2ag1 pramef11 pramef2ssx2 ssx3 and ssx4b had greater than orthologous mouse symbols symbols additional file table s5 of the human genes that we identified asmale reproductive tractspecific have not been previously identified in a transcriptomicsbased male reproductive tractspecific study [ ] the sum of ourhuman data confirms the findings of out of genes from djureinovic after reidentificationof gene symbols from reported affymetrix ids and consideration of orthologous genes mouse to human andrat to human our human data confirm the findings of out of genes from johnston out of genes from schultz out of genes fromjohnston out of genes from johnston 0crobertson bmc biology page of fig two hundred and thirtythree novel human reproductive tractspecific genes that each have mouse orthologous genes but with noreported knockout mouse models the listed genes were identified in one or more datasets as indicated in the venn diagram underlined geneswere also identified in our studies as reproductive tractspecific in mouse genes genes written in blue encode either enzymes kinasesgpcrs ogpcrs transporters transcription factors or proteins involved in epigenetic regulation genes genes written in dark red wereidentified in both testis testis andor testis cell and epididymis genes out of genes from johnston and out of genes from jelinsky of the genes that have a mouse gene ortholog have notbeen previously identified as male reproductive tractspecific and of these human genes currently lackmouse phenotype information based on data obtainedfrom ensembl biomart mgi impc and ncbihuman testisspecificthree hundred and eightysix genes were identified astestisspecific through either the reanalysis of djureinovic testis datasets genes identified analysis ofour de novo testis datasets genes identified orboth additional file table s5 three hundred andthirteen genes were congruent across both datasetswhile genes were uniquely identified through our reanalysis of djureinovic s datasets and only genes[ac1363524 ankrd20a1 ankrd62fam230aggtlc2iqcm potec prnt and utf1] wereuniquely identified through our de novo datasetsadditional file table s5 interestingly of the genes we identified through djureinovic s reanalyzed datasets were not previously identified in theirreport or any of the other previous reports [ ]of these genes we randomly verified of thesegenes as testisspecific in humans through conventionalpcr fig we also verified through rtpcr an additional genessuch as allc cdkl3 cox7b2or2h1 and sppl2cthat had been identified throughprevious studies additional file fig s7 of the genes identified through either testis datasets havenot been previously identified of these genes haveone or more mouse orthologs and of these genes arelacking reported phenotype information of the novelgenes lacking a reported mouse model genes encodeenzymes adam20 cpa5 dusp21 naa11 plscr2prss38 and triml1 encode transcription factorsbhmg1znf560znf729 encode transporters slc22a14 slc25a52and encode proteins of unknown drug target typetgif2lyfoxr2prdm9 0crobertson bmc biology page of fig representative novel reproductive tractspecific human a and mouse b genes without a reported mouse model the listed genes wereidentified through our studies as reproductive tractspecific in both humans and mice the digital pcr heatmap depicts the average transcriptsper million tpm value per tissue per gene from the indicated human and mouse rnaseq datasets as processed in parallel through ourbioinformatics pipeline the data was obtained from published and newly acquired datasets white tpm black ¥ tpm the expressionprofile of the human and mouse housekeeping genes gapdh and eif3l is included as reference for data obtained from published datasetssuperscript values succeeding the sample names reference the publications as follows djureinovic fagerberg guo zhu kumar browne consortium helsel da cruz and zimmermann such as etdb smim36 bend2 btg4 cnbd1dppa2 efcab5 erich6 fthl17 iqcm mroh2bms4a5 oosp2 pnma6e ppp4r3c rbmxl3 rtl9spdye4 spem2 all of these genes are listed in fig and many of these genes are listed in figs andor to the best of our knowledge no prior studies haveutilized purified human testis cells for the identificationof human testisspecific transcripts through our analysis we identified genes as human testisspecificthrough one or more of the human germ cell datasetsbut not through either of the human testis datasetsadditionalfile table s5 seventysix genes wereidentified exclusively through one or more of the fivehuman spermatogonia datasets genes such as anp32dc13orf42 dscr4 or13g1 or2d2 or52e4 ssx2tle7 while genes were identified exclusivelythrough the human spermatocyte datasets genes suchas h2bfm mageb17 mageb18 or2b6 tcp10 andznf709 and genes were identified exclusivelythrough the human spermatid datasets genes suchas ac0132691 clec20a or7e24 pramef2 spata31a3 tmem191c znf679 thirtyfour geneswere identified through all three cell types datasetsgenes such as ccdc166 eloa2 fam47a heatr9 and spata31a1 many of these genes are listedin figs andor of the genes identified as human testisspecificthrough one or more of the human germ cell datasets genes have not been previously identified ofwhich have one or more equivalent mouse orthologswith of these genes having not been knocked out inmouse of these novel genes with no mouse model encode enzymes glt6d1 prss48 satl1 sult6b1tmprss7 tpte2 triml2 and ttll8 encodes anepigenetic protein taf1l encode gpcrs gpr156tas2r13 tas2r30 tas2r46 tas2r50 vn1r2 encodes a kinase cdkl4 encode ogpcrs such asor2d3 or3a2 or52e5 or8g5 or10j1and 0crobertson bmc biology page of adrenal glandadiposecolonbrainskeletal musclesmooth musclesmall intestinesalivary glandleukocytespancreasstomachprostatethyroidkidneyspleentestisheartlungliverskincorpusuteruscaudaovarycaputaac0221675ac1876531adam20al6720431bhmg1bsph1bx2760929c1orf105c2orf92c4orf51c16orf95ccdc196ces5acpa5dcaf8l1efcab5erich6etdbfer1l5glt6d1iqca1llcn6magea11mageb6mroh2bnaa11pp2d1ppp4r3cprr23aprr23bprr23cprss38saxo1slc22a14spag11aspag11bspata31a1spata31d1spata31d3spata31d4spint3spint4tcp10l2tex13dtex44tex48tex51tle7tpte2triml1trpc5oswfdc8wfdc9wfdc10awfdc10bwfdc13gapdhskeletal musclesmooth musclesmall intestinestomachprostatethyroidspleentestislungskinkidneyheartlivercorpusuteruscaudaovarycaputadiposebladderbraincoloneyebgm5767gm5294adam201700042g07rikgm4969bsph14933412e24rik4930558k02rik4933424g06rik1700011l22rik1700018b08rikgm6657ces5acpa5pet2efcab5erich6etdfer1l5glt6d14931409k22riklcn6magea4mageb3mroh2bnaa11pp2d14932429p05rikprr23a3prr23a3prr23a3prss38saxo1slc22a14spag11bspag11bspata31spata31d1bspata31d1bspata31d1bspint3spint4spint5tcp10ctex13c1tex44tex48gm35060tle7tptetriml1trpc5oswfdc8wfdc9wfdc10wfdc10wfdc13hprtfig rtpcr confirmation of reproductive tract specificity in both humans a and mice b the genes listed in this figure are novel as identifiedthrough our studies and without a reported mouse model humans do not have an equivalent proteincoding equivalent to mouse spint5 gapdh and hprt are included as housekeeping genes 0crobertson bmc biology page of fig three hundred and two novel mouse genes with human orthologs without a reported mouse model the listed genes were identified inone or more mouse datasets as indicated in the venn diagram underlined genes were also identified through our studies as reproductive tractspecific in human genes genes written in blue encode either enzymes kinases gpcrs ogpcrs transporters transcription factors orproteins involved in epigenetic regulation genes genes written in dark red were identified in both testis testis andor testis cell andepididymis genesnkx24or14a2 encode transcription factorsznf99 znf648 znf705b znf705g and znf709 encode transporters abcc12 slc35g3 slc35g5 and encode proteins of unknown drug target type suchas ac0080733 ac1135541 aknad1 al0496342dnlz ervw1 etda fbxw10 fer1l5 lmntd1lrrc72 nms prr23a prr23b prr23c pxt1rfpl4b and ssx4b many of these genes are listed infigs andor fam236a figs and and obp2b met candidatethreshold through our analysis of human testes datasetsbut did not meet candidate threshold from any of thegerm cell or sertoli cell datasets indicating potential expression in peritubular myoid cells leydig cells or othercell outside of the seminiferous epithelium fam36a hasnot been previously identified and neither mouse orthologs 1700011m02rik gm9112 have been knocked outobp2b was previously identified through djureinovic and johnston however of the equivalent mouse orthologs lcn4 obp2a obp2b only obp2ahas been knocked out revealing abnormal coathair pigmentation ism2 and magec2 were identified through both human sertoli cell datasets while also identified throughtestis andor germ cell datasets both genes have beenpreviously identified ism2 magec2 ism2knockout mice display nonreproductive phenotypes consistent with this finding our mouse data do notidentify ism2 as reproductive tractspecific in micemagec2 lacks a mouse ortholog for functional analysis in micehuman sertoli cellspecifickrtap23 krtap412 lhx9 and psg5 were identified through one or both human sertoli cell datasets butwere not identified through any of the testis or germ cell 0crobertson bmc biology page of datasets indicating sertoli cellspecific expression in thetestes additional file table s5 none of these geneshave been previously identified as reproductive tractspecific in humans although lhx9 and psg5 havemouse orthologs that have been knocked out []human krtap23 has mouse orthologs krtap52gm4559 gm40460 and gm45618 and human krtap412 has mouse orthologs krtap47 and gm11555none of these mouse orthologs have been knocked outfig and additional file table s5knockin mousepsg5 knockout mice display nonreproductive phenotypes [] however lhx9 knockout mice display absent testes and sterility due to an essential requirementfor lhx9 during mouse gonad formation a lhx9gfpcreerbyknockingin gfpcreer at the endogenous lhx9 locuscrossed with the rosa26tdtomato reporter mouse linerevealed cre recombinase activity in retinal amacrinecells developing limbstestis hippocampal neuronsthalamic neurons and cerebellar neurons thuslhx9 is not reproductive tractspecific in mice ourmouse data confirm this findinglinegeneratedhuman epididymisspecificto the | 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 |
moringa oleifera l from the moringaceae family is a perennial tree widely cultivated in many tropic regions and easily grown even in adverse conditions m oleifera is also known as the miracle tree which for centuries has been indicated for traditional medicine with no reports of side effects in doses achievable by ingestion different parts of m oleifera is used to treat several conditions such as malnutrition diabetes blindness anemia hypertension stress depression skin arthritis joints and a0kidney stones disorders this plant also showed capacity of helping in maintenance of the cardiovascular system health bloodglucose levels and providing antioxidant antiinflammatory and anticancer activity as well as the regulation of urinary tract and lactation in nursing women the seed and leaves powder has water purification properties through flocculation it also supplements the food in the human diet and in the fortification of livestock feed especially in developing countries so m oleifera properties have also been applied to cosmetic and byproducts industries due to the high nutritive and protective properties of its seed oil according to the holistic or traditional medicine m oleifera has very relevant therapeutic properties and applications depending on the constitution somatic and psychological needs of patients it is usually referred as a natural product that can treat different physical and psychological health aspects offering an energetic action and structural rebuilder of the body and promoting emotions of highly positive attitudes towards life the high and specific immunological potential of m oleifera leads us to suggest an indepth study to assess the hypothesis of conferring a supportive effect against covid19 diseasekeywords moringa oleifera a0· drumstick tree a0· miracle tree a0· medicinal plant a0· cosmetics a0· food supplementdiana meireles and jo£o gomes authors contributed equally to this workdianameireleslivecompt diana meireles icbas institute of a0biomedical sciences abel salazar university of a0porto rua de je viterbo ferreira no a0porto portugal yidao acupuncture and a0tcm center porto portugal ciimar interdisciplinary centre of a0marine and a0environmental research university of a0porto porto portugal cbsin center of a0biosciences in a0integrative health porto portugalintroductionmoringa oleifera l moringa pterygosperma g wellknown as the drumstick or horseradish tree is native of northwest india its main producer but can also be found in south africa northeast africa madagascar tropical asia southwest asia and latin america the moringa genus comprises species m arborea m longituba m borziana m pygmaea m hildebrandtii m drouhardii m longituba m peregrina m stenopetala m rivae m ruspoliana m ovalifolia m concanensis and m olefera rani a0 from the moringaceae family m oleifera is the most known studied and used species anwar olson with human and animal applications the various resources obtained from this plantleaves flowers seeds pods bark and rootscan be used for cooking or in traditional medicine to treat several pathologies m oleifera has the capability to survive in humid or dry hot climates vol01234567891 0c d a0meireles and poor soils anwar et a0al mainenti m oleifera is a highly nutritious plant being ideal to treat malnutrition in developing countries zongo valdezsolana et a0al gopalakrishnan debajyoti et a0al m oleifera gained the title of miracle tree and commercial attention supported on several properties such as nutritional values amino acids and flavonols content which can be used in food supplements and cosmetic industry tables a0 and in fact when compared to other plants from a0g of dry leafs of m oleifera we can obtain times more vitamin c than from oranges times more vitamin a than from carrots times more calcium than in milk a0times more protein than in yoghurt times more potassium than from bananas and times more iron than the obtained from spinach oduro et a0al a0 a0rockwood saini et a0al table a0 shows the nutritional values for the edible parts of raw pods and leaves obtained from the united states department of agriculture usda database although it is table nutritional values per a0g of the edible portion of m oleifera pods and leavescomponentsper a0graw podsaraw leavesaenergy kcalwater gprotein gtotal lipid gcarbohydrate by difference gfibre total dietary gfatty acids total saturated gfatty acids total monounsaturated gfatty acids total polyunsaturated gfatty acids total trans gcholesterol mgvitamin a rae µgvitamin d d2 d3 µgvitamin d iuthiamin mgriboflavin mgniacin mgpantothenic acid mgvitamin b6 mgvitamin b12 µgvitamin e mgvitamin c total ascorbic acid mgfolate total µgfolic acid µgsodium mgpotassium mgcalcium mgphosphorus mgmagnesium mgiron mgzinc mgcopper mgmanganese mgselenium µga information obtained from united states department of agriculture nutrient database ndbnalusdagovndbfoods in june b average values and standard deviation published by witt only two values were found witt dried leavesb ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0ctable amino acids and flavonols per a0g of the edible raw portion of m oleifera leavesper a0graw leavescomponentsamino acids a0tryptophan g a0threonine g a0isoleucine g a0leucine g a0lysine g a0methionine g a0cystine g a0phenylalanine g a0tyrosine g a0valine g a0arginine g a0histidine g a0alanine g a0aspartic acid g a0glutamic acid g a0glycine g a0proline g a0serine gflavonols a0isorhamnetin mg a0kaempferol mg a0myricetin mg a0quercetin mginformation obtained from united states department of agriculture nutrient database ndbnalusdagovndbfoods in june known that the nutrient content varies according to the plantation site aslam and seasons witt the nutritional value of dried leaves not existent in usda database is presented as an average of values with standard deviation calculated from diverse papers that was compiled and published by witt from leaves to roots it is possible to obtain good quantities of important minerals proteins vitamins carotene amino acids and phenolic compounds anwar et a0al leone et a0al 2015a b saini et a0al fahey debajyoti et a0al divya et a0al m oleifera extracts have been studied with different medicinal purposes antiinflammatory antihypertensive diuretic antimicrobial antioxidant antidiabetic antihyperlipidemic antineoplastic antipyretic antiulcer and hepatoprotectant fahey abdull razis et a0al divya et a0al anwar et a0al published a table with various traditional medicinal uses of the different parts of m oleifera anwar et a0al the attractive properties of this plant led to studies of side effects and medical interactions in animal models and humans according the revision by stohs and hartman until this date none of the human in a0vitro studies or extrapolations of animal studies to humans reported adverse effects with doses of m oleifera leaves and leaf extracts achievable by oral ingestion although there was not any report of major adverse side effects there are some important information that should be registered in fact there are some studies suggesting that m oleifera cannot be used in combination with other modern medicines in humans a research by gholap et a0al concluded that m oleifera has been noted to be a good regulator of insulin thus according sileshi et a0al patients suffering from lack of insulin will probably have adverse reductions of sugar levels when using m oleifera for medicinal purposes suggesting that it could decrease the blood sugar to even lower levels when used in combination with other modern medications another study suggests that when treating thyroids m oleifera compounds of the leaf may improve thyroid function tahiliani et a0al this well proving evidence further suggests that it can possibly conflict with other thyroid medication triggering drug interactiona research work concerning the acceptability and safety of shortterm daily supplementation in a group of malnourished girls assessed the use acceptability and safety of m oleifera on children girls in zambia barichella et a0al with regards to safety concerns supplementation of a0g per day of m oleifera powder was deemed safe for children and adolescents both in the short and long term this research also noted that mild nausea was reported in of the children at various age groups when meals were supplemented with a0g of m oleifera daily showing to be still an inadequate and symptomatic dose in childrenother studies suggest that m oleifera could adverse and slowly breaking down the pharmaceutical drugs in the liver and thus a0may develop cirrhosis and liver failure resulting in malnutrition and weight loss as well as decreased cognitive function das et a0al kelly sileshi et a0al despite the numerous positive health benefits associated with m oleifera phytochemicals there are suspicions that it contains harmful substances fahey et a0al annongu et a0al maizuwo it contains specific chemical compounds such as alkaloids and other phytotoxins which when consumed in high doses presents potentially nerveparalysing properties and other adverse effects maizuwo et a0al some of these phytochemicals include moringine moringinine estrogen pectinesterase and phenols including tannin fahey et a0al there are unconfirmed reports that m oleifera stems roots and flowers potentially contain harmful phytochemical constituents especially during pregnancy which may promote uterus contraction leading to miscarriages in pregnant women dutta it is also suspected that it can prevent implantation in women hence it must be avoided a review of a0properties nutritional and a0pharmaceutical applications of a0moringa oleifera¦ 0c d a0meireles table compilation of food supplements containing m oleifera tree parts or extracts in june tree partsproduct informationbrandproductnaturingamoringa capsulesleavesmoringa teamoringa kids multivitamin complexmoringa powderbioheradried seeds extract moringa capsulesleavesmoringa syrupleavesleavesmiracle treemoringa anic teaanic moringa superfood supplementscapsulesmoringa superfood powdermoringa superfood sticksiswarimoringa powder anicdrasanvileaves dry extract nutrabasicsmoringaregulates the gastrointestinal transit natural antiinflammatory lowers cholesterol levels improves diabetic conditiondelays the ageing process ensures proper digestion high antioxidant power helps healing process tonifies body and mindstrengthens the immune system rich in vitamins and minerals stimulates natural defensesadds nutritional value source of fiber protein vitamins and minerals improves physical conditionstrengthens the immune system helps to reverse the aging process beautifies the skin reduces the appearance of wrinkles and fine lines maintains the normal glucose level stimulates brain function and concentration increases libidoit is a nutritionally complex whole food naturally rich in vitamins minerals and amino acids daily use of moringa can help to restore your imbalances in your dietthe moringa leaf boasts a vast array of beneficial nutrients making this tree one of the highest plant sources of vitamins and minerals aroundthe richness of its active ingredients helps maintain blood glucose levels provides flavonoids and polyphenols by those attempting to conceive as it functioning as an abortifacient nath et a0al dutta finally cytotoxicity was verified in experiments with human peripheral blood mononuclear cells only at a0mgkg of an aqueous leaf extract genotoxicity on blood rats cells was verified at a0mgkg asare et a0al however all mentioned side effects were verified with doses that far exceed the amounts used in food intake asare et a0al so research on the adverse side effects with doses achievable by oral ingestion should still go on since currently there are no scientifically confirmed clear toxic and harmful effects of m oleifera extracts and products on both human and animal models adedapo et a0al stohs et a0al many studies on nutrition phytotherapy disease treatment and prevention goals have been published thus supporting scientific basis about the efficiency of traditional uses of m oleifera fahey in fact records about symptoms signs and treatment strategies in different diseases are found in several ancient texts of traditional medicines such as ayurveda and traditional chinese medicine tcm karadi et a0al kasote et a0al debajyoti et a0al as an endemic source with highly digestible protein ca fe vitamin c and carotenoids is considered as a suitable natural product to be used by undernourishment populations dixit the resources obtained from a0m oleifera tree on a0a a0conventional approachleaves and a0podsin some countries leaves and fruits are commonly used in culinary as vegetables leaves can also be dried and used in infusions or grounded into powder allowing easier conservation and consumption moyo olson et a0al in all ways of use and conservation m oleifera does not lose nutritional value mahmood leaves and pods are low in calories and rich in minerals vitamins and natural antioxidants table a0 anwar rebufa et a0al phytochemicals like flavonoids are also present in leaves as well as a significant percentage of essential amino acids table a0 m oleifera leaves contain a high quantity of polyunsaturated fatty acids and low saturated fatty acids content moyo which combined with diuretic lipid and blood pressure lowering properties from leaves and pods contribute to the maintenance 0ctable compilation of cosmetics containing m oleifera tree parts or extracts as ingredients in june cosmetic ingredientsproduct informationproduct brandnameskinsecretantiwrinkle face creamantiaging moisturizer face creamhand creambody milklushafrican paradise body conditionerqueen bee hair honeymagical moringa facial moisturizercharity pot hand and body lotionpassion fruit lip balmgo faster feet foot lotiontwinkle toes foot powderlush gardener cold pressed soaplaboratoires s©robiologiquespurisoft®body shopmoringa range shower gel oil body butter body milk body sorbet hand cream soap body scrubmoringa eau de toilette moringa body mistpurifying and protective action against environmental stress such as smoke and pollutionmoisturizing nourishingdeodorizingremove dirt moisturizing nourishingskin cleansingpurification protects skin against pollution heavy metals cigarette smokeskin feels smooth and restoreddelicately scent your skin in a crisp floral aroma with moringaleavesm pterygosperma oilm pterygosperma leaf infusionoilm pterygosperma powderactive ingredient peptide from moringa seedsm pterygosperma oilm oleifera leaf extractm pterygosperma seed extractm oleifera seeds and oilm oleifera leaf extractoilm pterygosperma seed extractm pterygosperma extractclarinsextracomfort antipollution cleansing cream eliminates traces of pollution detoxifies the epidermis and protects the skin from the harmful effects of pollutionneutralizes the effects of pollution and purifies the skin to restore its natural radiancepurifies and refines while preserving your skins natural moisture balance neutralizes the harmful effects of pollutiononestep facial cleanserexfoliating body scrubonestep gentle exfoliating cleanserwater purifycomfort onestep cleanserdaily energizer cleansing gelnaturingamoringa soap biomoringa exfoliating face scrubmoringa o2herbal moisturizing lotionfacial tonersoapherbal shampooconditionershu uemuraantioxi pollutant and dullness clarifying cleansing oilurban moisture hydronourishing shampooconditionerdouble serumdeep treatment masquehigh antioxidant value slowing skin ageing exfoliate dead cells by regenerating the tissuemoringa seeds peel and exfoliate the skin while moringa oil moisturizes and regenerates the skinrejuvenate nourish and protects skinrepairs strengthens reduces hair fallenhanced power to remove micro impurities and stubborn makeup antipollution breakthroughhighly concentrated in nutrients vitamins and antioxidants intensely hydrates deep within strandsa review of a0properties nutritional and a0pharmaceutical applications of a0moringa oleifera¦ 0c table continuedcosmetic ingredientsm oleifera seed extractproduct brandnamebiobeaut©antipollution micellar cleansing watergentle cleansing foamcleansing oil gelgentle exfoliating geldualphase waterproof eye makeup removerd a0meireles product informationremoves makeup pollution particles and excess sebum while leaving the skin well moisturized the seed of moringa extract selected contains purifying peptides which on the surface limit the adhesion of the pollution particles and in depth activate their elimination this extract acts as a protective shield capable of preserving the good bacteria from the cutaneous flora against the aggression of pollutionof cardiovascular health anwar et a0al table a0 in dried m oleifera leaves it was also found a high content in calcium and iron which is normally residual in other plants used in our diet in the leaves is found greater a variety and quantity of proteins when comparing to other tree parts rebufa et a0al wang et a0al due to its nutritional rich values m oleifera can be a good enriching food additive to human diet and also an animal feed fortifier moyo adding fresh or dried leaves to the feed of milk cows increased milk production and respectively that fact would be of great importance in developing countries to fight deficiencies in nutrition bhargave studies of acceptance by the consumer of enriched foodssnacks with m oleifera have been obtaining good results ellis jung m oleifera can also help lactating mothers produce more milk and help to treat malnutrition in young children phytosterols from m oleifera increase estrogen production that enhance the activity of the mammary glands ducts gopalakrishnan doses of a0mgg of body weight given to mice result in increased milk production also the pup weight augment with increasing doses of m oleifera leaf powder intake titi et a0al titi and nurjanah studies of toxicity in animals show that m oleifera dried leaf extract might be safe for consumption although in high doses and prolonged intakes m oleifera may cause toxicity by accumulation of some elements a0ali et a0al the amount of a0g of m oleifera dried leaf per day is the maximum recommended doseage asiedugyekye et a0 al table a0 compiles some food supplements based on m oleifera tree parts or extracts a hydroalcoholic extract of green pods increased liver enzymes involved in the detoxification of xenobiotic substances in mice table a0 suggesting a chemo preventive potential of a drumstick extract against chemical carcinogenesis bharali et a0al m oleifera pods are also valuable to treat digestive and obesity problems and thwart colon cancer gopalakrishnan et a0al carotene the major component reported from the drumsticks of the m oleifera plant as well as the presence of vitamin a and c suggest an action in the induction of antioxidant and antiinflammatory profiles geervani and devi bharali et a0al praengam et a0al it was suggested that carotene and sterols present in the plant pods acts as potent inhibitors on the formation of reactive oxygen intermediates a prerequisite for tumorigenesis and so inducing apoptosis in the mouse colon carcinoma model gupta et a0al kraiphet et a0al studies in rats showed that m oleifera leaves extract might act as potential neuroprotectant via decreased oxidative stress and the enhanced cholinergic function kirisattayakul et a0al and function as a cognitive enhancer hence being used in dementia cases sutalangka et a0al it was also found an antidepressant activity in mouse models of depression when giving orally a a0mgkgday of a m oleifera alcoholic extract plus a0mgkgday fluoxetine for a0days kaur et a0al this effect can be increased when combined with fluoxetine as a selective serotonin reuptake inhibitorssri according to sutalangka et a0al and kaur et a0al 2015the influence of m oleifera may be due to the action of antioxidants and flavonoids through radical scavenging since its action is verified in other studies on animal models with cerebrovascular diseases exerting a multiplicity of neuroprotective actions within the brain and suppressing neuroinflammation and thus suggesting a great potential to promote memory learning and cognitive function vauzour et a0al other studies with consumption of m oleifera leaf powder revealed properties in human an animal models such as decreased blood glucose levels on diabetic type two subjects william reduction on post prandial blood glucose ghiridhari increased insulin secretion in healthy subjects anthanont et a0al decreased total plasma cholesterol and increased hdl nambiar the presence of sitosterol in m oleifera leaves may be one of the reasons for decreasing plasma cholesterol since phytosterols cause less intestinal absorption of dietary cholesterol and increase its excretion on feces jain mbikay 0cnoitcudorpmubes niks ni noitcuderledom laminadna ortiv ni la a0te amrev a0 a0 pytivitca tnadixoitnaledom laminaan yehafnoitcefni la a0te ruak la a0te akgnalatuselfiorp dipil no tcapmi evitisopledom lamina a0 a0 p raibman ledomnamuh a0 a0 pyattasiri yehaf la a0te lukak la a0te ruozuav la a0te roknod la a0te la a0te eednodnesodu hanajrundna iti t la a0te itit la a0te inoras la a0te nanhsirkalapogledom lamina a0 a0 p ledomnamuh a0 a0 p la a0te tnonahtna mailliw irahdirihgnilusni esaercni doolb laidnarp tsop no noitcuder ni slihportuen gnitalucric esaercniseiduts ortiv ni a0 a0 pesoculgledom namuh a0 a0 p la a0te eurdledom laminasserts etuca a0 a0 pledom lamina a0 a0 p lariv uehritna recnac ni romutitnadna lairetcab itna msitamrsshti serpeditna wnoitanibmoc ni tnas recnahne evitingoc tnatcetorporuen ralucsavorberecairetsyh sa sredrosid suovren airalamdna diohpit fo tnemtaertsesaesid negortse noitcudorp rosrucerpdna noitatcal esaercniselpicnirp lanoitnevnoc ot gnidrocca krab dna stoor srewofl sdees sdop sevael su hcus strap eert arefielo m tnereffid fo snoitca lacigolocamrahp fo elbat yrammus elbatarefielo m fo snoitca lacigolocamrahp ledomnamuh a0 a0 pmsidioryhtrepyh etaluger ledomnamuh a0 a0 pamehtyre niks ni ecuderseiduts ortiv ni a0 a0 pa ila la a0te ila la a0te dammahumgnigaitna niksstcartxe suoeuqa fognilaehdnuow tsaerb tnadixoitna yrotammaflniitnaicrac noloc ni sisotpopa ecudni la a0te ilarahb la a0te tehpiark la a0te inailihatledom lamina a0 a0 pnoitacfiixoted dnaledom lamina a0 a0 p ledomnamuhc ila a0 a0 pamonytivitca laiborcimitnaledom lamina a0 a0 psamonicrac latceroloc tsniaga ytivitca recnacitnadna la a0te nanhsirkalapog la a0te iramsalaseiduts ortiv niselknirwitna a0 a0 pniap tnioj dna aehrraid taert la a0te ilaledom namuh a0 a0 p la a0te nanhsirkalapog a0 a0 pan raelc dna paos ot lauqe pu lortnoc eht fognihsaw tnereffiddnah ni seitreporp lairetcabitna la a0te lednorotnoitardyh niks ni esaercni ledomnamuh a0 a0 p ni laitnetop evitneverpomehcrecnac neelps dna citapeh nanhsirkalapog la a0te ilarahbseiduts ortiv ni a0 a0 p ledomnamuhb ila a0 a0 p dna tnadixoitna citebaiditnaseitreporp citirhtraitnagnisnaelc evitcetorp gnizirutsiom gnihsiruonniks tnadixoitna la a0te yebar l ednaledom lamina a0 a0 p ledomnamuhanammaflni gnul etuca fo esaerced la a0te thginkcmledom lamina a0 a0 pnoit dna setaidemretni noitadixorep dipil fonegyxo evitcaer esaerced ilarahb eddna atpugsad la a0teledom lamina a0 a0 p yebar l edna iklamlaledom lamina a0 a0 pdik lla detaroilema dna desaercnisretemarap snoitcnuf yen ecuder sisorbfi revil detaroilemaesadixorepoleymytivitca citapeh yticixototapeh decudni tibihniledom lamina a0 a0 pseiduts ortiv ni a0 a0 p azmah la a0te areiv iklamlanajaham narmi dna meedansevaelsdopsdeesa review of a0properties nutritional and a0pharmaceutical applications of a0moringa oleifera¦ 0c detneverp dna elfiorp la a0te reugiugledom lamina la a0te reugiugledom lamina inamkcurledom lamina hzimahtabni la a0te nanhsirkalapogseiduts ortiv niandipil niagdevorpm thgiewi dna ytilitom lanitsetni etomorpstceffe evitaxalyticixototapeh decudnilomatecarap no evitcetorpotapeh etatsorp fo sledom tnednepedni negordna ni ytivitca recnacitnarecnac eruc dna cimeloretselohcopyhsmelborp yraniruarefielo m fo snoitca lacigolocamrahpdeunitnoc elbat eht dna etalaxo syendik eht ni noitisoped enotsyraniru eht ecuder la a0te idarak dna ytivitca evitcetorpotycyrotercesitna recluitna la a0te yrahduohcan a0p a0evitcetorpotapehipilitna dna cimeretselohcitnaledom lamina a0 a0 p amrukledom lamina a0 a0 pledom lamina a0 a0 p ahcenesledom lamina a0 a0 pcimed tcart yraniru fo tnemeganamsmotpmys snoitcefni hgni sdna ayruam la a0te alkuhsledom lamina a0 a0 p ledomnamuh a0 a0 p serec¡cledom laminalairetcabitna a0 a0 p la a0te reffazseiduts ortiv niantceffe noitatnalpmiitna a0p a0yrotammaflniitna a0p a0yticixototapehdecudni lomatecarapno evitcetorpotapeharopsoruen tsniaga lagnufitna inamkcurledom lamina a0 a0 pseiduts ortiv ni ahj a0 a0 pd a0meireles several m oleifera studies with leaf powder or extracts on animals revealed other properties beyond the previously referred antioxidant chemoprotectant and antihypertensive stohs and hartman the antioxidant activity derives from the high amounts of polyphenols leone et a0al 2015a b verma et a0al leaves extracts have revealed anticancer properties with antineoproliferative activity by inducing reactive oxygen species ros production only in cancer cells which leads to cell apoptosis gopalakrishnan the active compounds present in extracts from leaves and bark revealed anticancer activity against breast and colorectal cancer cell lines through diverse mechanisms as decreased cell mobility decreased colony formation low cell survival high apoptosis and g2m enrichment alasmari et a0al some extract fractions with anticancer activity have already been isolated characterized and tested in a0vitro and in a0vivo rat model krishnamurthy et a0al table a0in traditional medicine a paste made of leaves is applied externally in wounds siddhuraju and becker some scientific studies have shown that leave extracts have beneficial properties in skin aqueous leaves extract increased human dermal fibroblasts proliferation leading to faster wound healing muhammad et a0al a m oleifera leave extract fraction with ethyl acetate in low concentration a0µgml showed in a0vitro effect in skin healing by increasing proliferation of human dermal fibroblasts gothai et a0al a hydroalcoholic extract of m oleifera leaves used in a cream showed antiaging characteristics due to phenolic compounds sunscreen and photo protective characteristics were studied very recently baldisserotto et a0al when applying a cream with this extract it was also verified a reduction in sebum production ali et a0al 2013a b c and in transepidermal water loss allowing to increase hydration ali et a0al 2013a b c wrinkles and other signs of lack of skin vitality where improved during a0months of using the same topic formulation with m oleifera leaf extract ali et a0al the compounds responsible for this improvement in skin surface appear to be phenolics eg kaempferol and quercetin and antioxidants such as vitamins a c and b jadoon et a0al m oleifera leaf extract cream was also tested for potential skin irritation by a a0h semioccluded patch test and proved to be nonirritant and well accepted by the volunteers also reducing skin erythema ali et a0al 2013a b c table a0 m oleifera leaf powder can be used to clean hands when four grams of wet more efficient or dried powder are applied on hands and rubbed torondel et a0al the efficacy results were the same as for nonmedicated soap revealing potential to help in hand hygiene and prevent pathogen transmission in developing countries where hygiene products are scarcea leave extract sprayed in plant crops revealed another utility for this plant having beneficial effects on the growing anyb detneserper era seulav dnuof tonelbacilppanon a pyb detneserper era eulav tnacfiingis yllacitsitats htiw seidutssrewolfstoorkrab 0crate size and resistance on those plants and fruits bhargave m oleifera leaf tea studies demonstrated alterations in blood circulating neutrophils and conclude that moringa tea has adaptogenic capabilities in cases of stress drue et a0al table a0 previous studies using dried moringa leaves tea in mouse model with acute lung inflammation showed that mice that had decreased lung inflammation marked by alterations in cytokine production leukocyte migration and neutrophil apoptosis mcknight et a0al an ethanolic extract of moringa leaves has antianxiety effect in swiss albino mice the ethanolic extract of m oleifera leaves may have produced its anxiolytic effects via multiple mechanisms bhat seedsseeds collected from pods can be eaten raw or cooked from m oleifera seeds a rich vegetable oil can be produced m oleifera seed oil or behenben oil is produced through the cold pressing of the m oleifera seeds m oleifera oil can be used to cook as a source to prepare biodiesel as a lubricant and in the cosmetic industry rashid et a0al the oil name comes from its high content on behenic acid which confers more resistance to oxidative degradation comparing to other vegetable oils ben oil is rich in oleic acid up to palmitic but also stearic behenic and arachidic anwar it is used in various cosmetic formulations as emollient and confers nourishing moisturizing antioxidant and protective properties it is also a good skin cleansing product nadeem and imran table a0 details some cosmetic brands that use m oleifera leaves oil or active extracts as ingredients in the composition of their products this oil is also used in the enfleurage process allowing the extraction of fragrances and active compounds from difficult sources as flower petals milled m oleifera seed shells can be used as a natural exfoliating agentmoringa oleifera seeds can also help diabetic patients table a0 some studies by almalki and el rabey showed its antidiabetic activity by reducing the blood glucose level when rats where treated with or a0mg of m oleifera seeds powderkg body weight during a0weeks at the same time ingestion lead to an increase in antioxidant enzymes and consequently the compound content such as glucomoringin phenols and flavonoids moreover the same authors treating these diabetic rats significantly increased and ameliorated all kidney functions parameters in fact m oleifera seeds ameliorated liver fibrosis in rats reducing liver damage and symptoms of liver fibrosis decrease the ccl4induced elevation of serum aminotransferase activities and globulin level as well as reduce the elevated hepatic hydroxyproline content and myeloperoxidase activity hamza improving the indices of hepatoxicity in rats such as malonialdehyde level and total antioxidant capacity glutathione content catalase and superoxide dismutase activities hamza treatment with m oleifera seeds also altered oxidative stress in relation to its antiinflammatory activity histopathological observations showed mild or less infiltration of lymphocytes angiogenesis and synovial lining thickening from all above results and observations it can be concluded that the seeds possess promising antiarthritic property mahajan et a0al these seeds have others appeals to the daily life and industry seed powder showed capacity to purify water and remove heavy metals and anic compounds sharma et a0al through low molecular weight cationic proteins mediated precipitation kansal and kumari there was a reduction of in the turbidity of the water and of bacterial reduction bhargave lea the remaining paste after the oil extraction still has the same flocculation properties serving both purposes and adding value lea compounds such as pterygospermin moringine and benzyl isothiocyanate isolated from m oleifera seeds showed antimicrobial effect viera et a0al accordingly there is applicability for m oleifera seeds in the prevention of microbial diseases table a0 m oleifera oil has also been tested for its potential to produce biodiesel contributing as an alternative to the conventional diesel fuel rashid et a0al flowers and a0rootsm oleifera flowers are used directly as part of the diet but also to make infusions which ha | 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 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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 |
" child maltreatment leads to enormous adverse short and longterm health outcomes the aim ofthis study is to estimate the burden of disease and the cost of illness attributable to child maltreatment in japanmethods an incidencebased topdown cost of illness analysis was conducted to estimate medical costs andburden of disease attributable to child maltreatment based on a societal perspective the assessment includedshortterm and longterm medical costs and burden of disease measured by disabilityadjusted life years dalysthat generates mortality and morbidities based on several national surveys and systematic review we consideredthe main types of child maltreatment as exposure for which the incidence was obtained from literature reviewbased on population attributable fractions pafs burden of disease of physical and mental health consequencesattributable to child maltreatment were estimated then dalys were converted into monetary value the lifetimeeconomic burden was finally estimated by combining with medical costs and subject to sensitivity analysisresults the lifetime disease burden expressed in dalys was estimated at dalys ci dalys for the cohort victims in based on the incidence according to literature review the overall lifetimeeconomic burden was billion usd equivalent to million times of gross domestic product gdp per capitaamong the total economic burden costs of suffering and pain based on dalys were accounting for theseestimates were times of conservative estimates which used incidence data from official reported casess this study found that the national lifetime cost was huge and equivalent to million gdp percapita and its burden of disease was approximately equal to that of colon and rectum cancers or stomach cancerour findings particularly in terms of revealed the considerable burden of disease in long term and potential effectsof the strengthened maternal and child care as the preventive strategykeywords child maltreatment burden of disease study lifelong health consequences disabilityadjusted life yeardaly costofillness correspondence gairuoyanipssgojp1department of health policy national center for child health anddevelopment tokyo japan3department of empirical social security research national institute ofpopulation and social security research uchisaiwaicho chiyodakutokyo 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 0cmo bmc public health page of child maltreatment is a raising concern in public healthand social welfare in japan the reported number ofsuspected cases of child maltreatment is increasing from in to in according to theministry of health labour and welfare mhlw ofjapan child maltreatment is categorised into four essential types physical sexual or psychological includingwitnessing domestic violence wdv abuse and neglect exposure to multiple types and repeated episodes ofmaltreatment during childhood is associated with highrisks to enormous adverse health outcomes causing asignificant social and economic burden on individualsfamilies and societies those adverse outcomes duringchildhood include child death injuries and disabilitiesdevelopmental and behavioural problems moreover therelated physical and mental health conditions persistinto adulthood leading to the onset of chronic diseasesdepression drug alcohol misuse and risk sexual behaviour suicide ideation [ ]the number ofthe related analysis ofthe government has introduced a couple of protectivemeasures with increasing public budget [] assessment of costs and burden of disease helps developmentof resource allocation and priority setting in public sector paralleling with growing concerns on child maltreatmenttheprevalence health consequences and economic burdenis increasing so far for the economic burden there aretwo typical research frameworks one is a comprehensively costs evaluation from healthcare social educa[]tional areas and loss in productivity another one is to measure related economic and diseaseburden [ ] wada reported the socialcosts of child abuse in japan included direct costs ofdealing with abuse and the indirect costs related to longterm damage from abuse during the fiscal year onthe other hand the first framework is likely to underestimate longterm deleterious effects of child maltreatmenton which evidence derived from longitudinal studies isless available compared to that on the shortterm counterpart by integrating previous evidence our costofillness study aimed to assess lifetime economic anddisease burden of mortality and morbidities attributed tochild maltreatment based on the later frameworkinorder to address the evidence gap we extended cost calculations for monetary values converted from disabilityadjusted life years dalys covering related mortalityand morbidities methodsan incidencebased victims estimated by incidencetopdown approach or attributable risk approach measuring the proportion of a disease that is due to exposureto risk factor was applied in this study from a societalperspective we employed the following steps to estimate the total economic burden constituted by directand indirect costs population attributable fraction paf wasgenerated to estimate longterm impactscosts attributed to child maltreatment shortterm and longterm direct medical costs wereassessed by using national expenditure databasesindirect costs measured include productivity losscaused by abusive head trauma and economicburden deriving from dalys finally sensitivity analyses were performed for theplausible range of the discount rate and theincidence prevalenceestimating pafin the topdown approach paf for each disease i measured that how health outcomes and their associatedcosts may be attributed to child abuse using the following formula [ ]pafi ¼ p rri °p rri ¾¾ ¾ °p prevalence of child abuse rr the relative risk of theoutcome i in those who experienced child abuse compared with those who did notrisk ratio rr or odds ratio orseveral previous related systematic reviews and metaanalyses summarised the relevant health consequences[ ] as adverse childhood experiences acesoften intertwine with child maltreatment cluster in childrens lives and cumulatively lead to poor health outcomes we pooled the ors from a recent systematicreview and metaanalysis for the effect of multiple aceson health rather than that for each category of childmaltreatmentin japan thethe pooled prevalencea literature review was performed to synthesize the evidence on epidemiological characteristicsthe consequencesreview focused on thosepublished between december and march onmedline pubmed web of science scopus and ciniis japanese literature details of the search strategy search terms used and inclusion and exclusion criteria are provided in the additional file we combinedour review results with those studies in japan includedin an existing systematic review and calculated thesimple sizeweighted mean incidenceprevalenceinthe median value was also calculated toaddition 0cmo bmc public health page of examine the robustness supplementary table theannual incidence rate was obtained by the formula incidence rate ¼prevalenceaverage durationdue to the lack of local data on the average duration we adopted that published in australia theaverage years for physical abuse and years forsexual abuse based on this findingthe weightedaverage of years was used for other categories ofabusedirect medical costsshortterm medical costsfor abusive head trauma aht is the leading causeof death due to child abuse among children youngerthan years old we estimated its hospitalizationcosts as shortterm medical costs by multiplying theincidence of aht under years old the agespecific population in and admission medical fee per case there were two reported incidences one is the possibleincidence considering countable possibility ofaht cases at most and another one is the presumptiveincidence representing victims had intracranial injuriesor intentionalinjuries with certain icd10 code weused the possible incidence for the general calculationand the latter one in sensitivity analysis the total possible aht cases aged under years was about times ofthe presumptive counterparts longterm medical costsfor longterm medical costs we used national healthcare expenditures and patient survey tosimulate disease burden ofrelevant health consequences by sex and age group above and then multiplied with pafs to calculatethe attributable costs in the victim cohort of [ ] on the other hand we did not include selfharm and collective violence because of the limitationto distinguish the two in the reported overallinjurycasesindirect costsin this study we considered differential and loss of earning as a result of human capital depreciation is causedby mortality and morbidities it was presented as a monetary value of dalys and gdp per capita [ ]dalys and its monetary valuethe disease burden indicator daly aggregates yearsof life lost for premature death and years lost due todisability for morbidities related data wereobtained from the who global burden of diseasegbd using the pooled ors as described bykaren we matched each relatedhealth outcome with the cause of disease burdenin the who gbd categories though it was difficultto match some outcomes with the cause of gbdsupplementary table then monetary value was converted from daly attributable to child maltreatment by multiplying dalyand gdp per capita with adjustment of purchasingpower parity in productivity losses due to aht fatal casesproductivity loss due to fatal cases of child maltreatment was calculated based on the reported fatal caseswhich figure was obtained from official data andthe average lifetime income subject to discounting in there were abuserelated deaths reported injapan not including family suicide with the averageonset age of years the discounted lifetime income from to years old was calculated by assuming the longterm growth in labour productivityto be per year dalys losses of survival ahtfor disease burden due to survival aht we considered sequelae such as vision loss brain damage andreduced life span and longterm health consequences as developing diseases in adulthood we calculated the disease burden of aht in bymultiplying average cases and the estimated meanlifetime daly loss per case at different severity mildmoderate and severe longterm dalys losses of other diseasesthen the longterm health consequences were calculatedusing the following formuladaly losses ¼ Ï pafi 03original dalyi°i different child abuse related health outcome°¾¾sensitivity analysesa discount rate of is generally performed which wasrecommended in the domestic guideline for costeffectiveness analysis whereas especially in the usa discount rate of has been selected and applied inthe cost estimate reports of centers for disease controland a best practices for the social return on investmentanalysis recommended by experts and guidelines assuch the parameter potentially affects the finally resultswe adopted a plausible range of to for sensitivityanalysis 0cmo bmc public health page of in addition we also calculated costs and diseaseburden using the incidenceprevalence data based onofficially reported child abuse cases to calculate theconservative incidence of child abuse by categorieswe obtained the official data of victim cases reportedby child consultation facilities in and thendivided them by the total population number in corresponding age data by sex were not availablecocurrentinformation was not available and theoverlapped cases were not considered supplementarytable the initial victim age is assumed to be years old according to an ageweighted incidence calculation based on official reported cases we assumed the probable abuserelated death cases to be times of the reported cases based on the ratio of thepresumptive and the possible incidence of aht casesamong children aged under years resultsthe main results showed in tables were discounted at and conservative estimates were given for sensitivityanalysesthe pooled incidence prevalence and disease burdenthe estimations on different types of child maltreatment incidence draw from literature reviews variedregarding differences between sex except physicalabuse girls suffered more than boys in sexual abuseand witnessing domestic violence table the estimated lifetime disease burden associated with childmaltreatment onset in was considerable dalys with a ci of dalys to dalys table the top causes of totaldisease burden due to child abuse were suicide attempts cardiovascular disease and depression cancercostofillness analysis for child maltreatmenttable demonstrates lifetime costs attributed to childabuse onset in the total direct cost was estimatedtable estimated incidenceprevalence of child abuse in japanestimates aincidence bmalefemalephysical abuse sexual abuse psychological abusewdv c other d prevalencemalefemaleneglect a sample sized weighted mean valueb incidence rate prevalence average durationc wdv witnessing domestic violenced not specified as wdv often expressed as emotionalpsychological abuseto be usd million 95ci million11 million while the total indirect cost was estimatedto be usd million 95ci million52 million accounting for of the total lifetimecosts which were almost million times gdp percapita economic loss initiated from dalys in longterm costs of suffering and pain accounted for ofthe overall estimatessensitivity analysesconservative estimates based on the reported cases incidence showed a tendency similar to that observed in thedisease burden based on the literature review amongwhich psychological abuse including wdv accountedfor the majority of reported child abuse cases however the incidence estimated from the review weremuch higher than those reported by child protectionagencies the conservative estimation leading to about times difference gap on child maltreat burden bydifference discount rate table discussionour results indicated that disease and economic burdenattributable to child maltreatment is substantial in particular that originated from the longterm health consequences accounts for the majoritybased on literature review the pooled incidence ofchild maltreatment in japan is much higher than officially reported which is consistent with the findingsof other studies [ ] because of difficulty toidentify the actual cases and a public attitude to consider child abuse as a private affair in the society theofficially reported cases are likely to represent the tipof an icebergthe fourpsychological abuse including wdv representedthe majority of reported cases the results of the literature review also showed a gender difference in theprevalence oftypes of child abuse sizeweighted mean values girls were found to be morelikely to experience the harmful practices comparedto boys particularly sexual abuse this tendency wasalso observed in other countries in east asia and pacific region comparing those living in othercountries in the east asia and pacific region [ ]japanese children tended to less likely to experiencephysical abuse boys vs girls vsalthough it is difficult to directly compare the results across different study settings due to the different methodologies parameters and target populationsadopted the ingredients of the lifetime economic anddisease burden considered in our studyincludingmedical costs and monetary value of disease burdenare similar to that adopted in previous studies [ 0cmo bmc public health page of table longterm daly lost attributable to child abuse in japandiseases attributed to child abuse asuicide attemptdalys confidence intervalcancercardiovascular diseasedepressionrespiratory diseaseliver or digestive diseaseanxietyproblematic drug useabusive head traumaproblematic alcohol usediabetessexually transmitted infectionsviolence victimisationviolence perpetrationtotaldalys monetary value billion usa simple size weighted mean prevalence at discounted rate] still our results showed that the disease burdenwas about times of the conservative estimationdue to the huge gap of incidence generated from literature and that officially reported the number isconsistent with an australian research that showed awide distribution ofthe annual prevalence rangingfrom to in the conservative lifetimecourse simulation the initial victim age is assumed tobe years old according to an ageweighted incidencecalculation based on official reported cases whichwas also consistent with previous studies our study in particular highlighted dalys in longterm attributable to child maltreatment accountingin the overallfor a relevant proportion lifetime costs the estimation of disease burden attributed to child maltreatment dalys wascomparable to the total dalys due to colon and rectum cancers dalys in or stomachcancer dalys in to our knowledge this is the first study to estimatelifetime economic burden of child maltreatmentinjapan based on an epidemiological model the idea ofthis method is to convert diseaseinduced losses ofwellbeing into economic terms by multiplying theannual number oflost life years due to disease bysubreginal per capita income so far few studies hadever taken this part of costs into account potentiallyleading to an underestimation of health and economictable lifetime costs attributable to child abuse for the first time in ciitems of the costs usd milliondirect costs medical costsshortterm ahtlongterm other diseasesindirect costsabuse death a productivity lossessurvival aht dalys blongterm loss of other diseases btotal costsaht abusive head traumaa we used times of base line data for range costs of child abuseb costs of suffering and pain dalys converted into monetary value by multiplying a gross domestic product per capita million gdp per capita 0cmo bmc public health page of table sensitivity analyses on incidence resource and discounted ratesensitivity analysisliterature based estimation adisease burden in dalys 95cieconomic burden usd million 95cidr dr dr conservative estimation bdr dr dr dr discounted ratea estimated based on literature review simple size weighted average prevalenceb estimated based on the number of consultation cases disposed about child abuse at child guidance centres probable estimate of abuse death was assumedabout times confirmed aht casespossible cases of the costs of conservative estimatechild maltreatmentimpacts ofin addition weadopted conservative calculation methodology in thesensitivity analyses to estimate the burden of childmaltreatment for more reliable range estimationsthere are several limitations to this study first thecooccurrence of multiple types of child abuse isprevalent resulting in difficulties to identify theadverse effects separately in order to minimize possible consequent overestimation we used the pooledors of multiple adverse childhood health experiencesinstead of each types of child maltreatment and itsseverity second we focused on the economic burdendue to the mortality and morbidity of child maltreatment but did not consider nonhealth human capitalaspectslikeother economic burden estimation studies the availability of data on the related medical costs were limited wehealthconsequences and explored their unit costs for the estimates to address the knowledge gap thirdtargeted majorneverthelessthereproductiverecently in japan a continuum ofintensive supports to mothers and childrearing families encompassingcycle has been widelyimplemented in most local authorities such an integral approach serves as an essential preventive strategy against child maltreatment and other harmfulpractices by early detection and intervention of highrisk households in pregnancy postpartum and childrearing periods thisstudy can provide decisionmakers information on the economic burden of childmaltreatment as well as an important input in futureeconomic evaluations costeffectiveness analysis oncurrently ongoing intervention and policy in additionour results hint an emphasis on preventive interventions on suicide attempts and depression which aretop causes of the attributable disease burden due tochild maltreatmentour study demonstrated that lifetime disease and economic burden due to child maltreatment in japan is substantial its disease burden was approximately equal tothe burden of colon and rectum cancers or stomach cancer in particular it is important to include the longterm disease burden in future studies related to diseaseburden and cost of illness for both technical and policyperspectivessupplementary informationsupplementary information accompanies this paper at httpsdoi101186s12889020093978additional file table a1 studies included in the quantitativesynthesis table a2 health outcomes and pooled ors used in this studyaht not included table a3 incidence rate by age and average onsetage based on the number of consultation cases disposed about childabuse at child guidance centersadditional file systematics review 2018520findpossible literature including japanese studies on risk of health outcomesattributable to child maltreatment figure a1 study selection prismaflow diagramabbreviationsdalys disabilityadjusted life years pafs population attributable fractionsgdp gross domestic product mhlw ministry of health labour and welfarewdv witnessing domestic violence aht abusive head traumaicd international classification of diseases gbd global burden of diseaserr risk ratio or odds ratio aces adverse childhood experiencesacknowledgementswe are grateful thank members of health informatics department kyotouniversity of public health school for their kind supportauthors contributionsmx and gr designed the study mx did the calculation and draft themanuscript gr and ty takahashi contributed to the revise ty tachibanatb and nt critically reviewed and provided important intellectual feedbackon the revise all authors have read and approved the manuscriptfundingthis study is granted by health labour sciences research grant japanagency for medical research and development and as part of an ipss 0cmo bmc public health page of project on the realization of japans plan for dynamic engagement of allcitizens the funders did not have any role in the study design datacollection and analysis interpretation of data or in writing the manuscriptavailability of data and materialsall the raw data is publicly accessible from respective official website asreference national healthcare expenditures and patient survey the datasets analysed during the current study are available from thecorresponding author on reasonable requestethics approval and consent to participatenot applicableconsent for publicationnot applicablecompeting intereststhe authors declare no conflict of interestauthor details1department of health policy national center for child health anddevelopment tokyo japan 2department of health informatics kyotouniversity school of public health kyoto japan 3department of empiricalsocial security research national institute of population and social securityresearch uchisaiwaicho chiyodaku tokyo japan4maternalchild psychiatry department of psychosocial medicine nationalcenter for child health and development tokyo japan 5faculty ofeconomics saitama university sakuraku japanreceived march accepted august referencesgilbert r widom cs browne k fergusson d webb e sjtl j burden andconsequences of child maltreatment in highincome countries lancetnumber of consultation cases disposed about child abuse at child guidancecenters in japan in japanese [httpswwwestatgojpstatsearchfilespage1layoutdatalisttstat000001034573cycle8tclass1000001108815tclass2000001108820second21]definition and present condition of child abuse in japanese [httpswwwmhlwgojpseisakunitsuitebunyakodomokodomo_kosodatedvabouthtml] accessed july currie j spatz widom c longterm consequences of child abuse andneglect on adult economic wellbeing child maltreatment hughes k bellis ma hardcastle ka sethi d butchart a mikton c jones ldunne mp the effect of multiple adverse 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psychiatry ment health mccarthy mm taylor p norman re pezzullo l tucci j goddard c thelifetime economic and social costs of child maltreatment in australia childyouth serv rev wada i igarashi a the social costs of child abuse in japan child youth servrev miller tr steinbeigle r wicks a lawrence ba barr m barr rgjp disabilityadjusted lifeyear burden of abusive head trauma at ages pediatrics20141346e1545fang x fry da brown ds mercy ja dunne mp butchart ar corso psmaynzyuk k dzhygyr y chen y the burden of child maltreatment inthe east asia and pacific region child abuse negl corso ps fertig ar the economic impact of child maltreatment in theunited states are the estimates credible child abuse negl macroeconomics and health investing in health for economicdevelopment [httpwhqlibdocwhointpublications2001924154550xpdf]accessed july segel je costofillness studiesa primer rtiunc center of excellence inhealth promotion economics jo c costofillness studies concepts scopes and methods clin molhepatol metrics population attributable fraction paf [httpwwwwhointhealthinfoglobal_burden_diseasemetrics_pafen] accessed july norman re byambaa m de r butchart a scott j vos t the longtermhealth consequences of child physical abuse emotional abuse and neglecta systematic review and metaanalysis plos med 2012911e1001349kalmakis ka chandler ge health consequences of adverse childhoodexperiences a systematic review j am assoc nurse pract unicef child maltreatment prevalence incidence and consequences inthe east asia and pacific region new york unicef rothman kj epidemiology an introduction oxford university press joyce t huecker mr pediatric abusive head trauma shaken babysyndrome [updated aug ] in statpearls [internet] treasure islandfl statpearls publishing available from httpswwwncbinlmnihgovbooksnbk499836 yamaoka y fujiwara t fujino y matsuda s fushimi k incidence and agedistribution of hospitalized presumptive and possible abusive head traumaof children under months old in japan j epidemiol httpsdoi102188jeaje20180094japanese population projection [httpwwwstatgojpdatajinsui2016np] accessed july summary of patient survey [httpswwwmhlwgojpenglishdatabasedbhsssps_2014html] accessed july kirigia jm mburugu gn huka gs the indirect cost of disability adjusted lifeyears lost among the elderly in kenya int arch med httpsdoi1038232483 mortality and global health estimates [httpwwwwhointghomortality_burden_diseaseen] accessed july japan gdp gross domestic product [httpscountryeconomycomgdpjapanyear2016] accessed july the results of verification of death cases caused by child abuse threport [httpswwwmhlwgojpstfseisakunitsuitebunya0000173329_00001html] accessed july miller tr steinbeigle r wicks a lawrence ba barr m barr rg disabilityadjusted lifeyear burden of abusive head trauma at ages pediatrics20141346e154550 httpsdoi101542peds20141385shiroiwa t fukuda t ikeda s takura t moriwaki k development of anofficial guideline for the economic evaluation of drugsmedical devices injapan value health moore se scott jg ferrari aj mills r dunne mp erskine he devries kmdegenhardt l vos t whiteford ha burden attributable to childmaltreatment in australia child abuse negl publishers notespringer nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations 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 |
during the last decade green synthesized cerium oxide nanops ceo2 nps attracted remarkable interest in various fields of science and technology this review explores the vast array of biological resources such as plants microbes and other biological products being used in synthesis of ceo2 nps it also discusses their biosynthetic mechanism current understandings and trends in the green synthesis of ceo2 nps novel therapies based on green synthesized ceo2 nps are illustrated in particular their antimicrobial potential along with attempts of their mechanistic elucidation overall the main objective of this review is to provide a rational insight of the major accomplishments of ceo2 nps as novel therapeutics agents for a wide range of microbial pathogens and combating other diseases keywords nanotechnology green synthesis cerium oxide nanops antimicrobial infections biomedicalintroductionnanotechnology has got a remarkable interest in every field of science and technology and is presently considered among one of the leading research avenues it has a multitude of applications in the field of electronics imaging industry and healthcare14 mostly in healthcare it has been exploited in diseases diagnostics treatment delivery and formulations of novel drugs14 it exploits nano size structures with size ranges from nm known as nanop nps these nano scale entities have unique physiochemical properties and have been utilized in various fields of physics biology and chemistry5among other nps cerium oxide ceo2 nps have been mostly exploited due to their unique surface chemistry high stability and biocompatibility67 it is mostly used in the fabrication of sensors cells catalysis therapeutics agents drug delivery careers and antiparasitic ointments figure presently ceo2 nps is mostly synthesized via two methods such as physical and chemical79 however these methods utilize toxic reducing solvents posing several threats to the biodiversity and ecosystem moreover the nps obtained with such approaches are toxic and unstable making them less efficient910 thus recently a safe less toxic method has been used by researchers known as green synthesis this method utilizes various biological resources such as plants microbes or any other biological derivative1115 these biological extracts have a rich source of phytochemicals international of nanomedicine nadeem this work is published and licensed by dove medical press limited the full terms of this license are available at wwwdovepresscomterms php 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 0cnadeem dovepresslatter is extensively utilized for its biomedical pharmacological and food applications due to their safe and biocompatible nature9 moreover features like high yield everlasting stability and better morphologies can be obtained using a greener approach79green synthesis from plantsgreen syntheses of ceo2 nps have been reported using plant extracts microbial and other biological derivatives plants in this regard have been the most efficient source due to their abundance safe nature and rich source of reducing and stabilizing agents2629 various parts of plants such as leaves flower and stem have been used for the synthesis of ceo2 nps163031 till date the majority of green synthesis studies have been conducted on leaves extracts as it is a rich source of metabolites11163233 a broad variety of metabolitesphytochemicals in plant extracts such as ketones carboxylic acids phenols and ascorbic acid are used as reduction and stabilizing agents figure plants based ceo2 nps are produced through a simple approach in which bulk metal salt is mixed with the extract and the reaction completes in minutes to a few hours in ordinary lab conditions282934 the metallic salt solution is reduced into respective nanops via the phytochemicals whose synthesis is confirmed firstly through color change from colorless to yellowish brownish or whitish and then characterized through various spectroscopic and imaging techniques162935leaf extract of moringa oleifera l was used to synthesize ceo2 nps with spherical morphologies and size of nm the synthesized nps showed potential antimicrobial and wound healing properties36 gloriosa superba leaf extract was used as a reducing and stabilizing agent in synthesis of ceo2 nps and has shown potential antibacterial properties37 hibiscus sabdariffa natural extract yielded crystalline ceo2 with a diameter of nm30 spherical shaped nanops of size nm synthesized from gel extract of medicinally important plant aloe barbadensis38 the resultant ceo2 nps showed high antioxidant potential green synthesis of ceo2 nanops was demonstrated using jatropha curcus leaf extract and a monodispersed shape of nm10 spherical shaped cerium oxide nanops are synthesized using leaf extract of oleo europaea with a size of nm having high antimicrobial activity against both gramnegative and positive strains of bacteria16 origanum majorana extracts were used to synthesize ceo2 nps having pseudo photocatalytic activity having high figure general applications of ceo2 nanopssuch asketones amines enzymes and phenols which are believed to be responsible for the reduction and stabilization of bulk salts into respective nanops nps1619to date various applications of green synthesized ceo2 nps have been reported such as antimicrobial anti cancer antilarvicidal photocatalysis and antioxidant therapies162022 among other biomedical applications the antimicrobial potential is certainly the most exploited previously it has been reported that ceo2 nps to display their antimicrobial actions through various mechanisms9 but mostly ceo2 nps kill microbes via triggering the production of an excess of reactive oxygen species in cells916 however further studies need to be conducted to fully elucidate the complete mechanism of action here in this review we aim to focus on the following topics arrays of biological resources have been exploited to date for the synthesis of ceo2 nps moreover the synthesis mechanisms along their biomedical applications are discussed with special emphasis on the antimicrobial activitysynthesis of ceo2 npsnanops are synthesized through various physicochemical methods5 however both methods require toxic solvents high temperature and pressure which pose threats to the environment2325 moreover higher cost laborious downstream processing lesser biocompatibility instability and low yield make them further inefficient710 there is a growing need to fabricate nanostructures which have the potential to solve these problems59 presently researchers have exploited the green method to overcome all these challenges5 for instance plants microbes and other biological products have been used as reducing and or stabilizing agents in the fabrication of ecofriendly nps25 ceo2 nps have also been synthesized using various physical chemical and biological methods9 the submit your manuscript wwwdovepresscom dovepress international of nanomedicine 0cdovepress nadeem figure biosynthesis reduction stabilization and characterization of ceo2 nanopsspherical shape nm ftir confirmed that the reduction is attributed to the presence of different phenolic and flavonoids compounds in the extract18 ceo2 was synthesized using rubia cordifolia leaf fusions spectroscopic and microscopic analysis revealed hexagonal shaped nps having a size of nm the biogenic ceo2 nps also showed excellent anticancer potential33 nano rod size ranges from nm ceo2 nps resulted when pedalium murex l was added to the aqueous solution of salt at room temperature having high antibacterial activity32 china rose petal was used as a robust bio template for the facile fabrication of novel ceria nano sheet with a size of about nm39 the deviation in size and morphology noticed among the reported studies might be due to the different influence of reaction temperature ph time concentration of salt precursor or plants extracts and part of the plant being used13272840 moreover plants based ceo2 nps showed excellent stability at diverse conditions for instance green mediated ceria nps remain stable at liquid solution changes were observed1336 similarly biogenic ceo2 nps also showed high thermal stability at high temperature and remained stable for a longer period of time which indicates their long durability and everlasting stability27283341 until now various plants have been used in the biogenic synthesis of ceo2 nps and are shown in table physiochemical and no green synthesis from microbesmicrobes also have an intrinsic potential to synthesize nanops as they are a rich source of secondary metabolites23 among other nanops ceo2 nps with various shapes and sizes have been synthesized in recent years from microbes table green synthesis of ceo2 from microbial species is a simple reliable costeffective and ecofriendly approach42 microbial metabolites such as enzymes proteins and heterocyclic derivatives play a crucial role in reducing and stabilizing of ceo2 bulk salt into respective nps4243 moreover microbiogenic ceo2 nps exhibited improved stability water dispensability and showed high fluorescent properties and were less agglomerated43aspergillus niger extract yielded cubic fluorite nps with spherical morphology and an average size of nm ftir analysis revealed the presence of an hydroxyl group carboxylic group and phenol group which are supposed to be involved in the reduction of nps21 curvularia lunata extract has also been used to synthesize spherical shaped ceo2 nps with size ranges from nm color change from white to yellow brown indicated initial reaction the nps were tested against microbial pathogens and showed excellent antibacterial potential spherical shaped ceo2 nps of size ranges from nm were made using fusarium solani extract which showed effective growth inhibition and biofilm formation of pathogenic bacterial strains42 shadab ali khan observed the biosynthesis of spherical shaped nm ceo2 nps by using a thermophilic fungus humicola capping agent43 the resultant nps were characterized by uv xps pl spectroscopy tem ftir and xrd moreover these nps showed excellent potential in treatment of neurodegenerative disorders such as alzheimers and parkinsons diseases bacterial extract has also been exploited in the fabrication of ceo2 nps for instance bacillus shaped nps with an average size of nm the bacterial mediated nps also showed excellent antioxidant potential in vitro44 despite all these applications the microbial route of synthesis has certain shortcomings such as the high probability of pathogenicity laborious culturing and contamination issues however it offers a lot of promise in the field of nanotechnology and could become a leading avenue in subtilis extract yielded spherical international of nanomedicine submit your manuscript wwwdovepresscom dovepress 0cnadeem dovepresstable ceo2 nanops made from various plants speciesplant namepartcharacterizationshapesize nmftir groupoh ceoh hoh ocoleafxrd xps tem ftir uv vissphericalflower hrtem sem xrd xps eds ftirceohleafleafleafsem xrd ftir tgasphericalxrd sem tem uv vis ftir tgasphericaluvvis psa ftir xrd xps hrtemrtesphericalco oh hhoh ch nooh ceoseedxrd uvvis ftir fesem tgasphericalceo oceo ch co []acalypha indicaleafxrd sem tem eds ftiraloe barbadensisleafxrd tem ftir psasphericalspherical oh ceoh ceocech co ch cf ch cclaloe veraleafftir xps hrtemsphericalch ccrubia cordifolialeafuv vis xrd xps sem ftir edaxhexagonalprosopis farctaaerialuvvis pxrd tem fesem ftirsphericalchina rosepetalfesem fetem afm xrd xpsnanosheetcentella asiaticauvvis dls sem hrtem edssphericaloh ceooh ceo and no__ walnutshellleafxrd sem tem eds ftiruvvis xrd xps fesem tem hrtem eds uvdrs ftirsphericalsphericalceo and ohceoc oh chstempxrd sem tem ftir plflakyoh coo and chs nogloriosa superbahibiscus sabdariffaoliveoleo europaeaprosopis jujliflorasalvia macrosiphon boissazadirachta indicaeuphorbia tirucallipetroselinum crispummoringa oleiferaref[][][][][][][][][][][][][][][][][][][][]lemon grassgrassxrd pl tem saedleucas asperaleafpxrd sem uvvis tem saedmicrosphereuvvis ir xrd sem temsphericalpeeluv vis ftir xrd hrtemspherical watermelonfruit juicecarrageenanpxrd ftir uvvis semirregularpxrd ftir fesem uvvis and tga dtaspherical___ceo ceoceceo co co ch ohso3 ceo hoh co []continuedsubmit your manuscript wwwdovepresscom dovepress international of nanomedicine 0cdovepress nadeem table continued plant namepartcharacterizationshapeleafxrd fesem tem ftirsphericalxrd fesem eds vsmsphericalsize nmftir group__ceratonia siliquastevia rebaudianasalvadora persicamorus nigraannona muricatajusticia adhatodas nopxrd ftir uvvis tem fesem edsspherical0h ch cc ceo co cfruitfruittem xrd uvvisxrd ftir uvdrs fesemirregular_ ___leafxrd sem tem ftir uvdrssticklikeoh ch co no cc co cncjatropha curcusleafxrd tem uvvismonodispersed _origanum majoranapedalium murexelaeagnus angustifoliaeuphorbia amygdaloidesleaftem fesem xrd ftirsphericaloh ch coleafxrd ftir uvvis drs semnanorodoh ch co co cn cn ceoleafxrd sem tem ftirsphericalch cotem sem xrd uvvissphericalorangepeelxrd tem ftir uvviscubic structurecoh or corpiper betleleafxrd ftir sem eds xps temnh no cnref[][][][][][][][][][][][][]nanomedicine but is yet to be explored particularly these microbial based nps can be used in designing novel fertilizers fabricating sterile surfaces polymers and medical accessories moreover these biogenic nps can also be exploited in disease management drug synthesis and deliverytable ceo2 nanops synthesized from various fungus speciess nomicrobe namecurvularia lunatacharacterizationshapesize nmftir grouptgdta xrd raman pl ftir uvvis and temspherical_humicola spuvvis xps pls tem ftir xrdsphericalceo ceocefusarium solaniftir pl tgdta fesem xrd edax tem xps saed clsmsphericaloh cn cocaspergillus nigeruvvis ftir xps xrd tgdta pl temsphericalhoh oco ce oref[][][][]international of nanomedicine submit your manuscript wwwdovepresscom dovepress 0cnadeem dovepressgreen synthesis from biological productsapart from the synthesis of nanomaterial from eukaryotes and prokaryotes anisms nps have also synthesized from biological derivatives23 they also play a crucial role in the reduction and stabilization of nps2325 in contrast to plants and the microbial approach bioproduct based ceo2 are much safer scalable and have shown excellent biocompatibility4547 for instance egg white protein was used in order to synthesize ceo2 nps having size ranges from nm with spherical morphologies48 these nps were characterized and confirmed by uvs ftir tgadta and pxrd ftir analysis revealed that the phenol ether hydroxyl and amide groups were responsible for the reduction of these nps it also showed a good in vitro cytotoxicity effect towards human periodontal fibroblasts cells agarose is a natural matrix and has been used as a stabilizing and capping agent for ceo2 nps nps obtained have spherical morphologies with a diameter of nm nps were characterized using various methods including uv fesem ftir tgadta pxrd and tgadta techniques as revealed by ftir analysis it was found that the hydroxyl ether phenol and amide groups were involved in biosynthesis45 starch has also been exploited as a novel source for the synthesis of nanoceria with the results revealing spherical shape nps with a diameter of nm12spherical shaped ceo2 nps with a size of nm were synthesized from dextran the resultant nps exhibited strong anticancer potential46 gum tragacanth reported by darroudi 49 was used in the biosynthesis of ceo2 nps these nps were monodispersed shape with an average size range from nm the ceo2nps exhibited very low cytotoxic effects on neuro2a cell lines making them suitable candidates for various biomedical and pharmacological applications49 some other biological products which have been used for synthesis of ceo2 nps are listed in table despite their biological applications these biogenic nps could be used as a promising candidate in diseases treatment drug delivery and packing food some other products have also been explored for the synthesis of ceo2 nps which are shown in table biological activity of greenmediated cerium oxide nanopsantimicrobial activity of greenmediated cerium oxide nanopsin the last few years nanotechnologybased therapies have been exploited in disease diagnostics treatment and table biomediated ceo2 nanops from different sources of biological productss nonamecharacterizationshapeegg proteinuvvis fesem ftir tgadta pxrdsphericalsize nmhoneyuvvis fesem ftir tgadta eds pxrdsphericalagaroseuvvis fesem ftir tgadta pxrdsphericalstarchdextranpolyethylene glycoluvvis pxrd temtem dls xps uvvishrtem tem uvvis slsdlschitosanxrd hrtem ftir tgadta uvvispectindls fesem edss xrd ftir nmr pl fesem edss uvvissphericalsphericalsphericalsphericalspherical ¤ ftir groupoh ceo ceoce ch nooh ceo ceoce ceoc nooh ceo ceoce no____oh ch co och3 coc no ceotannic acidftir xps xrd hrtem uvvisiblepolycrystalline _ref[][][][][][][][][]submit your manuscript wwwdovepresscom dovepress international of nanomedicine 0cdovepress nadeem figure schematic representation of antibacterial activity of ceo2 nanops cell wall disruption cell membrane disintegration free radicals productions loss of protein peptides dna fragmentation vital enzymes inhibition loss of cellular fluids and disruption in electron transportformulations of novel drugs1 for instance the antimicrobial potential of nps has been mostly exploited and has showed substantial outcomes2324 presently ceo2 nps have attracted great interest as an antimicrobial agent in particular against bacterial pathogens151650 the exact mechanism of killing microbes is yet not clearly elucidated however it is proposed that ceo2 nps mostly kill microbes via a massive production of reactive oxygen species ros in cells as shown in figure the bactericidal potential of ceo2 nps is attributed to strong electrostatic properties distinctive morphologies small size and low band energy1652 due to strong electrostatic potential ceo2 nps interact with membrane proteins thiols groups which results in protein denaturation membrane impermeability eventually leads to microbial death4253 figure exposure to ceo2 nps kills microbes via membrane collapse by attachment with mesosomes malfunctioning of cellular compartments and bio anic molecules which ultimately lead to abnormal metabolism and physiology1642 similarly green mediated nps killpathogens in a similar fashion and various biological species have been exploited and tested against a wide variety of microbes1316 table however biogenic ceo2 nps unique morphologies small size and biocompatible nature were found to be more effcient and have the potential to range of pathogenic bacterial species1113151642 moreover it also has the potential to kill both grampositive and gramnegative bacteria but due to structural complexity of gramnegative bacterias membranes it is more sensitive against grampositive species1321374754 in antimicrobial in electrostatics between nps and the bacterial wall plant species and wall compositiondifference to differences treat a wide activity the is due international of nanomedicine submit your manuscript wwwdovepresscom dovepress 0cnadeem dovepresstable various microbes tested against biogenic ceo2 nanopss nosourcemicrobes testedolea europaeafungus aspergillus flavus fusarium solani and aspergillus niger bacterial sps staphylococcus aureus escherichia coli pseudomonas aeruginosa and klebsiella pneumoniamoringa oleiferas aureus and e colicurvularia lunatastaphylococcus aureus streptococcus pneumoniae and bacillus subtilis pseudomonas aeruginosa proteus vulgaris and klebsiella pneumoniaeleucas asperaklebsiella aerogenes escherichia coli pseudomonas desmolyticum and staphylococcus aureusacalypha indicaescherichia coli and staphylococcus aureusannona muricataenterococcusfaecalis staphylococcus aureus klebsiella pneumonia and escherichia coligloriosa superbastaphylococcus aureus and streptococcus pneumonia ecoli proteus vulgaris klebsiella pneumonia shigella dysenteriae and pseudomonas aeruginosaaspergillus nigerstreptococcus pneumoniae bacillus subtilis proteus vulgaris and escherichia colifusarium solanistaphylococcus aureus pseudomonas aeruginosa escherichia coli and klebsiella pneumoniajusticia adhatodastaphylococcus aureus and escherichia colieuphorbia amygdaloidesp acidilacticipectine coli and b subtilisref[][][][][][][][][][][][]resistance to confer due to rapid evolution of the bacterial genome bacteria have evolved to antimicrobial agents5556 thus in quest of a new treatment biogenic ceo2 nps has shown promising results in treating multi drug resistance bacteria and could be a promising candidate against such refractory pathogenesis26 ceo2 nps along other conjugates have been amalgamated with various anic and inanic hybrids to enhance the antimicrobial response1746 similarly biomediated ceo2 nps kills fungi by producing a mass number of free radicals and ros which causes distorted structure and physiology and leads to fungus death16 however a few studies have only been conducted on fungi despite the increasing knowledge on the antimicrobial activity of ceo2 nps much remains unknown about their exact mechanism of encountering bacteria toxicity in vivo studies and environmental concerns which needs to be addressed moreover the low band energy potential of ceo2 nps could be used in fabricating sterile surfaces at hospital or lab settings and will diminish nosocomial and other acquired infectionsother potential biomedical applicationsbeside antimicrobial therapies ceo2 nps have also been used in management of other ailments3557 for instance siliqua showed high biogenic ceo2 nps have been mostly used in treatment of various cancers such as osteosarcoma colon cervical and breast cancers1820274652 results indicated that these nps exhibited strong anticancer potential and could be used as a chemotherapeutic agent thanks to their minimal toxicity capacity to induce apoptosis andor necrosis in cancer cells46 ceo2 nps synthesized from origanum majorana and ceratonia antioxidant activity183157 results showed higher expression of antioxidant enzymes which in turn eradicated free radicals and improved cellular functions31 furthermore antioxidant potential was higher when compared to commercial synthetic antioxidants18 ceo2 nps synthesized from fruit extract of morus nigra exhibited excellent antidiabetic activity on l6 cell lines the treatment was dosage dependent and nps with lesser size resulted in higher uptake of glucose in vitro35 though biogenic ceo2 nps have shown excellent pharmacological potential however the mechanism of action minimum inhibitory concentration and best possible delivery system should need to be determined moreover cytotoxicity and genotoxicity should be tested in vivo models to evaluate the compatibility in both in vivo and in vitro modelssubmit your manuscript wwwdovepresscom dovepress international of nanomedicine 0cdovepress nadeem to their unique conclusions and future prospectsin this paper we have reviewed the recent trends and understandings of biogenic ceo2 nps and their pharmacological applications various sources such as plants microbes and other biological products have been discussed with the mechanism of synthesis and their biomedical applications due surface morphologies crystal small nature and biocompatible nature biogenic ceo2 nps have got phenomenal interest in biomedical and other fields for instance it has been used in treating various cancers antimicrobial and antioxidant therapies in particular the green synthesized nanops have shown significant antimicrobial potential against a wide range of bacterial species the mechanism of combating such pathogens have also been elucidated and supposed to be due to the mass production of reactive oxygen species and deactivation of scavenging enzymes the ros impedes the membranes disrupts the cellular compartments and disintegrates the bio anic molecules and hampers the associated functions and ultimately causes death it has also shown promising results against multidrug bacteria and could be a potential antimicrobial agent in future against such refractory pathogens however further studies should conduct in vivo models to reveal the full mechanism alongside any sideeffects moreover it has also shown excellent anticancer and antioxidant potential in vitro setups but their toxicity and dosage are yet unknown which needs to be addressed despite their role in various therapies their mechanism of synthesis needs to be optimized whereas in vivo evaluation as well as toxicity should be further screenedabbreviationsceo2 nps cerium oxide nanops dga differential thermal analysis dsl dynamic light scattering eds energydispersive xray spectroscopy fesem field emission scanning electron microscopy ftir fourier transform infrared spectroscopy hrtem highresolution transmission electron microscopy pl photoluminescence pxrd powder xray diffraction ros reactive oxygen species sls static light scattering tem transmission electron microscopy tga thermal gravimetric analysis uvvis uvvisible spectroscopy xps xray photoelectron spectrometry xrd xray diffractiondisclosurethe authors report no conflicts of interest for this workreferences kubik t boguniakubik k sugisaka m nanotechnology on duty in medical applications curr pharm biotechnol doi1021741389201053167248 bhushan b springer handbook of nanotechnology springer jianrong c yuqing m nongyue h et al nanotechnology and biosensors biotechnol adv doi101016j biotechadv200403004 smith dm simon jk baker jr jr jr applications of nanotechnology for immunology nat rev immunol doi101038nri3488 mohanraj v chen y nanopsa review trop j pharm res das s dowding jm klump ke cerium oxide nanops applications and prospects in nanomedicine nanomedicine doi102217nnm13133 he l su y lanhong j et al recent advances of cerium oxide nanops in synthesis luminescence and biomedical studies a review j rare earths doi101016s1002 walkey c das s seal s catalytic properties and biomedical applications of cerium oxide nanops environ sci doi101039c4en00138a rajeshkumar s naik p synthesis and biomedical applications of cerium oxide nanopsa review biotechnol rep doi101016jbtre201711008 magudieshwaran r ishii j raja kcn et al green and chemical synthesized ceo2 nanops for photocatalytic indoor air pollutant degradation mater lett doi101016jmatlet arunachalam t karpagasundaram m rajarathinam n ultrasound assisted green synthesis of cerium oxide nanops using prosopis juliflora leaf e | Colon_Cancer |
"objectives therapeutic radiographers trs are well placed to deliver health behaviour change advice to those living with and beyond cancer lwbc however there is limited research on the opinions of trs around delivering such advice to those lwbc this study aimed to explore trs practices and facilitators in delivering advice on physical activity healthy eating alcohol intake smoking and weight managementsetting and participants fifteen uk based trs took part in a telephone interview using a semistructured interview guide data was analysed using the framework analysis methodresults emergent themes highlighted that trs are mainly aware of the benefits of healthy behaviours in managing radiotherapy treatment related side effects with advice provision lowest for healthy eating and physical activity participants identified themselves as well placed to deliver advice on improving behaviours to those lwbc however reported a lack of knowledge as a limiting factor to ng so the trs reported training and knowledge as key facilitators to the delivery of advice with a preference for online trainings there is a need for education resources clear referral pathways and in particular training for trs on delivering physical activity and healthy eating advice to those lwbcintroductionit is estimated that of cancer cases are linked to unhealthy behaviours1 based on evidence from systematic literature reviews and meta analyses the world cancer research fund wcrf recommend that individuals are physically active limit consumption of energy dense foods salty foods red meat and avoid processed meat eat more plant foods maintain a healthy weight limit alcoholic drinks and avoid tobacco to reduce their risk of cancer2 those living with and beyond cancer lwbc are also advised to follow these guidelines due to increasing evidence that healthy behaviours may improve physical strengths and limitations of this study º this study provides an insight in therapeutic radiographers views on all key modifiable health behaviours for those living with and beyond cancer º the participants worked in different radiotherapy departments offering insight into the practices among therapeutic radiographers in the delivery of healthy behaviour advice from a wide range of hospitals º whilst data saturation was reached the sample size was small and therefore the findings may not be representative of the views of the wider therapeutic radiography workforce º the response rate was low therefore the participants might be more interested in the role of health behaviours in cancer survivorship which might bias the responses towards a positive view on the role of therapeutic radiographers in delivering advice within their roleand psychosocial outcomes after a cancer diagnosis2despite the potential benefits of healthy behaviours few people lwbc are meeting the wcrf recommended health behaviour recommendations9 those lwbc report one key reason for not adopting healthier lifestyle behaviours is lack of advice and support from their healthcare team11 healthcare professionals hcps are well placed to bring about positive health behaviour changes among cancer patients12 a trial of brief advice among breast cancer survivors showed that a simple physical activity recommendation from a hcp doubled the percentage meeting national exercise guidelines12 despite this research to date among both hcps and those lwbc consistently shows that few oncology hcps offer guidance to oncology patients on healthy lifestyle behaviours13reported barriers in providing health behaviour advice for those among hcps pallin a0nd et a0al bmj open 202010e039909 101136bmjopen2020039909 0copen access lwbc include believing that giving advice was not part of their role lack of time with patients lack of referral programmes lack of resources such as education leaflets for those lwbc and lack of knowledge regarding guidelines and research findings16 a recent qualitative study with oncology hcps identified that advice on health behaviours provided to those lwbc focussed on general health and controlling side effects with few hcps advising on health behaviours in the context of improving survival outcomes20 while these studies provide useful insight into the practices and barriers among oncology hcps the participants within these studies were primarily oncologists and nurses and focussed on the provision of physical activity and weight management advice there is limited research on the opinions of therapeutic radiographers trs in delivering advice on health behaviours to those lwbc despite at least of cancer patients receiving radiotherapy as part of their cancer treatment22trs are the only health professionals qualified to deliver radiotherapy and play a central role in supporting cancer patients23 in the uk the college of radiographers recognise the importance of trs in providing health behaviour advice to improve patient outcomes24 trs are also seen as an integral part of the health force in driving improvements in well being as outlined in the publication of ahps into action using allied health professionals to transform health care and well being which states that radiographers are key to implementing a preventative healthcare approach and that their expertise should be used to design and deliver health interventions25 trs are ideally placed to deliver health behaviour advice particularly through making every contact count mecc26 mecc is a strategy whereby health professionals use every appropriate opportunity and interaction with patients to promote healthy behaviours and signpost to relevant healthcare services using an ask advise act framework27 mecc fits extremely well within the trs role in which patient education is a key part of radiotherapy practice with trs providing care to the same patient every day often over a number of weeks23 trs therefore have the potential to make significant contributions in supporting positive health behaviour changes among those lwbchowever despite these opportunities one survey in the uk among trs identified that trs rarely advise patients on the key modifiable health behaviours including smoking alcohol healthy eating and exercise15 the findings also showed lack of knowledge and training as barriers among trs in delivering advice on these topics15 similarly focus group interviews with trs identified that lack of knowledge and training were barriers to the provision of smoking cessation advice28challenges remain in translating behaviour change interventions into existing care pathways and practices in a way that is appropriate for use by health professionals29 understanding trs practices and what support they need in delivering advice on the topics of physical activity healthy eating alcohol intake smoking and weight management could inform the development of interventions that will enable trs in delivering advice on improving health behaviours to those lwbc qualitative research is appropriate for exploring the beliefs experiences and motivations of individuals on specific matters and allow for more information and clarification30 limited qualitative data exists on trs practices and views on delivering advice on these health behaviour topics this study therefore aimed to address this and through a qualitative methodology explore trs practices in delivering health behaviour advice in addition to exploring the facilitators in delivering such advice preferences regarding training on delivering this advice were also exploredmethodsparticipants and recruitmentparticipants were trs working in the uk in a clinical role they had provided their contact details on a previous online survey investigating trs practices in delivering health behaviour advice agreeing to be invited for a follow up telephone interview an email was sent with an information sheet explaining the research and inviting these trs those who agreed to take part signed a consent form prior to the telephone interviewdata collectionsemi structured individual telephone interviews were carried out between april and may by a lecturer in therapeutic radiography with an msc who had completed qualitative interviewing as part of their training np the interviewer had no previous relationship with the study participants the topic guide see online supplementary material was based on the guide used within a previous study17 which explored oncology hcps views on the provision of lifestyle advice to cancer patients this guide was adapted for use among trs with additional questions added to assess preferences for training on delivering advice the topic guide was piloted with two participants to check for comprehension of the questions this data was included in the analysis because no substantial changes were required the interviews lasted approximately min range to min and were audio recorded anonymised and transcribed verbatim the transcripts were verified by np against each recording to confirm accuracy the aim was to carry out interviews until data saturation was reached it was anticipated that participants would be required to reach data saturation because it was a homogeneous group31 after interviews were carried out they were transcribed verbatim following familiarisation with the data np generated the initial codes and it appeared that saturation was reached after interviews as no new codes occurred in the 10th interview32 a further five interviews were carried out to confirm thispallin a0nd et a0al bmj open 202010e039909 101136bmjopen2020039909 0ctable participant identifier and demographic characteristicsparticipant identifierdemographic characteristicsprofessional gradegendertr tr tr tr tr tr tr tr tr tr tr tr tr tr tr femalefemalefemalefemalefemalefemalefemalemalefemalefemalefemalefemalemalefemalemaleband band band band band band band band band band band band band band band tr therapeutic radiographerpatient and public involvementpatient input was not used in the design of the research methods however the topic guide was piloted with trs in the academic setting additionally the topic guide was piloted with two participants and these were included in the analysisanalysisthe interview transcripts were analysed using the framework analysis method33 this method was chosen because it is an appropriate method for analysing homogeneous data and semi structured interview transcripts it is also appropriate when using inductive qualitative analysis33 a random selection of transcripts n3 were independently coded by af to check for reliability the researchers np af and rjb met and agreed on a final coding list in an iterative process af and rjb are both experienced qualitative researchers and health psychologists these agreed set of codes formed the analytical framework which was then applied to all of the transcripts and the data summarised in a matrix using microsoft excel themes were generated by reviewing the matrix connecting the data between the participants and the codes the completed consolidated criteria for reporting qualitative research checklist is available in the online supplementary material themes are presented in the results with supporting quotes and the participants identifier table resultsparticipantsthe radiotherapy radiography workforce census in the uk only reports the workforces professional grade and no other demographics35 in the uk trs level of open accessprofessional skills and knowledge are categorised by agenda for change professional band grades to therefore in this study the participants gender and professional grade were collected no other demographic information was collected table the response rate to taking part in the interview was seventy two trs were emailed and invited to take part in an interview returned consent forms and completed the telephone interview fifteen interviews were conducted with women and men the participants came from all regions of the uk including england wales scotland and northern irelandthemesfive main themes were identified trs provide behaviour change advice to manage radiotherapy related side effects trs make judgements about when it is appropriate to deliver health behaviour advice knowledge and training are key facilitators in the delivery of health behaviour advice trs feel patients undergoing radiotherapy treatment seek guidance on health behaviours and trs identify themselves as well placed to give health behaviour advice to patientstrs provide behaviour change advice to manage radiotherapyrelated side effectsmost respondents reported that they only provided advice on health behaviours that they believed would minimise radiotherapy related side effects this meant smoking cessation and alcohol intake were the two health behaviours trs mainly advised onwith head and neck patients we give advice particularly on smoking and drinking obviously get worse side effects tr the only thing we do generally say is about drinking plenty of fluids avoiding alcohol but thats more to do with prostate side effects bladder reactions and reducing gas tr radiographers are comfortable talking about alcohol when it comes to managing side effects tr no trs reported advising patients on healthy eating some trs mentioned advising patients on dietary intake but this is to patients who are at risk of losing weight for side effect management and potential impact on accuracy of radiotherapy treatment deliveryhealthy eating i dont tend to discuss too much a lot of patients have difficulty eating and we are encouraging maintaining weight while on treatment tr im not very sure if healthy eating is important any patients where were treating lower gi or pelvis we would advise them to avoid very high fibre foods spicy foods that might make them have very loose bowels but other than that we say more or less keep on your same diet we wouldnt generally discuss a healthy diet as a standard for all patients no tr pallin a0nd et a0al bmj open 202010e039909 101136bmjopen2020039909 0copen access some trs mentioned they advise patients to be physically active however this was only in the context of managing radiotherapy and cancer related fatigueso exercise is one of my main ones that i focus on with all patients particularly to help with their fatigue tr exercise i say thats its quite beneficial to help with fatigue tr i guess when we have patients come in fatigue is one of the side effects so we encourage our patients to remain active tr trs make judgements about when it is appropriate to deliver health behaviour advicetrs explained only discussing health behaviours particularly smoking and alcohol with patients if there were evident indications of a problem trs also often reported making a judgement of whether appropriate to advise a patient on a particular health behaviourso quite often you can tell if a patient is a smoker you can smell it or you can tell by their skin tr i tend to give advice when you make a judgement of when its appropriate an example might be if a patient smelt of smoke tr had patients come in and will smell of alcohol and at that time ill say to the patient that it can exacerbate side effects tr this meant trs did not provide advice on health behaviours to every patientbut for those patients where its not clearly going to benefit them to stop drinking you would just mention it very briefly not every patient will have that information tr knowledge and training are keys facilitator in the delivery of health behaviour advicedelivery of advice matched by knowledgethe reported delivery of advice on health behaviours appeared to be matched by knowledge of the benefits among those lwbcone participant explained how he only appreciated the importance of physical activity in cancer survivorship after attending a talk and being made aware of the evidencemy experience of appreciating the role exercise was from attending a talk i suppose it was really just highlighting in the studies the benefits obviously of a healthy lifestyle and introducing physical activity for patients on treatment tr healthy eating was a topic the participants felt particularly unqualified to deliver advice on and reported lack of knowledge as a barrier to the delivery of advice on healthy eatingits a difficult one diet i think its more a knowledge thing if you dont have the knowledge about what you can and cant say youre just not going to approach the subject tr a need for continuous postgraduate online trainingall interviewed said they would welcome postgraduate training on delivering health behaviour advice the majority expressed a preference for online training to help overcome the barrier of limited time among trs to attend trainingonline youre not having to take time out of clinical practice online is more accessible tr participants also mentioned that online training allows for yearly updates and continuous professional developmenti think itd be good online training because you can do it in your own time because i think thats sometimes the problem you have this training once and then maybe it never gets brought up again so it would be quite handy to have something small every year alongside all your other mandatory training tr participants did acknowledge that face to face training allows for further questioning thats not possible with online trainingi think one to one training because you can ask questions that may not be covered within the online training tr to overcome the barrier of not all staff being able attend face to face training participants suggested it would be useful to train some trs through face to face methods that they could then cascade to other trs within the radiotherapy departmentmaybe some face to face with some staff that they could cascade down might be useful as well tr a need for training in the undergraduate settingit was also suggested to incorporate training on delivering lifestyle advice into the undergraduate education programmecertainly get it into the undergraduate course to start with making them aware it is part of the role tr its still not something that i can say was primarily covered in the undergraduates training about the benefits of healthy lifestyle you know theres no real formal education that i can see tr trs reported knowledge of resources and referral pathways as facilitators in the delivery of adviceparticipants also felt knowledge of how to refer patients onto further support would enable them to have conversations on improving health behaviours with some pallin a0nd et a0al bmj open 202010e039909 101136bmjopen2020039909 0ctrs reporting that lack of knowledge of resources and referral pathways are barriers to initiating a conversation on behaviour changethere needs to be more information available to professionals of where exactly you can refer patients to whether that be website whether that be an app tr thats the only reason why they [therapeutic radiographers] dont want to open these conversations up because they dont know where to go with it or how to refer on tr they also acknowledge that in the short time they have with patients if they had a resource then would be more inclined to advisehaving something on a piece of paper education and having the resources if you can do it in min you should be able to slip that in tr you dont always have that information at hand so if it was readily available i think wed give out a bit more [health behaviour information] if it was just the case of pointing them in the right direction that would be a quick and easy thing to do tr the benefit of incorporating patients perspective into trainingparticipants also mentioned that getting patients perspectives on receiving advice on improving health behaviours should be incorporated into trainingi think that would be better coming from the patients themselves rather than just feedback from what journals and other literature says tr if thered even be patients that would be willing to maybe just even be involved with staff training tr trs feel patients undergoing radiotherapy treatment seek guidance on health behavioursmany of the trs also described that patients often ask them for guidance around health behaviour changes particularly on diet and exercise this shows that patients see trs as credible sources of information on health behaviourswe are getting asked the question more and more about weight loss healthy living wanting to exercise more tr it is quite a common thing to be asked at the end of treatment not so much the smoking and alcohol i have to say but diet and exercise is certainly something that people commonly ask tr trs identify themselves as well placed to give health behaviour advice to patientstrs acknowledged that they are a consistent healthcare member for patients undergoing radiotherapy and have many opportunities to deliver lifestyle advice therefore open accesstrs recognised that they are well placed to deliver health behaviour advice to patientswere in a unique position because we do see the same patient day after day and you do kind of start to develop a relationship with them tr i think were well placed to help influence patients behaviours and its something we should be seen to encourage and report tr were in the best position where we see the patients for a number of weeks every day to encourage any changes tr from the interviews it appeared that many patients undergoing radiotherapy excluding those at risk of malnutrition or significant weight loss are primarily reviewed and assessed by trs this highlights that trs are in an ideal position to deliver advice on health behaviours particularly when asked about nutrition advice deliverythey routinely see the specialist radiographer for the breast patients but they dont have a dietitian appointment tr prostate and breast are two tumour groups that are fully radiographer led review and about to of our work load they generally wouldnt be sent to a dietician tr only have a dietitian on board for the head and necks tr discussiontrs in this study saw themselves as well placed to deliver health behaviour advice but also reported that they do not routinely provide advice to all patients trs were particularly unlikely to provide advice on healthy eating and physical activity and were more likely to provide advice on those behaviours they believed would minimise radiotherapy or cancer related side effects this is in line with previous research among trs15 in one qualitative study a key facilitator reported among trs in delivering smoking cessation support to patients was knowledge of the link between smoking and toxicity28 another qualitative study that explored allied health professionals views regarding the provision of dietary advice to patients highlighted that trs report giving dietary advice to help counteract the side effects of radiotherapy37 additionally in our study if trs did provide dietary advice this tended to be general advice rather than cancer specific advice on healthy eatingin some studies oncology hcps have reported they do not self identify as the right person to provide lifestyle advice17 however in this study trs identified themselves as being well placed to deliver health behaviour advice and in a unique position as a consistent member of the multidisciplinary team providing care to patients however despite this they do not feel qualified to deliver pallin a0nd et a0al bmj open 202010e039909 101136bmjopen2020039909 0copen access advice particularly on the topic of healthy eating in the uk poor diet has the biggest impact on the national health service budget greater than alcohol consumption smoking and physical inactivity38 it has been noted that there are insufficient dietitians to provide dietary advice to all patients who may need dietary support39 in response to this all hcps are being asked to implement a preventative healthcare approach within their role and the delivery of healthy eating advice is fundamental to this23 key to achieving this is that trs will have the skills knowledge and behaviours to improve the health and well being of individuals24 as with other oncology hcp groups16 this study identifies the need for education and training among trs in delivering health behaviour advice particularly on healthy eating and physical activity this training should also address when and how to refer to other support if necessary as this was identified as a key facilitator in the delivery of advice on health behaviours particularly when time is a barrier to the delivery of this advice15all interviews demonstrated that trs would welcome training on delivering health behaviour advice and recommended it as a key facilitator in delivering advice in addition to incorporating it into the undergraduate setting the need for postgraduate training among trs in delivering health advice has also recently been reported by charlesworth et al28 in relation to the delivery of smoking cessation advice our findings from this study provide additional insight into trs preferences on the type of training on delivering lifestyle advice to those lwbc with trs demonstrating a preference of online training in the postgraduate setting among hcps online education has been reported to be as effective as face to face education42 additionally the use of online learning enables hcps to carry out training at time that fits in with clinical work43 trs in this study identified this benefit of online learning in overcoming the limited time available for trs to undertake continuous professional development and additional training interestingly trs in this study mentioned having patient input in the training would be helpful while hcps input is key to the development of interventions patient members play key advocacy roles and their input can enhance the outcomes of interventions45 patient input may also help overcome the reported barrier of fear of causing offence to a patient which has been reported as a barrier among oncology hcps in delivery of health behaviour advice17those lwbc wish to receive advice on health behaviours from their healthcare team13 and is of particular importance as the period following a cancer diagnosis has been shown be a teachable moment and an ideal opportunity to motivate patients around the importance of healthy eating and physical activity47 this was made apparent in this study whereby some trs mentioned that healthy eating and exercise were the health behaviours patients ask for advice on more often generally towards the end of their treatment this further highlights the importance of supporting trs in delivering evidence based health behaviour advice to meet patients needstrs have a responsibility to educate patients on the importance of following healthy behaviours given the increasing evidence showing implementing healthy behaviours improve a number of physical and psychosocial outcomes after a cancer diagnosis2 among pre menopausal and post menopausal women living with and beyond breast cancer a systematic literature review and meta analysis of follow up studies n213 breast cancer survivors identified that being overweight increases the risk of all cause and breast cancer mortality4 being physically active after a cancer diagnosis is also correlated with improved survival and reduced recurrence5 while data is limited emerging research suggests healthy dietary behaviours after a diagnosis may improve outcomes3 in a prospective observational study of patients with stage iii colon cancer a higher intake of a typical western diet was associated with a threefold increased risk of disease recurrence and a fold increased risk of all cause mortality8 additionally those lwbc are at increased risk for developing cardiovascular disease osteoporosis and diabetes and healthy behaviours can reduce the risk of developing these diseases51 of those interviewed in this study it appeared that those with breast prostate and colorectal cancer are primarily reviewed and assessed by trs therefore it is the responsibility of trs to deliver advice on improving health behaviours to these patients this is also particularly important because the strongest evidence for the benefits of diet and exercise is currently in breast prostate and colorectal cancer survivors53 these are also the most common cancers in the uk and radiotherapy plays a key role in managing these cancers22 therefore with the right skills and knowledge trs could deliver advice on improving health behaviours by supporting self efficacy among patients towards the end of their treatment which very often is in the radiotherapy department can be empowering for patients among those with prostate cancer implementing dietary changes brought psychological benefit as a method of coping and regaining control over their diagnosis46strengths and limitationsthis is the first qualitative study among trs to explore the provision of advice on all key modifiable lifestyle behaviours for those lwbc as per recommendations2 while the aim of qualitative research is not to generalise the findings the sample size was small and therefore the findings may not be representative of the views of the wider therapeutic radiography workforce however data saturation was reached likely due the homogeneous sample of participants additionally the participants worked in different radiotherapy departments and therefore provide insight into the practices among trs in the delivery of healthy behaviour advice from a wide range of hospitals also the participants worked in cancer centres in england wales scotland and northern pallin a0nd et a0al bmj open 202010e039909 101136bmjopen2020039909 0cireland providing insight into the practices across the uk another limitation of this study is the low response rate and that the participants might be more interested in the role of health behaviours in cancer survivorship which might bias the responses towards a positive view on this topic and the role of trs in delivering advice within their role despite this however provision of health behaviour change advice was low suggesting trs may be even less likely to educate patients around the importance of healthy behavioursfuture researchthis study highlights the need for training and education among trs on the delivery of health behaviour advice to cancer patients both in the undergraduate and postgraduate setting particularly on the topics of physical activity healthy eating and weight management higher education institutions have a responsibility in educating the allied health professional workforce on implementing health promotion within their role55 further research among pre registration tr students and lecturers within therapeutic radiography should therefore explore how best to address this need future research among trs should also use purposive sampling to identify the views and health promotion practices among those who may not have a primary interest in the area of health | Colon_Cancer |
nasopharyngeal carcinoma npc is an epithelial malignancy with high morbidity rates in the east and southeast asia the molecular mechanisms of npc remain largely unknown we explored the pathogenesis potential biomarkers and prognostic indicators of npcmethods we analyzed mrnas long noncoding rnas lncrnas and micrornas mirnas in the whole transcriptome sequencing dataset of our hospital five normal tissues vs five npc tissues and six microarray datasets normal tissues vs npc tissues downloaded from the gene expression omnibus gse12452 gse13597 gse95166 gse126683 and gse70970 gse43039 differential expression analyses gene ontology go enrichment kyoto encyclopedia of genes and genomes kegg analysis and gene set enrichment analysis gsea were conducted the lncrnamirnamrna competing endogenous rna cerna networks were constructed using the miranda and targetscan database and a proteinprotein interaction ppi network of differentially expressed genes degs was built using search tool for the retrieval of interacting genes string software hub genes were identified using molecular complex detection mcode networkanalyzer and cytohubbaresults we identified mrnas 14mirnas and lncrnas as shared degs related to npc in seven datasets changes in npc were enriched in the chromosomal region sister chromatid segregation and nuclear chromosome segregation gsea indicated that the mitogenactivated protein kinase mapk pathway phosphatidylinositol3 oh kinaseprotein kinase b pi3kakt pathway apoptotic pathway and tumor necrosis factor tnf were involved in the initiation and development of npc finally hub genes were screened out via the ppi networks several degs and their biological processes pathways and interrelations were found in our current study by bioinformatics analyses our findings may offer insights into the biological mechanisms underlying npc and identify potential therapeutic targets for npccorrespondence chenchuanben2010126com yuanji xu and xinyi huang contributed equally to this work department of radiation oncology fujian medical university cancer hospital fujian cancer hospital no fuma road fuzhou fujian peoples republic of 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 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 0cxu a0et a0al cancer cell int page of keywords nasopharyngeal carcinoma npc bioinformatics analysis gene expression omnibus geo differentially expressed genes degs gene ontology go competing endogenous rna cerna network nasopharyngeal carcinoma npc is an epithelial malignancy originating from the inner mucosal lining of the nasopharynx in there were an estimated new cases of npc worldwide accounting for of all cancer sites and deaths due to npc accounting for of all cancer sites the incidence of npc is geographically imbalanced with new cases mainly concentrated in east and southeast asia especially in south china [ ] the estimated agestandardized incidence rate of npc is per in china and only per in north america [ ] the oncogenesis and progression of npc are strongly associated with hereditary susceptibility environmental or random aspects and epsteinbarr virus ebv infection in the early stages of npc the main pathogenesis is related to ebv infection indeed the expression of ebvdna can be used for the monitoring and followup of npc patients and maybe a useful indicator for riskstratification strategies however despite the great advances in medical technology in recent years such as the application of intensitymodulated radiotherapy and optimized chemotherapy strategies the detection and treatment of npc remain challenging epidemiological investigations have shown that although the incidence and mortality rates of npc have been greatly reduced over the past decade even in endemic areas the survival rate of npc patients remains unsatisfactory due to local recurrence and distant metastasis especially in patients with advancedstage disease thus noninvasive cancerspecific biomarkers for early diagnosis and precision treatment are urgently requiredmicroarray and bioinformatics analyses have enabled researchers to screen the genetic alterations in npc and have proven to be convenient methods for identifying potential biomarkers in other diseases these analytic methods have uncovered several biomarkers with proven prognostic value and potential clinical applications in npc for example one study discovered a novel long noncoding rna lncrna named linc01385 involved in npc development and functional analysis demonstrated that linc01385 could serve as a therapeutic target in npc microarray and rnasequencing techniques have been used to identify differentially expressed genes degs and signaling pathways related to the oncogenesis and development of npc one study analyzed two microarray datasets to identify degs in normal tissue samples and npc tissue samples however the falsepositive rate for the two datasets was potentially high and the limited sample size may have led to unreliable results due to the substantial heterogeneity among the patients zhang et a0al investigated three microarray datasets to identify degs and hub genes that may serve as potential diagnostic biomarkers for npc both these studies analyzed only chip datasets and did not include sequencing data which would lead to offsets in the studies thus the precise molecular mechanisms and biological processes underlying npc remain largely unknown and must be urgently investigated to develop a precise curative treatment for npcin recent years competitive endogenous rna cerna has provided a new way to study the molecular mechanism of cancer he et a0al found that circgfra1 may serve as cerna to regulate gfra1 expression by sponging mi34a in triple negative breast cancer cerna is a transcript that can compete shared mirnas and regulate one another at the posttranscription level and the cerna networks link the function of mrnas to the function of micrornas mirnas lncrnas circular rnas and other rnas cerna can act as mirna sponges thereby affecting mirna expression cerna regulation network refers to the regulatory network with cerna participationtherefore in this study we aimed to explore the molecular pathogenesis potential biomarkers and prognostic indicators of npc by analyzing the full transcriptome sequencing data from fujian cancer hospital along with six microarray datasets acquired from the gene expression omnibus geo to identify degs between npc samples and normal tissue samples our findings may guide the precision treatment of npcmaterials and a0methodssample collection and a0preparationfresh nasopharyngeal tissues were collected from five npc patients who were treated in fujian cancer hospital between november and may normal nasopharyngeal tissues from five healthy donors were also collected all tissue samples were frozen using liquid nitrogen the five npc patients consisted of three men and two women with a median age of a0years the age and sex of five donors were matched to the npc patients two patients had stage iii npc while three patients had stage iva npc according to the 8th edition of the current international union against canceramerican joint committee on cancer guidelines for npc the 0cxu a0et a0al cancer cell int page of ethics committee of fujian cancer hospital approved the human tissue samples related to this work project ethics number sq201901801 the fresh tissue samples were removed from liquid nitrogen and subjected to total rna extraction using the trizol method the purity photometer® spectrophotometer implen ca usa of the extracted rna was determined using a nano the rna concentration was measured using the qubit® rna assay kit and a qubit® fluorometer life technologies ca usa and the rna integrity was evaluated using the rna nano assay kit of the bioanalyzer system agilent technologies ca usarna sequencinga total of a0μg rna from each tissue sample served as the somal rna was eliminated using epicentre ribozero¢ input material for the rna sample arrangements riborrna removal kit epicentre usa the rrnafree residue was removed using ethanol precipitation next depleted rna by using the nebnext® ultra¢ direcsequencing libraries were produced from the rrnational rna library prep kit for illumina® neb usa in brief fragmentations were implemented by bivalent cations below the high temperature in nebnext first strand synthesis reaction buffer5x the firststrand cdna thus obtained was compounded using a stochastic hexamer primer and mmulv reverse transcriptase rnase h then secondstrand cdna synthesis was carried out using dna polymerase i and rnase h in the reaction buffer dntps with dutp were substituted for dttp residual overhangs were turned into blunt ends through exonucleasepolymerase activities after the adenylation of the ² ends of the dna fragments the nebnext adapter carrying a hairpin loop structure was ligated to initiate hybridization to optimize the cdna fragments with a length of a0bp we purified the library fragments with the ampure xp system beckman coulter beverly usa next a0 μl user enzyme neb usa was applied with the sizeselected adaptorligated cdna at a0°c for a0min and then at a0°c for a0 min thereafter pcr was conducted using phusion highfidelity dna polymerase general pcr primers and index x primer finally the obtained products were refined ampure xp system and the library quality was evaluated on the agilent bioanalyzer system after the library was constructed qubit20 was used for preliminary quantification the library was diluted to a0ngμl then used the agilent system was used to determine the insert size of the library after the insert size was confirmed to be as expected qpcr was used to confirm the valid concentration a0 nm and accurate quantification of the library to ensure library quality when the library was deemed eligible varying libraries were pooled to meet the demands of valid concentration and enable offline data volume hiseq sequencing was conducted these wholetranscriptome sequencing data were termed the fjch datasetmicroarray datageo httpwwwncbinlmnihgovgeo is a public genomics dataset repository which collects highthroughput sequencing data chips and microarrays we downloaded the following six gene expression datasets from geo the mrna gene expression datasets gse12452 and gse13597 the mirna gene expression datasets gse43039 and gse70970 and the lncrna gene expression datasets gse95166 and gse126683 all six datasets were annotated using r software version via annotation documents all datasets were from the species homo sapiens and the dataset type was microarray expression profile details of every dataset study are provided in table a0identification of a0degsto identify degs between normal tissue samples and npc samples we analyzed the microarray data by using the limma package and a multivariate linear model of the adjusted tstatistic the cutoff criteria were as follows log fold change absolute value of log2 in table associated microarray datasets from a0the a0gene expression omnibus geo databasereferencepmidrecordtissue platformnormal cancerfjchdodd et al bose et al zheng et al unknownnpcnpc gse12452 gse13597npc gse126683 npcunknowm gse95166npcillumina hiseqtm2500miseqtm[hgu133_plus_2] affymetrix human genome u133 plus array[hgu133a] affymetrix human genome u133a arrayagilent045997 arraystar human lncrna microarray v3 probe name versionarraystar human lncrna microarray v20 agilent_033010 probe name versionlyu et al bruce et al gse43039 gse70970npcnpcccdtmmirna850version 4p14ncounter® human mirna assay v10 nanostring 0cxu a0et a0al cancer cell int page of the fold change of gene expression ¥ and adjusted p value ¤ enrichment analyses of a0npcgene ontology go is the main bioinformatics tool for gene annotation and analysis of the biological processes bps of genes and gene products which involves annotation of bps molecular functions mfs and cellular components ccs go analysis of degs was conducted using upsetr the kyoto encyclopedia of genes and genomes kegg is a bioinformatics database resource for determining the highlevel functions and uses of cells and anisms from their genomic information to investigate the functional and pathway enrichment in go and kegg we used upsetr to identify the modules involved in biological functions gene set enrichment analysis gsea is a knowledgebased method for the translation of genomewide expression profiles we analyzed pathways using gsea and identified each functional cluster using clusterprofiler the cutoff criteria were a false discovery rate and p value construction of a0the a0lncrnamirnamrna interaction networkthe lncrnamirna interactions were predicted using the mircode tool httpwwwmirco de which is described as a map of putative mirna target sites in the long noncoding transcriptome three convenient online databases namely mirdb httpwwwmirdb mirtarbas httpmirta rbase mbcnctuedutw and targetscan httpwwwtarge tscan were used to predict the target mrnas of the mirnas data with five or more binding sites were retained we selected the mrnas at the intersection of the three databases as the predictive targets of mirnas for the construction of lncrnamirnamrna cerna networks two separate cerna networks were constructed using upregulated and downregulated rnas and these were visualized using cytoscape https cytos cape version a popular online bioinformatics database proteinprotein interaction network constructionthe proteinprotein interaction ppi network was predicted using the search tool for the retrieval of interacting genes string httpstrin gdb version online database significant insights into the underlying mechanisms of npc can be provided by investigating the interactions between proteins all degs of mrnas were predicted using string and a comprehensive score over was regarded as statistically significant cytoscape version https cytos cape was used to visualize biological network and integrate the data the molecular complex detection mcode version algorithm of cytoscape was used for detecting densely connected regions in the ppi network which represented the most closely related gene sets among the degs networkanalyst https wwwnetwo rkana lystcafaces homexhtml a visual analysis platform for the networkbased metaanalysis of gene expression data was used to visualize the proportion of degs cytohubba a cytoscape plugin was used to filter out the top hub genes with the strongest connections to the other genes in the merged networkstatistical analysismost statistical analyses were conducted using the bioinformatics tools mentioned above and the version of r software is version differential expression levels of mrna mirna and lncrna were obtained using a twotailed students ttest for the identification of deg benjamini and hochberg false discovery rate method were performed to adjust pvalue functional and pathway enrichment analyses were analyzed by the hypergeometric test and bonferroni correction variables were expressed as mean ± standard deviation a p value was regarded as notably significantresultsdata collection and a0preprocessingto determine whether there was clustering or outliers in the sample set the differences between the clustering of the mrna fig a01ac lncrna fig a01df and mirna fig a01g h expression matrixes of the npc and normal tissue samples in different datasets were examined using threedimensional principal component analysis pca the results showed that npc was well distinguished from the normal tissue samplesidentification of a0degs in a0npcto identify the degs in npc the mrna lncrna and mirna expression profiles were analyzed using the limma package the results showed that mrnas lncrnas and mirnas were dissimilarly expressed logfc ¥ adjusted p ¤ fig a0 between the npc and normal tissues of these mrnas lncrnas and mirnas were significantly upregulated while mrnas lncrnas and mirnas were significantly downregulated in total degs were shared among the three mrna datasets fig a02d differentially expressed lncrnas delncrnas were shared among the three lncrna datasets fig a02h and differentially expressed mirnas demirnas were shared among the two mirna datasets fig a02k those degs may provide new insight into the biological mechanisms of npc and serve as 0cxu a0et a0al cancer cell int page of fig principal component analysis pca showing the clustering of mrna long noncoding rna lncrna and microrna mirna expression matrices in different samples and different datasets ac pca of mrna expression between the nasopharyngeal carcinoma npc cluster and normal tissue cluster in the fjch a gse12452 b and gse13597 c datasets the purple dots represent the npc samples and the blue dots represent the normal tissue control samples df pca of lncrna expression between the npc cluster and normal cluster in the fjch d gse95166 e and gse126683 f datasets the blue dots represent the npc samples and the red dots represent the normal tissue control samples g h pca of mirna expression between the npc cluster and normal cluster in the gse70970 g and gse43039 h datasets the green dots represent the npc samples and the blue dots represent the normal tissue control samplespotential therapeutic targets for npc functional roles of delncrnas shared among the three lncrna datasets are provided in additional file a0 table a0s1 and functional roles of demirnas shared among the two mirna datasets are provided in additional file a0 table a0s2construction of a0the a0cerna networkto explore the role of mirnas and corresponding target mrnas as well as corresponding lncrnas in npc we predicted the target mrnas of the demirnas and lncrnas that may have interrelations with mirnas the results may help to better explain the 0cxu a0et a0al cancer cell int page of fig volcano plots of the distributions of degs in different datasets ac volcano plots of the distributions of demrnas in the fjch a gse12452 b and gse13597 c datasets eg volcano plots of the distributions of delncrnas in the fjch e gse126683 f and gse95166 g datasets i j volcano plots of the distributions of demirnas in the gse43039 i and gse70970 j datasets differentially expressed genes degs were those with a fold change of and a pvalue of in the mrna expression matrix lncrna expression matrix and mirna expression matrix upregulated degs are mapped as red spots and downregulated degs are mapped as green spots genes without notable variation are labelled as black spots d venn diagram of the degs among the mrna expression datasets fjch gse12452 and gse13597 h venn diagram of the degs among the lncrna expression datasets fjch gse43039 and gse70970 k venn diagram of the degs among the mirna expression datasets gse43039 and gse70970critical regulatory functions of mirnas mrnas and lncrnas the interaction of upregulated and downregulated mirnas with delncrnas was predicted based on mircode the prediction of target mrnas of upregulated and downregulated mirnas was performed using three databases mirdb mirtarbas and targetscan lncrnas mirnas and mrnas were included in the upregulated and downregulated lncrnamirnamrna cerna networks respectively fig a0 3a b the blue red and green nodes represent mirnas lncrnas and mirnas respectively additional files tables s3 s4 shows the details of the interactions of the upregulated and downregulated mirnas and mrnas respectively additional file a0 tables s5 and s6 shows the details of the interactions of the upregulated and downregulated mirnas and lncrnas respectivelygo and a0kegg analyses of a0degsto further analyze the possible functions of the degs we conducted biological analyses by using clusterprofiler and upsetr the results suggested that the degs were significantly enriched in go and kegg terms the go analysis showed that the following bps were notably enriched among the degs chromosome segregation nuclear chromosome segregation sister chromatid segregation mitotic sister chromatid segregation negative regulation of chromosome anization fig a0 4a the following mfs were largely enriched in atpase activity protein serinethreonine kinase activity atpase activity coupled tubulin binding catalytic activity acting on dna dna dependent atpase activity dna helicase activity and singlestranded dna dependent atpase activity fig a04b finally the following ccs were found to be largely enriched in the chromosomal region condensed chromosome chromosome centromeric region 0cxu a0et a0al cancer cell int page of fig interaction networks of mrnamirnalncrna in nasopharyngeal carcinoma npc a a cerna network of upregulated genes b a cerna network of downregulated genes the blue red and green nodes represent predictive mirnas predictive long noncoding rnas lncrnas and predictive micrornas mirnas respectivelyfig upsetr plots showing the distributions of the gene ontology go annotations associated with the differentially expressed genes degs in nasopharyngeal carcinoma npc a biological processes b molecular functions c cellular components d upsetr plot showing the distribution of pathways associated with the degs in npc based on kyoto encyclopedia of genes and genomes kegg analysiscondensed chromosome centromeric region nuclear chromosome telomeric region and condensed chromosome kinetochore fig a04c the kegg pathway analysis suggested that degs in npc were largely enriched in the cell cycle dna replication and small cell lung cancer fig a0 4d the results suggested that chromosomal 0cxu a0et a0al cancer cell int page of dysfunction was closely related to the development of npcgsea of a0npcrelated genesto explore the biological functions of the degs involved in npc gsea was applied the mrna expression profile of the fjch dataset was subjected to gsea by means of clusterprofiler the analysis showed that the following biological pathways were overrepresented in the npc tissues as compared to the normal tissues the mitogenactivated protein kinase mapk signaling pathway the phosphatidylinositol3 oh kinaseprotein kinase b pi3kakt signaling pathway fig a0 5a the apoptotic pathway and the tumor necrosis factor tnf signaling pathway fig a0 5b the pathways found our study were involved with cancer progression metastasis and apoptosisppi network analysis of a0degsthe string database was used version to explore the ppi network based on the correlations among the degs in npc the obtained data were then examined using cytoscape software the ppi network of degs was constructed using mcode to obtain the vital gene module the networkanalyzer plugin was applied to further analyze the ppi network according to the scores the cytohubba plugin was used to analyze the hub genes associated with npc and the following genes with the top grades were deemed to be hub genes nusap1 racgap1 prc1 kif4a top2a pbk kif2c tpx2 cenpu oip5 ttk mad2l1 ndc80 birc5 melk cenpf foxm1 tyms cdk1 and cep55 fig a0 those genes may contribute to the investigation of biological mechanisms and uncover underlying therapeutic targets for npcfig gene set enrichment analysis gsea of the gene expression profiles of the fjch dataset a gsea shows that the mitogenactivated protein kinase mapk pathway and the phosphatidylinositol 3kinaseprotein kinase b pi3kakt pathway are concentrated in nasopharyngeal carcinoma npc b gsea reveals that the apoptosis pathway and the tumor necrosis factor tnf pathway are concentrated in npc 0cxu a0et a0al cancer cell int page of fig proteinprotein interaction ppi networks a a ppi network of differentially expressed genes degs constructed using string software b most relevant gene sets in the ppi network extracted using mcode c further analysis of degs using networkanalyzer d the top hub genes with the most correlations identified using cytohubbago and a0kegg analyses of a0hub genesto analyze the functions of the top hub genes we again conducted biological analyses by using clusterprofiler and upsetr the results suggested that the hub genes were significantly enriched in go and kegg terms go analysis showed that changes in the following bps of hub genes were notably enriched in chromosome segregation nuclear chromosome segregation sister chromatid segregation mitotic sister chromatid segregation microtubule cytoskeleton anization involved in mitosis and regulation of chromosome segregation fig a07a in addition the changes in the following mfs were mainly enriched in protein serinethreonine kinase activity tubulin binding microtubule binding and protein cterminus binding fig a0 7b finally changes in the following ccs of degs were enriched in the chromosomal region spindle condensed chromosome chromosome centromeric region kinetochore microtubule midbody condensed chromosome centromeric region condensed chromosome kinetochore and mitotic spindle fig a0 7c kegg pathway analysis indicated that the degs in npc were mainly enriched in the cell cycle cellular senescence oocyte meiosis progesteronemediated oocyte maturation and platinum drug resistance fig a07d enrichment analyses of the hub genes were similar to the results of the analyses of the degs hence the findings obviously 0cxu a0et a0al cancer cell int page of fig upsetr plots showing the distributions of the gene ontology go annotations associated with the hub genes of nasopharyngeal carcinoma npc in the case of a biological processes b molecular functions and c cellular components and d upsetr plot showing the distribution of pathways associated with the hub genes of npc based on kyoto encyclopedia of genes and genomes kegg analysissuggested that chromosomal dysfunction was a vital contributor to the tumorigenesis of npcdiscussionin this work we performed a comprehensive analysis of the full transcriptome sequencing dataset of fujian cancer hospital and six microarray datasets downloaded from the geo repository to uncover degs between npc tissues and normal nasopharyngeal tissues we identified differentially expressed mrnas demrnas demirnas and delncrnas among the seven datasets and constructed a lncrnamirnamrna network of npc go enrichment analysis kegg enrichment analysis and gsea proved that the enriched components and pathways among the degs associated with npc were inseparable from the chromosome dysfunction mapk signaling pathway and pi3kakt signaling pathway discovered in npc we also identified the top hub genes in the ppi network related to npc and the results of the enrichment analysis of the hub genes were similar to those of the degsstudies have shown that lncrnamirnamrna networks play significant roles in the development and progression of tumors by constructing visual networks we can see the interaction between degs of different molecular types the lncrnamirnamrna network constructed in our study indicated that in npc mrnas could be regulated by lncrnas via corresponding mirnas li et a0al identified mirnas including highly expressed mirnas and lowly expressed mirnas from the serum of npc patients with different radiosensitivity these mirnas were found to have remarkable differences between the patients fold change ¥ or ¤ and p the highly expressed mirna hsamir6088 and the lowly expressed mirna hsalet7f13p from the above study were also found in our cerna networks we also identified hsamir29a3p and hsamir103a3p as demirnas which were recently found to act as circulating biomarkers of npc with fairly good diagnostic accuracy for detecting npc as compared with controls area under the curve the radioresistant npc cne2ir cell 0cxu a0et a0al cancer cell int page of line has been shown to overexpress jun guo et a0al identified junrelated mirnas by using mirdip software including hsamir200b3p hsamir1395p hsamir200c3p hsamir95p and hsamir92b3p thus jun could promote tumorigenesis and tumor development qing et a0 al found that inhibiting cjun expression could enhance radiosensitivity and induce cell cycle arrest and apoptosis the above results show that cerna networks can offer insights into the complex regulation patterns of npc and potentially facilitate the individualized treatment of npcgo analyses of the bps of the degs associated with npc showed that the negative regulation of chromosome segregation nuclear chromosome segregation sister chromatid segregation mitotic sister chromatid segregation and negative regulation of chromosome anization were closely associated with the oncogenesis of npc among the cc annotations chromosomal region condensed chromosome chromosome centromeric region condensed chromosome centromeric region condensed chromosome kinetochore and nuclear chromosome telomeric region were notably related to npc several studies have reported on the chromosomal aberrations involved in the carcinogenesis of npc including chromosomal gains or losses [ ] loss of heterozygosity chromosomal rearrangements and chromosomal imbalances in one study loss of heterozygosity on 3p was observed in of primary npc specimens and almost of precancerous lesions tan et a0al hypothesized that apoptosis induced by oxidative stress may lead to cadmediated chromosomal breakage after incorrect dna repair cells that survive apoptosis may carry chromosomal rearrangements leading to the tumorigenesis of npc to investigate common genetic variations in npc natasya et a0al screened out cases of npc in the malaysian population by the comparative genomic hybridization cgh technology and the results showed chromosomal changes in all npc cases enrichment analyses of the hub genes identified in our study were greatly compliant with the results of the enrichment analyses of the degs thus the above findings clearly implicate chromosomal dysfunction as an important contributor to the carcinogenesis of npcgsea showed that the mapk signaling pathway pi3kakt signaling pathway apoptotic pathway and tnf signaling pathway were the top four pathways associated with npc the enriched pathways identified in our investigation are related to tumor progression metastasis and | Colon_Cancer |
the heterogeneity of cancer cells is generally accepted and astem celllike subpopulation that is called cancer stem cellscscs has been identiï¬ed in various types of malignanttumors although the lack of consensus on the deï¬nitioncscs are widely recognized as a small subpopulation amongcancer cells with the properties of selfrenewal and tumor initiation as cscs play a critical role in the recurrence andmetastasis of cancer targeting the cscs is thought to bea promising approach for curing cancera large number of past studies have tried to identify andcharacterize the cscs as normal tissuespeciï¬c stem cellsare considered as the main origin of cancer the cscsare also thought to be inherited at least partially the characterization of normal tissuespeciï¬c stem cells thereforemany studies on the identiï¬cationpuriï¬cation of cscs havesimply shared markers of hematopoietic stem cells includingthe most popularly used cell surface markers of cd44 andcd133 [ ] cd44 is a type i transmembrane glycoproteinthat is expressed on hematopoietic ï¬broblastic and glial cellsand functionally known to mediate cellcell and cellmatrixinteractions previous studies have demonstrated that thecd44 is not only a biomarker but also plays critical roles inthe maintenance of cscs the resistance to various therapiesstresses and the metastasis of cancer cells []cd133 is originally identiï¬ed as protein expressing on thecell surface of hematopoietic stem cells and has subsequently been found to be critical in the maintenance ofstemness of stem cells in various tissues [] cd133has also been found in some csc [] which contributesto therapeutic resistance through the activation of akt bcl2and mapkpi3k signaling pathways [] although theexpressions of cd44 and cd133 in cancer cells likely associate with the resistances to radiotherapy chemotherapy andvarious stresses the diï¬erent signiï¬cance between cd44and cd133 has not yet been well understoodin this study we investigated whether the expression ofcd44 and cd133 in human colorectal cancer cells hct8diï¬erently contributed to drug resistance our data indicated 0cstem cells internationalthat the expression of cd133 rather than cd44 closely associated with doxorubicin dxr resistance at least partiallythrough drug excretion and redox regulation materials and methods cell culture human colorectal cancer hct8 cells werecultured in rpmi medium fujifilm wako purechemical japan supplemented with fbs gibco°thermo fisher scientiï¬c ma usa at c in a humidiï¬edatmosphere of air and co2 separation of cd44 and cd133positive cells fromhct8 cells we separated the parent hct8 cells intocd44positive cd44 and cd133positive cd133 cellsby a twostep magnetic cell sorting method as described previously [ ] brieï¬y hct8 cells were collected as a singlecell suspension by trypsinization and then incubated withmagnetic microbeadconjugated antihuman cd44 antibodymiltenyi biotec germany for min after washing cellswere separated into cd44 and cd44 subpopulations byusing the automacs¢ pro separator miltenyi biotecaccording to the manufacturers instruction the puriï¬edcd44 cells were further expanded and then harvested as asinglecellsuspension to be incubated with magneticmicrobeadconjugated antihuman cd133 antibody miltenyibiotec for min after washing the cd44cd133 andcd44cd133 subpopulations were separated as describedabove this twostep isolation enabled us to obtain a suï¬cientnumber of cd44 cd44 cd44cd133 and cd44cd133 cells for our experimentsto verify the purity of each subpopulation isolated cellswere stained with pelabelled mouse antihuman cd133clone ac133 miltenyi biotec and fitclabelled mouseantihuman cd44 clone db105 miltenyi biotec according to the supplied protocols flow cytometry analysis wasperformed using a facscalibur becton dickinson asdescribed previously mouse igg1pe miltenyi biotecand mouse igg1fitc miltenyi biotec were used as a negative control cytotoxicity assays cells were seeded in 96well cultureplates at a density of cells per well and cultured overnight the cells were then treated with various concentrationsof dxr fujifilm wako pure chemical in the absence orpresence of verapamil fujifilm wako pure chemicalcytotoxicity assays were performed using the cell proliferation kit i mtt roche applied science germany asdescribed previously the absorbance was measured at nm using a microplate reader multiskan fc thermofisher scientiï¬c analysis on the expression of abc transporters theexpressions of the atpbinding cassette subfamilies of bmember abcb1 or g member abcg2 were analyzedby ï¬ow cytometry brieï¬y cells were incubated with mouseprimary antibodies against human abcb1 and abcg2bd biosciences ca usa and then labeled by fitcconjugated antimouse igg bd biosciences according tothe manufacturers instruction respective isotype controlswere used as a negative control after washing ï¬ow cytometry analysis was performed using a facscalibur analysis of cellular accumulation of dxr the intracellular accumulation of dxr was analyzed by ï¬ow cytometrybrieï¬y cells were treated by μm dxr for hr in theabsence or presence of μm verapamil or μm buthionine sulfoximine bso sigmaaldrich mo usa cellswere then collected as a singlecell suspension and washedtwice with icecold phosphatebuï¬ered saline the accumulation of dxr within cells was evaluated by the intracellularï¬uorescence intensity using a facscalibur the nucleusaccumulation of dxr was analyzed by using cell pelletstreated with triton x100pbs as assay material asdescribed previously expressionlevelsanalysisimmunoblot detection of intracellular ros the intracellular roslevel based on the oxidation of ²²dichlorodihydroï¬uorescein diacetate h2dcfda molecular probes thermofisher scientiï¬c was measured to form the ï¬uorescent compound ²²dichloroï¬uorescein dcf using a facscaliburofphosphorylatedp38 map kinase phosphop38mapk totalp38mapk and nuclear factor erythroid 2related factor nrf2 in the cells were estimated by immunoblotting brieï¬ycell lysate μg of total protein was separated by sodiumdodecyl sulfatepolyacrylamide gel electrophoresis sdspage gel transferred to pvdf membranes biorad causa and then incubated with primary antibodies cell signaling technology ma usafollowed by appropriatehrplabeled secondary antibodies dako agilent pathologysolutions ca usa blots were developed by enhancedchemiluminescence using an ecl kit ge healthcare lifesciences pa usa semiquantitation was done by measuringthe density of bands using the image quant las minibiomolecular imager ge healthcare life sciences asdescribed previously sirna treatment smallinterfering rna sirnaspeciï¬c targeting to cd133 on targetplus sirnaand a scramble sirna on targetplus sirna negativecontrol were obtained from dharmacon horizon discovery cambridge uk cells were seeded in 6well plates cellswell and incubated for hr transfectionswere performed using dharmafect sirna transfectionreagents dharmacon according to the manufacturersinstructions analyses were done at hr after sirnatransfection statistical analysis all of the results are presented as themeans ± sd statistical signiï¬cance was determined by oneway analysis of variance anova followed by tukeys testdr spss ii chicago il diï¬erences were considered significant when p results hct8 cells were separated into various subpopulationsbased on their expressions of cd44 and cd133 first we 0cstem cells internationalparent cellshlffl1hdccd44hlfhlfcd44fl1hcd44fl1hahlfhlfcd44cd133fl1hcd44cd133fl1hparent cd44cd44 cd44133 cd44133enilesab fo noitarefilorp llecparent cd44 cd44 cd44cd44bcfigure continued 0cstem cells internationalenilesabstnuo0ccd44cd133 ± stnuo0ccd44cd133 ± fl2hfl2hsyad stnuo0c ± fl2hstnuo0cd ± fl2hfigure the separation of hct8 colorectal cancer cells into diï¬erent subpopulations based on the expression of cd44 and cd133 arepresentative dot plots of ï¬ow cytometry analysis show the purities of each subpopulation of isolated cells quantitative data in the dotplots are presented as the percentages of positive cells from three independent experiments b representative photos of morphologicalproperties upper and mtt assay on cell growth lower at hr after the initiation of culture data are presented as the mean ± sd fromthree independent experiments c d representative histograms of ï¬ow cytometry analysis showed the expressions of cd44 c andcd133 d at baseline and days after cell culture the dotted vertical lines through histograms indicate the diï¬erence in the expressionpeaks between the baseline and at days after culture quantitative data in the histograms are presented as the mean ï¬uorescentintensity from three independent experimentsseparated the hct8 cells into cd44 and cd44 subpopulations and compared their sensitivity to anticancer drugs ofdxr and cisplatin cisdiaminedichloroplatine cddphowever no diï¬erence in the sensitivity to the two drugswas observed between cd44 and cd44 cells data notshown we further tried to purify a small population ofcd133 cells from these cd44 cells cd44 cells almostnegatively expressed with cd133 figure 1a as a resultwe separated hct cells into diï¬erent subpopulationsincluding cd44 cd44 cd44cd133 and cd44cd133 cells the purities of isolated cells in each subpopulation were conï¬rmed to be around by ï¬ow cytometryfigure 1aandphenotype changein diï¬erent growthsubpopulations of cells the morphology and proliferationof these cells could not be found obviously diï¬erent amongsubpopulations figure 1b the expression of cd44 in allsubpopulations kept stable within days of reculturingfrom the frozen cells that stocked immediately after isolationinterestingly the expression of cd44 was a tendency todecrease with culture time in cd44 ï¬uorescence intensity ± at baseline vs ± at days p figure 1c and cd44cd133 cells ï¬uorescence intensity ± at baseline vs ± at days p figure 1c but still kept stable in cd44cd133 cells at days after reculturing ï¬uorescence intensity ± at baseline vs ± at days p figure 1cthe expression of cd133 in cd44cd133 cells kept verystable ï¬uorescence intensity ± at baseline vs ± at days p figure 1d and cd44cd133cells did not turn to express cd133 within days of reculturing ï¬uorescence intensity ± at baseline vs ± at days p figure 1d therefore we usedthe cells within days after reculturing from the frozenstocked cells in subsequent experiments dxr resistance of cd44cd133 cells next we evaluated the sensitivity of cells to dxr by mtt assay withthe addition of μm of dxr in medium we foundthat the survival of cd44cd133 cells was significantlyhigher than all other subpopulations of cells after hr of 0cstem cells international ytilibaiv llecðmparent cellscd44 cellscd44 cellscd44133 cellscd44133 cellsdxrbso003hct8doxb24 hfl3h006hct8 cd44doxb24 hfl3h009hct8 cd44doxb24 hsllec tnerapstnuocsllec dcstnuocsllec dcstnuocdxr001hct8dox24 hfl3hstnuocadxrverapamil002hct8doxv24 hstnuocfl3h004hct8 cd44dox24 h005hct8 cd44doxv24 hstnuocstnuocfl3hfl3h007hct8 cd44dox24 h006hct8 cd44doxv24 hstnuocstnuocfl3hfl3hsllec dcsllec dc006hct8 cd44cd133dox24 h006hct8 cd44cd133doxv24 hstnuocstnuocstnuocfl3hfl3h00hct8 cd44cd133dox24 h00hct8 cd44cd133doxv24 hstnuocfl3hstnuocstnuocfl3hbfigure continuedfl3h012hct8 cd133doxb24 hfl3h015hct8 cd44cd133doxb24 hfl3h 0cstnuocstem cells internationalabcg2abcb1stnuocfl1hparent cellscd44cellscd44cellscd44133 cellscd44133 cells ± ± ± ± ± parent cellscd44cellscd44cellscd44133 cellscd44133 cellscfl1h ± ± ± ± ± figure dxr resistance of diï¬erent subpopulations of cells a mtt assay was done to evaluate the cytotoxicity of dxr data are expressedas the percentile of baseline before dxr treatment from three independent experiments p vs all other subpopulations brepresentative histograms of ï¬ow cytometry analysis show the accumulation of dxr in cells hr after the treatment with μm dxrin the absence or presence of μm verapamil and μm bso the dotted vertical lines through histograms indicated the mean levels ofdxr accumulation in cd44cd133 cells for comparing with other subpopulations of cells the results were reproducible in threeindependent experiments c representative histograms of ï¬ow cytometry analysis show the expression of the abcb1 or abcg2 indiï¬erent subpopulations of cells quantitative data in the histograms are presented as the mean ï¬uorescent intensity from threeindependent experimentsculture p vs other groups at diï¬erent dxr concentrations figure2ato understand the relevant mechanism we measured theintracellular accumulation of dxr in cells by ï¬ow cytometrythe accumulation of dxr in cd44cd133 cells wasdetected as the lowest among these subpopulations at hrafter the exposure to μm dxr figure 2b we furtherfound that the intracellular accumulation of dxr in cd44cd133 cells was obviously increased by the treatment withverapamil an inhibitor for drug eï¬ux cell membrane transporters of abcb1 and abcg2 figure 2b however theintracellular accumulation of dxr in cd44cd133 cellsdid not change by the treatment with bso a glutathione synthesis inhibitor that indirectly regulates drug eï¬ux throughabcc1 figure 2b we also conï¬rmed that the expressionof abcb1 p vs other groups but not abcg2 wasenhanced in cd44cd133 cells figure 2c suggestingthe probable role of abcb1 on dxr resistance in cd44cd133 cellsto further conï¬rm the causal relationship between theenhanced drug eï¬ux and dxr resistance we evaluatedthe cytotoxicity of dxr in the presence or absence of verapamil unexpectedly verapamil only partially enhanced thecytotoxicity of dxr in either cd44cd133 or cd44cd133 cells figure 3ait is well known that dxr interacts with nuclear dna toinhibit macromolecular biosynthesis therefore we alsoestimated the eï¬ect of verapamil on the nuclear accumulation of dxr the nuclear accumulation of dxr wasobserved obviously less in cd44cd133 than cd44cd133 cells but tended to have comparable levels withverapamil treatment figure 3b cd44cd133 cells showed better stress tolerance thancd44cd133 cells it is well known that the stress responsekinase p38mapk can be activated by various extracellularstresses and plays critical roles in cell survival and apoptosis although the basal level of phosphorylated p38mapkwas detected very similar between cd44cd133 andcd44cd133 cells p figure lower expressionwas observed in cd44cd133 than cd44cd133 cellsafter dxr exposure even under verapamiltreatmentp figure this suggests a better tolerance tostress of cd44cd133 cells independent on the accumulation of dxr 0cstem cells international limaparev ytilibaiv llec limaparev ytilibaiv llec hours ðmðmcd44cd133 cellscd44cd133 cellsa hours limaparevstnuoccd44133 cd44133ðmfl3hðm limaparevstnuocfl3hbfigure dxr resistance and nuclear dxr accumulation in cd44cd133 and cd44cd133 cells in the absence or presence of drug eï¬uxinhibitor a mtt assay was done to compare the cytotoxicity of dxr in cd44cd133 and cd44cd133 cells with or without theaddition of μm verapamil data were expressed as a percent of baseline before dxr treatment from three independent experiments p vs cd44cd133 cells b representative histograms of ï¬ow cytometry analysis showed the nuclear accumulation of dxr incells hr after the treatment by μm dxr with or without the addition of μm verapamil the results were reproducible in threeindependent experimentsverapamil verapamil phosphop38 mapk totalp38 mapk noisserpxe evitalercd133 p p p control hours dxr treatmentp p p control hours dxr treatmentfigure diï¬erent expression of phosphorylated p38mapk between cd44cd133 and cd44cd133 cells representative blots andsemiquantitative data on the expression of phosphorylated p38mapk and total p38mapk in cells treated with μm dxr in theabsence or presence of μm verapamil the quantitative data are normalized to total p38mapk data are expressed as relative values tocd44cd133 cells without dxr treatment and presented as the mean ± sd from three independent experiments 0cstem cells internationaldxr dxr limaparevstnuoc limaparevstnuoccd44133cd44133stnuocfl1hfl1hstnuocfl1hafl1hverapamil verapamil nrf2ð½tubulin noisserpxe evitalercd133 p p p controldxr treatment hours bp p control dxr treatment hours figure diï¬erent antioxidant capacity between cd44cd133 and cd44cd133 cells a representative histograms of ï¬ow cytometryanalysis show the intracellular ros levels hr after the treatment by μm dxr in the absence or presence of μm verapamil theresults were reproducible in three independent experiments b representative blots and semiquantitative data on the expression of nrf2in cells treated with μm dxr in the absence or presence of μm verapamil the quantitative data are normalized to βtubulin dataare expressed as relative values to cd44cd133 cells without dxr treatment and presented as the mean ± sd from three independentexperiments cd44cd133 cells showed higher antioxidantcapacity than cd44cd133 cells it is also well known thatdxr generates ros and oxidative stress due to ros generation may induce the activation of p38mapk therefore weestimated the ros levels in cells with or without dxr exposure we observed a lower level of ros in cd44cd133 0cstem cells internationalstnuocstnuocstnuocstnuocstnuoc0nm ± stnuocfl2h5nm ± stnuocfl2h10nm ± stnuocfl2h15nm ± stnuocfl2h25nm ± stnuocfl2h0nm ± fl2h25nm ± ytilibaiv llecfl2h50nm ± fl2h75nm ± fl2h100nm ± fl2hðm cd44133 cd44133 cd44133 control sirna cd44133 cd133 sirna abfigure continued 0cstnuo0cstnuo0cexpression of abcb10nm ± 0nmstnuo0c ± fl1h5nm ± fl1h50nmstnuo0c ± fl1hfl1h25nmstnuo0c ± stnuo0cfl1h100nm ± fl1hstem cells internationalaccumulation of dxrcontrol sirna 5nm ± fl3hcd133 sirna 5nm ± stnuo0cstnuo0cfl3hcdfigure the eï¬ect of silencing cd133 expression on dxr resistance of cd44cd133 cells a representative histograms of ï¬owcytometry analysis on the expression of cd133 in cd44cd133 cells after silencing by diï¬erent dosages of targeted sirna quantitativedata in the histograms are presented as the mean ï¬uorescent intensity from three independent experiments b mtt assay was done toevaluate the cytotoxicity to dxr cells were treated with nm sirna for hr followed by dxr treatment for another hr data areexpressed as a percent of baseline before dxr treatment from three independent experiments p vs cd44cd133 cells crepresentative histograms of ï¬ow cytometry analysis on the expression of abcb1 in cells after silencing by diï¬erent dosages of targetedsirna quantitative data in the histograms are presented as the mean ï¬uorescent intensity from three independent experiments drepresentative histograms of ï¬ow cytometry analysis on the accumulation of dxr quantitative data in the histograms are presented asthe mean ï¬uorescent intensity from three independent experimentsthan cd44cd133 cells especially under dxr exposurebut verapamil did not obviously change the ros levelsfigure 5a based on these ï¬ndings we speculated thatthe enhanced antioxidant capacity in cd44cd133 cellsmight help to maintain a lower level of phosphorylatedp38mapknrf2 a transcription factor that is well known to beactivated by oxidative stress such as ros and electrophilicsubstances can protect cells against various stresses we alsocompared the expression level of nrf2 between cd44cd133 and cd44cd133 cells western blotting showeda higher expression of nrf2 in cd44cd133 than cd44cd133 cells especially under dxr exposure p figure 5b and the enhanced expression of nrf2 in cd44cd133 cells was not cancelled by verapamil treatmentp figure 5b sirna treatment to further conï¬rm the regulatory roleof cd133 in drug resistance we tried to silence cd133expression in cd44cd133 cells by sirna and then estimated cytotoxicity of dxr although the decrease ofcd133 expression was clearly observed by targeted sirnap vs nm figure 6a dxr resistance of cd44cd133 cells only partially improved figure 6b unexpectedlythe silencing of cd133 did not change theexpression of abcb1 in cd44cd133 cells even using 0cstem cells internationalexcessive concentrations of cd133 sirna p vs nmfigure 6c we also conï¬rmed that the silencing of cd133did not aï¬ect the accumulation of dxr in cd44cd133cells p vs control sirna figure 6dthis suggests that beyond the drug excretion and redoxregulation other complex mechanisms are also likelyinvolved in the dxr resistance in cd44cd133 cells discussionby using the wellrecognized cell surface markers of cd44and cd133 for csc identiï¬cation we tried to separate thehct8 human colon cancer cells into cd44 cd44 cd44cd133 and cd44cd133 subpopulations and then investigated how the expressions of cd44 and cd133 associatedwith drug resistance actually we checked several cancer celllines on the expression of cd44 and cd133 including helacells and a549 cells however both hela cells and a549 cellsshowed almost expression of cd44 only the hct8cells showed a partial expression of cd44 about anda rare expression of cd133 therefore we only isolateddiï¬erent subpopulations from hct8 cells for this studyfirst we found that the expression level of cd44 keptvery stable in the cd44cd133 cells but gradually declinedin cd44cd133 cells during a cell passaging process on theother hand some of cd44 cells shifted to express cd44 during a cell passaging process figure 1c these ï¬ndings suggested the plasticity of cd44 expression in hct8 cellsactually ohata et al reported that cd44 highexpressedcells from human intractable colon cancer patients can diï¬erentiate into cd44 lowexpressed cells and a fraction of cd44lowexpressed cells can also generate cd44 highexpressedcells in a xenograft mouse model however it is unclearwhy the cd44cd133 cells but not cd44cd133 cellsstably maintain the expression level of cd44 unlike theextensive expression of cd44 with high plasticity the expression of cd133 was only observed in very few of the hct8cells with poor plasticitya number of previous studies have demonstrated thatcscs are likely resistant to chemotherapeutic drugs thecd44cd133 cells but not the cd44 and cd44cd133cells showed dxr resistance figure 2a according to thisdata the expression of cd133 but not cd44 seems to beclosely associated with drug resistance actuallythesecd44cd133 cells showed the enhanced expression ofabcb1 and the decreased intracellular accumulation ofdxr figures 2b and 2c liu et al reported that nonsmallcell lung cancer cells treated with lowdose cddp aresuï¬cient to enrich cd133 cells and upregulate abcb1expression through notch signaling which thereforeincreases the crossresistance to dxr however theinhibition of abcb1 by verapamil only partially improvedthe dxr resistance of cd44cd133 cells in this studyto ï¬nd other potential mechanisms involving in thedxr resistance of cd44cd133 cells we investigatedseveral interesting aspects including the stress protectionand redox regulation we found that p38mapk one of themost popular protein kinases known to be activated byinï¬ammatory cytokines lipopolysaccharide osmotic shockultraviolet light and other stresses was more obviouslyinduced by dxr in cd44cd133 cells than cd44cd133cells figure moreover the activation of p38 mapk wasnot dependent on the intracellular accumulation of dxrfigure dxr is known to insert between the base pairs of dna oftumor cells and exhibits antitumor eï¬ects by suppressing thebiosynthesis of both dna and rna through the inhibitionof dna polymerase rna polymerase and topoisomeraseii reactions furthermore it is believed that dxr has theability to generate suï¬cient ros to raise oxidative stressindeed we observed dxrinduced ros generation in bothcd44cd133 and cd44cd133 cells butthe dxrinduced ros generation was detected even higher in cd44cd133 than cd44cd133 cells independent on the intracellular accumulation of dxr figures 5a 2b and 3bsuggesting the enhanced antioxidant capacity in cd44cd133 cellsthe keap1nrf2 control system plays a central role in theantioxidant defense mechanisms nrf2 is known as a transcription factor to activate various genes involving in biological defense mechanisms it has been reported that nrf2 isconstantly expressed in many cancer cells [] moreover the enhanced expression of nrf2 has been conï¬rmedto associate with poor prognosis of cancer patients []in our study nrf2 expression was detected higher in cd44cd133 than cd44cd133 cells and the diï¬erence in nrf2expression was observed even clearer between cells with dxradministrationindependent on the dxr accumulationfigure 5b these ï¬ndings also clearly indicate theenhanced antioxidant capacity in cd44cd133 cellsalthough the absence of direct evidence by interferenceexperiment pathways involving in the stress protection andredox regulation might at least partially contributed to thedxr resistance of cd44cd133 cellsvery strangely our data showed that the silencing ofcd133 expression in cd44cd133 cells by sirna couldonly partially increase the cytotoxicity of dxr figure 6bbut did not change the expression of abcb1 and the intracellular accumulation of dxr figure 6c other unknownmechanisms beyond the drug excretion and redox regulationare asked to be deï¬ned on the dxr resistance of cd44cd133 cellsbased on data from the present study the expression ofcd133 rather than cd44 more closely associated with theresistance of cancer cells to anticancer drug as complexmechanisms including the drug excretion and redox regulation are likely involved in the drug resistance of cscs multiple approaches may be needed to overcome the big problemof drug resistance in cancer patientsabbreviationsabcb1abcg2atpbinding cassette subfamily b member 1pglycoproteinmultidrug resistance protein1mdr1atpbinding cassette subfamily g member2breast cancer resistanceproteinbcrpcd388 0cabcc1bsocscsdxrmttatpbinding cassette subfamily c member1multidrug resistanceassociated protein1mrp1buthionine sulfoximinecancer stem cellsdoxorubicinadriamycin345dimethylthiazol2yl25diphenyltetrazolium bromidenuclear factor erythroid 2related factor nrf2p38mapk p38 map kinaserossirnareactive oxygen speciessmall interfering rnadata availabilitythe data that support the ï¬ndings of this study are availablefrom the corresponding author upon reasonable requestdisclosurethe funder played no role in the study design data collectionand analysis decision to publish or preparation of themanuscriptconflicts of interestthe authors indicate no potential conï¬icts of interestacknowledgmentsthis work was supported by a grantinaid for the ministryof education science sports culture and technology ofjapan grant numbers and 16k15622 and thecollaborative research program of the atomic bomb diseaseinstitute of nagasaki universityreferences r c elble the role of cancer stem cells in relapse of solidtumors frontiers in bioscience vol e4 no pp j e visvader cells of origin in cancer nature vol no pp h clevers the cancer stem cell premises promises andchallenges nature medicine vol no pp y kinugasa t matsui and n takakura cd44 expressed oncancerassociated ï¬broblasts is a functional molecule supporting the stemness and drug resistance of malignant cancer cellsin the tumor microenvironment stem cells vol no pp t ishimoto o nagano t yae et al cd44 variant regulatesredox status in cancer cells by stabilizing the xct subunit ofsystem xc and thereby promotes tumor growth cancer cellvol no pp m tamada o nagano s tateyama et al modulation ofglucose metabolism by cd44 contributes to antioxidant statusand drug resistance in cancer cells cancer research vol no pp r c bates n s edwards g f burns and d e fisher acd44 survival pathway triggers chemoresistance via lyn kinasestem cells internationaland phosphoinositide 3kinaseakt in colon carcinoma cellscancer research vol no pp l y w bourguignon k peyrollier w xia and e giladhyaluronancd44 interaction activates stem cell markernanogandankyrinregulated multidrug eï¬ux in breast and ovariantumor cells the journal of biological chemistry vol no pp stat3mediated mdr1expressiongene l y w bourguignon c earle g wong c c spevak andk krueger stem cell marker nanog and stat3 signalingpromote microrna21 expression and chemoresistance inhyaluronancd44activated head and neck squamous cellcarcinoma cells oncogene vol no pp k tajima r ohashi y sekido et al osteopontinmediatedenhanced hyaluronan binding induces multidrug resistance inmesothelioma cells oncogene vol no pp j ni p j cozzi j l hao et al cd44 variant is associatedwith prostate cancer metastasis and chemoradioresistanceprostate vol no pp u carling l barkhatov h m reims et al can we ablateliver lesions close to large portal and hepatic veins with mrguided hifu an experimental study in a porcine modelblood vol no pp y wu and p y wu cd133 as a marker for cancer stem cellsprogresses and concerns stem cells and development vol no pp u m gehling s erg¼n u schumacher et al in vitro diï¬erentiation of endothelial cells from ac133positive progenitorcells blood vol no pp m peichev a j naiyer d pereira et al expression ofvegfr2 and ac133 by circulating human cd34 cellsidentiï¬es a population of functional endothelial precursorsblood vol no pp n uchida d w buck d he et al direct isolation of humancentral nervous system stem cells proceedings of the nationalacademy of sciences of the united states of america vol no pp b j cummings n uchida s j tamaki et al human neuralstem cells diï¬erentiate and promote locomotor recovery inspinal cordinjured mice proceedings of the national academy of sciences of the united states of america vol no pp b bussolati s bruno c grange et al isolation of renal progenitor cells from adult human kidney the american journalof pathology vol no pp l riccivitiani d g lombardi e pilozzi et al identiï¬cation and expansion of human coloncancerinitiating cellsnature vol no pp c a obrien a pollett s gallinger and j e dick a humancolon cancer cell capable of initiating tumour growth inimmunodeï¬cient mice nature vol no pp l lin a liu z peng et al stat3 is necessary for proliferation and survival in colon cancerinitiating cells cancerresearch vol no pp n haraguchi m ohkuma h sakashita et al cd133cd44 population eï¬ciently enriches colon cancer initiating cellsannals of surgical oncology vol no pp 0cstem cells international d inoue t suzuki y mitsuishi et al accumulation ofp62sqstm1 is associated with poor prognosis in patientswith lung adenocarcinoma cancer science vol no pp j q ma h tuersun s j jiao j h zheng j b xiao anda hasim functional role of nrf2 in cervical carcinogenesis plos one vol no article e0133876 q yang h deng h xia et al high nfe2related factor expression predicts poor prognosis in patients with lung cancer a metaanalysis of cohort studies free radical researchvol pp s sarvi a c mackinnon n avlonitis et al cd133 cancerstemlike cells in small cell lung cancer are highly tumorigenicand chemoresistant but sensitive to a novel neuropeptideantagonist cancer research vol no pp q zhang s shi y yen j brown j q ta and a d le asubpopulation of cd133 cancer stemlike cells characterized in human oral squamous cell carcinoma confer resistanceto chemotherapy cancer letters vol no pp s ma t k lee b j zheng k w chan and x y guancd133 hcc cancer stem cells confer chemoresistance bypreferential expression of the aktpkb survival pathwayoncogene vol no pp s bao q wu r e mclendon et al glioma stem cells promote radioresistance by preferential activation of the dnadamage response nature vol no pp c yan l luo c y guo et al doxorubicininduced mitophagy contributes to drug resistance in cancer stem cells fromhct8 human colorectal cancer cells cancer letters vol pp s goto y ihara y urata et al doxorubicininduced dnaintercalation and scavenging by nuclear glutathionestransferase Ï the faseb journal vol no pp h ohata t ishiguro y aihara et al induction of the stemlike cell regulator cd44 by rho kinase inhibition cont | Colon_Cancer |
"protein phosphatase 2a pp2a is a serinethreonine phosphatase that serves as a key regulator of cellularphysiology in the context of apoptosis mitosis and dna damage responses canonically pp2a functions as atumor suppressor gene however recent evidence suggests that inhibiting pp2a activity in tumor cells mayrepresent a viable approach to enhancing tumor sensitivity to chemoradiotherapy as such inhibition can cause cellsto enter a disordered mitotic state that renders them more susceptible to cell death indeed there is evidence thatinhibiting pp2a can slow tumor growth following radiotherapy in a range of cancer types including ovarian cancerliver cancer malignant glioma pancreatic cancer and nasopharyngeal carcinoma in the present review we discusscurrent understanding of the role of pp2a in tumor radiotherapy and the potential mechanisms whereby it mayinfluence this processkeywords protein phosphatase 2a conventional tumor radiotherapy dna damage response radiosensitizationeffectstherapeutic outcomesintroductionwhile mainstays of tumor treatment efforts conventional radiotherapy and chemotherapy often yield unsat[] these poorisfactoryoutcomes are generally linked to tumor cell multidrugresistance and resistance to ionizing radiation [] inaddition while these treatments are welltailored to killing rapidly proliferating tumor cells they generally failto impact hypoxic and quiescent cells ultimately resulting in treatment failure and tumor recurrence [] understanding the mechanistic basiscellchemoresistance and radioresistance is thus vital interestingly recent research evidence suggests that radiosensitization can be achieved by accelerating cell cycleprogression in quiescent cells such that they becomeproliferative [ ] inhibiting proteins such as pp2acan drive quiescent tumor cells to enter mitosis in turnpotentially increasing tumor cell sensitivity to treatment[ ] inhibiting pp2a may therefore represent afortumor correspondence baolinqu301163com xiao lei na ma and lehui du contributed equally to this workthe first medical center of chinese pla general hospital department ofradiation oncology beijing p r chinavaluable new approach to promoting tumor radiosensitization in the present review we discuss current research progress pertaining to the role of pp2a in thecontext of tumor radiotherapythe role of pp2a in radiation therapypp2a is a serinethreonine phosphatase that functionsas a tumor suppressor gene it is a complex composed of a core enzyme and a regulatory subunit thecore enzyme pp2ad is a dimer comprised of a kdcatalytic subunit pp2a c and a kd regulatory subunit pr65 or subunit a pp2a has three subunits including subunit a and two subtypes of subunit c α and with each of these subunits exhibiting distinct structural and catalytic activities there are also multiple subtypes of subunit b that serve to control the specificityand localization of pp2a overall there are four familiesof regulatory b subunits capable of binding to the coreenzyme b pr55 b² b56 or pr61 b pr72 and b[] early research suggested thatpr93pr110pp2a functions as a classic tumor suppressor gene thatis downregulated or nonfunctional in many tumor typesincluding lung skin breast brain ovarian cervical 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 0clei hereditas page of colon cancers [] at a functional level pp2a inhibits a range of tumor signaling pathways preventing il2induced jak3 and stat5 activation which isnormally dysregulated in many malignancies pp2acan also interact with the erk2mek and rasraf signaling pathways through direct and indirect mechanisms soas to control their activation given that constitutive rasrafmekerk signaling is a characteristic of many malignant tumor cells [] these highlights another mechanism whereby pp2a can control oncogenesis pp2a canalso mediate proteasome dephosphorization and therebyimpact cmyc which is often constitutively active in thecontext of tumorigenic transformation [ ]tumor metastasis recurrence and radioresistance allrepresent major roadblocks to the effective treatment ofcancer patients [ ] following pp2a inhibitionmany tumors exhibit slower growth increased apoptoticcell death and greater sensitivity to ionizing radiation ashas been observed in the context of nasopharyngeal carcinoma ovarian cancer pancreatic cancer liver cancerand malignant glioma [] in malignant glioma forexample pp2a inhibition increases the frequency ofcells in the m phase of mitosis inhibiting tumor proliferation while driving increased radiosensitivity similarly pp2a inhibition in nasopharyngeal carcinomahas been linked to significant increases in the frequencyof apoptotic cells and g2m arrest likewise inhibiting pp2a in cancer significantly delayed dma damage repair and thereby facilitated more rapid cell deathfollowing irradiation potential mechanisms whereby pp2a influencesradiotherapy outcomesthe role of pp2a in mitosispp2a is a key regulator of normal mitotic processes greatwall kinase inhibited pp2a by small proteins ensaand arpp19 thereby attenuating pp2aregulated cdk1dephosphorylation and promoting mitosis whereas severemitotic defects occur in the absence of greatwall kinase[ ] pp2a also negatively regulates cdk1 activity viaactivating wee1myt1 and by inhibiting cdc25 inhibiting pp2a can also drive the upregulation of moleculesdownstream of cdk1 thereby promoting mitosis thisgreatwall kinasepp2a signaling pathway is thought to bea primary regulator of normal cdk1 functionality in thecontext of mitosis pp2a can also act on other mitoticmediators such as the mitosisspecific kinases plk1which is a key marker of g2m phase arrest followingpp2a inhibition and which interacts with centrosomesduring mitosis [ ] pp2a is also involved in the negative feedback inhibition of plk1 and aurora b therebyregulating the spindle collection checkpoint in order toensure that microtubules are properly connected to thecentromere inhibiting pp2a causes g2m cell cycle checkpointinactivation and alters dna damage repairradiationinduced dna damage can induce cell cycle arrest and dna damage repair that is mediated by dnadamage checkpoint activation irradiationassociateddna damage can lead to g2m checkpoint activation andconsequent g2m phase arrest enabling dna repair tooccur prior to cellular entry into mitosis cdc2cyclinb is a key regulator of this g2m checkpoint as cdc2cyclin b activation is required in order for cells to proceedfrom the g2 phase into the cleavage phase dnadamage is rapidly followed by the phosphorylation and activation of the atr and atm kinases which in turn activate chk2 and chk1 chk2 and chk1 function in partby suppressing the activation of cdc25 family proteinssuch that cdc2cyclin b activation is inhibited followingdna damage cdc2cyclin b activity is thus reducedresulting in cell cycle arrest following drug orradiationinduced dna damage pp2a dephosphorylationcan inhibit plk1 which phosphorylates and activatescdc25 and cyclins involved in the g2m checkpointthereby facilitating cell cycle progression pp2a may thusprevent cells from dividing by inhibiting plk1 moreover inhibition of pp2a showed that radiationinducedinactivation of atr and chk1 kinase phosphorylation ofcdc2tyr15 and inactivation of g2m phase checkpointswhich attenuated radiationinduced g2m arrest therebyenabling tumor cells to enter into mitosis via reducingdna damage repair efficiency and aggravating cellularmitotic disorders inhibiting pp2a promotes g0 stage tumor cell entry intomitosiscdk2 activity has recently been found to govern the proliferation of quiescent cells following mitosis such thatcells enter the g0 phase when cdk2 activity levels arelow regulating cyclin ecdk2 activity at the end of thecell cycle can promote cellular proliferation inadult anisms pp2a has been found to promote cellular quiescence in studies of drosophila eyes andwings researchers have determined that inhibiting pp2aat the end of the cell cycle can induce additional cell division and thereby impair such quiescence in these drosophila the pp2a subunit b56 family member wdb servesas an important regulator of pp2arelated cellular quiescence when pp2a activity is suppressed cells thatwould normally enter the stationary phase instead exhibit robust cdk2 activity ectopic dominant testinghas further revealed that abnormal cyclin ecdk2 activity can promote additional cell cycle progression in thecontext of pp2a inhibition reduced wdbpp2a activity results in abnormally elevated cyclin e levels enabling quiescent cells to pass through the g0 phase andto thereby enter into mitosis increasing cellular 0clei hereditas page of sensitivity to radiotherapy and chemotherapy given theimportant role of tumor cell quiescence as a driver oftumor radioresistance and recurrence in cancer patients inhibiting pp2a may represent a viable means ofpromoting tumor radiosensitivity by driving cells in theg0 phase of the cell cycle to undergo mitosispp2a as a regulator of apoptosispp2a can control apoptosis by influencing both pi3kakt pathway signaling and the expression and activity ofapoptosisassociated proteins in cells with functional bcl2 for example pp2a has been shown to promote bcl2 dephosphorization and to thereby promoteapoptotic cell death [] in contrast in cells that arehighly metabolically active pp2a can dephosphorylateand thereby activate camkii so as to exert an antiapoptotic effect pp2a also modulates the p53 pathway such that it can activate baxnoxapuma and inhibitbcl2 to drive apoptotic death []in the context of the dna damage response the atmsignaling pathway directly activates and stabilizes pp2a byphosphorylating the ubiquitin ligase mdm2 pp2a in turninhibits akt1 pathway activity and thereby suppressesmdm2 activation thus preventing the mdm2mediateddegradation of p53 in the presence of irreversibledna damage pp2a can also directly dephosphorylatep53 stabilizing this protein an inducing cell cycle arrestand apoptosis inhibiting pp2a may therefore be a viable therapeutic strategy in highly metabolically activetumor cells suppressing pp2a activity in cells exhibitingdna damage can also inhibit bax expression and promote the cell cycle studies of combination radiotherapy and pp2a inhibition have highlighted the consequentinhibition of interactions between p53 and pp2a reducingthe role of the p53 pathway in response to dna damageand promoting cellular proliferation and p53independentapoptotic cell death pp2a as a regulator of the wntcatenin signalingpathwaypp2a is capable of inhibiting wntcatenin signalingpathway activity which normally plays importantroles in governing the migration and proliferation ofcells after wnt ligands interact with specific cellsurface receptors the tcf family transcriptional coactivator catenin undergoes nuclear translocation interactswith tcf and modulates target gene expression thisprocess often becomes constitutively activated duringthe early stages of oncogenesis in tumor cells inwhich the wnt signaling pathway is not active cytoplasmic catenin is generally degraded a complexcomposed of apc dvl axin and 3glycogen synthesis kinase can target catenin for degradation however the pp2ac regulatory subunit has also beenshown to play downstream signaling roles in the contextof the wntcatenin signaling pathway aspirinhas also been found to downregulate wntcateninsignaling pathway activity via inhibiting pp2a positive pp2a feedback signaling has also been suggested toalter the wntcatenin signaling pathway in pancreatic cancer and colorectal cancer cell lines thereby stabilizing the activation of this pathway [ ]intestinal cellscurrent clinical approaches to inhibiting pp2a as anapproach to tumor radiosensitizationto date pharmacological inhibition of pp2a has largelybeen dependent upon the use of natural compoundssuch as okadaic acid and anthraquinone thesecompounds however exhibit varying degrees of toxicityin contrast lb100 is a watersoluble pp2a inhibitor thatis less toxic than these other compounds research suggests that while radiotherapy can enhance pp2a activitylb100 pretreatment prior to radiotherapy can suppresspp2a activation while simultaneously enhancing tumorsensitivity to irradiation lb100 has been leveragedin several clinical trials as a pp2a inhibitor owing to itsefficacy and low toxicity in one study of pancreaticcancerfor example lb100 was found to effectivelyradiosensitize pancreatic cancer cells without adverselyaffecting normal small lb100mediated pp2a inhibition has also been shown to prevent radiationinduced rad51 foci formation and homologous recombination repair thereby causing sustaineddna damage in cells following radiation exposure the presence of undifferentiated stemlike tumor cellscapable of undergoing selfrenewal is thought to be oneof the key mechanisms underlying tumor recurrence andtherapeutic resistance traditionalradiotherapy andchemotherapy efforts are largely unable to impact thesecancer step cells as they grow slowly and are largely quiescent [ ] there is recent experimental evidencethat the receptor corepressor protein complex is a primary determinant of the stemlike properties of cancerstem cells in glioma tumors this receptor corepressor protein complex is composed of the receptorcorepressor protein a deacetylase complex steroidshormone receptors and transcription factors that function to control transcription in the context of glial differentiation cytokineinduced ciliary neurotrophicfactor stimulation of glioma precursor cells has beenshown to inhibit receptor corepressor protein complexactivity via aktpi3kmediated phosphorylation of thereceptor corepressor protein inhibition of pp2ausing lb100 resulted in enhanced ak1 activity therebypreventing receptor corepressor protein complex formation and promoting cellular division rendering quiescent tumor cells more sensitive to irradiation 0clei hereditas page of perspectivesinhibiting pp2a has been conclusively shown to enhancetumor cell radiosensitivity however further research isnecessary in order to facilitate the optimal clinical implementation of these experimental findings for examplewhile many studies have assessed the impact of inhibiting pp2a in tumor cells following radiation exposurefew studies have assessed the effect of such inhibition onnormal tissues which may also undergo potential radiosensitization [] differences in pp2a expressionprofiles between normal and tumor tissues are also essential to ensure that tumor cells can be effectively killedwithout causing undue harm to healthy tissues atpresent there are also few specific inhibitors of pp2aavailable to leverage the potential clinical utility ofcombination pp2a inhibition and radiotherapy treatment it is vital that novel highly specific pp2a inhibitorsbe developed the identification of specific inhibitorsthat preferentially targettumor cells while leavinghealthy cells intact would further advance the clinicalapplications of pp2a inhibition it is also important tonote that many studies of pp2a inhibition have focusedonly on single factors [] whereas tumor resistanceand recurrence are multifactorial in nature at presentthere is a dearth of systematic or comprehensive studiespertaining to the mechanisms whereby pp2a inhibitionbolsters the efficacy of radiation therapyconclusionin summary inhibiting pp2a in combination with radiotherapeutic treatment may represent a viable approachto enhancing patient treatment outcomes and preventingtumor recurrence however further research regardingthe mechanisms underlying such combination efficacy isstill required in addition more specific pharmacologicalinhibitors of pp2a must be developed in order toachieve better clinical outcomesabbreviationspp2a protein phosphatase 2a cdk1 cyclin dependent kinase plk1 pololike kinase cdc2 cyclin dependent kinase atm ataxia telangiectasiamutated atr atm and rad3related pi3k phosphoinositide 3kinasemdm2 murine double minute2 dna deoxyribonucleic acidrna ribonucleic acidacknowledgementsnot applicableauthors contributionsxiao lei and na ma designed the study and made the manuscript yanjieliang did the perspective part yanan han and pei zhang helped participatein the review design baolin qu and lehui du participated in the writing ofpaper and revision of manuscript all authors read and approved the finalmanuscriptfundingno funding was receivedavailability of data and materialsthe datasets are available under reasonable requestethics approval and consent to participatenot applicableconsent for publicationnot applicablecompeting intereststhe authors declare that they have no competing interestsreceived july accepted august referencessun d chen j wang y ji h peng r jin l wu w advances inrefunctionalization of erythrocytebased nanomedicine for enhancingcancertargeted drug delivery theranostics verma p mittal p singh a singh ik new entrants into clinical trials fortargeted therapy of breast cancer an insight anti cancer agents medchem httpsdoi1021741871520619666191018172926 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withverthandirad21 to regulate mitosis and an development in drosophilasci rep qin s li j si y he z zhang t wang d liu x guo y zhang l li s li q liu pinto bs orrweaver tl drosophila protein phosphatases 2a b' wdb andy cucurbitacin b induces inhibitory effects via cip2app2aakt pathway inglioblastoma multiforme mol carcinog hein al brandquist nd ouellette cy seshacharyulu p enke ca ouellettemm batra sk yan y pr55alpha regulatory subunit of pp2a inhibits themob1lats cascade and activates yap in pancreatic cancer cellsoncogenesis stafman ll williams ap marayati r aye jm stewart je mroczekmusulmane beierle ea pp2a activation alone and in combination with cisplatindecreases cell growth and tumor formation in human huh6hepatoblastoma cells plos one 201914e0214469lv p wang y ma j wang z li jl hong cs zhuang z zeng yx inhibitionof protein phosphatase 2a with a small molecule lb100 radiosensitizesnasopharyngeal carcinoma xenografts by inducing mitotic catastrophe andblocking dna damage repair oncotarget wrd regulate meiotic centromere localization and function of the meis332shugoshin proc natl acad sci u s a cameron bd traver g roland jt brockman aa dean d johnson l boyd kihrie ra freeman ml bcl2expressing quiescent type b neural stem cells inthe ventricularsubventricular zone are resistant to concurrenttemozolomidexirradiation stem cells xiong y lan j huang k zhang y zheng l wang y ye q pp2acupregulates pi3kakt signaling and induces hepatocyte apoptosis in liverdonor after brain death apoptosis pagano ma tibaldi e molino p frezzato f trimarco v facco m zagotto gribaudo g leanza l peruzzo r szabo i visentin a frasson m semenzatog trentin l brunati am mitochondrial apoptosis is induced by alkoxyphenyl1propanone derivatives through pp2amediated dephosphorylationof bad and foxo3a in cll leukemia 0clei hereditas page of sudoh s kawakami h ohta m nakamura s ciliary neurotrophic factorinducedincrease in betaamyloid precursor protein mrna in rat c6 gliomacells biochem biophys res commun li xf li sy dai cm li jc huang dr wang jy pp2a inhibition by lb100protects retinal pigment epithelium cells from uv radiation via activation ofampk signaling biochem biophys res commun huang cy hung mh shih ct hsieh fs kuo cw tsai mh chang ss hsiaoyj chen lj chao ti chen kf antagonizing set augments the effects ofradiation therapy in hepatocellular carcinoma through reactivation of pp2amediated akt downregulation j pharmacol exp ther ho wsc sizdahkhani s hao s song h seldomridge a tandle a maric dkramp t lu r heiss jd lb100 a novel protein phosphatase 2a pp2ainhibitor sensitizes malignant meningioma cells to the therapeutic effectsof radiation cancer lett miao j shi r li l chen f zhou y tung yc hu w gong cx iqbal k liu fpathological tau from alzheimer's brain induces sitespecifichyperphosphorylation and sds and reducing agentresistant aggregationof tau in vivo front aging neurosci lai d chen m su j liu x rothe k hu k forrest dl eaves cj morin gbjiang x response to comment on pp2a inhibition sensitizes cancer stemcells to abl tyrosine kinase inhibitors in bcrabl human leukemia scitransl med 201911eaav0819 yu cq yin lq tu zt liu dw luo wp the regulatory role of dopaminereceptor d1 on pp2a via sumo1 modification eur rev med pharmacol sciliu l huang z chen j wang j wang s protein phosphatase 2a mediatesjskinduced apoptosis by affecting bcl2 family proteins in humanhepatocellular carcinoma hepg2 cells j cell biochem deng x gao f may ws protein phosphatase 2a inactivates bcl2'santiapoptotic function by dephosphorylation and upregulation of bcl2p53binding blood publishers notespringer nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations huang b yang cs wojton j huang nj chen c soderblom ej zhang lkornbluth s metabolic control of ca2calmodulindependent proteinkinase ii camkiimediated caspase2 suppression by the b55betaproteinphosphatase 2a pp2a j biol chem koma yi ito a watabe k kimura sh kitamura y a truncated isoform of thepp2a b56gamma regulatory subunit reduces irradiationinduced mdm2phosphorylation and could contribute to metastatic melanoma cellradioresistance histol histopathol azad a storey a chk1 activity is required for bak multimerization inassociation with puma during mitochondrial apoptosis cellcommunication and signaling ruvolo pp a functional role for the b56 alpha subunit of proteinphosphatase 2a in ceramidemediated regulation of bcl2 phosphorylationstatus and function j biol chem dung td day ch binh tv lin ch hsu hh su cc lin ym tsai fj kuoww chen lm pp2a mediates diosmin p53 activation to block ha22t cellproliferation and tumor growth in xenografted nude mice through pi3kaktmdm2 signaling suppression food chem toxicol li hh cai x shouse gp piluso lg liu x a specific pp2a regulatory subunitb56gamma mediates dna damageinduced dephosphorylation of p53 atthr55 embo j jin z wallace l harper sq yang j pp2ab56 a substrate of caspase3regulates p53dependent and p53independent apoptosis duringdevelopment j biol chem park ds yoon gh lee hs choi sc capsaicin inhibits the wntcateninsignaling pathway by downregulating pp2a biochemical biophysicalresearch communications ashrafizadeh m ahmadi z farkhondeh t samarghandian s resveratroltargeting the wnt signaling pathway a focus on therapeutic activities j cellphysiol oct httpsdoi101002jcp29327 | Colon_Cancer |
" tumor microenvironment tme plays an important role in malignant tumors our study aimed toinvestigate the effect of the tme and related genes in osteosarcoma patientsmethods gene expression profiles and clinical data of osteosarcoma patients were downloaded from the targetdataset estimate algorithm was used to quantify the immune score then the association between immune scoreand prognosis was studied afterward a differential analysis was performed based on the high and lowimmunescores to determine tmerelated genes additionally cox analyses were performed to construct two prognosticsignatures for overall survival os and diseasefree survival dfs respectively two datasets obtained from the geodatabase were used to validate signaturesresults eightyfive patients were included in our research the survival analysis indicated that patients with higherimmune score have a favorable os and dfs moreover genes were determined as tmerelated genes theunsupervised clustering analysis revealed two clusters were significantly related to immune score and t cells cd4memory fraction in addition two signatures were generated based on three and two tmerelated genesrespectively both two signatures can significantly divide patients into low and highrisk groups and were validatedin two geo datasets afterward the risk score and metastatic status were identified as independent prognosticfactors for both os and dfs and two nomograms were generated the cindexes of os nomogram and dfsnomogram were and respectively tme was associated with the prognosis of osteosarcoma patients prognostic models based on tmerelated genes can effectively predict os and dfs of osteosarcoma patientskeywords tumor microenvironment osteosarcoma prognosis immune features nomogram osteosarcoma is the most common bone tumor especiallyin children and adolescents it was reported that approximately of patients are between and yearsold and osteosarcoma is considered as the second leadingcause of death in this age group currently surgery and correspondence 407404159qqcom4wenzhou medical university wenzhou chinafull list of author information is available at the end of the chemotherapy are still major treatments for osteosarcomapatients and these therapies are constantly improving inrecent years however due to the susceptibility of localaggressiveness and lung metastasis in osteosarcoma patients the prognosis of osteosarcoma remains unfavorable previous studies indicated that the 5years survivalrates were and in metastatic and nonmetastaticpatients respectively thereforeit is necessary to 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 0chu bmc cancer page of investigate the mechanism of pathogenesis and progressionof osteosarcoma and accurately classify the risk of patientsrecently an increasing number of diagnostic and prognostic biomarkers of osteosarcoma patients have beenidentified for example chen reported that tumorsuppressor p27 is a novel biomarker for the metastasis andsurvival status in osteosarcoma patients moreover huang discovered that dysregulated circrnas serve asprognostic and diagnostic biomarkers in osteosarcomapatients and the relative potential mechanism mainly attributes to the regulation of downstream signaling pathwaysby sponging microrna in addition lncrna microrna and many clinical data were also identified asprognostic biomarkers for osteosarcoma patients however osteosarcoma is one of the malignant cancers entitiescharacterized by the high level of heterogeneity in humanstherefore it is necessary to find accurate biomarkers forosteosarcomain recent years researchers have paid more and moreattention to the role of the tumor microenvironmenttme in malignant tumors the function of tme inthe tumorigenesis progression and therapy of tumorshave been initially understood [ ] more importantly estimation of stromal and immune cells in malignant tumor tissues using expression data estimate an algorithm to quantify the score of immune cellsand stromal cells by analyzing the gene expression datawas developed in based on the algorithm theprognostic value of immune and stromal cells in bladdercancer acute myeloid leukemia gastric cancer cervicalsquamous cell carcinoma adrenocortical carcinomaclear cell renal cell carcinoma hepatocellular carcinomathyroid cancer and cutaneous melanoma have beenreported [] generally the above research indicatedthat tme can serve as the prognostic biomarker in tumorsand many tmerelated genes were determined as the prognostic genes however the role of tme and tmerelatedgenes in osteosarcoma patients remains unclearin the present study gene expression data and corresponding clinicopathologic data were obtained from thetherapeutically applicable research to generate effectivetreatments target dataset then the estimatealgorithm was performed to quantify the immune score ofosteosarcoma and the tmerelated genes were identifiedby the differential expression analysis subsequently theprognostic value of tme and tmerelated genes weredetermined by a series of bioinformatics methodsmethodsgene expression datasetslevel data of gene expression profiles and correspondingclinical data of osteosarcoma patients were downloadedfrom target dataset ocgcancergovprogramstarget accessed on oct the correspondingclinicopathologic data included in the present study wereage gender race ethnicity tumor site and metastaticstatus after data were extracted from the public domainthe estimate an algorithm inferring tumor puritystromal score and immune cell admixture from expression data was performed to evaluate the immune score byusing the estimate package in r software version meanwhile the messenger rnamrna expressionprofiles and clinical data ofincludinggse21257 and gse39055 were obtained fromthe gene expression omnibus as external validationcohortstwo cohortssurvival analysis and correlation analysisafter scores were obtained patients were divided intohighscore group and lowscore group according to themedian of the immune score the kaplanmeier survivalanalysis with logrank test was performed to estimatethe differences of overall survival os and diseasefreesurvival dfs between high and lowscore cohorts inaddition the association between clinicopathologic dataand tme score was also studied mannwhitney signedrank test was performed to compare the differences ofimmune score between each clinical group all statisticalanalyses in the present study were performed using rsoftware except for the special instructions p value twoside was identified as statistically significantin the present studydegexpressed genedifferentially expressed gene analysisdifferentiallyanalysis wasperformed by comparing the proteincoding genesexpression between the lowimmune score group andthe highimmune score group the limma package in rsoftware was used to perform the differential analysisand genes with log fc and adjusted pvalue qvalue were identified as degs to further understand the function of degs identifiedin the present study gene ontology goincludingbiological processes bp molecular functions mf andcellular componentscc and kyoto encyclopedia ofgenes and genomes kegg analysis were performedby clusterprofiler package in r software evaluation of association with immune cellsto further investigate the association between degs andimmune cells the cibersort package was used toestimate the relative proportions of types of immunecells meanwhile the consensusclusterplus package was used to cluster in an unbiased and unsupervisedmanner based on the overlapping degs cumulative distribution function cdf and relative change inarea under the cdf curve were used to determine theoptimal number of clusters k then mannwhitney 0chu bmc cancer page of signedrank test was performed to study the differenceof immune cells proportion between the clusters and theviolin plot was established to show the differences ofimmune cells among clusters survival analysis of degsbased on the degs the univariate cox analysis was performed to determine the prognostic value of immunerelated genes then the osrelated genes were validatedin the gse21257 dataset while the dfsrelated geneswere validated in the gse39055 dataset only genes successfully validated were selected for further analysis afterward based on the validated genes the multivariate coxanalysis was performed to establish the prognostic signature for predicting the prognosis of osteosarcoma patientsthe risk score for each patient was calculated based onthe coefficient from the multivariate cox analysis and thecorresponding gene expression meanwhile all patientswere divided into the high and lowrisk groups accordingto the median of the risk score the survival receiver operating characteristic roc curve was used to show the discrimination of signatures and the kaplanmeier survivalcurve with the logrank test was generated to show thedifferences of os and dfs between high and lowriskgroups in addition the risk score of patients in the validation cohort was also calculated according to the aforementioned risk signature the kaplanmeier survivalcurve and survival roc curve were generated to show thepredictive ability of the signature in the validation cohortdevelopment of a nomogram for osteosarcoma patientsnomogram is a tool to visualize the predictive model andconvenient for clinical practice therefore we attemptedto develop a nomogram based on the tmerelated genessignature and clinicopathologic data to predict the prognosis of osteosarcoma patients firstlythe univariatecox analysis was performed to filter prognostic variableswhich will be further included in the multivariate coxanalysis secondly based on independent prognostic variables two nomograms were established for predicting theos and dfs respectively the cindex was used to assessthe discriminatory performance of the nomogram whichrange from to a cindex of means agreement by chance and a cindex of represents perfectdiscriminatory performance the higher value of the cindex the better performance of the nomogram is furthermore the calibration curves of and 3year weredeveloped to evaluate the effectiveness of nomogramsresultsimmune significantly associated with the prognosis ofosteosarcoma patients osteosarcoma patients were included in the presentstudy including males and females the immunescore of the cohort range from to tostudy the relationship between the immune score and theprognosis of osteosarcoma patients patients wereincorporated into the lowimmune score group while theremaining patients were incorporated into the highimmune score group the survival analysis indicated thatpatients with higher immune score had a favorable osand dfs fig 1a and b after adjusted age tumor siteand metastatic status the immune score still was a prognostic variable for both os and dfsfig 1a and b inaddition the relationship between immune score and clinical features was also investigated however there was nosignificant relationship between immune score and clinicalvariables supplementary figure 1a1cdifferential expression analysisaccording to the median of the immune score patients were divided into highscore n and lowfig association between immune score and prognosis in osteosarcoma patients a kaplanmeier survival analysis of overall survival for patientswith high vs low immune score b kaplanmeier survival analysis of diseasefree survival for patients with high vs low immune score 0chu bmc cancer page of score group n there were differentiallyexpressed genes between two groups which include upregulated genes and downregulated genesfig 2a b and supplementary table to furtherunderstand the function of degs go analysisand kegg analysis were performed the top significant results of go analysis among three types wereillustrated in fig 2c interestingly we can find that theresults of go analysis are mostly associated with immunity which further verify that the immunerelated degsare associated with immune features in addition the results of kegg also confirmed it such as phagosomeautoimmune thyroid disease antigen processing andpresentation b cell receptor signaling pathway intestinal immune network for iga production inflammatorybowel disease primary immunodeficiency th1 andth2 cell differentiation th17 cell differentiation natural killer cell mediated cytotoxicity and nfkappa bsignaling pathway fig 2dconsensusunsupervisedevaluation of degs and immune cellsto further understand the molecular heterogeneity ofosteosarcomaanalysis wasperformed to divide patients into subgroups to explorewhether immunerelated genes presented discernable patterns based on the consensus matrix heat map patientswere clearly divided into two clustersfig 3a in additionby comprehensively analyzing the relative change in areaunder the cumulative distribution function two clusterswere determined fig 3bc the immune score betweentwo clusters was significantly different fig 3d in additionthe proportion of types of immune cells in osteosarcomapatients was illustrated in a barplot fig 3e interestinglywe can see that the t cells cd4 memory activated ofcluster is significantly higher than cluster fig 5fprognostic value of tmerelated genesprevious studies indicated that tmerelated genes canserve as the prognostic biomarker for tumor patientsfig differentially expressed genes with the immune score in osteosarcoma patients a heatmap of significantly differentially expressed genesbased on immune score b the volcano figure to show the upregulated and downregulated genes c go analysis of differentially expressedgenes d kegg of differentially expressed genes go gene ontology kegg kyoto encyclopedia of genes and genomes 0chu bmc cancer page of fig the immune landscape of the tumor microenvironment ac unsupervised clustering of all samples based on the overlapping degs dcomparison of immune score between two clusters e the distribution of types of immune cells in osteosarcoma patients f the comparisonof types of immune cells between clusters deg differentially expressed genehence we performed the univariate cox analysis toidentify prognostic degs the results showed that and genes were identified as os and dfsrelateddegs respectively supplementary table and afterward five osrelated genes were successfully validated inthe gse21257 data set and five dfsrelated genes were successfully validated in the gse39055 cohort furthermoremultivariate cox analysis was performed and two prognostic signatures were generated for predicting the os anddfs respectively the risk score for predicting the os wasasrisk score fcgr2b0766 gfap0702 mpp70387 in addition the risk score for predicting thedfs was as follows risk score cyp2s10574 icam3 the auc values of osrelated signature were follows 0chu bmc cancer page of and in and 3year respectively fig 4aand the auc values of dfsrelated signature were and in and 3year respectively fig 5amoreover survival curves showed that patients in the highrisk group had worse os and dfs compared with the lowrisk patients figs 4b and 5b heat maps risk score plotsand survival status were generated to show the distinctionbetween highrisk patients and lowrisk patients figs 4ceand 5ce then both signatures were validated in independent cohorts for os signature the auc values ofvalidation cohort were and at and3year fig 4f for dfs signature the auc values ofvalidation cohort were and at and3year fig 5f additionallyin both validation cohortssurvival curves showed that lowrisk patients were favorableprognosis than highrisk patients figs 4g and 5gheat maps risk score plots and survival status of validation cohorts were also generated to show the distinction between highrisk patients and lowrisk patientsfigs 4hj and f 5hjdevelopment of a nomogram for osteosarcoma patientsto generate a nomogram for clinical use the cox analysiswas performed to select the clinical prognostic variables infig establishment and validation of the prognostic model for overall survival based on significant degs a receiver operating characteristiccurves of prognostic signature in the training cohort b the survival curve showed the different overall survival status between high and lowriskpatients c the heat map showed the expression of prognostic genes in the training cohort d the risk curve of each sample reordered by riskscore e the scatter plot showed the overall survival status of osteosarcoma patients in the training cohort f receiver operating characteristiccurves of prognostic signature in validation cohort g the survival curve showed the different overall survival status between high and lowriskpatients h the heat map showed the expression of prognostic genes in the validation cohort i the risk curve of each sample reordered by riskscore j the scatter plot showed the overall survival status of osteosarcoma patients in the validation cohort 0chu bmc cancer page of fig establishment and validation of the prognostic model for diseasefree survival based on significant degs a receiver operatingcharacteristic curves of prognostic signature in the training cohort b the survival curve showed the different diseasefree status between highand lowrisk patients c the heat map showed the expression of prognostic genes in the training cohort d the risk curve of each samplereordered by risk score e the scatter plot showed the diseasefree status of osteosarcoma patients in the training cohort f receiver operatingcharacteristic curves of prognostic signature in validation cohort g the survival curve showed the different diseasefree status between high andlowrisk patients h the heat map showed the expression of prognostic genes in the validation cohort i the risk curve of each sample reorderedby risk score j the scatter plot showed the diseasefree status of osteosarcoma patients in the validation cohortthe univariate cox analysis risk score and metastatic status were identified as both os and dfsrelated variablesfig 6a and e afterward risk score and metastatic statuswere determined as both independent os and dfsrelated variables in the multivariate cox analysis fig 6band f based on independent variables two nomogramswere established for predicting the os and dfs in osteosarcoma patients respectively fig 6c and g the cindexvalues were and in os nomogram and dfsnomogram respectively the results of cindex mean thatboth two nomograms have good discrimination meanwhile to evaluate the calibration of nomograms six calibration curves were generated and the results showed thatthe predictive curves were close to the ideal curve fig 6dand h which indicated a good calibrationdiscussionthe relationship between tme and tumor have beenwidely studied in recent years in the present study estimate algorithm was utilized to quantify the immunescore based on gene expression profiles in osteosarcomapatients from target database we confirmed that thetme is significantly associated with the prognosis ofosteosarcoma patientsinadditionfunctional enrichment analyses of tmerelated genes indicated that immunerelated processesincluding os and dfs 0chu bmc cancer page of fig nomograms based on the tumor microenvironment related genes for osteosarcoma patients a univariate cox analysis of overall survivalrelated variables b multivariate cox analysis of overall survivalrelated variables c nomogram for predicting the overall survival in osteosarcomapatients d1 and 3year calibration curves of overall survival nomogram e univariate cox analysis of diseasefree survivalrelated variables fmultivariate cox analysis of diseasefree survivalrelated variables g nomogram for predicting the diseasefree survival in osteosarcoma patientsh1 and 3year calibration curves of diseasefree survival nomogramknown to contribute to tumor progression more importantly degs based on the tme were identified asimportant prognostic biomarkers for osteosarcoma patients and two nomograms were developed for predicting the os and dfs of osteosarcoma patientsrespectivelyin recent years an increasing number of studiesfocused on the carcinogenesis and progression of tumorsbased on the tme and the estimate algorithm is oneof the most important quantitative tools for this researchfield based on the estimate algorithm the association between the prognosis and tme has been initially 0chu bmc cancer page of elucidated in some tumors such as cervical squamouscell carcinoma gastric cancer cutaneous melanomaacute myeloid leukemia bladder cancer and clear cellrenal carcinoma [ ] however previousstudies indicated that tme scores serve as a differentrole in different tumors for example for hepatocellularcarcinoma gastric cancer acute myeloid leukemiabladder cancer and clear cell renal carcinoma patientswith high immune score have a worse prognosis [ ] however for cervical squamous cell carcinoma adrenocortical carcinoma and cutaneous melanoma patients with high immune score have a favorableprognosis [ ] therefore we can find great heterogeneity among different tumors from the perspectiveof tme for osteosarcoma patients the present studyindicated that patients with higher immune score had abetter os and dfs hence the present study indicatedthat immune cells infiltrating tumor tissue may play animportant role in suppressing tumor progressionin our research tmerelated genes were identified by comparing the highscore and lowscore osteosarcoma patients the functional enrichment includinggo and kegg analyses showed that tmerelated geneswere mainly involved in the immune features such asregulation of leukocyte activation mhc protein complex mhc protein and complex binding more importantly the unsupervised cluster analysis based on degswas performed and all patients were divided into twoclusters immune score and t cell cd4 memory activated fraction were significant difference between twoclusters which further elucidated the relationship between degs and immune featuresdue to the poor prognosis of osteosarcoma patientsidentifying robust prognostic biomarker is very importantthe tumor immune microenvironment is closely relatedto the prognosis of bone tumor patients emilie etal performed the first genomewide study to describe therole of immune cells in osteosarcoma and found thattumorassociated macrophages are associated with reduced metastasis and improved survivalin highgradeosteosarcoma recently the prognostic signature based ontmerelated genes have been established for many tumors [ ] but only one study focused on osteosarcoma patients compared with the study performedby zhang we think that our research have someadvantages firstly our signatures were established basedon several validated genes and both two signatures weresuccessfully validated in independent cohorts secondlythe outcome of dfs was not reported in the previousstudy as reported in published studies tumor recurrenceis a terrible medical problem for osteosarcoma patientsand the 5year survival rate for osteosarcoma patients withmetastasis or relapse remains disappointing [ ]hence the dfs nomogram can improve the managementof osteosarcoma patients finally two nomograms incorporated tmerelated signature and clinical variables wereestablished in our research which further facilitated theclinical application of our findingsin our research five genes were incorporated into thefinal prognostic signatures fcgr2b gfap and mpp7were identified and validated as osrelated biomarkerswhile cyp2s1 and icam3 were dfsrelated biomarkersthe role of these genes in tumor prognosis had beenwidely reported in previous studies [] fcgr2bhas been confirmed as an immunerelated gene previously although the relationship between fcgr2band prognosis in sarcoma patients had not been reported the prognostic value of fcgr2b had been widelyconfirmed in other cancerssuch as hepatocellularcarcinoma and glioblastoma [ ] in addition newm etal demonstrated that mpp7 is novel regulatorsof autophagy which was thought to be responsible forthe prognosis of pancreatic ductal adenocarcinomacyp2s1 described as cytochrome p450 family subfamily s member was reported significantly associatedwith colorectal cancer in primary colorectal cancercyp2s1 was present at a significantly higher level ofintensity compared with normal colon more importantly the presence of strong cyp2s1 immunoreactivity was associated with poor prognosis the roleof icam3 in cancer was also widely reported in published studies and the akt pathway plays an importantrole in the impact of icam3 on tumors yg kim etal reported that icam3 can induce the proliferationof cancer cells through the pi3kakt pathway additionally jk park etal showed that the icam3 can enhancethe migratory and invasive potential of human nonsmall celllung cancer cells by inducing mmp2 andmmp9 via akt pathway showed that the icam3can enhance the migratory and invasive potential ofhuman nonsmall celllung cancer cells by inducingmmp2 and mmp9 via akt pathwayalthough the role of tme and tmerelated genes inosteosarcoma patients have been initially studied by bioinformatic and statistical analyses in our research somelimitations should be elucidated firstly the treatmentinformation cannot be obtained from the target database which may influence the prognosis of osteosarcomapatients secondly two nomograms were generated andshowed good performance in our study however externalvalidation by a large cohort is needed thirdly many independent prognostic genes for osteosarcoma patients wereidentified in the present study but the potential mechanism to influence osteosarcoma remains unclear finally inthe training cohort and degs were identified asos and dfsrelated degs respectively however onlyfive os and five dfsrelated genes were identified in thevalidation cohort the different age structures smaller 0chu bmc cancer page of sample sizes and the platform covering only part of thegenes may contribute to this resultreceived february accepted july in tme plays an important role in osteosarcoma patients and related with the progression of thetumor moreover tmerelated genes can serve as prognostic biomarkers in osteosarcoma patients howeverfurther researches are needed to study the potentialmechanism and validate the nomogram that developedin our present studysupplementary informationsupplementary information accompanies this paper at doi101186s12885020072162additional file additional file additional file additional file abbreviationstme tumor microenvironment deg differentially expressed genesos overall survival dfs diseasesfree survival roc receiver characteristiccurve estimate estimation of stromal and immune cells in malignanttumor tissues using expression data target therapeutically applicableresearch to generate effective treatments go gene ontology bp biologicalprocesses mf molecular functions cc cellular components kegg kyotoencyclopedia of genes and genomes cdf cumulative distribution functionacknowledgementsnoneauthors contributionsc h l y sq t c l and yh w conceived of and designed the study c h r sand c l performed literature search r s l y and b c generated the figuresand tables l y hl r x y and jy l analyzed the data c h wrote themanuscript and sq t and l y critically reviewed the manuscript l ysupervised the research all authors have read and approved the manuscriptfundingwe received no external funding for this studyavailability of data and materialsthe data of this study are from target and geo databaseethics approval and consent to participatethe research didnt involve animal experiments and human specimens noethics related issuesconsent for publicationnot applicablecompeting intereststhe authors declare that they have no competing interestsauthor details1department of joint surgery the affiliated hospital of qingdao universityqingdao china 2department of medical oncology the first hospital ofchina medical university shenyang china 3department of nursing sir runrun shaw hospital affiliated to zhejiang university hangzhou china4wenzhou medical university wenzhou chinareferencesjaffe n bruland os bielack s pediatric and adolescent osteosarcoma vol new york springer science 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j peng m guo y wen z miningtcga database for tumor microenvironmentrelated genes of prognosticvalue in hepatocellular carcinoma biomed res int zhao k yang h kang h wu a identification of key genes in thyroid cancermicroenvironment med sci monit xu wh xu y wang j wan fn wang hk cao dl shi gh qu yyzhang hl ye dw prognostic value and immune infiltration of novelsignatures in clear cell renal cell carcinoma microenvironment agingalbany ny chen b chen w jin j wang x cao y he y data mining of prognosticmicroenvironmentrelated genes in clear cell renal cell carcinoma a studywith tcga database dis markers li x gao y xu z zhang z zheng y qi f identification of prognostic genesin adrenocortical carcinoma microenvironment based on bioinformaticmethods cancer med pan xb lu y huang jl long y yao ds prognostic genes in the tumormicroenvironment in cervical squamous cell carcinoma aging albany ny wang h wu x chen y stromalimmune scorebased gene signature aprognosis stratification tool in gastric cancer front oncol huang s zhang b fan w zhao q yang l xin w fu d identification ofprognostic genes in the acute myeloid leukemia microenvironment agingalbany ny yan h qu j cao w liu y zheng g zhang e cai z identification ofprognostic genes in the acute myeloid leukemia immunemicroenvironment based on tcga data a | Colon_Cancer |
"objectives therapeutic radiographers trs are well placed to deliver health behaviour change advice to those living with and beyond cancer lwbc however there is limited research on the opinions of trs around delivering such advice to those lwbc this study aimed to explore trs practices and facilitators in delivering advice on physical activity healthy eating alcohol intake smoking and weight managementsetting and participants fifteen uk based trs took part in a telephone interview using a semistructured interview guide data was analysed using the framework analysis methodresults emergent themes highlighted that trs are mainly aware of the benefits of healthy behaviours in managing radiotherapy treatment related side effects with advice provision lowest for healthy eating and physical activity participants identified themselves as well placed to deliver advice on improving behaviours to those lwbc however reported a lack of knowledge as a limiting factor to ng so the trs reported training and knowledge as key facilitators to the delivery of advice with a preference for online trainings there is a need for education resources clear referral pathways and in particular training for trs on delivering physical activity and healthy eating advice to those lwbcintroductionit is estimated that of cancer cases are linked to unhealthy behaviours1 based on evidence from systematic literature reviews and meta analyses the world cancer research fund wcrf recommend that individuals are physically active limit consumption of energy dense foods salty foods red meat and avoid processed meat eat more plant foods maintain a healthy weight limit alcoholic drinks and avoid tobacco to reduce their risk of cancer2 those living with and beyond cancer lwbc are also advised to follow these guidelines due to increasing evidence that healthy behaviours may improve physical strengths and limitations of this study º this study provides an insight in therapeutic radiographers views on all key modifiable health behaviours for those living with and beyond cancer º the participants worked in different radiotherapy departments offering insight into the practices among therapeutic radiographers in the delivery of healthy behaviour advice from a wide range of hospitals º whilst data saturation was reached the sample size was small and therefore the findings may not be representative of the views of the wider therapeutic radiography workforce º the response rate was low therefore the participants might be more interested in the role of health behaviours in cancer survivorship which might bias the responses towards a positive view on the role of therapeutic radiographers in delivering advice within their roleand psychosocial outcomes after a cancer diagnosis2despite the potential benefits of healthy behaviours few people lwbc are meeting the wcrf recommended health behaviour recommendations9 those lwbc report one key reason for not adopting healthier lifestyle behaviours is lack of advice and support from their healthcare team11 healthcare professionals hcps are well placed to bring about positive health behaviour changes among cancer patients12 a trial of brief advice among breast cancer survivors showed that a simple physical activity recommendation from a hcp doubled the percentage meeting national exercise guidelines12 despite this research to date among both hcps and those lwbc consistently shows that few oncology hcps offer guidance to oncology patients on healthy lifestyle behaviours13reported barriers in providing health behaviour advice for those among hcps pallin a0nd et a0al bmj open 202010e039909 101136bmjopen2020039909 0copen access lwbc include believing that giving advice was not part of their role lack of time with patients lack of referral programmes lack of resources such as education leaflets for those lwbc and lack of knowledge regarding guidelines and research findings16 a recent qualitative study with oncology hcps identified that advice on health behaviours provided to those lwbc focussed on general health and controlling side effects with few hcps advising on health behaviours in the context of improving survival outcomes20 while these studies provide useful insight into the practices and barriers among oncology hcps the participants within these studies were primarily oncologists and nurses and focussed on the provision of physical activity and weight management advice there is limited research on the opinions of therapeutic radiographers trs in delivering advice on health behaviours to those lwbc despite at least of cancer patients receiving radiotherapy as part of their cancer treatment22trs are the only health professionals qualified to deliver radiotherapy and play a central role in supporting cancer patients23 in the uk the college of radiographers recognise the importance of trs in providing health behaviour advice to improve patient outcomes24 trs are also seen as an integral part of the health force in driving improvements in well being as outlined in the publication of ahps into action using allied health professionals to transform health care and well being which states that radiographers are key to implementing a preventative healthcare approach and that their expertise should be used to design and deliver health interventions25 trs are ideally placed to deliver health behaviour advice particularly through making every contact count mecc26 mecc is a strategy whereby health professionals use every appropriate opportunity and interaction with patients to promote healthy behaviours and signpost to relevant healthcare services using an ask advise act framework27 mecc fits extremely well within the trs role in which patient education is a key part of radiotherapy practice with trs providing care to the same patient every day often over a number of weeks23 trs therefore have the potential to make significant contributions in supporting positive health behaviour changes among those lwbchowever despite these opportunities one survey in the uk among trs identified that trs rarely advise patients on the key modifiable health behaviours including smoking alcohol healthy eating and exercise15 the findings also showed lack of knowledge and training as barriers among trs in delivering advice on these topics15 similarly focus group interviews with trs identified that lack of knowledge and training were barriers to the provision of smoking cessation advice28challenges remain in translating behaviour change interventions into existing care pathways and practices in a way that is appropriate for use by health professionals29 understanding trs practices and what support they need in delivering advice on the topics of physical activity healthy eating alcohol intake smoking and weight management could inform the development of interventions that will enable trs in delivering advice on improving health behaviours to those lwbc qualitative research is appropriate for exploring the beliefs experiences and motivations of individuals on specific matters and allow for more information and clarification30 limited qualitative data exists on trs practices and views on delivering advice on these health behaviour topics this study therefore aimed to address this and through a qualitative methodology explore trs practices in delivering health behaviour advice in addition to exploring the facilitators in delivering such advice preferences regarding training on delivering this advice were also exploredmethodsparticipants and recruitmentparticipants were trs working in the uk in a clinical role they had provided their contact details on a previous online survey investigating trs practices in delivering health behaviour advice agreeing to be invited for a follow up telephone interview an email was sent with an information sheet explaining the research and inviting these trs those who agreed to take part signed a consent form prior to the telephone interviewdata collectionsemi structured individual telephone interviews were carried out between april and may by a lecturer in therapeutic radiography with an msc who had completed qualitative interviewing as part of their training np the interviewer had no previous relationship with the study participants the topic guide see online supplementary material was based on the guide used within a previous study17 which explored oncology hcps views on the provision of lifestyle advice to cancer patients this guide was adapted for use among trs with additional questions added to assess preferences for training on delivering advice the topic guide was piloted with two participants to check for comprehension of the questions this data was included in the analysis because no substantial changes were required the interviews lasted approximately min range to min and were audio recorded anonymised and transcribed verbatim the transcripts were verified by np against each recording to confirm accuracy the aim was to carry out interviews until data saturation was reached it was anticipated that participants would be required to reach data saturation because it was a homogeneous group31 after interviews were carried out they were transcribed verbatim following familiarisation with the data np generated the initial codes and it appeared that saturation was reached after interviews as no new codes occurred in the 10th interview32 a further five interviews were carried out to confirm thispallin a0nd et a0al bmj open 202010e039909 101136bmjopen2020039909 0ctable participant identifier and demographic characteristicsparticipant identifierdemographic characteristicsprofessional gradegendertr tr tr tr tr tr tr tr tr tr tr tr tr tr tr femalefemalefemalefemalefemalefemalefemalemalefemalefemalefemalefemalemalefemalemaleband band band band band band band band band band band band band band band tr therapeutic radiographerpatient and public involvementpatient input was not used in the design of the research methods however the topic guide was piloted with trs in the academic setting additionally the topic guide was piloted with two participants and these were included in the analysisanalysisthe interview transcripts were analysed using the framework analysis method33 this method was chosen because it is an appropriate method for analysing homogeneous data and semi structured interview transcripts it is also appropriate when using inductive qualitative analysis33 a random selection of transcripts n3 were independently coded by af to check for reliability the researchers np af and rjb met and agreed on a final coding list in an iterative process af and rjb are both experienced qualitative researchers and health psychologists these agreed set of codes formed the analytical framework which was then applied to all of the transcripts and the data summarised in a matrix using microsoft excel themes were generated by reviewing the matrix connecting the data between the participants and the codes the completed consolidated criteria for reporting qualitative research checklist is available in the online supplementary material themes are presented in the results with supporting quotes and the participants identifier table resultsparticipantsthe radiotherapy radiography workforce census in the uk only reports the workforces professional grade and no other demographics35 in the uk trs level of open accessprofessional skills and knowledge are categorised by agenda for change professional band grades to therefore in this study the participants gender and professional grade were collected no other demographic information was collected table the response rate to taking part in the interview was seventy two trs were emailed and invited to take part in an interview returned consent forms and completed the telephone interview fifteen interviews were conducted with women and men the participants came from all regions of the uk including england wales scotland and northern irelandthemesfive main themes were identified trs provide behaviour change advice to manage radiotherapy related side effects trs make judgements about when it is appropriate to deliver health behaviour advice knowledge and training are key facilitators in the delivery of health behaviour advice trs feel patients undergoing radiotherapy treatment seek guidance on health behaviours and trs identify themselves as well placed to give health behaviour advice to patientstrs provide behaviour change advice to manage radiotherapyrelated side effectsmost respondents reported that they only provided advice on health behaviours that they believed would minimise radiotherapy related side effects this meant smoking cessation and alcohol intake were the two health behaviours trs mainly advised onwith head and neck patients we give advice particularly on smoking and drinking obviously get worse side effects tr the only thing we do generally say is about drinking plenty of fluids avoiding alcohol but thats more to do with prostate side effects bladder reactions and reducing gas tr radiographers are comfortable talking about alcohol when it comes to managing side effects tr no trs reported advising patients on healthy eating some trs mentioned advising patients on dietary intake but this is to patients who are at risk of losing weight for side effect management and potential impact on accuracy of radiotherapy treatment deliveryhealthy eating i dont tend to discuss too much a lot of patients have difficulty eating and we are encouraging maintaining weight while on treatment tr im not very sure if healthy eating is important any patients where were treating lower gi or pelvis we would advise them to avoid very high fibre foods spicy foods that might make them have very loose bowels but other than that we say more or less keep on your same diet we wouldnt generally discuss a healthy diet as a standard for all patients no tr pallin a0nd et a0al bmj open 202010e039909 101136bmjopen2020039909 0copen access some trs mentioned they advise patients to be physically active however this was only in the context of managing radiotherapy and cancer related fatigueso exercise is one of my main ones that i focus on with all patients particularly to help with their fatigue tr exercise i say thats its quite beneficial to help with fatigue tr i guess when we have patients come in fatigue is one of the side effects so we encourage our patients to remain active tr trs make judgements about when it is appropriate to deliver health behaviour advicetrs explained only discussing health behaviours particularly smoking and alcohol with patients if there were evident indications of a problem trs also often reported making a judgement of whether appropriate to advise a patient on a particular health behaviourso quite often you can tell if a patient is a smoker you can smell it or you can tell by their skin tr i tend to give advice when you make a judgement of when its appropriate an example might be if a patient smelt of smoke tr had patients come in and will smell of alcohol and at that time ill say to the patient that it can exacerbate side effects tr this meant trs did not provide advice on health behaviours to every patientbut for those patients where its not clearly going to benefit them to stop drinking you would just mention it very briefly not every patient will have that information tr knowledge and training are keys facilitator in the delivery of health behaviour advicedelivery of advice matched by knowledgethe reported delivery of advice on health behaviours appeared to be matched by knowledge of the benefits among those lwbcone participant explained how he only appreciated the importance of physical activity in cancer survivorship after attending a talk and being made aware of the evidencemy experience of appreciating the role exercise was from attending a talk i suppose it was really just highlighting in the studies the benefits obviously of a healthy lifestyle and introducing physical activity for patients on treatment tr healthy eating was a topic the participants felt particularly unqualified to deliver advice on and reported lack of knowledge as a barrier to the delivery of advice on healthy eatingits a difficult one diet i think its more a knowledge thing if you dont have the knowledge about what you can and cant say youre just not going to approach the subject tr a need for continuous postgraduate online trainingall interviewed said they would welcome postgraduate training on delivering health behaviour advice the majority expressed a preference for online training to help overcome the barrier of limited time among trs to attend trainingonline youre not having to take time out of clinical practice online is more accessible tr participants also mentioned that online training allows for yearly updates and continuous professional developmenti think itd be good online training because you can do it in your own time because i think thats sometimes the problem you have this training once and then maybe it never gets brought up again so it would be quite handy to have something small every year alongside all your other mandatory training tr participants did acknowledge that face to face training allows for further questioning thats not possible with online trainingi think one to one training because you can ask questions that may not be covered within the online training tr to overcome the barrier of not all staff being able attend face to face training participants suggested it would be useful to train some trs through face to face methods that they could then cascade to other trs within the radiotherapy departmentmaybe some face to face with some staff that they could cascade down might be useful as well tr a need for training in the undergraduate settingit was also suggested to incorporate training on delivering lifestyle advice into the undergraduate education programmecertainly get it into the undergraduate course to start with making them aware it is part of the role tr its still not something that i can say was primarily covered in the undergraduates training about the benefits of healthy lifestyle you know theres no real formal education that i can see tr trs reported knowledge of resources and referral pathways as facilitators in the delivery of adviceparticipants also felt knowledge of how to refer patients onto further support would enable them to have conversations on improving health behaviours with some pallin a0nd et a0al bmj open 202010e039909 101136bmjopen2020039909 0ctrs reporting that lack of knowledge of resources and referral pathways are barriers to initiating a conversation on behaviour changethere needs to be more information available to professionals of where exactly you can refer patients to whether that be website whether that be an app tr thats the only reason why they [therapeutic radiographers] dont want to open these conversations up because they dont know where to go with it or how to refer on tr they also acknowledge that in the short time they have with patients if they had a resource then would be more inclined to advisehaving something on a piece of paper education and having the resources if you can do it in min you should be able to slip that in tr you dont always have that information at hand so if it was readily available i think wed give out a bit more [health behaviour information] if it was just the case of pointing them in the right direction that would be a quick and easy thing to do tr the benefit of incorporating patients perspective into trainingparticipants also mentioned that getting patients perspectives on receiving advice on improving health behaviours should be incorporated into trainingi think that would be better coming from the patients themselves rather than just feedback from what journals and other literature says tr if thered even be patients that would be willing to maybe just even be involved with staff training tr trs feel patients undergoing radiotherapy treatment seek guidance on health behavioursmany of the trs also described that patients often ask them for guidance around health behaviour changes particularly on diet and exercise this shows that patients see trs as credible sources of information on health behaviourswe are getting asked the question more and more about weight loss healthy living wanting to exercise more tr it is quite a common thing to be asked at the end of treatment not so much the smoking and alcohol i have to say but diet and exercise is certainly something that people commonly ask tr trs identify themselves as well placed to give health behaviour advice to patientstrs acknowledged that they are a consistent healthcare member for patients undergoing radiotherapy and have many opportunities to deliver lifestyle advice therefore open accesstrs recognised that they are well placed to deliver health behaviour advice to patientswere in a unique position because we do see the same patient day after day and you do kind of start to develop a relationship with them tr i think were well placed to help influence patients behaviours and its something we should be seen to encourage and report tr were in the best position where we see the patients for a number of weeks every day to encourage any changes tr from the interviews it appeared that many patients undergoing radiotherapy excluding those at risk of malnutrition or significant weight loss are primarily reviewed and assessed by trs this highlights that trs are in an ideal position to deliver advice on health behaviours particularly when asked about nutrition advice deliverythey routinely see the specialist radiographer for the breast patients but they dont have a dietitian appointment tr prostate and breast are two tumour groups that are fully radiographer led review and about to of our work load they generally wouldnt be sent to a dietician tr only have a dietitian on board for the head and necks tr discussiontrs in this study saw themselves as well placed to deliver health behaviour advice but also reported that they do not routinely provide advice to all patients trs were particularly unlikely to provide advice on healthy eating and physical activity and were more likely to provide advice on those behaviours they believed would minimise radiotherapy or cancer related side effects this is in line with previous research among trs15 in one qualitative study a key facilitator reported among trs in delivering smoking cessation support to patients was knowledge of the link between smoking and toxicity28 another qualitative study that explored allied health professionals views regarding the provision of dietary advice to patients highlighted that trs report giving dietary advice to help counteract the side effects of radiotherapy37 additionally in our study if trs did provide dietary advice this tended to be general advice rather than cancer specific advice on healthy eatingin some studies oncology hcps have reported they do not self identify as the right person to provide lifestyle advice17 however in this study trs identified themselves as being well placed to deliver health behaviour advice and in a unique position as a consistent member of the multidisciplinary team providing care to patients however despite this they do not feel qualified to deliver pallin a0nd et a0al bmj open 202010e039909 101136bmjopen2020039909 0copen access advice particularly on the topic of healthy eating in the uk poor diet has the biggest impact on the national health service budget greater than alcohol consumption smoking and physical inactivity38 it has been noted that there are insufficient dietitians to provide dietary advice to all patients who may need dietary support39 in response to this all hcps are being asked to implement a preventative healthcare approach within their role and the delivery of healthy eating advice is fundamental to this23 key to achieving this is that trs will have the skills knowledge and behaviours to improve the health and well being of individuals24 as with other oncology hcp groups16 this study identifies the need for education and training among trs in delivering health behaviour advice particularly on healthy eating and physical activity this training should also address when and how to refer to other support if necessary as this was identified as a key facilitator in the delivery of advice on health behaviours particularly when time is a barrier to the delivery of this advice15all interviews demonstrated that trs would welcome training on delivering health behaviour advice and recommended it as a key facilitator in delivering advice in addition to incorporating it into the undergraduate setting the need for postgraduate training among trs in delivering health advice has also recently been reported by charlesworth et al28 in relation to the delivery of smoking cessation advice our findings from this study provide additional insight into trs preferences on the type of training on delivering lifestyle advice to those lwbc with trs demonstrating a preference of online training in the postgraduate setting among hcps online education has been reported to be as effective as face to face education42 additionally the use of online learning enables hcps to carry out training at time that fits in with clinical work43 trs in this study identified this benefit of online learning in overcoming the limited time available for trs to undertake continuous professional development and additional training interestingly trs in this study mentioned having patient input in the training would be helpful while hcps input is key to the development of interventions patient members play key advocacy roles and their input can enhance the outcomes of interventions45 patient input may also help overcome the reported barrier of fear of causing offence to a patient which has been reported as a barrier among oncology hcps in delivery of health behaviour advice17those lwbc wish to receive advice on health behaviours from their healthcare team13 and is of particular importance as the period following a cancer diagnosis has been shown be a teachable moment and an ideal opportunity to motivate patients around the importance of healthy eating and physical activity47 this was made apparent in this study whereby some trs mentioned that healthy eating and exercise were the health behaviours patients ask for advice on more often generally towards the end of their treatment this further highlights the importance of supporting trs in delivering evidence based health behaviour advice to meet patients needstrs have a responsibility to educate patients on the importance of following healthy behaviours given the increasing evidence showing implementing healthy behaviours improve a number of physical and psychosocial outcomes after a cancer diagnosis2 among pre menopausal and post menopausal women living with and beyond breast cancer a systematic literature review and meta analysis of follow up studies n213 breast cancer survivors identified that being overweight increases the risk of all cause and breast cancer mortality4 being physically active after a cancer diagnosis is also correlated with improved survival and reduced recurrence5 while data is limited emerging research suggests healthy dietary behaviours after a diagnosis may improve outcomes3 in a prospective observational study of patients with stage iii colon cancer a higher intake of a typical western diet was associated with a threefold increased risk of disease recurrence and a fold increased risk of all cause mortality8 additionally those lwbc are at increased risk for developing cardiovascular disease osteoporosis and diabetes and healthy behaviours can reduce the risk of developing these diseases51 of those interviewed in this study it appeared that those with breast prostate and colorectal cancer are primarily reviewed and assessed by trs therefore it is the responsibility of trs to deliver advice on improving health behaviours to these patients this is also particularly important because the strongest evidence for the benefits of diet and exercise is currently in breast prostate and colorectal cancer survivors53 these are also the most common cancers in the uk and radiotherapy plays a key role in managing these cancers22 therefore with the right skills and knowledge trs could deliver advice on improving health behaviours by supporting self efficacy among patients towards the end of their treatment which very often is in the radiotherapy department can be empowering for patients among those with prostate cancer implementing dietary changes brought psychological benefit as a method of coping and regaining control over their diagnosis46strengths and limitationsthis is the first qualitative study among trs to explore the provision of advice on all key modifiable lifestyle behaviours for those lwbc as per recommendations2 while the aim of qualitative research is not to generalise the findings the sample size was small and therefore the findings may not be representative of the views of the wider therapeutic radiography workforce however data saturation was reached likely due the homogeneous sample of participants additionally the participants worked in different radiotherapy departments and therefore provide insight into the practices among trs in the delivery of healthy behaviour advice from a wide range of hospitals also the participants worked in cancer centres in england wales scotland and northern pallin a0nd et a0al bmj open 202010e039909 101136bmjopen2020039909 0cireland providing insight into the practices across the uk another limitation of this study is the low response rate and that the participants might be more interested in the role of health behaviours in cancer survivorship which might bias the responses towards a positive view on this topic and the role of trs in delivering advice within their role despite this however provision of health behaviour change advice was low suggesting trs may be even less likely to educate patients around the importance of healthy behavioursfuture researchthis study highlights the need for training and education among trs on the delivery of health behaviour advice to cancer patients both in the undergraduate and postgraduate setting particularly on the topics of physical activity healthy eating and weight management higher education institutions have a responsibility in educating the allied health professional workforce on implementing health promotion within their role55 further research among pre registration tr students and lecturers within therapeutic radiography should therefore explore how best to address this need future research among trs should also use purposive sampling to identify the views and health promotion practices among those who may not have a primary interest in the area of health | Colon_Cancer |
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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 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" 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 |
" micrornas mirnas have been reported to have important regulatory roles in the progression of several types of cancer including cervical cancer cc however the biological roles and regulatory mechanisms of mirnas in cc remain to be fully elucidated the aim of the present study was to examine the functions of mirnas in cc and the possible mechanisms using a microarray it was identified that mirna15a5p mir15a5p was one of the most downregulated mirnas in cc tissues compared with adjacent noncancerous tissues the low expression of mir15a5p was observed in cc tumor tissues with distant metastasis and in cc cell lines in addition the effects of mir15a5p upregulation on cell viability apoptosis invasion and migration of cc cells were investigated using cck flow cytometry transwell and wound healing assays respectively it was demonstrated that upregulation of mir15a5p significantly suppressed the viability migration and invasion and promoted the apoptosis of siha and c33a cells furthermore yesassociated protein yap1 a wellknown oncogene was confirmed to be directly targeted by mir15a5p and was found to be negatively regulated by mir15a5p further correlation analysis indicated that mir15a5p expression was negatively correlated with yap1 expression in cc tissues notably overexpression of yap1 abrogated the tumor suppressive effects of mir15a5p in cc cells taken together these present findings indicated that the mir15a5pyap1 axis may provide a novel strategy for the clinical treatment of cccorrespondence to professor xu chen department of obstetrics and gynaecology huashan hospital north fudan university jingpohu road baoshan shanghai pr chinaemail xuchenccx163comcontributed equallykey words cervical cancer microrna15a5p cell viability migration invasion yesassociated protein introductioncervical cancer cc is a type of malignant tumor commonly presenting in women in cc cases are diagnosed each year and it accounts for of all female cancerassociated mortalities each year worldwide despite advances in the therapeutic strategies for cc including targeted therapies and immunotherapy the prognosis of cc remains poor due to the abnormal growth of epithelial cells thus it is imperative to clarify the molecular interactions occurring during the initiation and progression of ccmicrornas mirnas are a family of short noncoding rnas with an average length of nucleotides which negatively regulate target gene expression through either translation repression or rna degradation accumulating evidence has indicated that mirnas may function as oncogenes or tumor suppressors depending on their target mrna in various types of cancer including cc for example yang reported that mir214 inhibits the growth of cc cells by the regulation of its target enhancer of zeste homolog dong demonstrated a suppressive role of mir217 in the development of cc cells via targeting rhoassociated protein kinase chen reported that mir499a promotes the proliferation cell cycle progression colony formation migration and invasion of cc cells by targeting srybox transcription factor in addition several mirnas serve as diagnostic biomarkers in patients with cc such as mir152 and mir365 despite the aforementioned findings the roles of mirnas in the development of cc require further investigationin the present study a mirna microarray was performed to investigate the expression profiles of mirnas in cc tissues and the most downregulated mirna identified mir15a5p was selected for further analysis the potential role and underlying mechanism of mir15a5p in cc cells were also investigated the present results suggest that mir15a5p may serve as a therapeutic target for ccmaterials and methodspatients and samples in total paired cervical samples tumor tissues and adjacent noncancerous tissues were 0cchen mir15a inhibits cervical cancer cell growthobtained from female patients with cc who underwent cervical surgical resection without preoperative systemic therapy at the department of obstetrics and gynecology huashan hospital north of fudan university shanghai china between may and december the median age of the patients was years range years among all patients there were patients with metastatic cc and with nonmetastatic cc the matched nontumor adjacent tissue was obtained cm beyond the boundary of cc tissue all tissue samples were immediately snapfrozen in liquid nitrogen and stored at Ëc until use the experimental protocols were approved by the ethics committee of huashan hospital north of fudan university written informed consent for participation in the study was obtained from all patientsmirna expression profiling total rna from cc tissues three randomly selected paired tumor tissues and adjacent noncancerous tissues was extracted using mirneasy mini kit qiagen gmbh the samples were assessed using the mircury lna¢ array v180 agilent technologies inc the procedure and imaging processes were performed as described previously cell culture human cc cell lines hela c33a caski and siha 293t cells and normal cervical epithelial cells ect1e6e7 were obtained from the american type culture collection all cells were cultured in dmem sigmaaldrich merck kgaa supplemented with vv fbs sigmaaldrich merck kgaa plus uml penicillinstreptomycin at Ëc with co2reverse transcriptionquantitative pcr rtqpcr total rna was extracted from tissues or cell lines using trizol reagent invitrogen thermo fisher scientific inc for mirna rt cdna was generated from ng total rna samples using taqman¢ microrna reverse transcription kit applied biosystems thermo fisher scientific inc at Ëc for min for mrna rt cdna was synthesized using primescript rt reagent kit takara bio inc at Ëc for min qpcr for mirna and mrna was performed using the sybrgreen i realtime pcr kit applied biosystems thermo fisher scientific inc on an abi system applied biosystems thermo fisher scientific inc the reaction was performed under the following conditions Ëc for min followed by cycles at Ëc for sec and Ëc for sec and a final extension at Ëc for sec the primers for qpcr analysis were as follows mir15a5p forward 'aat gtt gcc cgt aat gcc3' and reverse 'ccc aag cgg aga aag gaa3' u6 forward 'gct tcg gca gca cat ata cta aaa t3' and reverse 'cgc ttc acg aat ttg cgt gtc at3' yesassociated protein yap1 forward 'cgg tcc act tca gtc tcc3' and reverse 'gag tgt ggt gga cag gta ctg3' and gapdh forward 'gtg gtg aag acg cca gtg ga3' and reverse 'cga gcc aca tcg ctc aga ca3' the expression levels of mir15a5p and yap1 were normalized to the expression of u6 and gapdh respectively the relative expression of each gene was calculated using the cq method cell transfection the mir15a5p mimic mimic negative control nc mir15a5p inhibitor inhibitor nc yap1 overexpression plasmid pcdnayap1 and pcdnavector were all provided by guangzhou ribobio co ltd when c33a and siha cells 5x105 cellswell in 6well plates grew to confluence mir15a5p mimic nm mimic nc nm mir15a5p inhibitor nm inhibitor nc nm pcdnayap1 µg or pcdnavector µg were transfected into cells at Ëc for h using lipofectamine® invitrogen thermo fisher scientific inc the sequences were as follows mir15a5p mimic 'uag cag cac aua aug guu ugu g3' mimic nc 'uuc ucc gaa cgu guc acg utt3' mir15a5p inhibitor 'cac aaa cca uua ugu gcu gcu a3' and inhibitor nc 'cag uac uuu ugu gua gua caa3'in addition small interfering rna targeting yap1 siyap1 and the negative control targeting a nonspecific sequence siscramble were provided by thermo fisher scientific inc siha and c33a cells were transfected with the sirnas nmoll using lipofectamine invitrogen thermo fisher scientific inc the sequences of siyap1 and siscramble were as follows siyap1 'ctc agg atg gag aaa ttt a3' and siscramble 'ttc tcc gaa cgt gtc acg t3' at h posttransfection the cells were harvested for further analysis and the inhibition efficiency was determined by western blottingcell viability the c33a and siha cells were seeded in 96well plates at a density of 5x103well overnight following transfection the cell viability was measured using a cck8 assay briefly µl cck solution was added to each well and cultured for h at Ëc the absorbance of the samples at nm was detected using a microplate reader biorad laboratories inccaspase activity following transfection c33a and siha cells were harvested and the caspase3 activity was measured using a caspase3 activity assay kit beyotime institute of biotechnology according to the manufacturer's protocolcell apoptosis the apoptosis of c33a and siha cells was examined using flow cytometry following transfection c33a and siha cells were collected and the apoptotic cells were identified using an annexin vfitc apoptosis detection kit abcam according to the manufacturer's protocol after washing with cold pbs the cells were resuspended in binding buffer followed by staining with annexin v and propidium iodide for min in the dark at room temperature the fluorescence was measured using a facscan flow cytometer beckman coulter inc and then analyzed by flowjo v871 software flowjo llcimmunofluorescence assay following transfection c33a and siha cells were fixed in absolute ethyl alcohol for min at room temperature after washing twice with pbs the fixed cells were stained with primary antibody targeting cleavedcaspase3 cat no c ell signaling technology inc for h at room temperature subsequently an antirabbit conjugated antibody with fitc cat no f0382 sigmaaldrich merck kgaa was added for h in the 0cinternational journal of molecular medicine dark fluorescence images were obtained using an inverted fluorescence microscope magnification x200cell invasion assays transwell chambers 8µm pore bd biosciences coated with matrigel bd biosciences were used for the invasion assay briefly c33a and siha cells 8x104 were seeded in the top chamber with serumfree medium while the lower chamber contained culture medium with fbs following incubation for h the cells were fixed in paraformaldehyde solution beyotime institute of biotechnology for min and stained with crystal violet beyotime institute of biotechnology for min at room temperature images were captured with an inverted microscope olympus corporation magnification x100wound healing assay for the wound healing assay c33a and siha cells were seeded onto 12well plates 2x105 cellswell and h after transfection a scratch was made using a 10µl pipette tip in the confluent cell monolayer then cells were washed twice with pbs and incubated in dmem without fbs the wound healing images were captured at and h after scratching using an inverted light microscope olympus corporation magnification x100 the wound healing rate was calculated using imagej software v146 national institutes of healthdualluciferase reporter assay mirna target prediction tools including miranda httpmirandaorguk and targetscan httptargetscanorg were used to search for the putative targets of mir15a5p pgl3yap1 widetype or pgl3yap1 mutant type pgl3yap1mut promega corporation were cotransfected with mir15a5p mimics into 293t cells in 24well plates 2x105well using lipofectamine invitrogen thermo fisher scientific inc at h posttransfection the luciferase activities were analyzed using the dualluciferase reporter assay system promega corporation with renilla luciferase activity as an internal control western blot analysis western blotting was performed as previously described briefly cells were lysed using radio immunoprecipitation assay buffer beyotime institute of biotechnology and the protein concentration was determined using the bicinchoninic acid assay total protein µglane was separated by sdspage and electrophoretically transferred onto a polyvinylidene difluoride membrane emd millipore subsequently membranes were blocked with skim milk for h at Ëc overnight each membrane was probed with primary antibodies against yap1 cat no and βactin cat no at Ëc overnight all primary antibodies were obtained from cell signaling technology inc subsequently the membrane was incubated with horseradish peroxidaseconjugated goat antirabbit igg cat no abcam at room temperature for h βactin served as the loading control and for normalization of protein expression the protein bands were developed using ecl kit ge healthcare and expression levels were quantified using imagej v146 national institutes of healthstatistical analysis all data are presented as mean ± standard deviation the correlation between mir15a5p and yap1 levels was evaluated using spearman's correlation analysis pairwise comparisons were performed by student's ttest and comparisons among groups were analyzed by oneway anova followed by tukey's posthoc test p005 was considered to indicate a statistically significant differenceresultsmir15a5p is downregulated in cc to examine the potential involvement of mirnas in the development of cc microarray analysis was performed to evaluate the mirna expression profiles between cc tissues and adjacent noncancerous tissues of differently expressed mirnas identified in the tumor group mirnas exhibited decreased expression and mirnas demonstrated increased expression compared with that in adjacent noncancerous tissues fig 1a among the aberrant mirnas the present study focused on mir15a5p for subsequent experiments due to its suppressive role in a variety of other cancer types such as endometrial cancer and chronic myeloid leukemia subsequently rtqpcr was performed to detect the expression of mir15a5p in pairs of tumor tissues and adjacent noncancerous tissues the results revealed that the level of mir15a5p was significantly lower in tumor tissues compared with that in adjacent noncancerous tissues fig 1b it was also observed that mir15a5p was expressed at a significantly lower level in tumor tissues with distant metastasis compared with in tumors tissues without distant metastasis fig 1c indicating that mir15a5p downregulation is associated with cc metastasis in addition rtqpcr was used to examine the mir15a5p level in four cc cell lines hela c33a caski and siha and the normal cervical epithelial cell line ect1e6e7 which was used as a control as expected mir15a5p was significantly lower in the four cc cell lines compared with ect1e6e7 cells fig 1d siha and c33a cells were selected for further experiments as they demonstrated the lowest expression of mir15a5p among all cell lines examinedupregulation of mir15a5p inhibits cell viability and promotes cell apoptosis in an attempt to understand the biological function of mir15a5p mir15a5p expression was upregulated or downregulated in the cultured siha and c33a cells by transfection with mir15a5p mimic or inhibitor respectively mir15a5p expression was significantly increased after mir15a5p mimic transfection whereas it was significantly decreased following mir15a5p inhibitor transfection in both siha and c33a cells fig 2a the present study then investigated the effect of mir15a5p expression on cell viability and the results demonstrated that the viability of siha and c33a cells was significantly inhibited by overexpression of mir15a5p whereas it was significantly enhanced by knockdown of mir15a5p compared with the negative control group fig 2b and c to assess the effects of mir15a5p upregulation on the apoptosis of siha and c33a cells caspase3 expression level and activity were analyzed by immunofluorescence and caspase activity assays respectively as presented in fig 2d and e the expression of cleaved caspase3 and caspase3 activity was increased in siha and c33a cells transfected with 0cchen mir15a inhibits cervical cancer cell growthfigure mir15a5p is downregulated in cc tissues and cell lines a heat map presents significant differentially expressed mirnas in cc tissues and matched adjacent noncancerous tissues n3 green indicates downregulation and red indicates upregulation b mir15a5p expression was measured by rtqpcr in pairs of cc tissues and matched adjacent noncancerous tissues c mir15a5p expression was measured in tumor tissues with distant metastasis and tumors tissues without distant metastasis by rtqpcr d mir15a5p expression was detected in four cervical cancer cell lines hela c33a caski and siha and the normal cervical epithelial cells ect1e6e7 data are expressed at the mean ± standard deviation n3 of one representative experiment p001 vs ect1e6e7 cells mir microrna cc cervical cancer rtqpcr reverse transcriptionquantitative pcr mir15a5p mimic compared with the mimic nc groups furthermore the results of flow cytometry demonstrated that the extent of apoptosis was significantly increased after mir15a5p mimic transfection compared with the mimic nc groups fig 2f taken together these results indicate that overexpression of mir15a5p inhibits cell viability by inducing cell apoptosisupregulation of mir15a5p inhibits the invasion and migration of cc cells the present study further investigated whether overexpression of mir15a5p could reduce the invasiveness and migratory potential of cc cells using a transwell assay it was identified that the invasive capacities of siha and c33a cells were significantly inhibited by mir15a5p mimic whereas they were increased by mir15a5p inhibitor compared with the nc groups furthermore the wound healing assay results also demonstrated a significant reduction of cell migration in siha and c33a cells following mir15a5p overexpression however the migration of siha and c33a cells was significantly enhanced by mir15a5p inhibition fig 3c and d collectively the present data suggest that overexpression of mir15a5p suppresses the invasive and migratory abilities of cc cellsyap1 is a direct target of mir15a5p using the targetscan and miranda algorithms yap1 was found to have a putative target site of mir15a5p in its 'utr fig 4a to validate the possibility that yap1 is a direct target gene of mir15a5p a luciferase reporter assay was then performed the data revealed that mir15a5p mimic significantly inhibited the luciferase activity in the constructs containing the wildtype 0cinternational journal of molecular medicine figure overexpression of mir15a5p suppresses cell viability and promotes cell apoptosis siha and c33a cells were transfected with the mir15a5p mimic or inhibitor for h and then cells were used for analysis a transfection efficiency was assessed by reverse transcriptionquantitative pcr cell viability was measured by cck8 assay at indicated times for b siha and c c33a cells d the expression of cleaved caspase3 was determined by immunofluorescence assay magnification x200 e the caspase activity was detected by a commercial caspase activity kit f cell apoptosis was measured by flow cytometry data are expressed at the mean ± standard deviation n3 of one representative experiment p005 p001 vs mimic nc p005 p001 vs inhibitor nc mir microrna nc negative control od optical density pi propidium iodidebinding site of yap13'utr while it had no evident effects on the activity of yap13'utrmut by contrast mir15a5p inhibitor significantly increased luciferase activity without any evident effects on yap13'utrmut activity fig 4b subsequently to further detect the potential regulation of yap1 by mir15a5p the expression of yap1 protein was measured in cc cells by western blotting as presented in fig 4c the expression of yap1 was significantly decreased upon ectopic expression of mir15a5p suggesting that high expression of yap1 was partly due to the downregulation of mir15a5p in cc cells in addition it was identified that the mrna level of yap1 was significantly increased in cervical cancer compared with the control and inversely correlated with mir15a5p expression levels in cancer tissues fig 4d and e these results indicated that yap1 is a downstream gene of mir15a5p in cc 0cchen mir15a inhibits cervical cancer cell growthfigure overexpression of mir15a5p suppresses cell invasion and migration siha and c33a cells were transfected with the mir15a5p mimic or inhibitor for h and then cells were used for analysis invasion of a siha and b c33a cells was measured by a transwell assay magnification x200 the migration of c siha and d c33a cells was assessed by a wound healing assay the images were taken at and h after gaps were generated wound healing was quantified by the distance of the wounded region with an absence of cells data are expressed at the mean ± standard deviation n3 of one representative experiment p001 vs mimics nc p001 vs inhibitor nc mir microrna nc negative controlyap1 inhibition suppresses cell viability promotes cell apoptosis and inhibits invasion and migration previous evidence has shown that yap1 exerts an oncogenic function in several types of human cancer such as breast and lung cancer as the findings of the present study revealed that yap1 is upregulated in cc it was hypothesized that yap1 may act as an oncogenic gene in cc to confirm this hypothesis siha and c33a cells were transfected with siyap1 or siscramble western blot assay revealed that yap1 was notably downregulated following transfection with siyap1 fig 5a functionally yap1knockdown significantly suppressed the cell viability and induced cell apoptosis compared with the siscramble group fig 5b and c furthermore knockdown of yap1 significantly suppressed the invasive and migratory abilities of siha and c33a cells fig 5d and e suggesting that yap1 may play an oncogene role in the development of ccoverexpression of yap1 moderates the negative functions of mir15a5p on cell viability migration and invasion to ascertain whether yap1 is involved in the inhibitory effects of mir15a5p on cc cells the present study cotransfected pcdnayap1 andor mir15a5p mimic as well as their controls into siha and c33a cells the overexpression efficiency was verified by western blotting as shown in fig 6a yap1 was notably increased in siha and c33a cells after pcdnayap1 transfection subsequently the cell viability apoptosis invasion and migration were evaluated overexpression of yap1 significantly abolished the inhibitory effects of mir15a5p upregulation on the viability of siha and c33a cells fig 6b the increased apoptosis induced by mir15a5p overexpression was also reversed by overexpression of yap1 fig 6c furthermore overexpression of yap1 significantly reversed the inhibitory effects of mir15a5p on cell invasion and migration fig 6d and e in addition it was identified that overexpression of yap1 alone significantly promoted cc cell viability inhibited cell apoptosis and enhanced the invasion and migration compared with blank control group suggesting the oncogenic role of yap1 in cc cells these results indicate that mir15a5p exerts its tumor suppressive role in cc at least partially through yap1 0cinternational journal of molecular medicine figure yap1 is a direct target of mir15a5p a schematic of the yap1 'utr containing the mir15a5p binding sites b luciferase assay of 293t cells cotransfected with firefly luciferase constructs containing the yap1 wt or mut 'utrs and mir15a5p mimics mimics nc mir15a5p inhibitor or inhibitor nc as indicated n3 p001 c siha and c33a cells were transfected with the mir15a5p mimic and mimic nc for h and the expression levels of yap1 protein were determined by western blotting p001 vs mimic nc d yap1 expression was measured by reverse transcriptionquantitative pcr in cc tissues and matched adjacent noncancerous tissues n40 p001 e spearman's analysis was used to analyze the correlation between the expression of yap1 and the expression of mir15a5p expression in cervical cancer tissues r p001 data are expressed at the mean ± standard deviation n3 of one representative experiment yap1 yesassociated protein mir microrna 'utr 'untranslated region wt wildtype mut mutant nc negative controldiscussionin the present study mir15a5p was shown to be decreased in cc tissues and cell lines and associated with cc metastasis furthermore overexpression of mir15a5p inhibited the cc cell viability invasion and migration and promoted cell apoptosis while inhibition of mir15a5p demonstrated the opposite effects additionally yap1 was confirmed as a functional target of mir15a5p ectopic expression of which significantly reversed suppression of mir15a5p the present data indicated that mir15a5p may function as a tumor suppressor in cc progression by inhibiting yap1 expressiona number of studies have shown that mirnas participate in the development of cc for example xia reported that mir374b overexpression suppresses cell proliferative and invasive abilities via affecting forkhead box m1 expression yao also demonstrated that mir641 upregulation restricts cc cell growth in vitro and in vivo xu reported that mir2185p suppresses the progression of cc via the lynnfκb signaling pathway yuan demonstrated that overexpression of mir138 suppresses cc cell growth in vivo these findings suggest that targeting mirnas may be an effective therapeutic strategy for cc in the present study based on microarray expression data it was identified that mir15a5p is one of the most markedly downregulated mirnas in cc tissues notably previous studies have reported that mir15a5p functions as a tumor suppressor in several human cancer types although mir15a5p has been found to be downregulated in cc to the best of our knowledge the tumorigenic role and mechanism remain unknown therefore the present study focused on mir15a5p in cc for molecular analyses in the 0cchen mir15a inhibits cervical cancer cell growthfigure yap1 inhibition suppresses cell viability promotes cell apoptosis and inhibits invasion and migration siha and c33a cells were transfected with siyap1 or siscramble and then cells were harvested for further study a the expression of yap1 was measured by western blotting b cell viability was measured by cck assay c the cell apoptosis was assessed by flow cytometry d cell invasion was measured by transwell assay e cell migration assessed by a wound healing assay data are expressed at the mean ± standard deviation n3 of one representative experiment p001 vs siscramble yap1 yesassociated protein mir microrna si small interfering rnafigure mir15a5p inhibits cell viability and induces cell apoptosis by targeting yap1 a siha and c33a cells were transfected with the pcdnayap1 plasmid for h and then the protein expression of yap1 was measured by western blotting subsequently siha and c33a cells were cotransfected with the pcdnayap1 plasmid and mir15a5p mimic for h and then cells were used for analysis b viability of siha and c33a cells was measured by cck8 assay at indicated times c the cell apoptosis was assessed by flow cytometry d cell invasion was measured by transwell assay e cell migration was measured by a wound healing assay data are expressed at the mean ± standard deviation n3 of one representative experiment p005 p001 vs blank group p001 mir microrna yap1 yesassociated protein microarray expression data the expression levels of numerous mirnas exhibited significant changes such as mir137 which demonstrated the most significant upregulation in cc tissues miao reported that mir137 upregulation inhibits cc cell invasion migration and epithelialmesenchymal transition by suppressing the tgfβsmad pathway 0cinternational journal of molecular medicine notably mir15a3p has also reported to exhibit differential expression and induce apoptosis in human cc cells although the present study did not detect the expression change of mir15a3p in the microarray expression data the expression of mir15a3p in four cc cell lines was examined and the results demonstrated that mir15a3p was also downregulated in cc cells compared with ect1e6e7 cells data not shown however the role and regulatory mechanisms of mir15a3p on invasion and migration remain unclear the function of more mirnas in cc will be investigated in the futureprevious studies have reported that mir15a5p has the potential to suppress cell growth and inhibit the progression of human cancers by regulating its downstream target genes for example luo demonstrated that overexpression of mir15a5p causes cellular growth inhibition and suppression of migration by targeting cyclin e1 in breast cancer wu and guo found that mir15a overexpression suppressed the cell proliferation and invasion by suppression of bmi1 translation in gastric cancer gc as well as pancreatic cancer pc of note several studies have reported aberrant expression of mir15a5p in cc tissues or cells however the role and mechanism of mir15a5p in cc remain largely unknown the present results demonstrated that overexpression of mir15a5p inhibited cell viability cell migration and invasion and induced cell apoptosis in siha and c33a cells while inhibition of mir15a5p demonstrated the opposite effects indicating that mir15a5p may serve as tumor suppressive role in cc yap1 a transcriptional coactivator and oncogene has been found to play an important role in different types of carcinoma for example liu reported that yap1 overexpression promotes the invasion migration and growth of colon cancer cells yu demonstrated that knockdown of yap1 causes a significant inhibition of the growth and migration of renal cell carcinoma cells in vitro and in vivo notably yap1 has been verified to target mir15a5p to suppress cell growth and metastasis in gastric adenocarcinoma and colon cancer however whether yap1 is a target of mir15a5p in cc remains unclear in the present study yap1 was confirmed to be a target of mir15a5p and its protein expression levels were negatively regulated by mir15a5p further investigation indicated that yap1 was significantly increased in cc tissues and inversely correlated with mir15a5p in cc tissues furthermore yap1 was confirmed to act as an oncogene gene in cc cells and its overexpression partly abrogated the inhibitory effect induced by enhanced expression of mir15a5p in cc cells taken together the present study demonstrates that mir15a5p exerts its tumor suppressive role in cc cells by targeting yap1due to the limitation in experimental conditions and funds further research in the future is required to investigate whether mir15a5p serves its role via other downstream targets in addition the present study investigated the cellular function of mir15a5p and its underlying mechanism in cc however in vivo studies and clinical trial data are required to validate the preliminary in vitro results obtained therefore the function of mir15a5p in cc needs to be further investigated in vivoin conclusion the present results demonstrated that mir15a5p suppresses the viability migration and invasion of cc cells by directly targeting yap1 based on these findings it is proposed that the mir15a5pyap1 axis may serve as a novel biomarker for new targets in cc therapyacknowledgementsnot applicablefundingfunding was received from the scientific research project of shanghai science and technology commission grant nos and availability of data and materialsthe datasets used andor analysed during the current study are available from the corresponding author on reasonable request authors' contributionsrc hl tz xy and sx performed the experiments contributed to data analysis and wrote the paper rc hl tz xy and sx analysed the data xc conceptualized the study design and contributed to data analysis and experimental materials all authors read and approved the final manuscriptethics approval and consent to participateall individuals provided written informed consent for the use of human specimens for clinical research the experimental protocols were approved by the ethics committee of huashan hospital north of fudan university patient consent for publicationnot applicablecompeting intereststhe authors declare that they have no competing interests references alldredge jk and tewari ks clinical trials of antiangiogenesis therapy in recurrentpersistent and metastatic cervical cancer oncologist tsikouras p zervoudis s manav b tomara e iatrakis g romanidis c bothou a and galazios g cervical cancer screening diagnosis and staging j buon fang j zhang h and jin s epigenetics and cervical cancer from pathogenesis to therapy tumour biol wang j liu y wang x li j we j wang y song w and zhang z mir1266 promotes cell proliferation migration and invasion in cervical cancer by targeting dab2ip biochim biophys acta mol basis dis zhu l zhu l s | 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 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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 |
" 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 |
methods results and discussion sections yz drafted the methods and results sections of the initial manuscript mvk assisted in manuscript revisions and proof reading all authors provided feedback and insights into the manuscript srt jkv msa yz mvk and sat edited and revised the manuscript and all authors read edited and approved the final version of the manuscriptcompeting interests the authors declare no competing interestsadditional informationsupplementary information is available for this paper at 101038s4159 xcorrespondence and requests for materials should be addressed to jkvreprints and permissions information is available at wwwnaturecomreprintspublishers note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliationsopen access 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 authors scientific reports 101038s4159802071102xvol0123456789wwwnaturecomscientificreports 0c' | Colon_Cancer |
" it is well established that retrieved lymph node rln counts were positively correlated with betteroverall survival in gastric cancer gc but little is known about the relationship between rln count and shorttermcomplications after radical surgerymethods a total of consecutive gc patients between january and december at nanjing drumtower hospital were retrospectively analyzed univariate analyses were performed to elucidate the associationbetween rln count and postoperative complications we further identified clinical factors that might affect the rlncountresults among all of the patients postoperative complications occurred in patients the mean rlncount was and patients were diagnosed with lymph node metastasis univariate analyses showedno significant difference between rln count and postoperative complications both overall and stratified by cdcgrade univariate and multivariate analyses further revealed that type of resection tumor invasion and lymph nodemetastasis were associated with rln counts the current study demonstrated that rln count was not associated with postoperative shorttermcomplications following gastrectomy of gc which provided a rationale for the determination of a proper rlncount of curative gastrectomykeywords retrieved lymph nodes postoperative complications gastric cancer there are approximately one million new cases of gastriccancer gc each year worldwide and half of them occurin eastern asia including china japan and south korea despite advances in early screening and comprehensive treatment of gc it remains the third most commoncause of cancerrelated death in the world for advanced gc a consensus has been reached of radical gastrectomy with d2 lymphadenectomy however there correspondence medguanwenxian163com wangmeng001263net feng sun song liu and peng song contributed equally to this workdepartment of gastrointestinal surgery nanjing drum tower hospital theaffiliated hospital of nanjing university medical school nanjing chinais still controversy over the number of retrieved lymphnodes rlns for accurate pathological stagingseveral studies have reported that rln count waspositively correlated with better overall survival in gceven in lymph nodenegative gc [] an rln countof ¥ has been recommended by the 8th edition tnmclassification for gc to guarantee the accurate pn stage moreover okajima suggested an optimal rlncount of ¥ for nodal staging recently by stratumanalysis of patients deng proposed an optimal rln count of ¥ for lymph nodenegative gc and for lymph nodepositive gc these abovestudies are all conducted by comparing the rln count 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 world of surgical oncology page of table demographic and clinical features of patientscharacteristicsage yearsgender nn ± malefemalebmi kgm2preoperative comorbidities nprevious abdominalsurgerydiabetes mellitushypertensionpreoperative laboratory dataserum albumin glcrp glasa ¥ mode of surgical approach nlaparoscopicopentype of resection ndistal gastrectomyproximal gastrectomytotal gastrectomyoperation time minblood loss mltumor invasiont1t3tumor sitecardiafundusbodypylorusantrumrln countlymph node metastasispositivenegativelnrloddsptnm stage iiiiiiivlauren subtypeintestinaldiffusemixedunknownpostoperative complicationspositive ± ± ± ± ± ± ± ± table demographic and clinical features of patientscontinuedcharacteristicsnegativepostoperative stay daystotal hospital charges ¥n ± ± bmi body mass index crp creactive protein asa american society ofanesthesiologists rlns retrieved lymph nodes lnr lymph node ratio loddslog odds of positive lymph nodeswith longterm survival but little is known about the relationship between the rln count and shortterm complications after radical surgerypostoperative complications of gc pose a significantimpact on the length of postoperative stay and hospitalcharges which further affect the quality oflife thereforeinvestigating the relationship between rlncount and postoperative shortterm complications wouldprovide more comprehensive evidence for selecting theappropriate rln countmethodspatientsa total of consecutive gc patients between january and december at nanjing drum tower hospital were retrospectively reviewed all patients underwent curative r0 gastrectomy and were histologicallyconfirmed the exclusion criteria were as follows multivisceral resection patients accepting preoperative radiotherapy or chemotherapy patients with previous stomach surgery and patients with incompleteclinical data this study was approved by the ethicscommittee of nanjing drum tower hospitalcharacteristicsfor preoperativedata collectiondataintraoperativeindex and postoperative features were extracted preoperative characteristics included age gender body massindex bmi comorbidities and laboratory data the intraoperative index involved the american society of anesthesiologists asa grade surgical approach type ofresection operation time and blood loss postoperativefeatures included depth of tumor invasion tumor site retrieved lymph node count lymph node metastasis lymphnode ratio lnrlog odds of positive lymph nodeslodds ptnm stage lauren subtype shortterm complications postoperative stay and total hospital chargeslnr was defined as the ratio of positive to retrievedlymph nodes lodds was calculated by log [positivelymph nodes 05total lymph nodes positive lymphnodes ] the postoperative shortterm complications occurring in the hospital or within days werecollected all complications were evaluated according tothe claviendindo classification system 0csun world of surgical oncology page of statistical analysisstatistical analyses were conducted by spss chicago il usa continuous variables were shown asmeans ± sd students t test was applied for normallydistributed data mannwhitney u test was applied fornonnormally distributed data categorical variable datawere presented as numbers and analyzed using the chisquared test or the fisher exact test univariate andmultivariate analyses were performed to analyze the riskfactors associated with the postoperative complicationsor retrieved lymph node count the optimal cutoffvalues of lnr and lodds were determined by receivertable univariate and multivariate analyses of characteristics associated with postoperative complicationscharacteristicsunivariateormultivariateor cipreferenceage ¥ gendermalefemalebmi kgm2preoperative comorbiditiesprevious abdominal surgerydiabetes mellitushypertensionpreoperative laboratory dataserum albumin glcrp ¥ glasa ¥ mode of surgical approachlaparoscopicopentype of resectiontotal gastrectomydistal gastrectomyproximal gastrectomyoperation timeblood losstumor sitecardiafundusbodypylorusantrumtumor invasion t3rlnslymph node metastasislnr lodds ptnm stage ¥ iiilauren subtypeintestinaldiffusemixedunknown cireferencereferencereferencep 0csun world of surgical oncology page of operating characteristic roc analysis all statisticaltests were conducted twosided and statistical differences were termed as p value resultspatient characteristicsthe characteristics of the patients enrolledin this study were presented in table there were gc patients in all including men and women the median age was years with arange from to years a total of patients underwent open gastrectomy while underwent laparoscopic surgery the type of resectionwas distal gastrectomy in patients proximalgastrectomy in and total gastrectomy in the mean operation time was min and themean intraoperative blood loss was ml pathologicalresults were stage iiiiiiiv in patientsrespectively the mean rln count was range and patients were tested with lymphnode metastasis overall postoperative shortterm complications occurred in patients the meanpostoperative stay was days and the mean total hospital charges were à ¥association between perioperative characteristics andpostoperative complicationsas presented in table univariate and multivariate analyses indicated that postoperative shortterm complications were significantly correlated with age gender levelof preoperative serum albumin and operation timestratified analyses by type of resection revealed thatcomplications occurred frequently in proximal gastrectomy compared with total gastrectomy while there wasno significant difference between distal gastrectomy andtotal gastrectomy no significant association was observed between rln count and overall postoperativecomplicationsimpact of rln count on postoperative complicationsof the patients developed complications of encountered a single complication and of encountered multiplecomplications the details of patients with shorttermcomplications based on the claviendindo classification are for grade i for grade ii frade iii for grade iv and for grade vthe rate of major complications cdc grade ¥ iiiwas the median rln count in this study was so we divided all patients into two groups basedon the median rln count univariateanalysesshowed no significant difference between rln countand postoperative complicationsboth overall andstratified by cdc grade table table univariate analyses of postoperative complicationsassociated with rln countcharacteristicsallrln count ¥ overall ngrade i nfever °cemesispainabdominopelvic collectionpleural effusiongrade ii nblood transfusionsearly postoperative bowel obstructiongastroparesisliver function abnormalitieswound infectionpneumoniaintraabdominal infectionsurinary tract infectionenteritisbacteremiagrade iii nanastomotic leakagelymphatic leakagepancreatic fistulabiliary fistulableedingabdominopelvic collectionpleural effusionintraabdominal abscesswound disruptiondelayed wound healinggastroparesisearly postoperative bowel obstructionsplenic necrosisgrade iv nheart failurekidney failurebrain infarctionmodspvaluegrade v ngrade ¥ iii nrlns retrieved lymph nodes mods multiple an dysfunction syndrome 0csun world of surgical oncology page of factors associated with rln countwe further explored the potential factors associated withrln count univariate analyses revealed that preoperative serum albumin type of resection tumor invasionlymph node metastasis and ptnm stage were associatedwith rln count p table stratification bytype of resection showed that rln count in either distalgastrectomy or proximal gastrectomy was significantlyin total gastrectomy multivariatelowerthan thatanalyses further indicated that type of resection tumorinvasion and lymph node metastasis were still significantly associated with rln count p table discussionnodal involvement significantly affected the prognosis ofgc patients because it is the major root of tumor relapse after surgery [ ] thus standardized lymphnode dissection is the basic requirement for curativetable univariate and multivariate analyses of factors associated with rln count ¥ characteristicsunivariateormultivariateor cipreference age ¥ gendermalefemalebmi kgm2preoperative comorbiditiesprevious abdominal surgerydiabetes mellitushypertensionpreoperative laboratory dataserum albumin glcrp ¥ glasa ¥ mode of surgical approachlaparoscopicopentype of resectiontotal gastrectomydistal gastrectomyproximal gastrectomyoperation timeblood losstumor sitecardiafundusbodypylorusantrumtumor invasion t3lymph node metastasisptnm stage ¥ iiilauren subtypeintestinaldiffusemixedunknown cireferencereferencereferencep bmi body mass index crp creactive protein asa american society of anesthesiologists rlns retrieved lymph nodes or odds ratio ci confidence interval 0csun world of surgical oncology page of r0 gastrectomy curative gastrectomy with d2 lymphadenectomy has been considered as the standard fashionfor decades in eastern asia especially in japan [ ]this procedure has been gradually accepted by westerncountries in recent years [ ] as for the rln countthe 8th edition tnm classification for gc recommendeddissecting at least lymph nodes moreover emergingevidence revealed the positive correlations between rlncount and overall survival of gc patients [ ] bycomparing rln count to survival time okajima suggested an optimal rln count of ¥ deng proposed an optimal rln count of ¥ for lymphnodenegative gc and for lymph nodepositive gcby stratum analysis of patients sano reported that rln count preferably achieved or moreby a multicenter study enrolling patients additionally lnr and lodds were also reported to[] thesebe associated with gc prognosisabove studies mainly focused on the relationship between rln count and longterm prognosis howeverlittle is known aboutits effects on postoperativeshortterm complicationsin this study we concentrated on the association betweenrln count and shortterm prognosis univariate analysesshowed no significant difference between rln count andpostoperative complications both overall and stratified bycdc grade therefore more lymph nodes were encouragedto be dissected from the perspective of shortterm prognosisalthough curative gastrectomy with d2 lymphadenectomy is considered a pivotal strategy for advanced gcthere are international and institutional differences in thenumber of rln count [ ] various factors were reported to influence the rln count including the confidence and enthusiasm of doctors both surgeons andpathologists surgical situation and innate lymph nodecount in each patient [ ] in our study we concludedthat rln count was related to the type of resection tumorinvasion and lymph node metastasis of note rln countwas positively correlated with the lymph node metastasisrate which underlined the importance of rln count foraccurate stagingactuallyfor a thorough pathological examinationrlns should be individually divided from a completetissue sample after surgery owing to much time andeffort was required during this procedureit has notbeen widely implemented clinically therefore the examined lymph node count by pathologists might belower than the dissected lymph node count multipleattempts have been conducted to improve the detection rate of lymph nodes [] li elucidatedthat the mean number of rlns could be significantlyelevated by injecting carbon nanops before surgery compared with controls vs markl and colleagues reported a twofold lymph nodepick up rate utilizing methylene blue staining thanunstained groups vs several dye materials were also used to increase the number of lymphnodes dissected during surgery such as fluorescentindocyanine green icg and 5aminolevulinic acid5ala [ ]we acknowledge that this study had some potentialit was a retrospective singlecenterlimitations firststudy so the results might be flawed because of residualconfounding factors second the rln count was closelyrelated to the quality of surgeons and pathologists theperioperative variables might differ in different doctorstherefore multicenter studies are needed to confirmour resultssin the current study demonstrated thatrlns\\ count was not associated with postoperativeshortterm complications following gastrectomy of gctherefore our analysis encouraged more lymph nodesto be dissected for accurate pathologic stagingabbreviationsbmi body mass index crp creactive protein asa american society ofanesthesiologists rlns retrieved lymph nodes lnr lymph node ratiolodds log odds of positive lymph nodesacknowledgementsthe authors gratefully acknowledge all the investigators for theircontributions to the trialauthors contributionsfs worked on the study design collected the data and drafted themanuscript sl contributed to the study design and data collection ps wasinvolved in the data collection and extraction cz helped collect the datawg was involved in the study design and data extraction mw revised themanuscript all authors have read and approved the final manuscriptfundingthere is no funding supporting this workavailability of data and materialsaccess to the data and the calculation method can be obtained from theauthors by email medsunfeng163comethics approval and consent to participatethis retrospective study was approved by the ethics committee of nanjingdrum tower hospital medical school of nanjing university due to theretrospective nature the requirement for informed consent was waived bythe irbs from nanjing drum tower hospital medical school of nanjinguniversityconsent for publicationnot applicablecompeting intereststhe authors declare that they have no competing interestsreceived june accepted august referencesstewart b wild cp world cancer report public health 0csun world of surgical oncology page of degiuli m de manzoni g di leo a ugo dd galasso e marrelli d gastric cancer current status of lymph node dissection world jgastroenterol son t hyung wj lee jh kim ym kim hi an jy clinical implication ofan insufficient number of examined lymph nodes after curative resectionfor gastric cancer cancer li z ao s bu z wu a wu x shan f clinical study of harvestinglymph nodes with carbon nanops in advanced gastric cancer aprospective randomized trial world j surg oncol markl b kerwel tg jahnig hg oruzio d arnholdt hm scholer c methylene blueassisted lymph node dissection in colon specimens aprospective randomized study am j clin pathol aoyama t yoshikawa t morita s shirai j fujikawa h iwasaki k methylene blueassisted technique for harvesting lymph nodes after radicalsurgery for gastric cancer a prospective randomized phase iii study bmccancer he m jiang z wang c hao z an j shen j diagnostic value of nearinfrared or fluorescent indocyanine green guided sentinel lymph nodemapping in gastric cancer a systematic review and metaanalysis j surgoncol koizumi n harada y murayama y harada k beika m yamaoka y detection of metastatic lymph nodes using 5aminolevulinic acid inpatients with gastric cancer ann surg oncol publishers notespringer nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliationsbray 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 van cutsem e sagaert x topal b haustermans k prenen h gastric cancerlancet zhang w zhangyuan g wang j jin k liu y wang f effect of lymphnodes count in nodepositive gastric cancer j cancer chu x yang zf impact on survival of the number of lymph nodes resectedin patients with lymph nodenegative gastric cancer world j surg oncoljiang l yang kh guan ql zhao p chen y tian jh survival and recurrencefree benefits with different lymphadenectomy for resectable gastric cancera metaanalysis j surg oncol deng j yamashita h seto y liang h increasing the number of examinedlymph nodes is a prerequisite for improvement in the accurate evaluationof overall survival of nodenegative gastric cancer patients ann surg oncolamin mb greene fl edge sb compton cc gershenwald je brookland rk the eighth edition ajcc cancer staging manual continuing to build abridge from a populationbased to a more personalized approach tocancer staging ca cancer j clin okajima w komatsu s ichikawa d kosuga t kubota t okamoto k prognostic impact of the number of retrieved lymph nodes in patients withgastric cancer j gastroenterol hepatol deng j liu j wang w sun z wang z zhou z validation of clinicalsignificance of examined lymph node count for accurate prognosticevaluation of gastric cancer for the eighth edition of the american jointcommittee on cancer ajcc tnm staging system chin j cancer res kim th suh ys huh yj son yg park jh yang jy the comprehensivecomplication index cci is a more sensitive complication index than theconventional claviendindo classification in radical gastric cancer surgerygastric cancer wang j hassett jm dayton mt kulaylat mn the prognostic superiority oflog odds of positive lymph nodes in stage iii colon cancer j gastrointestsurg dindo d demartines n clavien pa classification of surgical complicationsa new proposal with evaluation in a cohort of patients and results of asurvey ann surg hirabayashi s kosugi s isobe y nashimoto a oda i hayashi k development and external validation of a nomogram for overall survivalafter curative resection in serosanegative locally advanced gastric cancerann oncol tóth d bÃró a varga z török m árkosy p comparison of different lymphnode staging systems in prognosis of gastric cancer a biinstitutional studyfrom hungary chin j cancer res de steur wo dikken jl hartgrink hh lymph node dissection in resectableadvanced gastric cancer dig surg maruyama k kaminishi m hayashi ki isobe y honda i katai h gastric cancer treated in in japan data analysis of nationwide registrygastric cancer liang h deng j evaluation of rational extent lymphadenectomy for localadvanced gastric cancer chin j cancer res degiuli m sasako m ponti a vendrame a tomatis m mazza c randomized clinical trial comparing survival after d1 or d2 gastrectomy fastric cancer br j surg sano t coit dg kim hh roviello f kassab p wittekind c proposal ofa new stage grouping of gastric cancer for tnm classification internationalgastric cancer association staging project gastric cancer zhao e zhou c chen s prognostic nomogram based on log odds ofpositive lymph nodes for gastric carcinoma patients after surgical resectionfuture oncol alatengbaolide lin d li y xu h chen j wang b liu c lu p lymph noderatio is an independent prognostic factor in gastric cancer after curativeresection r0 regardless of the examined number of lymph nodes am jclin oncol wang j dang p raut cp pandalai pk maduekwe un rattner dw comparison of a lymph node ratiobased staging system with the 7th ajccsystem for gastric cancer analysis of patients from the seer databaseann surg 0c" | Colon_Cancer |
"piglet diarrhea is one of the most severe diseases aï¬ictingpiglets leading to their delayed growth and developmentlow feed returns and even death which has seriously damaged the economic development of pig industries globally recently clostridium perfringens the important pathogenic microanism that causes diarrhea in piglets wasdivided into ï¬ve toxinotypes a b c d and e theclostridium perfringens type c c perfringens type c is agastolerant bacterium widely distributed in nature thatmay cause various diseases in animals including cellulitisgas gangrene intestinal toxemia and necrotic enteritis importantly c perfringens type c can produce alphaand beta toxins which are known to play critical roles inintestinal epithelial cell damage and necrosis as well as intestinal ammatory responses [ ]the mitogenactivated protein kinase mapk signalingpathway is known to participate in various biological processesincluding innate immunity cell growth stress responseapoptosis and diï¬erentiation the mammalian mapkfamily includesthree subfamilies namely extracellularsignalregulated kinases erks cjun nterminal kinasesjnks and p38 mapks the mapk signaling pathwayis one of the major pathways activated by cells following infection and intoxication the c perfringens alpha toxin caninduce the release of cytokine il8 by activating the erk12and p38 mapk signaling pathways while the c perfringens beta toxin can cause the phosphorylation of p38 andjnk it has been reported that p38 jnk12 and 0cbiomed research internationalerk12 may be activated in the course of ammatory boweldisease ibd []long noncoding rnas lncrnas are a type of noncoding rna molecules longer than nucleotides which playan important role in many physiological and pathologicalprocesses [ ] lncrna h19 can promote the development of bronchopulmonary dysplasia by regulating themapk signaling pathway and the mapk signaling pathwaycan be used as a potential target for the treatment of bronchopulmonary dysplasia jiang found that lncrnamalat1 can promote high glucoseinduced apoptosis ofrat cartilage endplate cells through the p38mapk signalingpathway at present studies have conï¬rmed thatlncrna h19 lncrna neat1 and lncrnabc012900 play an important role in ibd by regulatingthe intestinal epithelial barrier identifying lncrnas relatedto mapk signaling pathway genes is very necessary to studypiglet diarrhea caused by c perfringens type ccurrently there are no published literature reports ondiï¬erential expression and regulation of genes related to themapk signaling pathway in diarrhea piglets caused by cperfringens type c in our preliminary transcriptome studywe have identiï¬ed mrnas and lncrnas in theileum tissues of piglets infected with c perfringens type c building on this the present work was designed tofurther investigate the expression patterns of mapk signaling pathway genes in the ileum tissues of infected pigletsusing quantitative realtime polymerase chain reactionqrtpcr in addition we screened diï¬erentially expressedlncrnas related to the mapk signaling pathway based onan integrated analysis of lncrnas and mrnas collectivelythese results will reveal the expression patterns of the mapksignaling pathway genes in the diarrheastricken ileum ofpiglets infected with c perfringens type c which provides avaluable basis for further breeding of diarrhearesistantpiglet strains materials and methods ethics statement all experimental procedures usinganimals were performed in accordance with the regulationsfor the administration of aï¬airs concerning experimentalanimals ministry of science and technology china revisedin june this study was approved by the ethics committee of the college of animal science and technologygansu agricultural university approval number all eï¬orts were taken to minimize suï¬ering in theanimal subjects the c perfringens type c culture animal treatmentand sample collection thirty 7dayold suckling pigletsyorkshire sowlandrace boar from dingxi city in gansuprovince china were selected as the experimental subjectsthese piglets were notinfected with escherichia colisalmonella orc perfringens as determined by commercialenzymelinked immunosorbent assay elisa kits jiancheng bioengineering institute nanjing china twentyï¬ve experimental pigs were randomly selected to serve asthe infected group while the remaining ï¬ve formed thecontrol group ic the c perfringens type c strain cvcc was purchased from the china veterinary culture collection center beijing china bacteria were cultured usingthe methods described in huang each piglet wasfed ml of the c perfringens type c culture medium cfuml daily for days fecal symptoms weremonitored and recorded daily during the infection periodusing a previously described method [ ] they werejudged and scored as follows normal solid feces slightdiarrhea soft and loose feces moderate diarrhea semiliquid feces and severe diarrhea liquid and unformed fecesaccording to the summed diarrhea scores piglets wereranked from high to low the top ï¬ve piglets and the bottomï¬ve were designated as the susceptibility is and resistanceir groups respectively the ileum tissues from the ir isand ic groups were collected and ï¬ushed cleanly with apbs buï¬er ph then quickly frozen in liquid nitrogenand stored at °c until rna extractions rna extraction and highthroughput rna sequencingthe total rna was extracted from ileum tissues using thetrizol reagent invitrogen carlsbad ca usa the purityof rna samples was assessed using a nanophotometer spectrophotometer implen westlake village ca usa ileumtotal rna quantity and integrity were measured using aqubit fluorometer life technologies carlsbad causa and rna nano assay kit of the bioanalyzer system agilent technologies santa clara ca usarespectively approximately μg rrnadepleted rna ribozero rna was acquired from total rna by an epicentreribozero¢ rrna removal kit epicentre usa and cleanedup by ethanol precipitation the cdna libraries were constructed and sequenced on a hiseq platform illuminasan diego ca usa identiï¬cation of diï¬erentially expressed genes degsand diï¬erentially expressed lncrnas all of the raw sequencing data were deposited into a sequence read archive sraunder accession number prjna399620 at the nationalcenter for biotechnology information ncbi based on previous gene expression proï¬les obtained from rnaseq atotal of mrnas were identiï¬ed in the ileum tissues ofpiglets among these we screened genes with fold changeof ¥ p value and fpkm value from ir vs icand is vs ic as diï¬erentially expressed genes we selectedthe degs from ir vs ic for subsequent analysistable s11 in the sequencing data we screened thelncrnas of diï¬erentiallyexpressed mapk signalingpathway genes by trans and then selected the diï¬erentiallyexpressed lncrnas with p value as standard pathway and clustering analyses of degs pathwayenrichment analysis for degs was performed with keggdatabase using david online software davidncifcrfgov we used fishers exact test to screen out significantenrichment pathways related with immunity p thegenes detected in a candidate immune systemrelated pathwaysus scrofa mapk signaling pathway were subjected to 0cbiomed research internationalrigilike receptor signaling pathwaynfkappa b signaling pathwaynodlike receptor signaling pathwaytolllike receptor signaling pathwaycolorectal cancerchemokine signaling pathwayt cell receptor signaling pathwaytnf signaling pathwayb cell receptor signaling pathwaycytokinecytokine receptor interactionmapk signaling pathwaycamp signaling pathwaywnt signaling pathwayjakstat signaling pathwayneglog10_p value gene ratiocountfigure the bubble plot showing the immunerelated kegg signaling pathways significantly enriched by degs all degs in the ileumtissue of c perfringens type cinduced piglets were subjected to a comparative kegg database search to identify their involvement inimmune systemrelated pathways the gene ratio is on the xaxis and the kegg pathway names are on the yaxis a dots size isproportional to the number of target genes and its coloring indicates diï¬ering neglog10p valueshierarchical clustering using the omicshare tools httpwwwomicsharecomtools construction of proteinprotein interaction ppinetworks of genes associated with the mapk signalingpathway to assess the interactions among genes associatedwith the mapk signaling pathway the ppi network ofproteins coded by the obtained degs was built by using thesearch tool for the retrieval of interacting genesproteins string database stringdb inthe string database we chose sus scrofa as the anismwhile setting the edge of the network as conï¬dence we chosetextmining experiments databases coexpression neighborhood gene fusion and cooccurrence as the active interactionsource and chose a medium conï¬dence level thethickness of the line connecting any two genes indicates thestrength of the data support expression levels of lncrnas and genes associated withthe mapk signaling pathway based on the results above genes and lncrnas associated with the mapk signalingpathway were randomly selected for further quantitativedetermination by qrtpcr the rna samples used forqrtpcr were derived from the samples used for sequencing one microliter of total rna ngμl was reversetranscribed into cdna using a primescript¢ rt reagentkit takara dalian china primers were designed for eachgene using the blast online software provided by the ncbidatabase and then synthesized by genewiz co ltdtianjin china table s2 the qrtpcr was performedon a lightcycler ii platform roche basel switzerlanda ï¬nal volume of μl for the qrtpcr reaction systemconsisted of μl of 2x sybr green realtime pcr mastermix takara dalian china μl of forward and reverseprimers μmol μl of cdna ngμl and μlof rnasefree ddh2o the cycling conditions includedan initial activation phase at °c for min followed by cycles at °c for s denaturation and at ° ± °c for s annealing with an extension phase at °c for sthe mrna and lncrna abundances were calculated usingδδct method three technical replicates werethe performed for each sample statistical analysis all qrtpcr experimental data wereanalyzed using oneway analysis of variance statistical signiï¬cance was determined using the twotailed students ttest method the results are expressed here as mean ± sdstandard deviation a p value and fold change were considered statistically significant a p value and fold change were interpreted as highly significant results acquisition of degs and screening of mapk signalingpathwayrelated genes based on the results of the kegganalysis we selected the ï¬rst types of significant enrichment pathways related to the immune system table s12such as the mapk signaling pathway nfkappa b signalingpathway t cell receptor signaling pathway nucleotidebinding oligomerization domaincontaining protein nodlike receptor signaling pathway retinoic acidinducible gene protein rigi like receptor signaling pathway andtolllike receptor signaling pathway figure furthermorea total of degs from the infected piglet groups ir andis consisting of upregulated and downregulatedgenes were involved in the mapk signaling pathway whencompared with the ic group table hierarchical 0cbiomed research internationaltable list of degs in the ileum of c perfringens type cinfected piglets and involved in the mapk signaling pathwaytranscript_idensssct00000014692ensssct00000008273ensssct00000018048ensssct00000010506ensssct00000035948ensssct00000011362ensssct00000006417ensssct00000005604ensssct00000017223ensssct00000036577ensssct00000035592ensssct00000027163ensssct00000011849ensssct00000006665ensssct00000019500ensssct00000011433ensssct00000032467ensssct00000008863ensssct00000028838ensssct00000010840ensssct00000015453ensssct00000002650ensssct00000012777ensssct00000006548ensssct00000019534ensssct00000008861ensssct00000014068ensssct00000005290ensssct00000003320ensssct00000017958ensssct00000011540ensssct00000012903ensssct00000011042ensssct00000027425ensssct00000035014ensssct00000035374ensssct00000036028ensssct00000035439gene_idgene_namegene_locationgadd45gstk3arrb2fasdusp6il1aelk4mknk2pdgfaflnctab1mapk8traf2ppm1afgfr1fgfr4ikbkgtradddusp10enssscg00000013448enssscg00000007541enssscg00000016578enssscg00000009585enssscg00000000087enssscg00000010380enssscg00000005838enssscg00000005084enssscg00000015815enssscg00000014047enssscg00000012825enssscg00000024182enssscg00000010831enssscg00000006074enssscg00000017918enssscg00000010448enssscg00000022284enssscg00000008090enssscg00000025182enssscg00000009890 mapkapk5enssscg00000014149enssscg00000002383enssscg00000011672enssscg00000005965enssscg00000017950enssscg00000008088enssscg00000012871enssscg00000004791enssscg00000002989enssscg00000016494enssscg00000010548enssscg00000011791 map3k13enssscg00000010081mapk1mecomenssscg00000011743enssscg00000014878enssscg00000010301enssscg00000012163enssscg00000030957pak1ppp3cbrps6ka3nfkb1rasa2myctp53il1b1fgf19akt2brafchukmef2cfosrasgrp1chr2chr3chr18chr14chr5chr14chr1chr1chr15chr2chrxgl8965011chr10chr4chr12chr14gl8947002chr3chr9chr14chr2chr7chr13chr4chr12chr3chr2chr1chr6chr18chr14chr13chr14chr13chr9chr14chrxchr8upupupupupupupupupupupupir_fpkm is_fpkm ic_fpkm iris vs icdowndowndowndowndowndowndowndowndowndowndowndowndowndowndowndowndowndowndowndowndowndowndowndowndowndownclustering of the degs in ileum tissues from ir is and icshowed hat the two infected groups were clustered figure among these degs tnf receptorassociated factor traf2 mitogenactivated protein kinase mapk8ï¬broblast growth factor receptor fgfr1 and growtharrest and dna damage inducible gamma gadd45gwere all upregulated in the ir and is groups whereas theconserved helixloophelix ubiquitous kinasechukmitogenactivated protein kinase mapk1 ap1 transcription factor subunit fos and tumor protein p53tp53 genes were downregulated figure distribution positions and ppi network of degs locatedin the mapk signaling pathway the map of the sus scrofamapk signaling pathway in the kegg database was used asa template and the location of each of the degs in thispathway was conï¬rmed figure many degs were locatedin a key position of this pathway and diï¬erentially expressedin the infected piglets versus the control group such asmapk1 mapk8 traf2 gadd45g and braf figure given the same expression trends for the degs in the irgroup and the is group these genes were presented togetherin a single graph figure 0cbiomed research internationalnfkb1dusp6akt2rps6ka3map3k13fosmecomfasppp3cbarrb2pak1il1afgf19chukrasa2mapk1dusp10mef2cstk3mapkapk5rasgrp1brafelk4il1b1myctp53flncppm1aikbkgpdgfatab1fgfr1mapk8fgfr4gadd45gtraf2mknk2traddicirisfigure clustering of genes involved in the mapk signaling pathway hierarchical clustering of degs in the ileum tissue of cperfringens type cinduced piglets ir and is relative to the control group ic that are involved in the mapk signaling pathwayupregulated and downregulated genes relative to the mean are respectively colored red and blue rows represent the mrnas whilecolumns represent diï¬erent treated groupsppi network analysis revealed that except for individualgenes stk3 mecom rasa2 and rasgrp1 most of thegenes had strong relationships to each other with mapk1tp53 mapk8 myc and chuk lying at the core of theppi network figure potential lncrnas targeting mapk signaling pathwaygenes in the ileum tissues of c perfringens type cinfectedir and is piglets we ï¬ltered those diï¬erentially expressedlncrnas identiï¬ed in the ileum tissues between the infectedgroups and the control group a total of degs from themapk signaling pathway were predicted to be targets of delncrnas table s3 speciï¬cally we found that aldbssct0000008940 lnc_000486 aldbssct0000002407lnc_000796 lnc_000477 aldbssct0000003968 andlnc_000686 had common target braf ikbkg was thecommon target of aldbssct0000002686 lnc_001291aldbssct0000008223 lnc_001496 lnc_000556 andaldbssct0000006650 in addition aldbssct0000004760aldbssct0000002686 aldbssct0000006510 aldbssct0000006650 and aldbssct0000004038 were shown to bethe targets of mapk8 mapkapk5 traf2 ikbkg andchuk respectively the interactions between mrnas andlncrnas are shown in figure quantitative pcr validation as shown in figure qrtpcr results showed that the expression trends of allgenes and lncrnas in the ic group ir group and is groupwere consistent with the results of rnaseq expressiontrends were consistent for all transcripts in both analyseswith a coeï¬cient of determination r2 for the irgroups mrnas and r2 for the is groups mrnasfigure the expression trends of lncrna obtained bythe above two analytical methods were also consistent withan r2 for the ir groups lncrnas and r2 for the is groups lncrnas these results demonstrated thatc perfringens type c infection greatly aï¬ected the expressionof these mapk signaling pathwayrelated genes in ileum tissues of piglets discussionc perfringens type c can cause many diseases in animalssuch as hemorrhagic enteritis necrotic enteritis diarrheaand even death alpha and beta toxins secreted by cperfringens type c can enhance target cell toxicity by activating the mapk signaling pathway [ ] in rabbit neutrophils the alpha toxin induces the generation of superoxidesthrough activation of the erkmapk signaling pathway as 0cbiomed research internationalpdgfafgf19fgfr1fgfr4grb2sosrasgrp1raspbrafmapksmek12ppchukikbkgnfkb1mknk2rps6ka3pproliferationinflammationantiapoptosiscrebmapk1pelk4mycfosdnadnaproliferationdifferentiationg12rasa2caspstk3mekk1ptnftnfrtraddtraf2pak1falsfasdaxxask1pdna damagegadd45gmekk4akt2ppppm1ail1ail1b1il1rirak14traf6tab1tak1pmkk3mkk6pp38dusp6dusp10ppp3cbpmkk4mkk7parrb2flncmapk8pfospdusp6dusp10mecomppptp53elk4mef2cmapkapk5nfat2nfat4dnadnaproliferationdifferentiationinflammationapoptosisfigure localization of degs in the mapk signaling pathway these respective positions were marked in the sus scrofa mapk signalingpathway as retrieved from the kegg database the red green and gray boxes indicate upregulated downregulated and nonregulated genesrespectivelystk3stk3stk3pak1pak1pak1mecommecommecomrasa2rasa2rasa2map3k13map3k13map3k13gadd45ggadd45ggadd45gil1bil1bil1bfosfosfosil1ail1ail1afasfasfasnfkb1nfkb1nfkb1mapk8mapk8mapk8ikbkgikbkgikbkgakt2akt2akt2mycmycmyctp53tp53tp53mapkapk5mapkapk5mapkapk5traf2traf2traf2traddtraddtraddchukchukchukdusp10dusp10dusp10brafbrafbraftab1tab1tab1ppm1appm1appm1aflncflncflncmef2cmef2cmef2crasgrp1rasgrp1rasgrp1fgfr4fgfr4fgfr4fgf19fgf19fgf19rps6ka3rps6ka3rps6ka3pdgfapdgfapdgfafgfr1fgfr1fgfr1dusp6dusp6dusp6mknk2mknk2mknk2ppp3cbppp3cbppp3cbmapk1mapk1mapk1elk4elk4elk4arrb2arrb2arrb2figure interactions among degs involved in the mapk signaling pathway in this ppi network proteins were represented as nodesand the interactions between two proteins denoted as edges active interaction sources textmining experiments databases coexpressionneighborhood gene fusion and cooccurrence the thickness of the line connecting two genes indicates the strength of the data support 0cbiomed research internationallnc_001539lnc_000841elk4lnc_000378lnc_000854gadd45galdbssct0000000346il1b1lnc_000157lnc_001171il1aaldbssct0000002553mapkapk5fgfr1mef2caldbssct0000008856aldbssct0000002686lnc_000528lnc_000556ensssct00000033304aldbssct0000008940aldbssct0000003968flncfgf19lnc_001291pak1aldbssct0000006650lnc_000486ikbkglnc_000686brafaldbssct0000002407lnc_000796aldbssct0000000192lnc_001496aldbssct0000008223lnc_000477aldbssct0000011852ppm1amapk1mapk8chuktraf2lnc_000048aldbssct0000007662ppp3cbmecomaldbssct0000011950aldbssct0000003606aldbssct0000004760aldbssct0000004038aldbssct0000006510lnc_000767aldbssct0000010255figure lncrnamrna association network the interaction network between the degs in the mapk signaling pathway and the lncrnas targeting themreported by oda in human lung adenocarcinomaepithelial cell lines c perfringens phospholipase c cpplccontributes to the production of il8 by activating theerk12nuclear factor kappa b nfκb system and thep38 mapk system the beta toxin also induces the phosphorylation of p38 and jnk this study assessed the diï¬erential expression patterns ofmapk signaling pathway genes in the ileum of pigletsinfected by c perfringens type c using rnaseq qrtpcr and bioinformatics as one of the ancient signal transduction pathways mapk is widely used for studying theevolution of many physiological processes mapks area family of serthr protein kinases conserved evolutionarilyacross all eukaryotic anisms which become activatedin response to stimuli to participate in the regulation of avariety of cellular activities such as gene expression mitosismetabolism motility survival apoptosis and diï¬erentiationthe mapk signaling pathway plays crucial roles in theoccurrence and development of ammatory bowel diseaseibd [ ] for instance waetzig and schreiber reported that erk1erk2 jnk and p38mapk from themapk signaling pathway were crucially involved in theintestinal mucosal injury from ibd studies have shown thatinhibiting the expression of jnk [ ] or increasing that oferk12 can reduce intestinal ammation and epithelial cell apoptosis furthermore jnk and erk12 may beused eï¬ectively as therapeutic targets against ibd [ ]there were pathways associated with immuneresponses of which the nfκb signaling pathway tolllike receptor signaling pathway and jakstat signaling pathway are known to be associated with diarrheacaused by c perfringens infection in animals the aboveresults indicated that c perfringens type c elicited a strongimmune response in the ileum tissue of the diarrheal pigletsin addition degs in the mapk signaling pathway weresignificantly enriched our clustering analysis for geneexpression showed two infected groups namely ir and isclustered together hence the established model of pigletdiarrhea was successfulthirtyeight degs screened in this study are located atkey positions in the mapk signaling pathway thus suggesting this pathway may play a crucial role in piglet diarrheacaused by c perfringens type c based on the ppi networkof degs associated with the mapk signaling pathwaythree hub genes mapk1 tp53 and mapk8 were identiï¬edas an important member of the mapk system erk plays 0cbiomed research internationalelk4dusp10dusp6chukbrafegnahc doflegnahc doflegnahc doflegnahc doflegnahc dofleganhc doflic ir ismapk1ic irisgadd45gic ir ismycic ir istab1ic ir isaldbssctic ir isegnahc doflegnahc doflegnahc doflegnahc doflegnahc doflegnahc doflic irisfgf19ic ir ismapk8ic ir isikbkgic ir istp53ic ir isaldbssctic ir isegnahc doflegnahc doflegnahc doflegnahc doflegnahc doflegnahc doflic ir isfgfr4ic ir ismapkapk5ic ir isnfkb1ic ir istraf2ic ir isaldbssctic ir isegnahc doflegnahc doflegnahc doflegnahc doflegnahc doflegnahc doflic ir isfgfr1ic ir ismecomic ir ispak1ic ir istraddic ir isaldbssctic ir isegnahc doflegnahc doflegnahc doflegnahc doflegnahc doflegnahc doflic ir isfosic ir ismef2cic ir ispdgfaic ir isrps6ka3ic ir isaldbssctic ir isfigure veriï¬cation of qrtpcr for some diï¬erentially expressed genes examined here are candidate mapk signaling pathway genes andlncrnas targeting them that were also diï¬erentially expressed in the ileum tissue of c perfringens type cinduced piglets bars are themean ± sd n and expressed the fold change in gene expression p and fold change p and fold change multiple roles in regulating ammatory responses the production of ammatory cytokines and the proliferation anddiï¬erentiation of epithelial cells further erk can inhibitapoptosis of intestinal epithelial cells in ibd patientswaetzig reported that downregulated erk12expression was capable ofinhibiting proliferation and 0cbiomed research internationalrcptrqyb cf golir mrna y ¨¯ r2 rcptrqyb cf golis mrnay ¨¯ r2 log2fc by rnaseqlog2fc by rnaseqlog2fc by rnaseq ir lncrna y ¨¯ r2 rcptrqyb cf gollog2fc by rnaseq is lncrna y ¨¯ r2 rcptrqyb cf golfigure correlations of log2fc values between the candidate mapk signaling pathway genes and the lncrnas targeting theminducing apoptosis of intestinal mucosal cells in our studymapk1 erk2 expression was significantly downregulatedin the infected piglets suggesting mapk1 could result inintestinal mucosal cell apoptosis in piglets with diarrheaexperimentally induced by c perfringens type cthe protooncogene myc is one of the transcription factors involved in the occurrence and development of manytypes of cancer and plays a key role in cell proliferation yamaguchi showed that myc can regulate cellproliferation of intestinal mucosa and participate in the control of cell cycle progression in colorectal cancer theupregulated expression of myc in cancer tissues has beenwell determined in our study the expression of mycin the ir group and the is group was significantly lower thanthat in the control group which was consistent with theresults of intestinal cell apoptosis caused by diarrhea thisindicates that myc is related to piglet diarrhea caused byc perfringenstp53 is an important transcription factor that participates in stressinduced responses by regulating the expression of genes associated with cell cycle arrest apoptosisaging dna repair and metabolic changes for examplewang and friedman found that shortchain fatty acidscfa mixtures can promote apoptosis of colonic epithelialcells by limiting the tumor suppressor protein tp53s expression in patients with ulcerative colitis rosmanurbach documented that tp53 gene expression was unstable inthe colonic mucosa and low in the serum indicating thattp53 is closely related to colonic mucosal ammation asan antiapoptotic gene a decreased expression level of tp53should lead to increased activity of the preapoptotic gene caspase thereby initiating an intracellular apoptosis programand causing apoptosis our results showed that the expression of tp53 was lower in the infected groups than that inthe control groups which suggested that the low expressionof tp53 might induce the apoptosis process of the intestinalcells during c perfringens type c infections recent workby girnius and davis demonstrated that jnk can promote the apoptosis of exfoliated epithelial cells in this studycompared with the ic group mapk8 was upregulated in theis and ir groups though more so in the former than in thelatter which indicated that jnk might participate in intestinal cell damage caused by c perfringens type c continuousactivation of jnk1 during intestinal cell apoptosis can elicit amarked decrease in the expression of tp53 which is consistent with the changed expression levels of jnk and tp53 inour study herethe tnfr1related death domain protein tradd is amajor adaptor molecule one crucially involved in the formation of signaling complexes the induction of apoptosis andnecrosis and the activation of mapk and nfκb importantly tradd is engaged in mediating both cell deathand proammatory signals in this study the expression of tradd was significantly upregulated in ileum tissuesfrom infected piglet groups being most expressed in the isgroup our ï¬ndings thus suggest that tradd might promote the apoptosis of intestinal cells along with having anadverse eï¬ect on host defense against infection by c perfringens type ctraf2 a key gene in the upper part of the mapk signaling pathway participates in regulating the activation of jnkinduced by tnfα traf2 may protect the apoptosis ofintestinal epithelial cells mediated by tnfα thereby hindering the ammation response since traf2 shows 0cbiomed research internationalmarked expression in the colon tissue of patients with ibdthere is a potential role for this gene in ibd consistentwith previous research in this study we found that traf2sexpression increased in the ileum tissues of piglets frominfected groups relative to the control group for infectedgroups the expression level of traf2 was higher in the irthan the is group these results suggest traf2 may help piglets resist c perfringens infection by regulating their immuneand ammatory responsesconserved helixloophelix ubiquitous kinase chuk islocated downstream of the mapk signaling pathway linkingthe mapk signaling pathway and the nfκb signaling pathway chuk plays an important role in the negative feedbackof nfκb canonical signaling to limit the activation ofammatory genes rengaraj reported that compared with the control group the expression of chuk inchicken necrotic enteritis caused by c perfringens is downregulated in this study the chuk in the ir group andthe is group were significantly downregulated comparedwith the normal group which is consistent with the aboveresearch resultsthe growth arrest and dna damageinducible gene gadd45g functions as a stress response protein havingbeen implicated in various biological processes such asdna repair cell growth cell diï¬erentiation and apoptosis gadd45g may participate in the regulation of cellapoptosis by activating the mapk signaling pathway yan found that gadd45g was diï¬erentially expressedin the spleen tissue of piglets infected with c perfringens in our study gadd45g had the highest expression in the isgroup indicating this gene is closely related to intestinaldamage and enterocyte death in the ileum of those pigletssensitive to c perfringens type cwe uncovered lncrnas which could somehow participate in regulating the expression of genes located within themapk signaling pathway lncrna is an important regulatorin host defense against bacterialinfection diseases aldbssct0000006510 could target expression of the traf2gene which participates in regulating the activation of jnkinduced by tnfα aldbssct0000004760 targets theexpression of mapk8 a key gene in the mapk signalingpathway which was overexpressed in the ir and is groupscompared with the uninfected group therefore albsssct0000004760 may participate in the mapk signaling pathway by regulating the expression of mapk8 all in all thelncrnas reported in this study may crucially participate inthe development of piglet diarrhea caused by c perfringenstype c however the speciï¬c mechanisms underpinning thisregulation still need further investigation conclusionin conclusion this study is the ï¬rst to screen degsinvolved in the mapk signaling pathway in piglet ileum tissues infected with c perfringens type c most of the degsare at key positions of that pathway of which mapk1tp53 mapk8 myc and chuk belong to the core of theppi network in addition we also identiï¬ed diï¬erentiallyexpressed lncrnas targeting mapk signaling pathwaygenes collectively this work will add to our knowledge ofhow mapk signaling pathway genes respond to diarrhea disease in the ilea of piglets infected with c perfringens type cdata availabilityall the raw sequencing data have been deposited into an sraprjna399620 at the ncbi other relevant data involved inthis study are presented in the results and supplementarymaterialsconflicts of interestthe authors declare no conï¬icts of interestauthors contributionsthis work was conceived and designed by ruirui luo andzunqiang yan qiaoli yang xiaoyu huang wei wangand kaihui xie collected samples ruirui luo pengfei wangand xiaoli gao performed the experiments and analyzed thedata ruirui luo wrote the paper and shuangbao gun guidedthe execution of the study and revised the manuscript allauthors read and approved the ï¬nal manuscriptacknowledgmentswe thank charlesworth publishing services ltd for providing us with language editing this research was supported bythe scientiï¬c research startup funds for openlyrecruiteddoctors of gansu agricultural university gaukyqd and the national natural science foundation ofchina grant numbers and supplementary materialssupplementary materials additional ï¬le table s1 list of differentially expressed genes and immunerelated keggenrichment pathways in the ileum of piglets infected withclostridium perfringens type c table s2 primer sequencesfor the realtime pcr analysis of genes and lncrnas associated with mapk signaling pathway genes table s3 list of lncrnas targeting mapk signaling pathway genes anddiï¬erentially expressed in the ileum of c perfringens typecinfected piglets supplementary materialsreferences x y huang q l yang j h yuan eï¬ect of geneticdiversity in swine leukocyte antigendra gene on piglet diarrhea genes vol no l petit m gibert and m r popoï¬ clostridium perfringens | Colon_Cancer |
cancer is the second leading cause of death in the united states cancer screenings candetect precancerous cells and allow for earlier diagnosis and treatment our purpose was tobetter understand risk factors for cancer screenings and assess the effect of cancer screenings on changes of cardiovascular health cvh measures before and after cancer screenings among patientsmethodswe used the guideline advantage tgaamerican heart association ambulatory qualityclinical data registry of electronic health record data n patients to investigateassociations between timeseries cvh measures and receipt of breast cervical and coloncancer screenings long shortterm memory lstm neural networks was employed to predict receipt of cancer screenings we also compared the distributions of cvh factorsbetween patients who received cancer screenings and those who did not finally we examined and quantified changes in cvh measures among the screened and nonscreenedgroupsresultsmodel performance was evaluated by the area under the receiver operator curve aurocthe average auroc of curves was for breast for cervical and for coloncancer screening distribution comparison found that screened patients had a higher prevalence of poor cvh categories cvh submetrics were improved for patients after cancerscreeningsa1111111111a1111111111a1111111111a1111111111a1111111111open accesscitation guo a drake bf khan ym langabeer iijr foraker re timeseries cardiovascularrisk factors and receipt of screening for breastcervical and colon cancer the guidelineadvantage one e0236836 101371 pone0236836editor antonio palazo´nbru universidad miguelhernandez de elche spainreceived 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 pone0236836copyright guo 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 are ownedby a third party and the authors do not havepermission to share the data requesting access tothe guideline advantage tga data must be done one 101371 pone0236836 august one 0ccardiovascular risk factors and receipt of cancer screeningsby contacting the american heart association viaemail qualityresearchheart the python coderelated to the analyses can be found in githubrepository githubcomaixiaguopythoncodefunding the authors received no specificfunding for this workcompeting interests the authors have declaredthat no competing interests existdeep learning algorithm could be used to investigate the associations between timeseriescvh measures and cancer screenings in an ambulatory population patients with moreadverse cvh profiles tend to be screened for cancers and cancer screening may alsoprompt favorable changes in cvh cancer screenings may increase patient cvh healththus potentially decreasing burden of disease and costs for the health system eg cardiovascular diseases and cancersintroductioncancer is the second leading cause of death for both men and women in the united statesus breast cancer is the second leading cause of cancer death among women colorectal cancer ranks second among men and third among women while cervical cancer ranksas a major cause of cancer death among women regular cancer screenings for breast cervical and colorectal cancers can help to diagnose cancers early and reduce cancer deaths for example in the past years the number of deaths caused by cervical cancer has significantly decreased thanks to pap tests which can find abnormal cervical cells before they turn tocancer similarly colonoscopy removes noncancerous colon polyps before becomingmalignant and regular mammography screening can identify breast cancer in an earliermore treatable stage thus breast cancer screening bcs cervical cancer screening cecsand colorectal cancer screening cocs are very important for early detection and treatmentfactors associated with cancer screenings include demographic factors health insurancecoverage education level smoking status obesity and cholesterol testing for example receiptof mammography is associated with modifiable factors such as weight smoking and other lifestyle factors [] receipt of cecs is associated with healthier weight lower cardiovascular disease occurrence and lower cholesterol some studies suggest that smokingsedentary lifestyle high body mass index and high comorbidity are associated with a higherpercentage of cocs participation [] traditionally data for such studies originate fromquestionnaires claims data and telephone surveys and statistical analysis methods such aslogistic regression models are applied to examine the associations between the risk factors andcancer screenings electronic health records ehr contain longitudinal healthcare information and data including diagnoses medications procedures lab tests and images andtherefore can be used to discover new patterns and relationships from the rich data deeplearning algorithms have been widely and successfully used in bioinformatics and healthcarefields as they can effectively capture features and patterns in longitudinal data in this study we investigated associations between longitudinal cvh risk factors and thereceipt of cancer screenings using ehr data by the long shortterm memory lstm model we then studied the distribution of cvh factors between patients who did and did notreceive cancer screenings to further investigate the associations finally we compared measures of cvh longitudinally within those who did and did not receive screening to betterunderstand the effect of cancer screenings on cvh measuresmaterials and methodsethics statementall the data were fully anonymized before we accessed them our study was approved by theinstitutional review board at the washington university school of medicine in st louis we one 101371 pone0236836 august one 0ccardiovascular risk factors and receipt of cancer screeningsobtained a written acknowledgement of proprietary rights and nondisclosure and data useagreement from the american heart association the washington university_nda_dua_contractid 158065_20190426_kdata source and study populationthe guideline advantage tga is a clinical data registry established in by the americancancer society the american diabetes association and the american heart associationaha ehr data has been collected from over clinics across the us by the tga totrack and monitor disease management and outpatient preventative care we used longitudinal tga data to predict three types of cancer screenings among unique patientswe used a 6year range to identify female patients in the year oldage group who received bcs female patients in the year old age group who receivedcecs and patients in the year old age group who received cocs if patientsreceived multiple types of cancer screening we only considered the first using the same criteria for gender and age we randomly selected a comparison group of patients who did notreceive cancer screenings for bcs for cecs and for cocswe utilized the following cvh measures defined by the aha smoking status body massindex bmi blood pressure bp hemoglobin a1c a1c and cholesterol lowdensitylipoprotein ldl in our dataset we then classified them into three categories ideal intermediate or poor according to table we utilized the multum drug database as a template to convert the drug names in our dataset to their corresponding drug classes thelevenshtein distance algorithm was employed for the conversion by comparing the drugnames in our dataset to the multum drug database template the conversion was consideredsuccessful and medications were considered as treatments for bp a1c or ldl table if thedistance between the two compared strings was less than five all cvh measurements prior tothe date of cancer screening were considered in the analysis for those who received screeningand all cvh measurements in the data set were considered in the analysis for those who didnot receive screeningfor the primary analysis we selected patients who had at least one measure of cvh for bcs for cecs and for cocs in the comparison groups there were availabledata for bcs cecs and cocsstatistical analysiswe first studied the lstm prediction of cancer screening from timeseries cvh factors wedivided each cvh factor into its submetric of ideal intermediate or poor according totable for example if a patient had a measure of ideal blood pressure then that featuretable measures of cvh which are available in the tga adapted from lloydjones poor healthintermediate healthideal healthhealth behaviorssmoking statusyesformer � monthsbody mass index� kgm2 kgm2health factorsnever or quit months kgm2ldl� mgdl mgdl or treated to goal mgdlblood pressurefasting plasmaglucosesystolic � mm hg or diastolic � mmhgsystolic mm hg or diastolic mm hg or treated togoalsystolic mm hgdiastolic mm hg� mgdl mgdl or treated to goal mgdl101371 pone0236836t001 one 101371 pone0236836 august one 0ccardiovascular risk factors and receipt of cancer screeningswas called blood pressure ideal all features were then embedded to a 32dimensional vectorspace by word2vec for each type of cancer screenings the python genism word2vecmodel used the following hyperparameters size embedding dimension was window themaximum distance between a target word and all words around it was min_count theminimum number of words counted when training the model was sg the training algorithm was cbow the continuous bag of words time information for each measure wasadded and was calculated by the difference in days between each visit date and the most recentvisit date thus each feature was associated with its own time point in the unit of daysthe resulting embedded vectors and associated time points were fed to the lstm modeldue to the comparison group being much larger than the number of patients with cancerscreening we randomly selected patients for bcs patients for cecs and patientsfor cocs and repeated this process for times to account for the imbalance betweenscreened and unscreened groups each time the data set for each type of cancer screening wassplit into a training data set and a test data set we trained the lstm model onthe training data and tested the trained model on the test data we utilized the average of thearea under the receiver operator curve auroc to evaluate the performance of our lstmmodel for each type of cancer evaluatedour lstm model comprised an input layer one hidden layer with dimensions andan output layer the hyperparameter used in the model was as follows a sigmoid function wasused as the activation function in the output layer a binary crossentropy was used as the lossfunction adam optimizer was used to optimize the model with a minibatch size of sampleswe then investigated whether distributions of cvhcounts and percentages for each submetricdiffered between patients who did and who did not receive cancer screenings by chisquared test finally we studied changes in cvh factors within screening group for the samepatients who received screening and for those who did not within screening group we compared cvh measures from before and on the day of the screening to the cvh measures collected after the screening for the patients who did not receive screening we compared cvhmeasures before and after the midpoint of the visit dates if patients only had a single visitthen they were not included in the before and after analysis analyses were conducted by usingthe libraries of scikitlearn scipy matplotlib with python version in resultsthe majority of our study population was white with a mean of age of approximately yearsfor bcs years for cecs and years for cocs table the nonwhite study populationwas predominantly africanamerican the average number of measures avg amongpatients who received screening was higher than that of patients who were not screened forexample the average number of bp measurements for patients with bcs was for cecsand for cocs compared to for bcs for cecs and for cocs for patients who werenot screenedfig displays the performance of lstm cancer screening predictions in terms of repeated aurocs for each type of screening the average auroc of curves was forbcs for cecs and for cocstable lists the numbers and proportions of patients in ideal intermediate and poor categories for each submetric for the comparison between patients who received cancer screeningand those who did not we applied a chisquared test to check if the frequencies herepercentages between screening groups were significantly different from one other within eachcvh submetric as shown in table patients who received cancer screening had a higher one 101371 pone0236836 august one 0ctable characteristics [mean sd or n ] of the study population by receipt of cancer screeningcardiovascular risk factors and receipt of cancer screeningscancer screeningsbcsdemographicsage mean std yearwhite race n nonwhite race n unknown race n cvh factors mean std avg measuresa1cldl mgdlbmi kgm2systolic blood pressure sbp mmhgdiastolic blood pressure dbp mmhgcurrent smoking n cecsdemographicsage mean std yearwhite race n nonwhite race n unknown race n cvh factors mean std avg measuresa1cldl mgdlbmi kgm2systolic blood pressure sbp mmhgdiastolic blood pressure dbp mmhgcurrent smoking n cocsdemographicsage mean std yearwhite race n nonwhite race n unknown race n cvh factors mean std avg measuresa1cldl mgdlbmi kgm2systolic blood pressure sbp mmhgdiastolic blood pressure dbp mmhgcurrent smoking n n n n yesnon � n n avg avg avg avg avg avg � the percentages may not add up to due to rounding101371 pone0236836t002prevalence of poor a1c for bcs for cecs and for cocs compared topatients who did not receive screening for bcs for cecs and for cocsfig shows changes in cvh submetrics within the same patient screening groups fig a2c show the changes in cvh submetrics for the patients who were screened while fig2e and 2f show the changes in cvh for patients who were not screened one 101371 pone0236836 august one 0ccardiovascular risk factors and receipt of cancer screenings one 101371 pone0236836 august one 0ccardiovascular risk factors and receipt of cancer screeningsfig the area under the curve auc are shown for lstm cancer screening predictions from timeseries cvh factors which were repeated times withdifferent comparison patients for bcs a cecs b and cocs c101371 pone0236836g001from the first column of fig we can see that the prevalence of poor submetricsdecreased after cancer screenings for example all five submetrics improved after bcs fig a while bp and a1c improved after cecs fig 2b and bp a1c and smoking improvedafter cocs fig 2c notably for the prevalence of poor a1c decreased for all patients whoreceived cancer screenings in bcs in cecs and in cocs on the other handfrom the second column of fig we can see that the prevalence of poor a1c increased forall comparison patientsdiscussionin this study we demonstrated associations between timeseries cvh risk factor measuresand receipt of three types of cancer screenings ie breast cervical and colon cancer screenings by using a nationally representative datasettga data the tga data enabled us toexamine multiple sites cvh submetrics and types of cancer screenings using advanced deeplearning models an advantage of our study was that all cvh submetrics were investigatedsimultaneously for an association with different cancer screenings on a unique nationallyrepresentative dataset of patients ie the large tga data set which contains longitudinaltable comparison cvh factors between patients with cancer screening or without [n ]patients with bcs n chisquared pvalueidealintermediatepoorpatients without bcs n idealintermediatepoorpatients with cecs n chisquared pvalueidealintermediatepoorpatients without cecs n idealintermediatepoorpatients with cocs n chisquared pvalueidealintermediatepoorpatients without cocs n idealintermediatepoorbmi bmi bmi 101371 pone0236836t003bp bp bp a1c a1c a1c ldl ldl ldl smoking smoking smoking one 101371 pone0236836 august one 0ccardiovascular risk factors and receipt of cancer screenings one 101371 pone0236836 august one 0ccardiovascular risk factors and receipt of cancer screeningsfig the plots of percentages for poor cvh factors for the same patients before and after time points of cancer screening for patients with screeningsac and before and after middle time points for patients without cancer screenings df the first row is for bcs second row is for cecs andthe third is for cocs101371 pone0236836g002cvh measurements and cancer screening patterns from more than different clinics in theusthe comparison of different cvh measure distributions between patients who receivedcancer screenings and those who did not showed that patients with poorer cvh especiallypoor a1c were more likely to receive cancer screenings specifically patients with poorera1c were more likely to receive cancer screenings some recent studies have showed that individuals with diabetes had higher incidence of certain cancers and also were more likely tobe diagnosed with advancedstage tumors [] thus providers might be more likely torecommend patients with diabetes to uptake cancer screenings for early prevention of developing cancers which may lead to more individuals with diabetes to participate in cancerscreeningsmoreover we investigated the effects of cancer screenings on the changes of cvh measuresof the patients to better understand if the screenings had potential associations with theimprovement of cvh measures our results indicated that patients who received cancerscreenings appeared to have better control of cvh factors especially a1c than patients whodid not receive cancer screenings specifically a1c levels were improved after patientsreceived any type of screening while a1c levels worsened among patients who did not receivecancer screening a similar trend could be observed for bmi it became better after patientsreceived any type of screening while bmi became worse among patients without bcs orcocs levels of bp were improved after patients received bcs or cocs screenings and worsened among patients without bcs or cocs poor levels of ldl decreased among patientsafter receipt of bcs and among those without bcs however ldl improvements were muchgreater among patients after receipt of bcs decrease in ldl than those without bcs decrease in ldl after receipt of bcs and cocs current smoking declined comparedto the increase observed among those without the screeningsin summary our analyses showed that patients with poor cvh measures were more likelyto receive cancer screenings patients with receipt of cancer screenings appeared to haveimproved cvh measures after the screening as compared to before one possible reason forthis was that patients might receive more attention and through care from providers to detectand manage cvh by virtue of reviewing cancer screening and other risk factor data at thepopulation level better cvh is associated with a lower risk of cardiovascular disease cvdand cancers thus cancer screenings may indirectly decrease burden and cost on thehealth system eg cvd and cancers by improving patient cvh healthlimitationsthere were some limitations in our analyses we used values of auroc to evaluate associations between timeseries cvh measurements and receipt of cancer screenings higherauroc values indicated stronger associations between predictors and the binary outcomes however our observed auroc values were relatively low and thus have limited clinicalutility at this time cancer screenings are potentially affected by cvh and other factors weacknowledge that we had relatively few patients with receipt of cancer screening specificallythere were relatively few patients who received cancer screenings compared to patients whodid not within the same age and gender groups this limitation likely affected the accuracy of one 101371 pone0236836 august one 0ccardiovascular risk factors and receipt of cancer screeningsour prediction models the prediction accuracy of our models could be improved if morepatients in our data set had received cancer screeningswe demonstrated that deep learning lstm models can effectively predict the associationsbetween timeseries cvh measures and receipt of cancer screening poor cvh especiallypoor a1c may prompt providers to recommend cancer screening for their patients andpatients who received cancer screening may also receive better care for andor have improvedselfmanagement of cvh especially a1c overall these findings suggest that unhealthierpatients are screened for cancers and that cancer screening may also prompt favorablechanges in cvhauthor contributionsconceptualization randi e forakerformal analysis aixia guosupervision randi e forakerwriting original draft aixia guowriting review editing bettina f drake yosef m khan james r langabeer ii randi eforakerwwwmedicalnewstodaycoms282929phpreferences humphrey ll helfand m chan bks woolf sh breast cancer screening a summary of the evidencefor the us preventive services task force annals of internal medicine 107326000348191375_part_120020903000012 american cancer society cancer facts figures am cancer soc 10 pmid wwwcdcgovcancercervicalstatisticsindexhtmwwwcdcgovcancerdcpcpreventionscreeninghtmwwwcdcgovcancercervicalstatisticsindexhtm1 edwards qt li ax pike mc kolonel ln ursin g henderson be ethnic differences in the use ofregular mammography the multiethnic cohort breast cancer res treat 101007s1054900800497 pmid bynum jpw braunstein jb sharkey p haddad k wu aw the influence of health status age andrace on screening mammography in elderly women arch intern med 101001archinte165182083 pmid lipscombe ll hux je booth gl reduced screening mammography among women with diabetesarch intern med 101001archinte165182090 pmid berz d sikov w colvin g weitzen s weighing in on screening mammography breast cancer restreat 101007s105490080037y pmid cook nr rosner ba hankinson se colditz ga mammographic screening and risk factors for breastcancer am j epidemiol 101093ajekwp304 pmid fontaine kr heo m allison db body weight and cancer screening among women j womenshealth gend based med 101089152460901300233939 pmid hsia j kemper e kiefe c zapka j sofaer s pettinger m the importance of health insurance asa determinant of cancer screening evidence from the womens health initiative prev med baltim 101006pmed20000697 pmid hueston w stiles m the papanicolaou smear as a sentinel screening test for health screening inwomen arch intern med pmid one 101371 pone0236836 august one 0ccardiovascular risk factors and receipt of cancer screenings robb ka miles a wardle j demographic and psychosocial factors associated with perceived risk forcolorectal cancer cancer epidemiology biomarkers and prevention robb ka miles a wardle j perceived risk of colorectal cancer sources of risk judgments cancer epidemiol biomarkers prev 10115810559965epi060151 pmid gimeno garca az factors influencing colorectal cancer screening participation gastroenterologyresearch and practice 1011552012483417 pmid jensen pb jensen lj brunak s mining electronic health records towards better research applications and clinical care nature reviews genetics 101038nrg3208 pmid goodfellow i bengio y courville a deep learning mit press 101533 rajkomar a oren e chen k dai am hajaj n hardt m scalable and accurate deep learning withelectronic health records npj digit med 101038s4174601800291 pmid hochreiter s s long shortterm memory neural comput bufalino v bauman ma shubrook jh evolution of the guideline advantage lessons learnedfrom the front lines of outpatient performance measurement ca cancer j clin 103322caac21233 pmid wwwscrippssparkleassetsdocumentsheart_rhythm_factspdf shickel b tighe pj bihorac a rashidi p deep ehr a survey of recent advances in deep learningtechniques for electronic health record ehr analysis ieee j biomed heal informatics 101109jbhi20172767063 pmid levenshtein vi binary codes capable of correcting deletions insertions and reversals sov phys dokl citeulikeid311174lloydjones dm hong y labarthe d mozaffarian d appel lj van horn l defining and settingnational goals for cardiovascular health promotion and disease reduction circulation 101161circulationaha109192703 pmid mikolov t corrado g chen k dean j word2vec proc int conf learn represent iclr kingma dp ba j adam a method for stochastic optimization corr 2015abs14126 pearson k on the criterion that a given system of deviations from the probable in the case of a correlated system of variables is such that it can be reasonably supposed to have arisen from random sampling philos mag tsilidis kk kasimis jc lopez ds ntzani ee ioannidis jpa type diabetes and cancer umbrellareview of metaanalyses of observationlal studies bmj online 101136bmjg7607 pmid lipscombe ll fischer hd austin pc fu l jaakkimainen rl ginsburg o the associationbetween diabetes and breast cancer stage at diagnosis a populationbased study breast cancer restreat 101007s1054901533235 pmid bhatia d lega ic wu w lipscombe ll breast cervical and colorectal cancer screening in adults withdiabetes a systematic review and metaanalysis diabetologia 101007s00125 pmid wilkinson je culpepper l associations between colorectal cancer screening and glycemic control inpeople with diabetes boston massachusetts prev chronic dis wang yq wang cf zhu l yuan h wu lx chen zh ideal cardiovascular health and the subclinicalimpairments of cardiovascular diseases a crosssectional study in central south china bmc cardiovasc disord 101186s1287201706979 pmid foraker re abdelrasoul m kuller lh jackson rd van horn l seguin ra cardiovascularhealth and incident cardiovascular disease and cancer the womens health initiative am j prev med 101016jamepre201507039 pmid yin j using the roc curve to measure association and evaluate prediction accuracy for a binary outcome biometrics biostat int j 1015406bbij20170500134 one 101371 pone0236836 august one 0c' | 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 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" 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 |
bousmalis leveraged cgan with the contentsimilarity loss to generate realistic target images and jointly trained the gan discriminator with the task network unidirectional translation has been applied to remove dataset variations for example bentaieb et al designed a stain normalization approach using a task conditional gan to translate he images to a reference stain madani et al proposed a semisupervised approach for cardiac abnormality classification using using gan discriminator for abnormality classification in minimally labeled xray images and showed that the adversarial loss could reduce domain overfitting mahmood translated real endoscopy images to graphicallyrendered synthetic colon images with groundtruth annotations for depthestimation during surgical navigation unidirectional translation has also been applied to crossmodality scenario for instance zhao proposed a modified unet to translate paired brain ct to mri bidirectional translationbidirectional image translation also known as reconstructionbased dt leverages two gans constraining the mapping space by enforcing semanticconsistency between the original and reconstructed images cyclegan by zhu is one of the most popular architectures for bidirectional translation cyclegan utilizes cycleconsistency to constrain the translation mapping and improve the quality of generated images cyclegan has been expanded to handle larger domain shifts with semanticconsistency loss functions cycada multidomain translation stargan and translation between two domains with multimodal conditional distributions munit in supervised learning bidirectional translation expands the training data to make the segmentation task model imia yearbook of medical informatics 2020choudhary 0crobust the translation and segmentation network can be trained either independently two stages or jointly zhang et al presented a onestage framework with an additional shapeconsistency loss in cyclegan to achieve better segmentation masks and lower failures chartsias used a twostage framework to segment mri images using ct images cai combined segmentation loss on generated images as an additional shape constraint for 3d translation and leveraged mri for pancreas segmentation in ct images in the unsupervised setting image translation is used to create labeled data for the target domain huo proposed a joint optimization approach for the synthesis and segmentation of ct images using labeled mri their framework achieved comparable performance in comparison to the fully labeled case there are a few observations about gans a cyclegan does not guarantee consistent translation of minor anatomical structures and boundaries and thus needs additional constraints like gradient and shape consistency for instance jiang incorporated tumorshape and featurebased losses to preserve tumors while translating ct data to mri data b attention networks can account for varying transferability of different image regions for instance liu proposed a novel attentionbased unet as a gan generator to translate hardtogenerate textured regions from mri to ct for alternate scenarios such as 3d2d paired images or semisupervised dadt zhang segmented xray images by using synthetic xray images created from accessible 3d ct annotations nguyen et al used semisupervised da with paired ct images to constrain cyclegan to generate more realistic images pan leveraged mri to generate missing pet images for patients for alzheimers disease diagnosis chen proposed stateoftheart unsupervised segmentation method using bidirectional dadt between mri and ct combining cyclegan with shared feature encoder layers between domains their method resembled cycada and showed the efficacy of combining dt with featurebased alignment c dadt can be used for singlemodality medical imaging chen leveraged a cyclegan with semanticaware adversarial loss to perform lung segmentation across different chest xray datasets latent feature space transformation in domain adaptation unlike the imagetoimage translation in dtda the lfstda transforms the source domain and target domain images to a shared latent feature space to learn a domaininvariant feature representation the goal is to minimize domainspecific information while preserving the taskrelated information the lfstda can be trained in an unsupervised fashion to obtain a domaininvariant representation or in a concurrent manner where the representation network and the task network eg image classification network are trained simultaneously to improve the performance lfstda is used in three basic implementations divergence minimization adversarial training and crossdomain reconstruction compared to dtda lsftda is more computationally efficient because it focuses on translating relevant information only instead of the complete image also featurebased domain alignment outperforms dtda by preserving taskcritical features divergence minimizationa simple approach to learn domaininvariant features and remove distributionshift is to minimize some divergence criterion between source and target data distributions common choices include maximum mean discrepancy mmd correlation alignment coral [ ] contrastive domain discrepancy cdd and wasserstein distance mmd coral and wasserstein distances are classagnostic divergence metrics and do not discriminate class labels when aligning samples cddbased da aligns samples based on their labels by minimizing the intraclass discrepancy and maximizing the interclass discrepancy mmd and coral are two of the most utilized divergence metrics that match the firstorder moment mean and the secondorder moment covariance of distributions however the represented hidden features can be complicated in the real world and may not be fully characterized by mean and covariance wasserstein distance aligns feature distributions between domains via optimal transport theory compared to the adversarialbased approaches divergencebased da has not been as widely explored in medical imaging for crossmodality da zhu utilized maximum mean discrepancy to map mr and pet images to a common space to mitigate missing data several works have used samemodality da to mitigate dataset variations in xray retinal fundus and electron microscopy images adversarial traininginstead of minimizing a divergence metric adversarial methods train a discriminator typically a separate network in an adversarial fashion against the feature encoder network the goal of the feature network is to learn a latent representation such that the discriminator is unable to identify the input sample domain from the representation for medical imaging featurebased adversarial domain adaptation has been widely utilized for various applications for example in crossmodality adaptation zhang applied a domain discriminator to adapt models trained for pathology images to microscopy images lsftda is also used for singlemodality adaptation to overcome dataset variations in pathology images mr images and ultrasound images for example lafarge have utilized a domain discriminator to mitigate the color variations of histopathology images for mitosis detection in breast cancer kamnitsas have applied a domain discriminator to mr images from different scanners and imaging protocols to improve the brain lesion segmentation performance reconstructionbased adaptationthe reconstructionbased adaptation maximizes the interdomain similarity by encoding images from each domain to reconstruct images in the other domain the reconstruction network decoder performs feature alignment by recreating the feature extractors input while the feature extractor encoder transforms input image into latent representation ghifary proposed imia yearbook of medical informatics 2020advancing medical imaging informatics by deep learningbased domain adaptation 0cdrcn for object detection using only target domain data reconstruction while bousmalis et al proposed a domain separation network that extracts image representations in two subspaces the private domain features and the shareddomain features the latter being used to reconstruct input image for medical imaging reconstructionbased methods are less developed and are usually combined with adversarial learning for samemodality adaptation oliveira et al have combined imagetoimage translation with a featurebased discriminator to mitigate the variations in xray images and improve segmentation performance for crossmodality adaptation ouyang combined variational autoencoder vae with adversarial training to adapt mr to ct scans challenges and opportunities domain selection and direction of domain adaptationselecting related domains for effective knowledge transfer is an openresearch area in ml in medical imaging domains are often selected based on the type of imaging technique eg radiology anatomy availability of labeled data and whether the modalities are complementary for the underlying task regarding whether da could be performed symmetrically across domains the potential information loss in a particular direction is critical for assessing task performance for example for unsupervised da from ct to mri reverse da may sometimes be needed to preserve tumors for supervised da between multiple he stained images tellez showed that mitosisdetection and cancer tissue classification in a particular color space leads to higher accuracy typically to assess domain relationship and da direction it is necessary to use a largescale empirical studies such as exploring bidirectional da across multiple datasets b a representationshift metric to roughly quantify the risk of applying learnedrepresentations from a particular domain to a new domain or c multisource da which automatically explores latent source domains in multisource datasets and quantifies the membership of each target sample however such experimentation requires extensive benchmarking studies that are lacking in medical imaging transferability of individual samplesmost da studies for medical imaging assume that all samples are equally transferable across two domains thus they focus on globally aligning domain distributions however the ability to transfer or align varies across clinical samples because of a intradomain variations eg in multimodal da between mri and ct each modality can have contrast variations b noisy annotations due to human subjectivity c target label space being a subset of source label space and d varying transferability among different image regions eg tumors are difficult to translate and could be missed during ct to mri imagetranslation some samples in the source domain may be less useful and can lead to negative transferring which adversely impacts da selecting relevant samples or reducing the impact of outlier samples using transferability frameworks is a potential solution some strategies include weighting samples based on classifier discrepancy downweighting outlier classes using the classification probability for target data leveraging openset based optimization and leveraging an attention mechanism to focus on hardtotransfer samples or using a noise coadaption layer recent medical imaging studies have explored sample selection and transferability assessment using reverse classification accuracy attentionbased unet and transferable semantic representations limitations of domain adaptation in medical imagingfor medical imaging most dlbased da uses adversarial methods primarily gan for unsupervised da adversarial methods are prone to errors because the discriminator can be confused and there is no guarantee that the domain distributions are sufficiently similar moreover the generator in gan is prone to hallucinating content to convince the discriminator that data belongs to the target distribution as such cyclegan could be trained to synthesize tumors in images of healthy patients beyond applying consistency constraints during image translation artifacts which are not directly visible in synthesized images are also important for consideration for example cyclegans incorporate highfrequency information in the intermediate representation used by the second generator to translate the image back to the source domain this high frequency information can interfere with downstream tasks dtda approaches require translating the entire image increasing the complexity of the models for largesized medical images like whole slide images few studies [ ] have compared adversarial da methods for mrict translation however a comprehensive comparison of various featurebased da approaches is lacking future studies could explore combining dtda and lfstda approaches moreover current frameworks typically focus on sourcetarget domain pair while many tasks such as stain normalization in histopathology images can be multidomain leveraging synthetic datada for medical imaging can be applied in relatively underexplored applications such as singleview 3d reconstruction or temporal disease analysis this could benefit imageguided surgery in which training data is very scarce and difficult to obtain one way is to leverage synthetic data with ground truth information adapting it to the real data this approach has been successfully applied in natural images reverse domain adaptation ie translating real data to synthetic data is also a promising solution mahmood generated synthetic endoscopy data with known depth information by using an anatomical colon model and a virtual endoscope this simulated data was used for 3d reconstruction of real endoscopic images pan translated mr data to generate synthetic pet images to infer missing patient scans for temporal analysis of alzheimers disease another area that could benefit from synthetic data is skin lesion detection imia yearbook of medical informatics 2020choudhary 0ctable summary of da studies in medical imaging categorized by da methodology task modality anatomy and learning scenarios s segmentation c classification 3dr 3d reconstructionda method framework task source target domains anatomy learning scenario publications single modality dt lfst unidirectional reconstruction s c s c c s s s s s s s divergence s reconstruction s adversarial crossmodality radiology xray different demographics xray different demographics synthetic data mri xray different demographics ultrasound different sources xray different disease states ultrasound different sources mri different flair sequences mri moderate to severe tbi mri different disease types demographics mri different vendors mri crossinstitutional xray different disease types demographics contrast lung xray different datasets breast lung lung brain lung fetal head lung heart brain brain brain brain brain spine reconstruction ct mri s ct xray s mri ct s mri ct s s ct mri s 3d ct mri s mri ct mri ct mri t1 t2 s ct mri s mri pet c mri ct s 3dr ct 3d rendered depthmap mri pet c ct mri s s mri ct reconstruction s 3d mri ct unidirectional divergence adversarial dt lfst single modality dt lfst adversarial unidirectional c c c s s divergence wsi different he stains wsi different stains wsi crossinstitutional wsi different stains microscopy different specimens crossmodality dt lfst reconstruction s s unidirectional adversarial c wsi ck pdl1 wsi he if wsi microscopy lung lung heart heart heart heart breast pancreas abdomen abdomen brain hip thigh pelvis brain brain colon brain abdomen heart heart pathology breast colon ovary breast prostate colon brain lung breast bladder lung colon opthalmology single modality dt unidirectional adversarial divergence lfst s s s s retinal fundus multiple datasets retinal fundus multiple datasets retinal fundus multiple datasets retinal fundus multiple datasets retina retina retina retina unsupervised semisupervised unsupervised semisupervised unsupervised chen tang gholami et a madani yang dong degel orbesarteaga kamnitsas novosad yan shanis venkataramani oliveira unsupervised supervised unsupervised supervised unsupervised unsupervised supervised unsupervised unsupervised unsupervised jiang zhang chen chartsias zhang cai huo liu hiasa pan zhao mahmood zhu yang dou ouyang bentaieb lafarge ren hou chacon kapil brieu zhang zhao javanmardi wang zhuang imia yearbook of medical informatics 2020advancing medical imaging informatics by deep learningbased domain adaptation 0c conclusions and future directionsdeep learning has been widely applied to medical imaging data analysis however the lack of wellannotated images and the heterogeneity of multicenter medical imaging datasets are two key challenges for dl performance da has emerged as an effective approach for minimizing domainshift and leveraging labeled data from distinct but related domains fstda and dtda are two popular approaches to minimize the distribution divergence in multiple medical imaging studies exploring samemodality or crossmodality scenarios they have proven to achieve good performance particularly in unsupervised da settings and organ segmentation tasks current approaches are primarily adversarial with domains being selected based on certain heuristics and underlying tasks extensive benchmarking studies are needed to quantify the domain relationship for different imaging modalities and to compare adversarial and nonadversarial approaches varying sample transferability and multimodal domains for medical imaging are two other major issues one strategy is to explore downweighting or attentionbased networks also alternative multimodal frameworks such as munit can be explored finally for certain application areas in medical imaging such as 3d reconstruction and temporal disease analysis where da is relatively unexplored synthetic data can be usedacknowledgmentsthe work was supported in part by grants from the national science foundation eager award nsf1651360 childrens healthcare of atlanta and georgia tech partnership grant giglio breast cancer research fund petit institute faculty fellow award and carol ann and david d flanagan faculty fellow research fund for professor may d wang this work was also supported in part by the scholarship from china scholarship council csc under the grant csc no the content of this article is solely the responsibility of the authors and does not necessarily represent the official views of the nihreferences mendelson ds rubin dl imaging informatics essential tools for the delivery of imaging services acad radiol oct20101195 litjens g kooi t bejnordi be setio aaa ciompi f ghafoorian m a survey on deep learning in medical image analysis med image anal dec4260 adlermilstein j jha ak sharing clinical data electronically a critical challenge for fixing the health care system jama apr sharma p shamout fe clifton da preserving patient privacy while training a predictive model of inhospital mortality arxiv preprint arxiv191200354 dec zhang y wei y zhao p niu s wu q tan m huang j collaborative unsupervised domain adaptation for medical image diagnosis arxiv preprint 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chronic respiratory diseases are highly prevalent worldwide and will continue to rise in theforeseeable future despite intensive efforts over recent decades the development of novel and effectivetherapeutic approaches has been slow however there is new and increasing evidence that communities ofmicroanisms in our body the human microbiome are crucially involved in the development andprogression of chronic respiratory diseases understanding the detailed mechanisms underlying this crosstalk between host and microbiota is critical for development of microbiome or hosttargeted therapeuticsand prevention strategies here we review and discuss the most recent knowledge on the continuousreciprocal interaction between the host and microbes in health and respiratory disease furthermore wehighlight promising developments in microbiomebased therapies and discuss the need to employ moreholistic approaches of restoring both the pulmonary niche and the microbial communityreceived nov accepted after revision april copyright ers this version is distributed under the terms of the creative commons attribution noncommercial licence 10118313993003023202019eur respir j 0crespiratory basic science r gosens introductionin the past decade we have learned that the lungs previously considered sterile in fact harbour a dynamicecosystem of diverse bacteria fungi and viruses this lung microbiota is detectable in health [ ]altered in disease predictive of disease outcomes [ ] and correlates with variations in host immunity[ ] recent insights based on studies in both humans and mice are that the baseline immunetone of the lungs even in health is closely linked to the local microbial milieu this hypothesis thatthe immune tone in the airways and alveoli is at least in part regulated by the microbiota is a radicaldeparture from our conventional dichotomous understanding of lung immunity dormant in healthactivated in infection next to known changes in the inflammatory milieu of the lungs most lung diseasessuch as asthma copd cystic fibrosis idiopathic pulmonary fibrosis ipf andrecently lung cancer have been associated with a microbial dysbiosis in the lung however it isunknown if changes in the bacterial composition drive disease pathogenesis or if they are rather areflection of alterations of the ecological niche [ ] thus it is of utmost importance to understand theunderlying molecular mechanisms at the hostmicrobiome interface to develop novel targeted therapies orpreventative approacheshere we discuss the impact of hostmicrobiome crosstalk on respiratory health and disease whilefocusing on how the local microbiota and the host interact at the epithelial surface furthermore wereview current therapeutic approaches and suggest a more holistic approach for treating lung disease inthe future this review is a followup of presentations and discussions at and after the ers researchseminar crosstalk in the lung microenvironment implications for understanding chronic lung diseaseberlin february the mucosal niche in the lungall external interfaces of the human body such as gut skin reproductive and respiratory tracts arecolonised by a distinct microbial flora the microbial communities differ at the various body sites dueto local factors eg oxygen carbon dioxide ph nutrients host defence factors inhaled pollutants thatshape the niche as examples the lumen of the lower gastrointestinal tract represents a lowoxygennutrientrich environment and is thus commonly populated by highabundance communities of anaerobicanisms by contrast the skin is a lownutrient environment directly exposed to environmental oxygenand is thus more commonly populated by relatively sparse communities of oxygentolerant bacteria thusthe local environment is a crucial determinant of the formation of microbial communities early indevelopment but also at later stages antimicrobial peptidesand otherclearance production ofconsequently the lung microenvironment creates a special ecological niche for microbes that differs fromother body sites and will presumably influence nichespecific colonisation accordingly airway epithelialcells are a principle contributor in shaping this niche as they are strategically located to be the first contacttissues various mechanisms arebetween inhaled substances including microanisms and hostemployed by the airway epithelium in host defences against infectionincluding its barrier functionmucociliaryandantiinflammatory mediators and ability to transport eg polymeric iga and igm from the basal to theapical side of the epithelium through the polymeric immunoglobulin receptor pigr [] it is likelythat similar mechanisms contribute to the formation of nichespecific communities along the respiratorytract and such local conditions are crucial for allowing beneficial microbiota to persist at epithelialsurfaces the involvement of such hostmicrobiota interactions in disease is wellillustrated in cysticfibrosis in cystic fibrosis mutational dysfunction of the cystic fibrosis transmembrane regulator cftrprotein results in a reduction of anion mostly bicarbonate and chloride exchange across the epithelialsurface resulting in a dehydrated surface and sticky mucus this mucus cannot be readily cleared from theairways which helps to explain why cftr mutations are associated with alterations of the residentmicrobiota including frequent colonisation with staphylococcus aureus and pseudomonas aeruginosa asrecently reviewed in however we are still lacking knowledge on the underlying mechanisms of theniche alterations in more complex genetic lung diseases such as asthma and copdsubstances proadditionallyallowing it to mount appropriate responses or develop tolerance [ ]the epithelium transmits environmental and microbial signals to the immune systemvarious mechanisms of sensing microbial presence include pattern recognition receptors such as thetolllike receptors and other processes not mediated via classical receptormediated signalling egthe integrated stress response upon sensing microbial environmental or endogenous challengesepithelial cells mount active responses by increasing defence molecules such as antimicrobial peptidescytokines and chemokines these enhance the defence against respiratory pathogens while simultaneouslychanging the ecological niche of complex microbial communities indicating that the properties of theecological niche encountered by microanisms changes as a result of environmental exposures10118313993003023202019 0crespiratory basic science r gosens anatomical differences along the respiratory tract such as differences in epithelial cell composition shapethis ecological niche while the epithelium contributes to local conditions that shape the microbiotaepithelial exposure to microbes and their products has marked effects on its function consequentlyhuman epithelia and the microbiota have developed interactions that are of mutual benefit responding topathogens and tolerating innocuous substances this concept is important to understand how inhaledenvironmental triggers regulate immunity since microbial components contribute to the response toenvironmental exposures such as farm and geogenic earthderived dust however how theepithelium integrates these microbial and nonmicrobial signals into a finetuned response is incompletelyunderstoodin order to transmit signals from the environment epithelial cells use sophisticated communicationsystems with other lung cell types the airway epithelium and that of gut and skin plays key roles intransmitting signals from microanisms and environmental stimulito instruct antigenpresentingdendritic cells to direct immunity towards inflammation or tolerance epithelial cells interact not onlywith other immune cells such as macrophages neutrophils innate lymphoid cells and tcells but alsowith structural cells such as fibroblasts airway smooth muscle cells and endothelial cells via a plethora ofdifferent mechanisms figure [ ] this array of interactions with environmental triggersmicroanisms and lung cells allows epithelial cells to orchestrate host defence and immunity and tomaintain epithelial integrity and mediate pathologic airway remodelling figure establishment and maintenance of the lung microbiotathe establishment of the lung microbiota likely occurs in the first days and weeks of life although earlyhighprofile reports suggested the presence of a placental microbiome that could influence prepartumlung development subsequent wellcontrolled studies have failed to detect bacterial signals distinctfrom contaminating dna present in negative controls the lung microbiota of newborn mice isbelow the limit of detection via quantitative pcr and increase in total burden in the following days andweeks [ ] in human infants the composition of the respiratory microbiome seems to mature in apredictable wellcharacterised pattern during the first year oflife besides the special nichecharacteristics of the lung earlylife respiratory microbiota are influenced by mode of delivery vaginalversus caesarean method of feeding breast versus bottle and exposures siblings daycare attendance provocatively earlylife respiratory microbiota may predict subsequent susceptibility to respiratorytract infections [ ] suggesting roles of the local microbes in immune homeostasis and resistance topathogens in contrast being exposed to an increased microbial diversity during childhood promotes thedevelopment of balanced immunity and is protective against inflammatory responses to allergens andasthma development [ ] this protection is partly associated with distinct farm dust which in vitroincreases epithelial barrier function and antiviral defences once established the composition of the lung microbiome is determined by three ecological factorsimmigration elimination and relative growth rates of community members figure in health lungcommunities are sparse and dynamic largely determined by the equilibrium between immigration viamicroaspiration and elimination via cough mucociliary clearance and immune defences littleevidence supports the presence of resident lung bacteria in health that reproduce and survive selectivepressures [ ] nevertheless the transient and dynamic communities detected in health are largelyviable and exert a detectable effect on the immune constitution of the lower respiratory tract [ ]the establishment of a diverse respiratory microbial flora is influenced by many different factors there isa finetuned balance between tolerance of commensal or beneficial bacteria at the epithelial surface andthe development of active immune responses to pathogens notably this balance is regulated by both hostand microbederived signals sudden shifts in this tightly controlled equilibrium such as outgrowth ofspecific pathogens or for example virally induced damage to the airway epithelium destroying the barrierand inducing local immune responses can have detrimental effects on both the host and the microbiomeand might therefore contribute to the pathogenesis of respiratory diseasesrole of the environment in shaping the lung microbiomeenvironmental influences play pivotal roles in the development of lung diseases this might be due to directeffects on the host epithelial barrier and immune responses but most known risk factors such as cigarettesmoke air pollution [ ] viral infections and di so directly impact the microbiota [ ]thus a combination of both might be critical for disease developmentfor example one of the most studied inhaled toxins cigarette smoke supposedly has a detrimental effecton both the host and microbial communities prolonged cigarette smoke exposure contributes to increasedbaseline inflammation in the airways and epithelial remodelling towards goblet cell hyperplasia and areduction in cilia and ciliary activity and can reduce the antimicrobial defence provided by the airway10118313993003023202019 0crespiratory basic science r gosens mucociliaryclearingmicroaspirationairway regionbacterial metabolitesouter membrane vesiclesquorumsensing moleculesalveolar regionantimicrobial peptidesciliary beatingmucuscytokineschemokinesgrowth factorsextracellular vesiclescritically altered in chronic lung diseasebacteriarespiratory virusclub cellciliated cell goblet cell alveolar type cell alveolar type cellfibroblastmucus layer basal celldendritic cell macrophagesmooth muscle cellcapillaryfigure hostmicrobiome crosstalk in the lung microenvironment the lung microenvironment consists of different cell types depending onthe location in the proximaltodistal airway tree the epithelial layer in larger airways is constantly exposed to a variety of different microbes ofthe local microbiota while the composition of the latter is influenced by host factors such as elimination via mucociliary clearance it alsodepends on the competition among the microbial inhabitants it is now evident that there is a complex crosstalk between host and microbes inthis environment accordingly bacterial metabolites or outer membrane vesicles can influence the host status while antimicrobial peptides orcytokines can shape the composition of the microbiota as virtually all of these single factors are altered in chronic respiratory disease it is ofutmost importance to appreciate and assess the complexity of this system in future studiesepithelium in patients with copd it has been shown to be associated with reduced lung bacilli andincreased haemophilus influenzae and streptococcus pneumoniae but the largest study to date inhealthy smokers has not found a significant effect of cigarette smoke on the lung microbiome thus it is intriguing to speculate that cigarette smoke primarily affects the host system which over timeand upon disease copd development in susceptible individuals may also affect the microbiomeanother example of environmental influence on respiratory disease is diet which has profound effects onboth microbiota and health even in the short term the intake of dietary fibre induces similarbeneficial microbiota changes in the lung and gut while lowfibre diets change the gut microbialcommunity over multiple generations in mice highfibre diets have beneficial effects in pregnant miceand suppress allergic airway disease aad in mothers and their offspring this also highlights theimportance of crosstalk between the gut and lung highfibre diets induce the production of shortchainfatty acids by gut bacteria which are transported systemically to the lung where they exertantiinflammatory actions and ameliorate aad in mice in contrast a lipidrich di ters themicrobiota and promotes metabolic inflammation and has been associated with premetastatic nichedevelopment in lung cancer however the detailed mechanisms of action remain unclearalong with bacteria common major respiratory viruses including respiratory syncytial virus rhinoviruscoronaviruses influenza and adenoviruses are part of the respiratory microbiome and contribute tothe pathogenesis of chronic respiratory diseases this may result from complex interactions of viruses withthe hosts immune system and the microbiota including other pathogens these interactions can influencethe prevalence of bacterial pathogens by increasing the expression of adhesion molecules on therespiratory epithelium damaging respiratory epithelial cells compromising barrier functionimpairingmucociliary clearance and altering host immunity and the lung microenvironment [] interestinglyit has been suggested that viruses and bacteriophages induce consistent and reproducible changes inrespiratory microbiomes in chronic disease but not the healthy state and have been shown to increasemicrobial diversity in the nasopharynx [ ] furthermore fungi such as aspergillus spp contributeto the pathogenesis of chronic respiratory diseases as pathogens on their own but also by activation of theimmuneinteractions with microbiota particularly withnontuberculous mycobacteria [ ]through theirsystemand probablyimportantly the absence of certain members from the microbiome can have detrimental influences on lunghealth this is illustrated by the relative disappearance of parasitic worms which have been a constant partof our gut microbiome and have even been found in fossils from the period of jawed fish in fact our10118313993003023202019 0crespiratory basic science r gosens modern immune system developed in the continuous presence of helminths which may explaintheir important regulatory role in immunity typically worms induce type2 immune responses which areconsidered instrumentalin host defence against these parasitic infections however worm infectionsinduce regulatory responses that are aimed to suppress immune responses directed at worm antigenswhich allows worms to live in their host for years additionally this regulatory response has a bystandereffect by promoting allergen tolerance this is illustrated by studies in mouse models in whichvarious helminth infections have been shown to provide protection against the development of allergicasthma the range of helminthinduced protective mechanisms includes the inhibition of interleukinil33 in the airways and the induction of regulatory tcells [ ] bcells [ ] andmacrophages intriguingly the presence of helminths also affects the composition of the bacterialcommunity which might be important for the protection against allergic airway disease in mice niche and microbiome alterations in respiratory diseasein disease the ecology of the lower respiratory tract changes dramatically the airways and alveolinormally inhospitable to bacterial reproduction are radically altered by the influx of nutrientrichoedema and mucus establishment of stark oxygen gradients surge of bacterial growthpromotinginflammatory responses [ ] and impairment of local host defences [ ] thus it is unsurprisingthat crosssectional studies have identified altered lung microbiomes in established lung diseases aschanges in ecological niches supposedly result in different microbial communities due to selective pressurethe composition of the lung microbiome then becomes increasingly determined by the relative growthrates of its constituentswhile there are some studies on the role of the microbiome in ipf and lung cancer mostknowledge is based on studies of chronic inflammatory respiratory diseases such as asthma copd andcystic fibrosis generally those patients have increased susceptibility to infections and exacerbations thatagain affect the microbiome [ ] h influenzae moraxella catarrhalis and s pneumoniae areassociated with the development of severe asthma and copd they modify the lung microbiome anddrive inflammation oxidative stress symptoms and exacerbations [ ] which may form a viciouscircle perpetuating the disease m catarrhalis and hinfluenzae in particular induce neutrophilicinflammation more severe disease and steroid resistance [] furthermorein asthma alteredrespiratory microbiome profiles are associated with asthma phenotype and severity responses toallergens and treatment in cystic fibrosis and bronchiectasis haemophilus and pseudomonas spp increasingly dominate the lungmicrobiota [ ] genetic association studies in phe508del cystic fibrosisaffected homozygousindividuals have shown associations of single nucleotide polymorphisms with important disease featuresrelated to bacterial colonisation for example gene variants of human leukocyte antigen class ii genesimplicating a role for tcell and bcell immunity associate with age of onset of persistent p aeruginosainfection indicating the role of the hosts immune system in selecting a distinct microbiota like the bacterial composition ofthe lung microbiome antiviral responses are altered in chronicrespiratory diseases in asthma where airway epithelial repair in response to viral infection is aberrant dueto changes in genes regulating epithelial barrier function and repair the airway epithelium hasreduced innate immunity to common viruses such as rhinovirus [ ] this is due to reduced type iand iii interferon and increased mirna levels [ ] transforming growth factor tgfβ a cytokinecommonly upregulated in asthma suppresses airway epithelialinnate immune responses throughsuppressor of cytokine signalling socs1 and socs3 contributing to impaired antiviral immunity this impaired antiviral immunity may also alter the bacterial microbiome and contribute to coinfectionsby bacteria rhinovirus infection can also impair the phagocytosis of bacteria by alveolarmacrophages which can lead to bacterial outgrowth in copd murine studies showed thatinfluenzainfected epithelial progenitors of the distal lung exhibited severely impaired renewal capacity dueto virusinduced blockade of βcatenindependent fgfr2b signalling this could contribute toimpaired repair of the distal lung in copd although its involvement in human disease is still unclearthus viral and bacterial infections and the inability to resolve them may aggravate impaired airway andalveolar repair combined with a heightened airway andor alveolar immune tone aberrant repairmechanisms following pathogen exposures in vulnerable individuals might represent a mechanism for howhostmicrobiome interactions contribute to disease progression it is currently unclear if alterations in thelung microbiome can also precede lung structural changes and inflammation and thereby contribute toonset of diseaseas discussed earlier the relationship between lung dysbiosis and lung inflammation is bidirectionaldisordered lung communities trigger epithelial and luminal inflammation further altering growth conditionsof the lung microenvironment chronic inflammation and perpetuating dysbiosis [ ] a common10118313993003023202019 0crespiratory basic science r gosens isthe outgrowth oftheinflammatory lung conditionsfinding acrossinflammationassociatedproteobacteria phylum and in mice the lung microbiome has been shown to be associated withpulmonary levels of the innate cytokine il1α additionally alterations in the microbiome of the gutmight influence the immune tone of the lung which is shown by the development of more severeexperimental asthma in germfree mice [ ] or mice with disturbed microbiomes due to earlylifeantibiotic treatment [ ] in contrast the absence of microbiota in germfree mice or due toantibiotic treatment has recently been shown to protect mice with kras mutations and p53 loss from lungcancer development according to the authors this effect was due to the induction of il1β andil23 through the microbiota that led to il17 production by γδt cells and tumorigenesisin humans copd patients are two to three times more likely to have crohns colitis and of peoplewith inflammatory bowel disease also have pulmonary inflammation [] the gastrointestinal tracthas by far the highest microbial content and thus an interaction of gut microbiota and their metaboliteswith the gastric mucosa affects systemic immunity which may in turn impactthe lung consequently alterations in gut microbiota and physiology might contribute to respiratory disease andvice versa possibly via the gutlung axis however it is important to note that germfree breeding and tosome extent antibiotic treatment will affect both the gut and the lung microbiome so it is still notcompletely clear whether these effects can fully be explained by gutlung crosstalk or if they areinfluenced by aberrant local microbiotaimmune crosstalk along this line in mice the lung immune tonehas been suggested to be more correlated to lung bacteria than gut bacteria in contrast in mice colitisinduced pulmonary pathology was associated with increased inflammation andgramnegative bacterial endotoxins in the lung that probably emanate from the gut furthermorecigarette smokeinduced experimental copd models [] indicate that reduced gas exchange andhypoxia in the lung is associated with hypoxia in the gut causing colon remodelling cell deathinflammation and impaired barrier function additionally activation of nodlike receptors bybacteria in the gut increase production of reactive oxygen species by alveolar macrophages suggesting thatthe gastrointestinal microbiome contributes to oxidative stress in the lung conversely oralapplication of beneficial probiotic bacteria bifidobacterium and lactobacillusbased reduced airwayinflammation and emphysema in cigarette smokeexposed mice a large canadian study reported that four bacterial genera lachnospira veillonella faecalibacterium androthia were reduced in the faeces of human infants that subsequently developed asthma and inoculating ahuman faecal microbiome supplemented with these four taxa into germfree mice partially protected theirf1 progeny from experimentally induced aad thus there are encouraging studies in mousemodels highlighting cause and effect of the gutlung axis in respiratory disease however findings firstneed to be validated in humans before proposing this crosstalk as a treatable trait for respiratory diseasein summary at the hostmicrobiome interface disordered communities are probably both cause andeffects of host inflammation however given the close reciprocal interactions between the microbiome andhost at all times it might be impossible to determine the real cause of the very first changes in diseasedevelopment as host and microbiome factors are coinciding and closely intertwinedimplications for the development of novel therapiesdue to the strong and dynamic interdependency between host and microbiome in local niches it isunsurprising that most drugs used in clinical practice that were designed to target the host also affect themicrobiome accordingly inhaled corticosteroids and proton pump inhibitors affect the lung microbiota[] as well as subsequent pneumonia risk [ ] macrolide antibiotics broadly effective acrosschronic lung conditions such as copd affect both lung microbiota and host immunity perhapsmost provocatively baseline differences in lung microbiota appear to predict patient responsiveness totherapies such as inhaled corticosteroids suggesting that variation in lung microbiota may representan untapped phenotype of precision medicine in the lung this opens new possibilities to exploit thisimportant crosstalk in therapeutic interventions but in order to do so we first need to improve ourunderstanding of the molecular mechanismsincreasing evidence of the importance of the microbiome raises the concept of restoring diseasedmicrobiomes to prevent or treat diseases using microbiotadirected therapies or hosttargeted therapiessuch as probiotics metabolites lung microbiota transplantation or vitamin d therapy which we discusshere in more detailmany clinical studies have investigated the efficacy of probiotic bacteria which are supposedly beneficialfor the host to prevent chronic diseases such as asthma or allergic rhinitis however these studies havelargely produced contradictory outcomes which might be due to the fact that the used probioticswere not selected based on potential mechanistic effects and may not be ideal the microbiome is a10118313993003023202019 0crespiratory basic science r gosens complex ecosystem comprised of a variety of different inhabitants and influenced by many externalhostderived factors thus the addition of single strains may not make a profound difference thus thetransfer of whole microbiomes via faecal microbiota transplantation fmt could be a more promisingapproach the introduction of healthy microbiota into diseased hosts has restored immunity andphysiology demonstrating that intestinal microbiota and their products can modulate host immunitylocally and systemically and that fmt can replace diseaserelated microbiomes with healthy ones fmt is remarkably successfulin treating antibioticresistant clostridium difficileinducedcolitis and is now being used as treatment in selected patients [ ] the new microbiomeengrafts quickly and lasts for at least a month indicating a potential difficulty in inducing longtermbeneficial changes in the microbiome via only targeting the microbial side while there are encouragingdata questions remain whether fmt may also affectlung health and whether lung microbiotatransplantation is feasible furthermore recently severe complications of fmt have been reported due totransfer of drugresistant bacteria thus it is essential to determine whether such approaches that sofar only transiently change the microbiome can be used for the required longterm treatments of chroniclung diseases that coincide with a variety of structural changes aberrant mucociliary clearance and manymore such as[]specific microbial moleculeslipopolysaccharide lps andalong with living bacteriapeptidoglycan can induce or modulate inflammatory responsesin addition culturesupernatants of probiotic bacteria display antiinflammatory effects which have been ascribed to thepresence of secreted immunemodulatory metabolites for example culture supernatants of certainprobiotic bifidobacterium species decreased the secretion of type cytokines from immune cell lines andthe expression of costimulatory molecules on primary dendritic cells a likely mechanism is quorumsensing quorum sensing is a means of communication among bacteria of the same species to coordinateeffector functions such as biofilm formation sporulation or toxin secretion the bestdescribed quorumsensing molecules are acyl homoserine lactones ahls several ahls are targeted by the host tointerfere with growth of pathogens and are in turn exploited by bacteria to regulate host geneexpression for their benefit some ahls can bind to distinct bitter taste receptors expressed on the airwayepithelium and innate and adaptive immune cells [ ] thereby modulating barrier andimmune functions the moststudied ahl 3oc12hsl can activate phagocytesto increasephagocytosis expression of adhesion receptors and chemotaxis [ ] but is itself cleaved andinactivated by airway epithelial cells another example of bacterialderived modulators of the hosts immune responses are outer membranevesicles omvs omvs are spherical bilayered membrane vesicles released from the surface of bothgramnegative and grampositive bacteria and contain much of the biological material from the parentbacterium but in a nonreplicative form [ ] evidence suggests that the release of omvs providesbacteria with competitive advantages when exposed to acute and chronic hostassociated stressors innate and adaptive immune responses [] antimicrobialthey may protect bacteria againstpeptides and antibiotics [ ] moreover omvs contain factors eg siderophores aiding in theacquisition of nutrients in an environment devoid of crucial elements such as iron besidessupporting the survival of the parent bacteria omvs may also play role in the progression of pulmonarydiseases bacteria frequently associated with copd exacerbations are known to release omvs furthermore macrophages stimulated with omvs derived from prominent airway pathogens such asp aeruginosa h influenzae or m catarrhalis release higher amounts of tumour necrosis factorα and il6 legionelladerived omvs significantly enhanced bacterial replication in macrophages andbacteriafree p aeruginosa omvs have been shown to potently induce pulmonary inflammation in mice strengthening the idea that omvs exert diseasepromoting activities in addition omvs have beenshown to induce tolerance and hyporesponsivenessthereby facilitating bacterial adherence to andinternalisation by macrophages which may contribute to clearance of the infection [ ] thusdespite our increasing knowledge on omvs and their potential role in interkingdom communicationthere is a need for further research to better understand their pathogenic properties and possibletherapeutic or prophylactic implications eg novel vaccinesan interesting alternative to fmt or probiotic bacteria might be to use immunemodulatory microbialmetabolites or beneficial omvs such chemically defined bacterial substances could be produced at largescale under controlled conditions applied in defined effective doses both systemically or locally and mayhave fewer adverse sideeffects ie in immunocompromised patients compared to live bacteria several studies have already used defined bacterial metabolites to treat aad in mice lps from escherichiacoli o111 bacterial polysaccharide a oligodeoxynucleotides with bacterial cpg motifs flagellin b shortchain fatty acids dtryptophan and the neutro | Colon_Cancer |
" ovarian cancer is the second most common gynecologic cancer with high mortality rate andgenerally diagnosed in advanced stages the 5year diseasefree survival is below micrornas subset of thenoncoding rna molecules regulate the translation in post transcriptional level by binding to specific mrnas topromote or degrade the target oncogenes or tumor suppressor genes abnormal expression of mirnas were foundin numerous human cancer including ovarian cancer investigating the mirnas derived from the peripheral bloodsamples can be used as a marker in the diagnose treatment and prognosis of ovarian cancer we aimed to findbiological markers for early diagnosis of ovarian cancer by investigating brca1 gene mutation carrier monozygoticdiscordant twins and their high risk healthy family individuals mirnasmethods the study was conducted on monozygotic twins discordant for ovarian cancer and the liquid biopsyexploration of mirnas was performed on mononuclear cells that were isolated from the peripheral blood samplesthe mirna expression profile changes in the study were found by using microarray analysis mirna isolationprocedure performed from the lymphocyte in accordance with the kit protocol the presence and quality of theisolated mirnas screened by electrophoresis raw data logarithmic analysis was studied by identifying thethreshold normalization correlation mean and median values target proteins were detected for each mirna byusing different algorithmsresults after the comparison of monozygotic discordant twins for epithelial ovarian carcinoma upregulation of the mirnas mir6131 mir1305 mir1973p mir3651 and downregulation of mirnas mir3135b mir4430 mir664b5p mir7663p were found statically significantcontinued on next page correspondence hy2188istanbuledutrdepartment of cancer genetics istanbul faculty of medicine oncologyinstitute istanbul university istanbul turkey 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 0ctuncer of ovarian research page of continued from previous pages the detected mirnas out of mirnas might be used in the clinic as new biological indicatorsin the diagnosis and follow up of epithelial ovarian cancer with complementary studies the mirna expressionprofiles were identified to be statistically significant in the evaluation of ovarian cancer etiology brca1 mutationstatus and ovarian cancer risk in accordance with the obtained datathere is a need for validation of the mirnas which were particularly detected between monozygotic twins and itsassociation with ovarian cancer was emphasized in our study in wider cohorts including ovarian cancer patientsand healthy individualskeywords monozygotic twins mirna expression profiles brca1 and brca2 biomarkers ovarian cancer is a significant cause of mortality in gynecologic cancers and one ofthe leading cause ofcancerassociated mortality in turkey ovarian cancer is the 7th most common type of cancer in women inaccordance with worldwide globocan datas showthat each year more than women are diagnosedwith ovarian cancer oc worldwide and approximately women die from it the data of globocan for turkey shows that annually women are diagnosed with ovarian cancer and women die fromthis malignancy the 5year survival rate was given as these data revealed that ovarian cancer is an important reason of gynecologic cancer associated mortality rate the epithelial ovarian cancerseocoriginating from the ovarian surface epithelium constitutes approximately of ovarian malignancy themajority of eoc patients are diagnosed in advanced stages stage iii and iv and year freesurvivalrate is below the standard treatment for newlydiagnosed ovarian cancer is the combination of cytoreductive surgery and platinbased chemotherapy significant advances in radical surgery and chemotherapystrategies have improved clinical outcomes but unfortunately no progress has been made with relapse andtreatment resistance ninety percent of ovarian cancer occurs sporadically in the population whereas hereditary type appears of ovarian cancer patientsbrca1 and brca2 genes are the most common breastovarian cancer syndrome associated genes both brca1and brca2 have roles in the control of the genomic stability cell cycle and apoptosis the mutations occurringin these genes result with the inability of dna repairand therefore results in the accumulation of the mutations in the cell the rate of the breast cancer susceptibility of women with brca1 gene mutation until theage of years was and the rate of ovarian cancersusceptibility rate is breast cancer susceptibilitywomen with brca2 gene mutation until the age of years is and ovarian cancer development risk is twin studies became important on genetics by theendandcentury geneticnineteenthofthethe differentiated genesepidemiologic studies with monozygotic twins were accepted as highly useful investigation models in the pastdecades and have been used recently when a similarity for a disease or a quantitative feature betweenmonozygotic and dizygotic twins is compared variationsare excluded according to studies conducted in thepopulation and thereforeit is easier to identify andmake etiological differences visible via twin studies because the affected siblings and dizygotic twins share theapproximately ofthephenotypic differences between twins are known to beassociated with the genetic variation in addition diversity may be revealed with a very limited patient population therefore the results of the twin studies can beapplied to the population and can make valuable contributions to the genetic studies monozygotic twins aregenetically similar and generally expected to be compatible for congenital malformations chromosomal abnormalities and mendelian disorders there are numerousstudies conducted via discordant monozygotic twins revealing the genetic contribution therefore investigating the genetic variability in monozygotic twins ishighly important and the majority of the human geneticsassociated research focus on finding genetic variability indiscordant monozygotic twins phenotypically discordantmonozygotic twins are used as the model systems inidentification of the variable in understanding the pathogenesis of a disease the most striking study is the oneconducted with monozygotic twins in canada and evidencing that multiple sclerosis ms was a genetic disease micrornas are one of the subset of the noncoding rnas generally consisting of single strand in nucleotide length not transformed to protein havingroles in post transcriptional regulation or suppression oftranslation of the target mrnas [ ] the regulatoryroles of mirnas were demonstrated to occur in tumorigenesis cell differentiation proliferation and apoptosis[] mirna genes are known to locate in thechromosomal breaks this dna breaks cause chromosomal abnormalities frequently associated with cancersusceptibility and tumor development [] the noninvasive biologicalindicators have been used for the 0ctuncer of ovarian research page of treatment resistance of ovarian cancer the most common of this indicators are the cancer antigen125 ca and cancer antigen153 ca153 these biological indicators can be used in the followup of thetreatment response in the diagnosed patients but cannotbe used in the early diagnosis and in differentiation ofthe malignant disease therefore there is a need forspecial therapeutic agents customized for patients thatmay be used target specific therapies and in the earlydiagnosis of the ovarian cancer in identification of theefficacy of therapy and in the follow up period thusstudies investigating the target molecules and biologicalindicators are required that will enable the early diagnosis and in the development of the better therapy optionsdifferentially synthesize mirnas such as mir mir141 mir125b mir2223p or let7 family has beenshown in studies with ovarian cancer patients however the use of these mirnas as a biomarker in ovariancancer is not yet available in order to clearly define therole of mirnas in the pathogenesis of ovarian cancerwe planned to investigate the brca mutant monozygotic twins with the same genetic profile but with discordant for ovarian malignant transformation in thisstudy mirnas which are thought to have the potential biological indicator role were studied from bloodsamples of both discordant monozygotic twins andbrca wild type healthy siblingsmethodspatients recruitmentthe peripheral blood lymphocytes of monozygotic twinsdiscordant for ovarian cancer and healthy individuals inthe same family were used in the study the patient diagnosed with ovarian cancer and all family members applied to the cancer genetics clinic of oncologyinstitute of istanbul university for brca breast cancersusceptibility gene testing were examined for brcagene mutation all family members in the study consisted of highrisk individuals with hereditary breast andovarian cancer hboc syndrome and the people included in the study were given as br codes according topatient file number the monozygotic ovarian cancer patient healthy monozygotic twin healthy sisters and niece were found to have brca1 gene mutation c5266dupc pgln1756profs74 rs397507247 on exon thepatients brother and daughter were found negative forbrca1brca2 gene mutations in this study lymphocyte cells separated from peripheral blood belonging tototal of cases including younger age ovarian cancer patient and healthy monozygotic twin a patients daughter elder sisters a younger sister a nephew and a brotherwere examined by mirna microarray method thepedigree of the family included in the study and theirhierarchical cluster analaysis via euclidean method isshown on fig the study was approved by the ethics committee ofthe istanbul faculty of medicine following institutionalethics committee approval informed consents were obtained from all participants before enrollment into thestudy ethics committee approval number atstoredthey werelymphocyte and mirna isolationficoll sigmaaldrich darmstadt germany density gradient was used to separate white blood cells mononuclear cells from other blood components mirnaisolation procedure was performed from the lymphocytein accordance with the kit protocol using the mirneasymini kit qiagen cat noid the proceduresteps in accordance with the protocol are as follows μl qiazol solution was included on the cells storedin nitrogen tank cell fractionation was enabled by mixturing using the vortex for complete nucleoproteinfractionation °c roomtemperature for min by adding μl chloroformthey were shacked and mixed on hand the tubes wereincubated for min at °c room temperature thenwere centrifuged for min at °c and g thesupernatant formed after centrifugation was transferredto collection tube using a pipette and was mixed usingvortex by inclusion of μl ethanol the supernatant formed after the ethanol centrifuging was transferred to collection tube was removed with a pipettewas mixed with vortex by including μl ethanol seven hundred microliters was taken from the obtained mixture and was transferred to the rneasyminielute spin colon placed on ml collection tubethe tubes were centrifuged for s at g at °croom temperature seven hundred microliters rwt buffer was added to spin colons and the colons werewashed by centrifuging at g for sthe centrifuging procedure was repeated by including μl rpe buffer twice consecutively to colons the colons placed into clean tubes with ml were dried bycentrifuging min in maximum speed the colonsplaced in ml sterile tubes were included μl distilled water by centrifuging at g in min and themirnas were collectthe quality control of the mirnasthe presence and quality of the isolated mirnas werescreened by electrophoresis at v on agarose gelthen the purity and concentrations of the mirnaswere measured on thermo scientific nanodrop spectrophotometer nanodrop technologies wilmington de usa device the mirna purity for each persondevicemeasurement result were obtained with the comparisonaccordance withthe nanodropin 0ctuncer of ovarian research page of fig the pedigree of the family included in the study and their hierarchical cluster analysis legend the pedigree of the family and using thecorrelations between samples plotted a dendrogram for sample grouped by hierarchical clustering euclidean distance complete linkage br healthy brother brca1 negative nonbrca1 mutation carrier br healthy niece brca1 positive brca1 mutation carrier br healthy daughter brca1 negative br healthy monozygotic twin brca1 positive brca1 mutation carrier br monozygotic twindiagnosed with ovarian cancer brca1 positive brca1 mutation carrier br healthy sister brca1 positive brca1 mutation carrier br healthy sister brca1 positive brca1 mutation carrier br healthy sister brca1 positive brca1 mutation carrierof the measurements at spectrophotometrically at nm and nm wave lengths the measurement rates at nm wave lengths is a sign of quality of the purity of the samples therefore the samples in the idealvalue interval of and for rna measurementswere included in the study the purity of mirnas wereevaluated using a bioanalyser device bioanalyseragilent technologies santa clara ca usa agilentrna nano kit agilent technologies santa claraca usa for confirming whether the mirnas were appropriate and in adequate level for microarray analysisthe evaluated sample concentrations and results wereanalyzed the samples with rna concentrations between ngμl and rrna rate over and rna integrity number values between and were evaluated asthe appropriate samples for array study 0ctuncer of ovarian research page of microarray trial protocolmicroarray protocol was performed by preparing thespikein solution sample marking hybridization sampledephosphorylation sample denaturation sample ligationhybridization of the samples slide loading preparationof the hybridization unit and elution and scanning ofslides the slide scanning procedure was performedusing the agilent microarray scanner agilent microarray scanner with surescan high resolution technology agilent technologies santa clara ca usadevice the scanning procedure of the slides were performed on sureprint g3 human mirna microarrayrelease 8x60k agilent inc santa clara ca platform and using the agilent technologies g2600d scanning protocol the analysis of the tiff tagged imagefile format extensioned files obtained after scanningprocedure was performed using the agilent feature extraction v11011 programthe success levels of stages developed in all experiment process with this analysis program the quality ofthe levels the process were monitored and evaluatedthen bioinformatic analysis procedure was performeddata analysisraw data logarithmic analysis was studied by identifyingthe threshold normalization correlation mean and median values then the mirnas demonstrating differentexpression profile among the samples were filteredusing the fold change rates and independent twosample t test the possible difference between the compared groups were evaluated all evaluations were performed to enable the cutoff values as the foldchangerates fc ¥ and pvalue hierarchical clusteranalysis was performed using the euclidean method fig and complete linkage cluster method the control ofthe experimental errors and the detection of the erroneous finding rate were identified using the hochbergmethodbioinformatic analysistarget proteins were detected for each mirna by usingtwo algorithms targetscan71 httpswwwtargetscanvert_71 and mirdbv5 httpsmirdbmirdbthe targeted genes thought for each mirna wereconfirmed by also both algorithms and the mirnatarget relations were also experimentally confirmed mirtarbase70httpsmirtarbasembcnctuedutwphpindexphp databasecomparison groupsin the study mirna analysis was performed at the genome level withwithout mutation in cases withwithoutovarian cancer the mirna data was evaluated by comparing different groups in order to investigate the effectof brca mutation in ovarian malignancy developmentand determine the mirnas that can be important in theovarian cancer pathogenesis in group the monozygotic twins discordant for ovarian cancer were comparedin order to find the effects of mirnas in the formationof ovarian cancer in group the family members withbrca1 mutation were compared with family memberswithout brca1 mutation to identify the changes ofmirnas expression levels according to brca positivityin group the monozygotic ovarian cancer patient withbrca1 mutation carrier and the other healthy familymembers with mutation carrier were compared for investigate the effects of both ovarian cancer developmentand brca positivity on mirnas expression level ingroup all family members were compared with ovarian cancer monozygotic twin in order to find the mirnas that might be important in the predisposition ofovarian cancer the comparison groups also showed intable resultswe identified differentially expressed comparisonof mirnas between the groups the raw data obtainedafter experimental studies were filtered before the comparisons between the groups the upregulated or downregulated mirnas expression levels more than foldfc and smaller than the p value p wereconsidered in evaluation and the comparisons betweenthe groups were performed based on these values allthese comparisons were evaluated for ovarian cancer etiology brca1 mutation carriage and the ovarian cancerrisk hierarchical cluster analysis of the expression of mirnas represents sharp separations of upregulatedyellow from downregulated blue in fig mirnas total of mirnas were found statistically different after the comparison of phenotypicallydiscordant monozygotic twin siblings the mirnasmir1273 g3p mir1305 mir1973p mir3651 mir and mir92a3p expressions were found to haveupregulated and the other mirnas let7i 5p mir125a5p mir15b5p mir22 3p mir3135b mirtable comparison groups and cases in the groupscasegroup br1639controlbr1447group br1639br1447br1547br2030br1546br1861br1850br2028group br1639group br1639br1447br1447br1547br2030br1546br1861br1547br2030br1546br1861br1850br2028 0ctuncer of ovarian research page of fig hierarchical cluster analysis of the expression of mirnas legend br healthy brother brca1 negative nonbrca1 mutationcarrier br healthy niece brca1 positive brca1 mutation carrier br healthy daughter brca1 negative br healthymonozygotic twin brca1 positive br monozygotic twin diagnosed with ovarian cancer brca1 positive br healthy sister brca1positive br healthy sister brca1 positive br healthy sister brca1 positive the mirnas that may be effective in the etiology ofovarian cancer were identified after the comparison of monozygotic twins who were phenotypically discordant for ovarian cancer diagnosis ingroup 320d mir3423p mir4430 mir451a mir664b5pand mir7663p expressions were found to have downregulated after the bioinformatic analysis a total of upregulated and downregulated statistically significantmirnas and their target molecules are given in table and fig different mirnas level were compared between group in order to determine the effect of brca1 gene mutation group was consisted after the comparison of thebrca1 gene mutation carrier family members and individuals not carrying brca12 gene mutation accordingto mirnas expression profiles after the comparisonsdownregulated and upregulated mirnasrelated tobrca1 gene mutation carrier were determined the expression of a total of mirnas including mir4449mir46533p mir4865p mir5739 mir6165 andmir 8743p associated with the brca1 gene mutationcarrying were upregulated and the expression of a totalof mirnas including mir1263p mir320a mir320b mir320c mir320d mir320e mir3243p mir mir mir4428 mir4516 mir4741 mir mir564 mir6089 mir68695p mir68915pmir71075p and mir78473p were found downregulated after the bioinformatic analysis a total of upregulated and downregulated statistically significantmirnas and their target molecules are shown in table and fig aftercomparisondifferent mirna levels were compared between group in order to determine the relation with ovarian cancerdevelopment and brca positivity group consists ofcomparison of mirnas of brca1 positive ovarian cancer patient with all other brca1 positive healthy individualsanddownregulated mirnas related to mutation carriage inbrca1 gene and epithelial ovarian cancer etiology weredetermined the expression of mirnas includingmir1260a mir1260b mir165p mir175p mir181b5p mir 26b5p mir4281 mir4286 mir5100mir68403p mir71145p mir7975 and mir7977were found to have upregulated and the expression of mirnas including mir12255p mir1423p mir 26a5p mir mir29a3p mir30d5p mir3196upregulated 0ctuncer of ovarian research page of table ovarian cancer etiology related upregulated and downregulated mirnas and target proteins in monozygotic twinsmirnassequence of mirnamirnastatustarget genesfold change fcvaluesaccacugcacuccagccugagupregulatedznf138 tmem239 bmp3uuuucaacucuaaugggagagaupregulatedptpn4 prkaa1 papd7frat2 depdc1 fbxo41uucaccaccuucuccacccagcupregulatedcd82 pmaip1 mthfd1 check1 ago1 casp10cauagcccggucgcugguacauga upregulatedggcuggucagaugggagugupregulatedracgap1 ola1 tex261ptgs1 nfic znf200pagr1 igf2bp1 cacng8uauugcacuugucccggccuguugagguaguaguuugugcuguuucccugagacccuuuaaccuguga downregulated erbb3 cdkn1a tp53 erbb2 egfr stat3mycstat3 pten atm notch2 cdh1 nfkb1upregulateddownregulated tlr4 bmp4 eif2c1 neurog1 socs1 igf1vegfamir1273 g3pmir1305mir1973pmir3651mir6131mir92a3plet7i5pmir125a5pmir15b5puagcagcacaucaugguuuacamir223paagcugccaguugaagaacugudownregulated bcl2 vegfa ccnd1 ccne1 cdk1 cdk4 cdk6 e2f3mapk1downregulated cdkn1a wnt1 erbb3 mycbp hmgb1 e2f2 ptenpoted sod2mir3135bmir320dmir3423pmir4430mir451amir664b5pggcuggagcgagugcaguggugaaaagcuggguugagaggaucucacacagaaaucgcacccguaggcuggagugagcggagaaaccguuaccauuacugaguuugggcuaagggagaugauuggguadownregulated birc5 abl2 mapk1 mycndownregulated dctn5 syncrip fbxo28downregulated gemin4 dnmt1 id4 srebf1srebf2 bmp7downregulated znf485abl2 mapk1 msh5 ptendownregulated cpne3 rab5a il6r akt1 mmp2downregulated cd55msn rhobtb3 plag1mir7663pacuccagccccacagccucagcdownregulated cox1 mapk1 nf2 rad51 stk4 stk24 vegfcfig the upregulated and downregulated mirnas and foldchanges associated with ovarian cancer etiology in monozygotic twins 0ctuncer of ovarian research page of table brca1 gene mutation positivity related upregulated and downregulated mirnas and target molecules an additional tablefile shows this in more detail [see additional file ]mirnassequence of mirnatarget genesmirnastatusfold changefc valuesmir4449mir46533pmir4865pmir5739mir6165mir8743pmir1263pmir320amir320bmir320cmir320dmir320emir3243pmir3656mir4284mir4428mir mir4741mir484mir564mir mir6869 5pmir68915pmir71075pmir7847 3pcgucccggggcugcgcgaggcaupregulatedzfhx3uggaguuaaggguugcuuggagaupregulatedatg2a crebl2 mat2a frs2 tmed4 ubn2uccuguacugagcugccccgaggcggagagagaauggggagccagcaggaggugaggggagcugcccuggcccgagggaccgaupregulatedupregulatedupregulatedupregulatedolfm4cd40arhgap5 igf1r dock3 cadm1dlx6cd207chic1ppl2a plxdc1per1tfap2afads1 amer1luzp1 cox6b1hdac1 aqp3 stat3 cdk9ucguaccgugaguaauaaugcgdownregulatedtom1 crk vegfa sox2 twf1 pitpnc1 igfbp2 krasaaaagcuggguugagagggcgadownregulatedmcl1 banp itgb3 bmi1 nrp1 nfatc3 trpc5aaaagcuggguugagagggcaadownregulatedcdk6dctn5syncriparf1 bcl9l znf600aaaagcuggguugagaggguaaaagcuggguugagaggaaaagcuggguugagaaggacugccccaggugcugcuggggcgggugcggggguggdownregulateddownregulatedsyncripfbxo28smarcc npm3dctn5 syncrip fbxo28dctn5 npm3 znf275 ddx19a ncapd2 txnl1downregulateddownregulated wnt9b crebbp dvl2 wnt2bdownregulatedmrpl12 lsp1 mnt prdm2znf770 cecr1gggcucacaucaccccaudownregulatedbcl2l11rbbp5hnrnpa1 znf264 trib3 crtapcaaggagacgggaacauggagcdownregulatedmsl1mapre3myh14casp2 ccnd2 cdk14tp63gggagaagggucggggcdownregulatedstat3m6prgpr137cccnd2 ccnt1 cdkn1a scoc tp53cgggcuguccggaggggucggcudownregulatedddx39bmapk1 zbtb39 hmga1ucaggcucaguccccucccgaudownregulatedfis1 pagr1 zeb1 slc11a2smad2 anapc7 tbrg1aggcacggugucagcaggcdownregulatedgid4 cnbp e2f3 rcan3 akt2 appl1 slc1a2 gpr155ggaggccgggguggggcggggcggdownregulatednkx2 tpt1 kctd5 bbx sgcd cdh7 ccnb1gugaguaguggcgcgcggcggcdownregulatedtubb2amapk1nrbf2 wee1hmga2 mapk1 stag2uaaggagggggaugaggggdownregulatedchd4 cd207 ddx6 chrdl1ccnd2 tp63ucggccuggggaggaggaagggdownregulatedvav3 casp16 ccnd1 casp16 mapk14cguggaggacgaggaggaggcdownregulatedhavcr1 poted dnajc10 sod2 m6pr cdk19mir3423p mir3665 mir3960 mir4466 mir4530mir46873p mir47875p mir4943p mir50015pmir50065p mir5787 mir6068 mir6087 mir mir6090 mir6124 mir6125 mir638 mir65105p mir68005p mir7704 mir8063 and mir were found to have downregulated after bioinformatic analysis a total of upregulated and downregulated statistically significant mirnas and the targetmolecules are given in table and fig for identifying the ovarian cancer predisposition allfamily members were compared with ovarian cancermonozygotic twins in group the upregulated ordownregulated mirnas in association with the epithelialovarian cancer risk were identified after the comparisonthe expression of the mirnas consisting of let7a5plet7b5p mir181a5p mir1973p mir215pmir2233p mir23a3p mir27a3p mir36533pmir4255p mir572 mir5745p mir6127 and mir were detected to be upregulated the expression ofthe mirnas consisting of let7i5p mir 125a5pmir15b5p mir1505p mir22 3p mir3283pmir4430 mir451a mir46975p mir664b5p andmir7663p were detected to have downregulated atotal of upregulated and downregulated statisticallysignificant mirnas and the target molecules are givenin table and fig discussionwomen are diagnosed with ovarian cancer at an advanced stage due to limited number of biologicalmarkers for ovarian cancer patients although existingovarian cancer biomarkers cancer antigen125 and cancer antigen153 ca125 ca15 are sensitive in thefollowup of diagnosed gynecological cancers they have 0ctuncer of ovarian research page of fig the upregulated and downregulated mirnas and foldchanges associated with brca1 gene mutation positivityofearlysensitivityin the diagnosislessstagegynecological cancers and separation of malignant tumorformations from benign formations therefore tounderstand the underlying mechanisms of ovarian cancer and to explore targeting drugs and to improve newtreatment protocols for ovarian malignancy revealingsignificant genetic changes is necessary the genetic andepidemiologic studies conducted on monozygotic twinsare known to provide accurate and direct informationabout the gene and environment interaction with thedisease occurrence mechanism the changes in genesthat result in the occurrence of tumors such as mirnaexpression level among the monozygotic twins providesinformation on the etiology of disease and may have arole as a biological indicator in identifying the early stagedisease and in the follow up of the prognosis we aimedto identify the noninvasive biological markers that maybe used in the early diagnosis of ovarian cancer throughinvestigating the mirnas in the peripheral blood ofmonozygotic twin siblings discordant for ovarian cancerwith the mirna molecules of the other healthy members in the family thus that may cause less bias thanthe controls to be selected from the population ninetynine different mirna molecules presented in the studywere detected after the comparison of monozygotic twinsiblings who were discordant for ovarian cancer andwith the other healthy individuals seventeen differentmirnas were found that could be used for detectingearly diagnosis and prognosis of ovarian cancer betweenthe monozygotic twin siblings who were discordant forovarian cancer in our study the association between out of mirnas and ovarian carcinoma is beingreported for the first time in this study due to the highnumber of newly detected mirnas in our study the discussion and comparison were only made between thecandidate mirnas although mir1973p mir1305mir6131 mir3651 mir3135b mir4430 mir664b5p and mir7663p have not been shown to be associated with ovarian cancer in literature but limited number of studies have suggested the association with othercancerswang found the elevated level of mir1973pinthe same way as we do the upregulated mir1973p expression level was shown to promote the cellular invasion and metastasis in bladder cancer in that studyresearchers reported that linc00312 gene was responsible for invasion and metastasis mechanisms and thisgene inhibited the cellular migration and invasion bysuppressing the mir1973p expression similar resultswere detected in thyroid cancer in the study of liu [ ] jin reported that increased expressionlevel of mir1305 caused pluripotent stem cells to accelerate the cell cycle g1s transfer in addition to causingthe cellular differentiation with the increased mir1305expression the expression levels ofreducedmirna125a5p and let7i5p found in the scope of ourstudy have been shown to parallel with other studies inthe literature langhe suggested thatlet7i5pmight be described as a diagnostic indicator in ovariancancer the mirna125a5p expression was upregulated to inhibit the cancer proliferation and migrationin the in vitro study of qin in human cervical carcinomas and mir125a5p upregulated expressionlevel was demonstrated to inhibit the cervical cancer 0ctuncer of ovarian research page of table brca1 mutation carriage and epithelial ovarian cancer etiology related upregulated and downregulated mirnas targetmolecules an additional table file shows this in more detail [see additional file ]mirnassequence of mirnamirnastatustarget genesaucccaccucugccaccaaucccaccacugccaccauuagcagcacguaaauauuggcgupregulatedupregulatedupregulatedcaaagugcuuacagugcagguagupregulatedpsat1unc13a rps27 brd7sfrp1 dkk2 smad4 psat1unc13a rps27ccne1 arl2 bcl2 hmga1 cdk6 ccnd1vegfa reck prdm4tgfbr2 pten cdkn1a bcl2l11e2f1tp53stat3aacauucauugcugucgguggguupregulatedtcl1a timp3 plag1 bcl2rnf2vsnl1 atmfold changefc valuesmir1260amir1260bmir16 5pmir175pmir181b5pmir26b5pmir4281mir4286mir5100uucaaguaauucaggauagguupregulatedgggucccggggaggggggaccccacuccugguaccupregulatedupregulateduucagaucccagcggugccucuupregulatedptgs2 epha2 chordc1 ezh2ccne1abca1 gata4ncdn cdkn1a bcl3ldlr znf354b nsd1 rabgap1taok1 mknk2cox10 dek kcnn3 rab11fip1dynlt1 notch2slfn12l ctc1 gxylt2gde1fads1per1 atg9amir68403pgcccaggacuuugugcggggugupregulatedmir71145pucuguggaguggggugccuguupregulatedm6pr hnrnpul1 shmt1 znf529acvr2b paics taf8mir7975mir7977auccuagucacggcaccauucccagccaacgcaccaupregulatedupregulatedmir12255pguggguacggcccaguggggggdownregulatedkbtbd8gulp1 casz1 rad51hspa1b znf703 tmem185bsf3b3cox6b1 ccdc9 cdh7orc4 odf2l mtrnr2l7psmg2 mtrnr2l3mir1423pmir26a5pmir2861mir29a3pmir30d5pmir3196mir 3423pmir3665mir3960mir4466mir4530mir46873pmir47875pmir4943pmir50015pmir50065pmir mir6068mir6087mir6088mir6090mir6124uguaguguuuccuacuuuauggadownregulatedarntltgfbr1 rac1 rock2 ccnt2 tab2 ptpn23uucaaguaauccaggauaggcudownregulatedezh2rb1adam17 hmga2ccnd2 cpeb3 dnmt3bggggccuggcggugggcgguagcaccaucugaaaucgguuauguaaacauccccgacuggaagcggggcggcaggggccucdownregulateddownregulateddownregulateddownregulatedly6eccdc64 ccnd1 dars2 apaf1mcl1cdk6sparc dnmt3adnmt3b col4a1gnai2 tp53 casp3 snai1 ezh2 bcl9 notch1 smad1pou3f3tulp1 h2afxpcgf3 casp16atg2a ccdc64ucucacacagaaaucgcacccgudownregulatedgemin4 dnmt1 id4 srebf1srebf2 bmp7 rmnd5aagcaggugcggggcggcgggcggcggcggaggcggggggggugcgggccggcggggcccagcaggacgggagcguggcuguuggagggggcaggcdownregulateddownregulateddownregulateddownregulateddownregulated | Colon_Cancer |
" 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 |
moringa oleifera l from the moringaceae family is a perennial tree widely cultivated in many tropic regions and easily grown even in adverse conditions m oleifera is also known as the miracle tree which for centuries has been indicated for traditional medicine with no reports of side effects in doses achievable by ingestion different parts of m oleifera is used to treat several conditions such as malnutrition diabetes blindness anemia hypertension stress depression skin arthritis joints and a0kidney stones disorders this plant also showed capacity of helping in maintenance of the cardiovascular system health bloodglucose levels and providing antioxidant antiinflammatory and anticancer activity as well as the regulation of urinary tract and lactation in nursing women the seed and leaves powder has water purification properties through flocculation it also supplements the food in the human diet and in the fortification of livestock feed especially in developing countries so m oleifera properties have also been applied to cosmetic and byproducts industries due to the high nutritive and protective properties of its seed oil according to the holistic or traditional medicine m oleifera has very relevant therapeutic properties and applications depending on the constitution somatic and psychological needs of patients it is usually referred as a natural product that can treat different physical and psychological health aspects offering an energetic action and structural rebuilder of the body and promoting emotions of highly positive attitudes towards life the high and specific immunological potential of m oleifera leads us to suggest an indepth study to assess the hypothesis of conferring a supportive effect against covid19 diseasekeywords moringa oleifera a0· drumstick tree a0· miracle tree a0· medicinal plant a0· cosmetics a0· food supplementdiana meireles and jo£o gomes authors contributed equally to this workdianameireleslivecompt diana meireles icbas institute of a0biomedical sciences abel salazar university of a0porto rua de je viterbo ferreira no a0porto portugal yidao acupuncture and a0tcm center porto portugal ciimar interdisciplinary centre of a0marine and a0environmental research university of a0porto porto portugal cbsin center of a0biosciences in a0integrative health porto portugalintroductionmoringa oleifera l moringa pterygosperma g wellknown as the drumstick or horseradish tree is native of northwest india its main producer but can also be found in south africa northeast africa madagascar tropical asia southwest asia and latin america the moringa genus comprises species m arborea m longituba m borziana m pygmaea m hildebrandtii m drouhardii m longituba m peregrina m stenopetala m rivae m ruspoliana m ovalifolia m concanensis and m olefera rani a0 from the moringaceae family m oleifera is the most known studied and used species anwar olson with human and animal applications the various resources obtained from this plantleaves flowers seeds pods bark and rootscan be used for cooking or in traditional medicine to treat several pathologies m oleifera has the capability to survive in humid or dry hot climates vol01234567891 0c d a0meireles and poor soils anwar et a0al mainenti m oleifera is a highly nutritious plant being ideal to treat malnutrition in developing countries zongo valdezsolana et a0al gopalakrishnan debajyoti et a0al m oleifera gained the title of miracle tree and commercial attention supported on several properties such as nutritional values amino acids and flavonols content which can be used in food supplements and cosmetic industry tables a0 and in fact when compared to other plants from a0g of dry leafs of m oleifera we can obtain times more vitamin c than from oranges times more vitamin a than from carrots times more calcium than in milk a0times more protein than in yoghurt times more potassium than from bananas and times more iron than the obtained from spinach oduro et a0al a0 a0rockwood saini et a0al table a0 shows the nutritional values for the edible parts of raw pods and leaves obtained from the united states department of agriculture usda database although it is table nutritional values per a0g of the edible portion of m oleifera pods and leavescomponentsper a0graw podsaraw leavesaenergy kcalwater gprotein gtotal lipid gcarbohydrate by difference gfibre total dietary gfatty acids total saturated gfatty acids total monounsaturated gfatty acids total polyunsaturated gfatty acids total trans gcholesterol mgvitamin a rae µgvitamin d d2 d3 µgvitamin d iuthiamin mgriboflavin mgniacin mgpantothenic acid mgvitamin b6 mgvitamin b12 µgvitamin e mgvitamin c total ascorbic acid mgfolate total µgfolic acid µgsodium mgpotassium mgcalcium mgphosphorus mgmagnesium mgiron mgzinc mgcopper mgmanganese mgselenium µga information obtained from united states department of agriculture nutrient database ndbnalusdagovndbfoods in june b average values and standard deviation published by witt only two values were found witt dried leavesb ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0ctable amino acids and flavonols per a0g of the edible raw portion of m oleifera leavesper a0graw leavescomponentsamino acids a0tryptophan g a0threonine g a0isoleucine g a0leucine g a0lysine g a0methionine g a0cystine g a0phenylalanine g a0tyrosine g a0valine g a0arginine g a0histidine g a0alanine g a0aspartic acid g a0glutamic acid g a0glycine g a0proline g a0serine gflavonols a0isorhamnetin mg a0kaempferol mg a0myricetin mg a0quercetin mginformation obtained from united states department of agriculture nutrient database ndbnalusdagovndbfoods in june known that the nutrient content varies according to the plantation site aslam and seasons witt the nutritional value of dried leaves not existent in usda database is presented as an average of values with standard deviation calculated from diverse papers that was compiled and published by witt from leaves to roots it is possible to obtain good quantities of important minerals proteins vitamins carotene amino acids and phenolic compounds anwar et a0al leone et a0al 2015a b saini et a0al fahey debajyoti et a0al divya et a0al m oleifera extracts have been studied with different medicinal purposes antiinflammatory antihypertensive diuretic antimicrobial antioxidant antidiabetic antihyperlipidemic antineoplastic antipyretic antiulcer and hepatoprotectant fahey abdull razis et a0al divya et a0al anwar et a0al published a table with various traditional medicinal uses of the different parts of m oleifera anwar et a0al the attractive properties of this plant led to studies of side effects and medical interactions in animal models and humans according the revision by stohs and hartman until this date none of the human in a0vitro studies or extrapolations of animal studies to humans reported adverse effects with doses of m oleifera leaves and leaf extracts achievable by oral ingestion although there was not any report of major adverse side effects there are some important information that should be registered in fact there are some studies suggesting that m oleifera cannot be used in combination with other modern medicines in humans a research by gholap et a0al concluded that m oleifera has been noted to be a good regulator of insulin thus according sileshi et a0al patients suffering from lack of insulin will probably have adverse reductions of sugar levels when using m oleifera for medicinal purposes suggesting that it could decrease the blood sugar to even lower levels when used in combination with other modern medications another study suggests that when treating thyroids m oleifera compounds of the leaf may improve thyroid function tahiliani et a0al this well proving evidence further suggests that it can possibly conflict with other thyroid medication triggering drug interactiona research work concerning the acceptability and safety of shortterm daily supplementation in a group of malnourished girls assessed the use acceptability and safety of m oleifera on children girls in zambia barichella et a0al with regards to safety concerns supplementation of a0g per day of m oleifera powder was deemed safe for children and adolescents both in the short and long term this research also noted that mild nausea was reported in of the children at various age groups when meals were supplemented with a0g of m oleifera daily showing to be still an inadequate and symptomatic dose in childrenother studies suggest that m oleifera could adverse and slowly breaking down the pharmaceutical drugs in the liver and thus a0may develop cirrhosis and liver failure resulting in malnutrition and weight loss as well as decreased cognitive function das et a0al kelly sileshi et a0al despite the numerous positive health benefits associated with m oleifera phytochemicals there are suspicions that it contains harmful substances fahey et a0al annongu et a0al maizuwo it contains specific chemical compounds such as alkaloids and other phytotoxins which when consumed in high doses presents potentially nerveparalysing properties and other adverse effects maizuwo et a0al some of these phytochemicals include moringine moringinine estrogen pectinesterase and phenols including tannin fahey et a0al there are unconfirmed reports that m oleifera stems roots and flowers potentially contain harmful phytochemical constituents especially during pregnancy which may promote uterus contraction leading to miscarriages in pregnant women dutta it is also suspected that it can prevent implantation in women hence it must be avoided a review of a0properties nutritional and a0pharmaceutical applications of a0moringa oleifera¦ 0c d a0meireles table compilation of food supplements containing m oleifera tree parts or extracts in june tree partsproduct informationbrandproductnaturingamoringa capsulesleavesmoringa teamoringa kids multivitamin complexmoringa powderbioheradried seeds extract moringa capsulesleavesmoringa syrupleavesleavesmiracle treemoringa anic teaanic moringa superfood supplementscapsulesmoringa superfood powdermoringa superfood sticksiswarimoringa powder anicdrasanvileaves dry extract nutrabasicsmoringaregulates the gastrointestinal transit natural antiinflammatory lowers cholesterol levels improves diabetic conditiondelays the ageing process ensures proper digestion high antioxidant power helps healing process tonifies body and mindstrengthens the immune system rich in vitamins and minerals stimulates natural defensesadds nutritional value source of fiber protein vitamins and minerals improves physical conditionstrengthens the immune system helps to reverse the aging process beautifies the skin reduces the appearance of wrinkles and fine lines maintains the normal glucose level stimulates brain function and concentration increases libidoit is a nutritionally complex whole food naturally rich in vitamins minerals and amino acids daily use of moringa can help to restore your imbalances in your dietthe moringa leaf boasts a vast array of beneficial nutrients making this tree one of the highest plant sources of vitamins and minerals aroundthe richness of its active ingredients helps maintain blood glucose levels provides flavonoids and polyphenols by those attempting to conceive as it functioning as an abortifacient nath et a0al dutta finally cytotoxicity was verified in experiments with human peripheral blood mononuclear cells only at a0mgkg of an aqueous leaf extract genotoxicity on blood rats cells was verified at a0mgkg asare et a0al however all mentioned side effects were verified with doses that far exceed the amounts used in food intake asare et a0al so research on the adverse side effects with doses achievable by oral ingestion should still go on since currently there are no scientifically confirmed clear toxic and harmful effects of m oleifera extracts and products on both human and animal models adedapo et a0al stohs et a0al many studies on nutrition phytotherapy disease treatment and prevention goals have been published thus supporting scientific basis about the efficiency of traditional uses of m oleifera fahey in fact records about symptoms signs and treatment strategies in different diseases are found in several ancient texts of traditional medicines such as ayurveda and traditional chinese medicine tcm karadi et a0al kasote et a0al debajyoti et a0al as an endemic source with highly digestible protein ca fe vitamin c and carotenoids is considered as a suitable natural product to be used by undernourishment populations dixit the resources obtained from a0m oleifera tree on a0a a0conventional approachleaves and a0podsin some countries leaves and fruits are commonly used in culinary as vegetables leaves can also be dried and used in infusions or grounded into powder allowing easier conservation and consumption moyo olson et a0al in all ways of use and conservation m oleifera does not lose nutritional value mahmood leaves and pods are low in calories and rich in minerals vitamins and natural antioxidants table a0 anwar rebufa et a0al phytochemicals like flavonoids are also present in leaves as well as a significant percentage of essential amino acids table a0 m oleifera leaves contain a high quantity of polyunsaturated fatty acids and low saturated fatty acids content moyo which combined with diuretic lipid and blood pressure lowering properties from leaves and pods contribute to the maintenance 0ctable compilation of cosmetics containing m oleifera tree parts or extracts as ingredients in june cosmetic ingredientsproduct informationproduct brandnameskinsecretantiwrinkle face creamantiaging moisturizer face creamhand creambody milklushafrican paradise body conditionerqueen bee hair honeymagical moringa facial moisturizercharity pot hand and body lotionpassion fruit lip balmgo faster feet foot lotiontwinkle toes foot powderlush gardener cold pressed soaplaboratoires s©robiologiquespurisoft®body shopmoringa range shower gel oil body butter body milk body sorbet hand cream soap body scrubmoringa eau de toilette moringa body mistpurifying and protective action against environmental stress such as smoke and pollutionmoisturizing nourishingdeodorizingremove dirt moisturizing nourishingskin cleansingpurification protects skin against pollution heavy metals cigarette smokeskin feels smooth and restoreddelicately scent your skin in a crisp floral aroma with moringaleavesm pterygosperma oilm pterygosperma leaf infusionoilm pterygosperma powderactive ingredient peptide from moringa seedsm pterygosperma oilm oleifera leaf extractm pterygosperma seed extractm oleifera seeds and oilm oleifera leaf extractoilm pterygosperma seed extractm pterygosperma extractclarinsextracomfort antipollution cleansing cream eliminates traces of pollution detoxifies the epidermis and protects the skin from the harmful effects of pollutionneutralizes the effects of pollution and purifies the skin to restore its natural radiancepurifies and refines while preserving your skins natural moisture balance neutralizes the harmful effects of pollutiononestep facial cleanserexfoliating body scrubonestep gentle exfoliating cleanserwater purifycomfort onestep cleanserdaily energizer cleansing gelnaturingamoringa soap biomoringa exfoliating face scrubmoringa o2herbal moisturizing lotionfacial tonersoapherbal shampooconditionershu uemuraantioxi pollutant and dullness clarifying cleansing oilurban moisture hydronourishing shampooconditionerdouble serumdeep treatment masquehigh antioxidant value slowing skin ageing exfoliate dead cells by regenerating the tissuemoringa seeds peel and exfoliate the skin while moringa oil moisturizes and regenerates the skinrejuvenate nourish and protects skinrepairs strengthens reduces hair fallenhanced power to remove micro impurities and stubborn makeup antipollution breakthroughhighly concentrated in nutrients vitamins and antioxidants intensely hydrates deep within strandsa review of a0properties nutritional and a0pharmaceutical applications of a0moringa oleifera¦ 0c table continuedcosmetic ingredientsm oleifera seed extractproduct brandnamebiobeaut©antipollution micellar cleansing watergentle cleansing foamcleansing oil gelgentle exfoliating geldualphase waterproof eye makeup removerd a0meireles product informationremoves makeup pollution particles and excess sebum while leaving the skin well moisturized the seed of moringa extract selected contains purifying peptides which on the surface limit the adhesion of the pollution particles and in depth activate their elimination this extract acts as a protective shield capable of preserving the good bacteria from the cutaneous flora against the aggression of pollutionof cardiovascular health anwar et a0al table a0 in dried m oleifera leaves it was also found a high content in calcium and iron which is normally residual in other plants used in our diet in the leaves is found greater a variety and quantity of proteins when comparing to other tree parts rebufa et a0al wang et a0al due to its nutritional rich values m oleifera can be a good enriching food additive to human diet and also an animal feed fortifier moyo adding fresh or dried leaves to the feed of milk cows increased milk production and respectively that fact would be of great importance in developing countries to fight deficiencies in nutrition bhargave studies of acceptance by the consumer of enriched foodssnacks with m oleifera have been obtaining good results ellis jung m oleifera can also help lactating mothers produce more milk and help to treat malnutrition in young children phytosterols from m oleifera increase estrogen production that enhance the activity of the mammary glands ducts gopalakrishnan doses of a0mgg of body weight given to mice result in increased milk production also the pup weight augment with increasing doses of m oleifera leaf powder intake titi et a0al titi and nurjanah studies of toxicity in animals show that m oleifera dried leaf extract might be safe for consumption although in high doses and prolonged intakes m oleifera may cause toxicity by accumulation of some elements a0ali et a0al the amount of a0g of m oleifera dried leaf per day is the maximum recommended doseage asiedugyekye et a0 al table a0 compiles some food supplements based on m oleifera tree parts or extracts a hydroalcoholic extract of green pods increased liver enzymes involved in the detoxification of xenobiotic substances in mice table a0 suggesting a chemo preventive potential of a drumstick extract against chemical carcinogenesis bharali et a0al m oleifera pods are also valuable to treat digestive and obesity problems and thwart colon cancer gopalakrishnan et a0al carotene the major component reported from the drumsticks of the m oleifera plant as well as the presence of vitamin a and c suggest an action in the induction of antioxidant and antiinflammatory profiles geervani and devi bharali et a0al praengam et a0al it was suggested that carotene and sterols present in the plant pods acts as potent inhibitors on the formation of reactive oxygen intermediates a prerequisite for tumorigenesis and so inducing apoptosis in the mouse colon carcinoma model gupta et a0al kraiphet et a0al studies in rats showed that m oleifera leaves extract might act as potential neuroprotectant via decreased oxidative stress and the enhanced cholinergic function kirisattayakul et a0al and function as a cognitive enhancer hence being used in dementia cases sutalangka et a0al it was also found an antidepressant activity in mouse models of depression when giving orally a a0mgkgday of a m oleifera alcoholic extract plus a0mgkgday fluoxetine for a0days kaur et a0al this effect can be increased when combined with fluoxetine as a selective serotonin reuptake inhibitorssri according to sutalangka et a0al and kaur et a0al 2015the influence of m oleifera may be due to the action of antioxidants and flavonoids through radical scavenging since its action is verified in other studies on animal models with cerebrovascular diseases exerting a multiplicity of neuroprotective actions within the brain and suppressing neuroinflammation and thus suggesting a great potential to promote memory learning and cognitive function vauzour et a0al other studies with consumption of m oleifera leaf powder revealed properties in human an animal models such as decreased blood glucose levels on diabetic type two subjects william reduction on post prandial blood glucose ghiridhari increased insulin secretion in healthy subjects anthanont et a0al decreased total plasma cholesterol and increased hdl nambiar the presence of sitosterol in m oleifera leaves may be one of the reasons for decreasing plasma cholesterol since phytosterols cause less intestinal absorption of dietary cholesterol and increase its excretion on feces jain mbikay 0cnoitcudorpmubes niks ni noitcuderledom laminadna ortiv ni la a0te amrev a0 a0 pytivitca tnadixoitnaledom laminaan yehafnoitcefni la a0te ruak la a0te akgnalatuselfiorp dipil no tcapmi evitisopledom lamina a0 a0 p raibman ledomnamuh a0 a0 pyattasiri yehaf la a0te lukak la a0te ruozuav la a0te roknod la a0te la a0te eednodnesodu hanajrundna iti t la a0te itit la a0te inoras la a0te nanhsirkalapogledom lamina a0 a0 p ledomnamuh a0 a0 p la a0te tnonahtna mailliw irahdirihgnilusni esaercni doolb laidnarp tsop no noitcuder ni slihportuen gnitalucric esaercniseiduts ortiv ni a0 a0 pesoculgledom namuh a0 a0 p la a0te eurdledom laminasserts etuca a0 a0 pledom lamina a0 a0 p lariv uehritna recnac ni romutitnadna lairetcab itna msitamrsshti serpeditna wnoitanibmoc ni tnas recnahne evitingoc tnatcetorporuen ralucsavorberecairetsyh sa sredrosid suovren airalamdna diohpit fo tnemtaertsesaesid negortse noitcudorp rosrucerpdna noitatcal esaercniselpicnirp lanoitnevnoc ot gnidrocca krab dna stoor srewofl sdees sdop sevael su hcus strap eert arefielo m tnereffid fo snoitca lacigolocamrahp fo elbat yrammus elbatarefielo m fo snoitca lacigolocamrahp ledomnamuh a0 a0 pmsidioryhtrepyh etaluger ledomnamuh a0 a0 pamehtyre niks ni ecuderseiduts ortiv ni a0 a0 pa ila la a0te ila la a0te dammahumgnigaitna niksstcartxe suoeuqa fognilaehdnuow tsaerb tnadixoitna yrotammaflniitnaicrac noloc ni sisotpopa ecudni la a0te ilarahb la a0te tehpiark la a0te inailihatledom lamina a0 a0 pnoitacfiixoted dnaledom lamina a0 a0 p ledomnamuhc ila a0 a0 pamonytivitca laiborcimitnaledom lamina a0 a0 psamonicrac latceroloc tsniaga ytivitca recnacitnadna la a0te nanhsirkalapog la a0te iramsalaseiduts ortiv niselknirwitna a0 a0 pniap tnioj dna aehrraid taert la a0te ilaledom namuh a0 a0 p la a0te nanhsirkalapog a0 a0 pan raelc dna paos ot lauqe pu lortnoc eht fognihsaw tnereffiddnah ni seitreporp lairetcabitna la a0te lednorotnoitardyh niks ni esaercni ledomnamuh a0 a0 p ni laitnetop evitneverpomehcrecnac neelps dna citapeh nanhsirkalapog la a0te ilarahbseiduts ortiv ni a0 a0 p ledomnamuhb ila a0 a0 p dna tnadixoitna citebaiditnaseitreporp citirhtraitnagnisnaelc evitcetorp gnizirutsiom gnihsiruonniks tnadixoitna la a0te yebar l ednaledom lamina a0 a0 p ledomnamuhanammaflni gnul etuca fo esaerced la a0te thginkcmledom lamina a0 a0 pnoit dna setaidemretni noitadixorep dipil fonegyxo evitcaer esaerced ilarahb eddna atpugsad la a0teledom lamina a0 a0 p yebar l edna iklamlaledom lamina a0 a0 pdik lla detaroilema dna desaercnisretemarap snoitcnuf yen ecuder sisorbfi revil detaroilemaesadixorepoleymytivitca citapeh yticixototapeh decudni tibihniledom lamina a0 a0 pseiduts ortiv ni a0 a0 p azmah la a0te areiv iklamlanajaham narmi dna meedansevaelsdopsdeesa review of a0properties nutritional and a0pharmaceutical applications of a0moringa oleifera¦ 0c detneverp dna elfiorp la a0te reugiugledom lamina la a0te reugiugledom lamina inamkcurledom lamina hzimahtabni la a0te nanhsirkalapogseiduts ortiv niandipil niagdevorpm thgiewi dna ytilitom lanitsetni etomorpstceffe evitaxalyticixototapeh decudnilomatecarap no evitcetorpotapeh etatsorp fo sledom tnednepedni negordna ni ytivitca recnacitnarecnac eruc dna cimeloretselohcopyhsmelborp yraniruarefielo m fo snoitca lacigolocamrahpdeunitnoc elbat eht dna etalaxo syendik eht ni noitisoped enotsyraniru eht ecuder la a0te idarak dna ytivitca evitcetorpotycyrotercesitna recluitna la a0te yrahduohcan a0p a0evitcetorpotapehipilitna dna cimeretselohcitnaledom lamina a0 a0 p amrukledom lamina a0 a0 pledom lamina a0 a0 p ahcenesledom lamina a0 a0 pcimed tcart yraniru fo tnemeganamsmotpmys snoitcefni hgni sdna ayruam la a0te alkuhsledom lamina a0 a0 p ledomnamuh a0 a0 p serec¡cledom laminalairetcabitna a0 a0 p la a0te reffazseiduts ortiv niantceffe noitatnalpmiitna a0p a0yrotammaflniitna a0p a0yticixototapehdecudni lomatecarapno evitcetorpotapeharopsoruen tsniaga lagnufitna inamkcurledom lamina a0 a0 pseiduts ortiv ni ahj a0 a0 pd a0meireles several m oleifera studies with leaf powder or extracts on animals revealed other properties beyond the previously referred antioxidant chemoprotectant and antihypertensive stohs and hartman the antioxidant activity derives from the high amounts of polyphenols leone et a0al 2015a b verma et a0al leaves extracts have revealed anticancer properties with antineoproliferative activity by inducing reactive oxygen species ros production only in cancer cells which leads to cell apoptosis gopalakrishnan the active compounds present in extracts from leaves and bark revealed anticancer activity against breast and colorectal cancer cell lines through diverse mechanisms as decreased cell mobility decreased colony formation low cell survival high apoptosis and g2m enrichment alasmari et a0al some extract fractions with anticancer activity have already been isolated characterized and tested in a0vitro and in a0vivo rat model krishnamurthy et a0al table a0in traditional medicine a paste made of leaves is applied externally in wounds siddhuraju and becker some scientific studies have shown that leave extracts have beneficial properties in skin aqueous leaves extract increased human dermal fibroblasts proliferation leading to faster wound healing muhammad et a0al a m oleifera leave extract fraction with ethyl acetate in low concentration a0µgml showed in a0vitro effect in skin healing by increasing proliferation of human dermal fibroblasts gothai et a0al a hydroalcoholic extract of m oleifera leaves used in a cream showed antiaging characteristics due to phenolic compounds sunscreen and photo protective characteristics were studied very recently baldisserotto et a0al when applying a cream with this extract it was also verified a reduction in sebum production ali et a0al 2013a b c and in transepidermal water loss allowing to increase hydration ali et a0al 2013a b c wrinkles and other signs of lack of skin vitality where improved during a0months of using the same topic formulation with m oleifera leaf extract ali et a0al the compounds responsible for this improvement in skin surface appear to be phenolics eg kaempferol and quercetin and antioxidants such as vitamins a c and b jadoon et a0al m oleifera leaf extract cream was also tested for potential skin irritation by a a0h semioccluded patch test and proved to be nonirritant and well accepted by the volunteers also reducing skin erythema ali et a0al 2013a b c table a0 m oleifera leaf powder can be used to clean hands when four grams of wet more efficient or dried powder are applied on hands and rubbed torondel et a0al the efficacy results were the same as for nonmedicated soap revealing potential to help in hand hygiene and prevent pathogen transmission in developing countries where hygiene products are scarcea leave extract sprayed in plant crops revealed another utility for this plant having beneficial effects on the growing anyb detneserper era seulav dnuof tonelbacilppanon a pyb detneserper era eulav tnacfiingis yllacitsitats htiw seidutssrewolfstoorkrab 0crate size and resistance on those plants and fruits bhargave m oleifera leaf tea studies demonstrated alterations in blood circulating neutrophils and conclude that moringa tea has adaptogenic capabilities in cases of stress drue et a0al table a0 previous studies using dried moringa leaves tea in mouse model with acute lung inflammation showed that mice that had decreased lung inflammation marked by alterations in cytokine production leukocyte migration and neutrophil apoptosis mcknight et a0al an ethanolic extract of moringa leaves has antianxiety effect in swiss albino mice the ethanolic extract of m oleifera leaves may have produced its anxiolytic effects via multiple mechanisms bhat seedsseeds collected from pods can be eaten raw or cooked from m oleifera seeds a rich vegetable oil can be produced m oleifera seed oil or behenben oil is produced through the cold pressing of the m oleifera seeds m oleifera oil can be used to cook as a source to prepare biodiesel as a lubricant and in the cosmetic industry rashid et a0al the oil name comes from its high content on behenic acid which confers more resistance to oxidative degradation comparing to other vegetable oils ben oil is rich in oleic acid up to palmitic but also stearic behenic and arachidic anwar it is used in various cosmetic formulations as emollient and confers nourishing moisturizing antioxidant and protective properties it is also a good skin cleansing product nadeem and imran table a0 details some cosmetic brands that use m oleifera leaves oil or active extracts as ingredients in the composition of their products this oil is also used in the enfleurage process allowing the extraction of fragrances and active compounds from difficult sources as flower petals milled m oleifera seed shells can be used as a natural exfoliating agentmoringa oleifera seeds can also help diabetic patients table a0 some studies by almalki and el rabey showed its antidiabetic activity by reducing the blood glucose level when rats where treated with or a0mg of m oleifera seeds powderkg body weight during a0weeks at the same time ingestion lead to an increase in antioxidant enzymes and consequently the compound content such as glucomoringin phenols and flavonoids moreover the same authors treating these diabetic rats significantly increased and ameliorated all kidney functions parameters in fact m oleifera seeds ameliorated liver fibrosis in rats reducing liver damage and symptoms of liver fibrosis decrease the ccl4induced elevation of serum aminotransferase activities and globulin level as well as reduce the elevated hepatic hydroxyproline content and myeloperoxidase activity hamza improving the indices of hepatoxicity in rats such as malonialdehyde level and total antioxidant capacity glutathione content catalase and superoxide dismutase activities hamza treatment with m oleifera seeds also altered oxidative stress in relation to its antiinflammatory activity histopathological observations showed mild or less infiltration of lymphocytes angiogenesis and synovial lining thickening from all above results and observations it can be concluded that the seeds possess promising antiarthritic property mahajan et a0al these seeds have others appeals to the daily life and industry seed powder showed capacity to purify water and remove heavy metals and anic compounds sharma et a0al through low molecular weight cationic proteins mediated precipitation kansal and kumari there was a reduction of in the turbidity of the water and of bacterial reduction bhargave lea the remaining paste after the oil extraction still has the same flocculation properties serving both purposes and adding value lea compounds such as pterygospermin moringine and benzyl isothiocyanate isolated from m oleifera seeds showed antimicrobial effect viera et a0al accordingly there is applicability for m oleifera seeds in the prevention of microbial diseases table a0 m oleifera oil has also been tested for its potential to produce biodiesel contributing as an alternative to the conventional diesel fuel rashid et a0al flowers and a0rootsm oleifera flowers are used directly as part of the diet but also to make infusions which ha | 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 |
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 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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 |
" the popularization of health and medical informatics yields huge amounts of data extracting clinicalevents on a temporal course is the foundation of enabling advanced applications and research it is a structure ofpresenting information in chronological order manual extraction would be extremely challenging due to thequantity and complexity of the recordsmethods we present an recurrent neural network based architecture which is able to automatically extractclinical event expressions along with each events temporal information the system is built upon the attentionbased and recursive neural networks and introduce a piecewise representation we divide the input sentences intothree pieces to better utilize the information in the sentences incorporates semantic information by utilizing wordrepresentations obtained from bioasq and wikipediaresults the system is evaluated on the thyme corpus a set of manually annotated clinical records from mayoclinic in order to further verify the effectiveness of the system the system is also evaluated on the timebank_dense corpus the experiments demonstrate that the system outperforms the current stateoftheart models thesystem also supports domain adaptation ie the system may be used in brain cancer data while its model istrained in colon cancer data our system extracts temporal expressions event expressions and link them according to actuallyoccurring sequence which may structure the key information from complicated unstructured clinical recordsfurthermore we demonstrate that combining the piecewise representation method with attention mechanism cancapture more complete features the system is flexible and can be extended to handle other document typeskeywords clinical text mining event extraction temporal extraction relation extraction piecewise representationattention mechanism precision medicine is an emerging approach for diseasetreatment and prevention it becomes the whole worldbiomedicine domain the research hot spot which needsthe support of biomedical methods eg data mining it correspondence clixjtueducn zhijing li and chen li contributed equally to this work1school of computer science and technology xian jiaotong universityxian shaanxi china2shaanxi province key laboratory of satellite and terrestrial network techrd xian jiaotong university xian shaanxi chinaassociates with key information extracted from clinicalrecords eg symptoms over a disease course the associations are often statistically concluded from the evidencecollected from the clinical records the medical bigdata mostly exists in an unstructured form eg textwhich could store useful information very well aligningbiomedical events in clinical data along the events actually occurring time is a meaningful and efficient way ofstructuring such complex data the result may assistauxiliary diagnosistreatment scheme determinationepidemic prediction and side effect discovery etc 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 0cli bmc medical informatics and decision making page of many works have been devoted in the study of application in the medical era however large data analysis ofmedicaltreatment needs to map the correspondingmedical events in the clinical records the medical eventswith temporal information are very useful in medical erathese efforts will become the foundation of understanding disease facilitating the analysis of large medical dataas well for example the clinical record in fig may bepresented in a structured manner as the occurring eventsalong with temporal information it is easier for understanding the events and corresponding time point for example using the time point april as a referencethe entity bleeding is before the time point and the entitybolus chemotherapy and nausea is after the time pointin such case the actual events and their occurring consequence becomes clear at a glancein this paper we present a novel system which is builtupon deep neural networks to automatically extractevent expressions and their related temporal expressionsfrom clinical records the system has been evaluated onthe temporal histories of your medical event thyme corpus a corpus developed by a number of professionals according to characteristics of the corpusclinical data contains very long sentences that willundoubtedlythe difficulty of processingtherefore we do not simply use neural networks wewant to make full use of the contextualinformationour proposed method anically combines piecewiserepresentation and attention mechanism by a recurrentneural network rnn and achieves the stateoftheartperformance the results show improvements in automatic extraction of clinical event expressions along witheach events temporal informationincreaserelated workextracting clinical events along with temporal information is a complicated task and the existing systems oftenaccommodate several independent components each ofwhich retrieves different parts eg events and time andassemble them together each component may use a setof hand crafted rules or be based on a pretrained mlmodel velupillai develop the blulab systeminclude the cleartk support vector machine and conditional random fields classification approach and get thefirst place in semeval2015 task clinical tempeval macavaney present the system guir includeconditional random fields and decision tree ensemblesusing lexical syntactic semantic distributional and rulebased features guir receive the best score insemeval2017 task clinical tempeval in the way oftemporal expressions extraction tourille use aneural network based approach and achieve goodperformance for both event and relation extraction insemeval2017 task clinical tempeval lin propose a recurrent neural network with multiplesemantically heterogeneous embeddings within a selftraining framework for clinical temporal relation extraction task they achieve good results for both in andcrossdomainafter event and temporal expression extractionassigning each event with the right temporal expressioninvolves more complicated process some systemsmatch event with temporal expression by a set of syntactic rules crafted by experts wang use syntacticrulebased method for automatic pathway relation information extraction from biomedicalliterature these methods are fast but not flexible enough somefig the example of the medical information extraction the texts marked by the underscores ___ are the temporal expressions the textsmarked by the dash lines _ _ _ are the event expressions 0cli bmc medical informatics and decision making page of existing methods for medical relation information extraction are based on machine learning ml models[]conditional random field crf and supper vectormachine svm are often used in the task of relation extraction lu liu propose an svm model toextract the relations between the potential named entitypairs finkel propose a crfbased informationextraction system to determine relationships deeplearning revives the popularity of neural networkswhich can learn effective relation features from the givensentences withoutengineeringsocher is the first work that employs an rnnmodel to classify relation one early work proposed byluo is based on a recurrent neural network and ableto classify relations from clinical notes compared withthe rule based methods these methods are more flexibleneural network based methods take less time by quicklyscreening out the most unlikely candidate entitypairstherefore some approaches attempt to combine bothhence we think rnn is a good choice and we adopt thernn systemcomplicated featurein recent years attention mechanism has been widelyused in various tasks of nlp based on indepth learning li propose a model that combines abidirectional long shortterm memory network with amultiattention mechanism forrelation extractionzhou propose the attentionbased bidirectional long shortterm memory networksattblstmfor relation classification and the modelresults outperforms most of the existing methods withonly word vectors on the semeval2010 relation classification task so in this paper we also introduce theattention mechanismmethodologythe system consists of three components the first component extracts temporal expressions temporal expressions enable events to be chronologically annotatedthe second component identifies the relevant medicalevents event expressions any situation relevant to thepatients clinical timeline the third component detectsthe relations between the events and the temporalexpressionsannotating clinical records are very expensive frequently only a data of disease specific type is availablebesides the regular mlbased extraction we introducedomain adaption to allow the system to be able to extract the information from one type of disease eg braincancer while it is trained on another type eg coloncancerwe show the pipeline of our system in fig thesystem is built upon an annotating pipeline adoptingthemanagementunstructuredinformationfig the dataprocessing pipeline of the system we first extracttemporal expressions and event expressions respectively frommedical records then extract the relations between themarchitecture uima framework the preprocessingincludestokenization partofspeech tagging andlemmatization which used the stanford corenlp toolkit in both time and event extractions spans oftime and event expressions are represented by the offsets in texts the automatic annotations of event expressions temporal expressions and their relations arebased on three rnn models utilizing lexical syntacticand semantic features [] at the core of deeplearning techniques for nlp lies the vector basedword representation which maps words to an ndimensional space for the choice of the word embeddings corpus we do some research work only entities time and event expressions in the clinical records can be found in the wikipedia in comparisonthe bioasq corpus which is full of biomedical information contains more than of the entities weuse word embeddings from the european projectbioasq obtained by using word2vec on pubmed abstracts [ ] and include the vectorsof distinct words each word is representedas a 200dimensional vector if a word could not befound in the bioasq corpus the embedding is generated from wikipedia by word2vec mikolov as a complement [ ]thestateinternalof rnn can demonstratedynamic timing behavior the hidden state vector can be computed by the following formulaht ¼ f wht þ uxtðþin this formula xt is the input ht is the hidden stateu and w are the weight coefficients f is the nonlinearfunction such as tanh or relu 0cli bmc medical informatics and decision making page of extraction of temporal expressions and event expressionsindependent models are trained for the extractions oftemporal expressions and event expressions figure shows the infrastructure of the system there are twoforms of temporal expressions one is numeric temporalexpressions eg etc and the other is casualtemporal expressions eg day weeks during aperiod etc firstly we generalize all the numeric temporal expressions into for example both and become which can be easily recognizedby the regular expression the regular expression is useddue to the characteristics of the data the numeric temporal expressions are not well recognized by rnn if wedo not use it secondly the casual temporal expressionsare recognized by a rnn model the casual temporalexpressions in the training set are tagged to representthe tokens position in a particular expression there are four tags in our proposed method includingb i o and e which state that the token is at thebeginning on the inside on the outside or at the end ofthe entity respectivelyin this section we propose the system arnn whichis based on a recurrent neural network combining theattention mechanism we need to predict the tokensposition tag of each word before we can train the arnnmodel to predict the type of each temporal expressionwe treat each temporal expression as an entity and eachentity is treated as a unit input we use the average valueof all the word embeddings of an entity in the nextprocess the network in the fig shows the flow chartof arnn network to predict the type of the entitythere is an example sentence from the corpus we willget a ct enterography to rule out crohns disease inthis case the given entity is enterography and thecontext words are we will get a ct to rule outcrohn disease in order to better apply the context information we employ the attention mechanism to learnthe weighted score of each context word related to thegiven entitythe higher weight the higher semantic is bound upwith the given entity 01 00αi exp vti u vhtuv is the parameter that has to be learned from fig we can see that st is the state vector that integrates theother context words information with the given entity attime t st can be computed asst ¼xihαihiwe combine st and h3 to obtain h0sent the given entity which can repre ¼ h3 þ sth0in this process the prediction of entitys type will bepredicted from the given input entity vector the methodof using regular expression are also used to match themissing temporal expressions eg similar to temporal expressions extraction the eventexpressions extraction is built on another rnn unliketime expressions event expressions are all single wordsthere is no need to sign each tokens position we usethe softmax classifier to predict the label y² of the temporal and event expressions the state vector h0 is usedas input and y² could be computed by 10 11py ¼ softmax w h0 ¼ arg maxypyyeventtime relation erer extraction is the most important task in this paperin this section we propose the novel system aprnnwhich is based on a recurrent neural network combiningthe attention mechanism and the piecewise representation the eventtime relation are regarded as a classification problem it is divided into four categories based onsome wellknown communities such as semeval orbionlp the event time relation associates the identifiedevent expressions and temporal expressions and indeedindicates the what and when of a medical event inclinical records the four types are before after beforeoverlap and overlapthe shortestsyntactic path used includespiecewise representationthe dependency parsing of each sentence has beenobtained by utilizing stanford corenlp toolkit thepreviously trained word embeddings which representeach word by a 200dimension word vector and theshortest syntactic paths are fed into the rnn modelof er extraction the word embeddings and shortestsyntactic path are used as features the informationofthewords the poss and the length we add all thesevectors as the entity feature after adding up we stillget a 200dimensional vector for each entity if theentity include severaltemporal expressions we add the vectors of each token and get theaverage vector as the entity vector the whole is divided into sentences as input units we extractall entity pairs based on the annotations given a sentence x x1 x2 ¦ xt the words are projected intoa sequence of word vectors denoted by e1 e2 etwhere t is the number of words in this part wewould like to introduce the piecewise representationtokens eg 0cli bmc medical informatics and decision making page of fig architecture of the rnn model for the extraction of temporal expressions and event expressions we propose the attentionbased rnnmodel to do the entity extraction on the left side of the figure is the details of the attention mechanism the right part of the figure is thernn modelinformation as shown in fig in other wordsthe input sentence is divided intothree parts according to the entity pair we call thisprocess piecewise representation the purpose ofpiecewise representation is to better use of the contextthe examplesentence is divided into three parts according to theentity pair will enterography the whole sentence iswe will get a ct enterography to ruleout crohnsdisease the first part is the sequence before the entity pair we the second part is the sequence between the entity pair get a ct and the third partis the sequence after the entity pair to rule¦ thereare reasons for segmenting the sentence the firstone is that in many cases some studies may choosethe sentence between the entity pair as input insteadof using the whole sentence nevertheless thiscan miss some information and some of them maybe useful only several words cannot supply enoughcontextfeatures segmented sentences can be used to extract the effectiveinformation forextractingrepresentsinformation to the greatest extent of each sequenceand avoid the absence of contextualinformationanother reason comes from the network and our corpus in the corpus the longest sentence contains words however the average length of all sentenceshas words with the rnn structure since the information of a sentence is learned word by word thefeature vector produced at the end of the sentenceactuallysentence althoughrnn has the memory in learning process but thememory time is not long accumulation by recurrentconnectionslongterm informationquickly and the feature vector atthesentence is hard to carry the information of earlysteps in model training there are many longdistancesentences more than words in the training dataso the piecewise representation can help the systembetter use the information of the sentence the syntactic analysis and pos information of the examplesentence are also shown in the fig the end ofentirethetendsto fet 0cli bmc medical informatics and decision making page of fig the flow of our proposed model aprnn on the left side of the figure is the details of the attention mechanism the right part of thefigure is the rnn model which is divided into three partsmodelthe er model contained three parts the first component the feature layer the second component thehidden layer catch the information of word sequenceand produces wordlevel features representations andthen merges wordlevel features into a sentencelevelfeature vector by selecting the most valuable featureinformation among allfeatures weshow the whole process in the fig in this part weproposeattention mechanism to obtain thethe wordleveltherepresentation of the sentence not all the words inthe context describe the er relation each word inthe context has different effects on the given entitypair therefore we introduce the attention mechanism to learn the weighted score of each context wordrelated to the entity pair for the first part the attention mechanism is used to screen the most useful information we use the bilinear operator to computethe attention weight αi for each vector h1 and h2 toreflect how the information relevant to the first entity 0cli bmc medical informatics and decision making page of in the entity pair the current state h3 the calculation method of αi and st is the same as the description in the extraction of temporal expressions andevent expressions sectionwe combine st and h3 to obtain h0 which can represent the sequence before the entity pair for the secondpart we choose the state of the last entity in the sequence ht to represent the whole sequence for thethird part the same method is used as the first part wealso use the same attention mechanism to obtain therepresentation oft the singlesentencelevel feature vector need to be obtained to represent the entire sentence for the relation classificationwe introduce the maxpooling approach as in cnnmodels to obtain the single sentencelevel feature vector the maxpooling is formulated as followsthe sequence h0htf gm ¼ maxtnext the sentencelevel feature vector m is passed tothe output layer furthermore the output layer has classes we use the softmax classifier to predict the labely² from a set of labels y from the sentence the statevector m is used as input therefore y² could be computed bypy ¼ softmax wmð ¼ arg maxypyyþin addition there are two settings in er extraction inorder to better compare our approach the first is basedon our proposed method the second is only utilize thernn network without any piecewise representation orattention mechanism as shows in fig experiment and resultsdatathe major medical data are the data of medical institutions diagnosis and treatment collection of massiveclinical data and laboratory data produced every day atall levels of hospitala golden annotated corpus marked up with temporalexpressions events and relationship between them isneeded to allow us to evaluate by our methods thethyme corpus which has been used since isone of the suitable corpora consisting of clinical andpathological notes of patients with colon cancer andbrain cancer from mayo clinic unlike other datasetsthe events in this dataset are all single words which arevery suitable for our system the notes are manually annotated by the thyme project thymehealthnlpusing an extension of isotimeml for the annotation oftemporal expressions events and temporal relations of the corpus is used for training is forfig the flow of the comparison system this is a commonrnn modeldevelopment and is for testing the developmentset is used for optimizing learning parameters thencombine it with the training set to build the system usedfor reporting results table shows the distribution ofthe thyme corpus the colon cancer data are used astraining data and are tested on both colon cancer andbrain cancer data to demonstrate its effectiveness withtable the distribution of the thyme corpus in this table weshow the different types of data in the corpusdatacolon cancertrain dev testbrain cancertrain testdocumenttemporal expressions event expressions er 0cli bmc medical informatics and decision making page of or without domain adaptation and this can reflect thatour approach is not limited to a particular field inevaluation all methods have access to the same set oftraining and testing datatable the temporal expressions extraction results on braincancer we utilize different methods to do the task the resultsare shown in part the result of previously best system isshown in part resultsthe method has been evaluated on both colon cancerdata and brain cancer data to demonstrate the effectiveness with or without domain adaptation in order to dobetter research several methods are used to de the entityextractionsix methods of temporal expression extraction arulebased method a system based on crf a system based on general rnn without any attention mechanism or context rnn a system based on rnnwith easy attention mechanism but without any contextwords rnnatt our proposed method a rnn system with attention mechanism and context words asystem combines the crf and rnn network all the results are compared part in tables and for therulebased methods firstly we find all the prepositionsaccording to our experience and experimental statisticswe extract five tokens behind their own prepositionsthrough careful observation of data we found thatmany time expressions always show up behind a preposition we then judge whether those five words are relatedto time expressions we define a time dictionary to listthe words which we think can be a part of the time exlike month week day hour maypressionsmonday morning once and so on next we contrastthe five tokens with time dictionary and findwhether it can represent a date or a precise time finallywe extract all the continuous tokens that we thoughtmay relate to the time expressions if there is a definite before those tokens extract it as well there existsome expressions do not after a preposition and onlycontain one word and most of them have the same prefix like pre post peri so we use this prefix rule tofind the remain expressions the major feature we usedfor training the crf and svm classifier is simple lexicaltable the temporal expressions extraction results on coloncancer the part shows the results of six different methodsthat we used to do the temporal expressions extraction thepart shows the result of the previously best systempart methodrulebasedcrfrnnrnnattarnnpart crfarnnblulab run prf1part methodrulebasedcrfrnnrnnattarnncrfarnnpart guirprf1features word embeddings partofspeech tag numericlower case blulab run andtype capital typeguir system are the previously best system mentionedin the section two these results are shown in tables and part in both tables and rule based methods achievethe lowest result the recalls are relatively better thanthe precisions due to the welldefined dictionary theerror analysis shows that some pre post and periare considered as time expressions while they should notbe meanwhile the rulebased method often mistakestwo independent expressions as one if they are adjacentin table the rnn systems performances are lowerthan blulab run 3a cleartk svm pipeline usingmainly simple lexical features along with informationfrom rulebased systems the rulebased information iseffective but it has limitations it can extract rules according to the characteristics of data we do not addany rules to the rnn system the observation on theerror analysis shows that without any attention mechanism and context words rnn is not very effective forsimilar combinations of numbers and letters eg h days etc because the form of the corresponding wordvectors are generated randomly and the time series contains a large number of the above type so the modelcannot learn characteristics of time series so it cannotbe correctly extracted after adding the attention mechanism and context words the arnn system achieve therelatively good results because of the good results ofcrf we combine the crf with the arnn and achievethe best result from table we can see that the rnnoutperforms the guir system which is the current bestsystem it is an ensemble of crf and decision tree withlexical syntactic semantic distributional and rulebasedfeatures the guir system can not extract the previously unseen or atypical date formats very well it is obvious that their rules are not comprehensive enoughthis problem also exists in rnn system however whenadding the attention mechanismit can extract more 0cli bmc medical informatics and decision making page of new and otherwise unknown formats the arnn andcrfarnn system achieve the best results in this partwe have two test data sets one is colon cancer anotherone is brain caner we trained all the models on thesame training data and test them on two different testdata sets except for the different test data the parameters are exactly the same the experimental results provethat our model is effective on other test data setsmeanwhilefive methods of event extraction amethod based on svm a system based on crf asystem based on general rnn without any attentionmechanism or context rnn a system based onrnn with easy attention mechanism but without anycontext words rnnatt our proposed method arnn system with attention mechanism and contextwords all the results are evaluated part in tables and for event extraction the svm and crf modelobtain the relatively good results in colon data and perform poorly in brain colon data compared to the bestsystem limsi however rnn achieves preferably results in the two sets of test data even higher than thebest system limsi as shown in both tables and when adding the attention mechanism and contextwords the results are improvedas for the er extraction which is the key point of thepaper first we compare our proposed model with thefollowing methods a general rnn system withoutany attention mechanism or piecewise representationwe use the sentence between the entity pair as the inputrnn a general rnn system without any attentionmechanism or piecewise representation we use thewhole sentence as the input rnnwhole we can seethe results of rnnwhole is better than the results ofrnn it means that the sentence length can affect theperformance of the system therefore we use the sentence between the entity pair as the input for other system a general rnn system with attention mechanismbut without piecewise representation rnnatt ageneral rnn system without attention mechanism butwith piecewise representation rnnpie our proposed system aprnn but only use the word embeddings trained from wikipedia aprnnwiki ourtable the event extraction results on colon cancer different methods are utilized by us all the results are shown inpar1 the part shows the result of the previously best systemf1methodsvmpart prcrfrnnrnnattpart arnnblulab run table the event extraction results on brain cancer we adopt methods to do the task the results can be compared in part the result of the best system is shown in part part part methodsvmcrfrnnrnnattarnnlimsiprf1proposed system aprnn but only use the word embeddings trained from bioasq aprnnbioasq our proposed system which is based on a recurrentneural network combining the attention mechanism andthe piecewise representation all these results are evaluated part in tables and except for model and the word embeddings for other models are from bothwikipedia and bioasq from the results we can seethat both attention mechanism and piecewise representation are useful they can improve the results to someextent we can directly compare the value of attentionin two groups of experiments result and result result and result the result and result result and result can directly demonstrate the performance with and without segmentation the difference between model and is that model is missing thepiecewise representation and the difference betweenmodel and is without or with the attention mechanism the result has been improved with the piecewiserepresentation the experiment and are aboutlooking at the impact of word embeddings the result and result show that different word embeddings canlead to different results after combining the two corpuswikipedia and bioasq the results increase slightlytable the er classification results on colon cancer part shows the results of the relevant methods we used the otherrelated works which achieved the very good results are shownin part part part methodrnnrnnwholernnattrnnpieaprnnwikiaprnnbioasqaprnnblulab run svmattblstmprf1 0cli bmc medical informatics and decision making page of table the er classification results on brain cancer the resultsof our proposed methods are shown in part part shows theresults of other relatedpart part methodrnnrnn wholernnattrnnpieaprnnwikiaprnnbioasqaprnnlimsisvmattblstmprf1aprnn different factors that may affect the resultsare verified from experimental results eg piecewise representation attention mechanism word embeddings allthese factors are utilized to make better use of contextual informationwe compare our work with other related work thelimsi system which achieves the best score on the ertask in semeval2017 task li rao and zhang proposed the litway which is a system that has adopteda hybrid approach that uses the libsvm classifier with arulebased method for relation extraction theyachieve the best score in the seedev task of bionlpst thus we use their approach as a benchmark forour system the bilstmattention networks proposedby zhou were chosen as another benchmarking model attblstm which outperforms most of theexisting methods they designed a bidirectional attention mechanism to extract wordlevel features from thesentence the features for the attentionbased model include word vector | 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 |
malignant mesothelioma is an aggressive cancer associated with asbestos exposure with median survival time of to months following diagnosis given that mesothelial cells also line the peritoneum and pericardium malignant mesothelioma can present in unusual sites and in patients with nonrespiratory complaints a 73yearold male presented to the emergency department for worsening intermittent diffuse abdominal pain for the past months with associated unintentional 40pound weight loss early satiety and diarrhea he denied exposure to asbestos computed tomography imaging revealed multiple masses concerning for malignancy including the primary retroperitoneal mass a mass involving the terminal ileum and a mass in the right upper lung esophagogastroduodenoscopy demonstrated significant mass effect within the stomach without signs of endoluminal infiltration computed tomographyguided biopsy of the retroperitoneal abdominal and intramuscular paraspinal masses was performed stage iv epithelioid mesothelioma was confirmed when hematoxylin and eosin staining revealed pleomorphic malignancy nuclei containing a vesicular chromatin pattern and prominent nucleoli and immunohistochemical staining was positive for ck oscar cytokeratin gata3 calretinin ema and ck56 he was started on cisplatin pemetrexed and bevacizumab but developed severe abdominal pain with pneumoperitoneum and bowel perforation month later and expired shortly thereafter to our knowledge this represents a highly atypical presentation of malignant mesothelioma considering the involvement of the retroperitoneum with diffuse lesions in the abdominopelvic cavity and thorax sparing the lung pleurae this case also calls attention to the occurrence of malignant mesothelioma in patients without known asbestos exposure and the crucial role of pathology in diagnosing atypical presentationskeywordsepithelioid mesothelioma neoplasm asbestos esophagogastroduodenoscopy retroperitonealintroductionmalignant mesothelioma mm is a rare cancer originating from mesothelial cells forming the linings of the pleura to of mm cases12 peritoneum to of cases23 and pericardium of cases456 typically the pathophysiology of mm is believed to occur in patients with asbestos exposure leading to chronic inflammation of the pleura67 subsequently mm presents in a patient with shortness of breath with findings on imaging of nodular thickening of the pleura pleural effusion or a mass7 in cases involving the peritoneum a more common initial presentation would be ascites with computed tomography ct findings including irregular thickening of the peritoneum lymph node enlargement and possible metastasis to the chest table regardless of location published guidelines detail the importance of first looking at the histology of a biopsy prior to immunohistochemical ihc and molecular testing to confirm suspicion of mm from the histology mm is classified into epithelioid sarcomatoid or biphasic mixed with epithelioid mm remaining the most common with its polygonal cells resembling reactive mesothelial cells other histologic features include scalloped cell borders with increased cytoplasm prominent and enlarged nucleoli and nuclear atypia10 the presence of a solid mass separate from the pleura and peritoneum with histologic features of mm eliminates the requirement for stromal invasion for the diagnosis910immunohistochemical testing should use markers of mesothelial origin such as cytokeratin markers and alternative tumor markers to narrow the differential diagnosis based on histology10 this will vary by histology and location 1mercyone des moines medical center des moines ia usa2des moines university des moines ia usareceived may revised june accepted july corresponding authordustin j uhlenhopp mercyone des moines medical center 6th avenue des moines ia usa email duhlenhoppmercydesmoinescreative 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 of investigative medicine high impact case reportstable typical characteristics of malignant abdominal mesothelioma1819characteristicspercentage of casespathological subtypes epithelial sarcomatous mixedasbestos exposure male femalepresenting symptoms ascitesa abdominal pain asthenia weight loss anorexia fevera diarrhea vomitingact scan findings ascites abdominal mass peritoneal thickening mesenterial thickeningadditional laboratory findings thrombocytosis 000mm3 anemia male gdl female gdlmaletofemale ratiomedian overall survivalaverage age of onset months yearsabbreviation ct computed tomographyamost significant symptoms on presentation associated with deathof the mm epithelioid markers are needed to rule out carcinomas and peritoneal markers are needed to rule out adenocarcinomas and cancers causing peritoneal carcinomatosis810 key markers for mesothelioma are positive ihc staining for calretinin d240 podoplanin and cytokeratin in this case report we present an unusual case of mm with metastatic disease in a patient with no known asbestos exposure and a history of abdominal pain and weight loss found to have multiple lesions and diffuse lymphadenopathy on imagingcase descriptiona 73yearold male presented to the emergency department with worsening intermittent diffuse abdominal pain and 40pound weight loss over the past months associated with increased flatulence early satiety and frequent exive nonbloody diarrhea the patient described the pain as cramping that typically worsened in severity at night he denied feverchills dyspnea cough epistaxis hematemesis hemoptysis hematochezia and melena the patient was a longtime science instructor at a local community college prior to transitioning to the swedish importexport business with no significant exposure history including asbestos he had a prior history of right middle cerebral artery stroke and was a former smoker with a 60packyear smoking historyinitial testing revealed normal electrolytes and liver function panel elevated lactic acid mmoll leukocytosis white blood cell 12100mm3 and anemia hematocrit abdominal ct with oral and intravenous iv contrast revealed a homogenous retroperitoneal mass measuring cm with displacement of the gastroesophageal junction and lesser curvature of stomach as well as a soft tissue mass involving the terminal ileum measuring cm both were concerning for neoplasm additional findings included diffuse lymphadenopathy in the retroperitoneum likely signifying metastatic disease no evidence of obstruction was noted see figure esophagogastroduodenoscopy confirmed concerns for extrinsic compression of gastroesophageal junction and fundus of stomach suggesting an underlying mass no endoluminal gastric masslesions were appreciated see figure colonoscopy was attempted but scope was not able to advance past the sigmoid colon safely due to colonic stricturecomputed tomography chest with iv contrast identified moderate diffuse emphysema a right upper lung lobe spiculated mass measuring cm and indeterminate small left upper and lower lobe nodules see figure diffuse retrocrural and gastroesophageal lymphadenopathy was noted in addition to the retroperitoneal lymphadenopathygiven the concern for lymphoma versus alternative neoplastic disease ctguided biopsy was performed on the retroperitoneal abdominal mass and an intramuscular paraspinal mass hematoxylin and eosin staining revealed pleomorphic malignancy nuclei containing a vesicular chromatin pattern and prominent nucleoli see figure cells were predominantly polygonal with occasional spindling noted ihc staining was positive for ck oscar cytokeratin gata3 calretinin ema and ck56 staining was negative for pax8 tte1 p40 p63 d240 wt1 moc31 cd34 mari cd45 dog desmin cd117 sox10 actin ca9 and myogenthese findings were suggestive of epithelioid mm with metastasis to the terminal ileum and lung a diagnosis that was confirmed by mayo clinic laboratories noting this is a particularly atypical presentation of mesotheliomathe patient opted for treatment of stage iv mesothelioma with plans to pursue a 21day cycle of cisplatin pemetrexed and bevacizumab for up to cycles however he presented to the emergency department days after starting the second cycle with severe abdominal pain of hours duration he was found to have a moderate pneumoperitoneum with bowel perforation worsening intraabdominal mesothelioma and several small bilateral acute renal parenchymal infarcts the patient and his family opted for comfort care and he passed the following day 0cuhlenhopp et al figure abdominal computed tomography with oral and intravenous contrast demonstrating a homogenous retroperitoneal neoplastic mass measuring cm green arrows on left image with displacement of the gastroesophageal junction and lesser curvature of stomach a soft tissue mass involving the terminal ileum measuring cm and a 17cm midmesentery mass are also noted green arrows on right imagefigure esophagogastroduodenoscopy revealed extrinsic compression of the fundus suggesting an underlying mass or lesion no endoluminal gastric masseslesions were appreciated computed tomographyguided biopsy later revealed this to be epithelioid mesothelioma originating from the retroperitoneal spacediscussionthe diagnosis of mm carries a poor prognosis and requires careful review by pathology our case details the workup necessary to diagnosis mm in a patient with an atypical presentation of intermittent abdominal pain found to have figure computed tomography chest with intravenous contrast identified moderate diffuse emphysema a right upper lung lobe spiculated mass measuring cm green arrows and indeterminate small left upper and lower lobe nodules as this did not involve the pleura as is traditionally seen this was felt to be a metastasis of the primary retroperitoneal mass with metastasis to lungs and ileumsignificant metastatic disease prior to identification of a primary malignancyreview of the case reveals the importance of tissue analysis and the continued public health concern of mesothelioma 0c of investigative medicine high impact case reportsongoing research is investigating whether these genetic changes have a role in disease development in cases without asbestos exposure610 in the case of malignant peritoneal mesothelioma there is a higher fraction of mm without an identifiable environmental exposure which raises the question whether the disease process and role of genetics vary by the location of mm14 in the future these will hopefully lead to the development of immunotherapies that are able to target these pathways and improved disease prognosis6known asbestos exposure was not a factor in our case and should not be used to rule out mesothelioma10 over of mm cases are attributable to asbestos exposure with of malignant peritoneal mesothelioma attributed to asbestos1516 recent studies detail that despite the restriction of asbestos products in the united states for over years the role of asbestos in chronic inflammation and dna damage will continue to cause malignancy615 in the united states alone there were approximately new mm cases in with current estimates anticipating a peak incidence of mm worldwide in given the continued use of asbestos worldwide and time to disease development6715 this case highlights the importance of considering mesothelioma in the differential diagnosis of abdominal pain regardless of asbestos exposure historydeclaration of conflicting intereststhe authors declared no potential conflicts of interest with respect to the research authorship andor publication of this fundingthe authors received no financial support for the research authorship andor publication of this ethics approvalour institution does not require ethical approval for reporting individual cases or case seriesinformed consentverbal informed consent was obtained from the patient for their anonymized information to be published in this orcid idsdustin j uhlenhopp orcid0000000315092203ann saliares orcid0000000281484435tagore sunkara orcid0000000195369027references robinson bm malignant pleural mesothelioma an epidemiological perspective ann cardiothorac surg doi103978jissn2225319x20121104 hui m harshavardhana kr uppin sg pericardial mesothelioma presenting as chronic constrictive pericarditis a series of three cases from a single institution indian j pathol microbiol doi104103ijpmijpm_711_17figure hematoxylin and eosin stain of the retroperitoneal mass malignant mesothelioma that is somewhat pleomorphic but mostly epithelioid polygonal cells with some spindling are seen cells have pleomorphic nuclei occasionally multinucleated with moderate amount of eosinophilic cytoplasm nuclei have a vesicular chromatin pattern with distinct nucleoli there is no obvious glandular or squamous differentiationmm carries a median survival time of to months following diagnosis7 this poor prognosis is partially due to the fact that over of cases are not discovered until the distant metastasis stage11 while most cases of epithelioid mm have an improved prognosis compared with sarcomatoid mm it can possess pleomorphic features that indicate highly aggressive cancer and shortened expected survival time71012 treatment consists of chemotherapy radiation and surgery although no therapy is curative7in our case ihc staining had an important role in developing the diagnosis of epithelioid mm with pleomorphic features it distinguished the disease from reactive mesothelial hyperplasia and other malignancies with possibly better prognoses10 the right lung lobe mass is not the typical presentation for mm involving the thorax and complicates the clinical picture the presentation of the retroperitoneal mass coupled with diffuse metastatic lesions and atypical involvement of the thorax make this patients presentation particularly unique calretinin gata3 and ck56 were positive in the tissue obtained from the retroperitoneal and paraspinal muscle masses which implicated a mesothelial origin for the malignancy calretinin is a strong indicator of epithelioid mm and is useful in distinguishing mm from adenocarcinoma10 recent reports estimate a positive staining for gata3 in mesothelioma in of cases and note a presence in epithelioid mm in one third of cases1013 another important ihc markers for mm is d240 which was negative in our patientwhile not completed in our patient other markers present in mm include the deletion of p16 occurring in of epithelioid mm and the presence of a bap1 mutation101214 0cuhlenhopp et al lev½ m boublkov¡ l b¼chler t Å¡imÅ¡a j treatment of malignant peritoneal mesothelioma klin onkol doi1014735amko2019333 sardar mr kuntz c patel t et al primary pericardial mesothelioma unique case and literature review tex heart inst j mutsaers se the mesothelial cell int j biochem cell biol napolitano a carbone m malignant mesothelioma time to translate trends cancer bibby ac tsim s kanellakis n et al malignant pleural mesothelioma an update on investigation diagnosis and treatment eur respir rev doi10118316000617 liang yf zheng gq chen yf song h yin wj zhang l ct differentiation of diffuse malignant peritoneal mesothelioma and peritoneal carcinomatosis j gastroenterol hepatol gill rr imaging of mesothelioma in a tannapfel ed malignant mesothelioma recent results in cancer research vol springer husain an colby tv ordonez ng et al guidelines for pathologic diagnosis of malignant mesothelioma update of the consensus statement from the international mesothelioma interest group arch pathol lab med howlader n noone am krapcho m et al seer cancer statistics review accessed february seercancergovcsr1975_2016 arif q husain an malignant mesothelioma diagnosis arch pathol lab med miettinen m mccue pa sarlomorikala m et al gata3 a multispecific but potentially useful marker in surgical pathology a systematic analysis of epithelial and nonepithelial tumors am j surg pathol attanoos rl churg a galateausalle f gibbs ar roggli vl malignant mesothelioma and its nonasbestos causes arch pathol lab med craighead je epidemiology of mesothelioma and historical background in a tannapfel ed malignant mesothelioma recent results in cancer research vol springer spirtas r heineman ef bernstein l et al malignant mesothelioma attributable risk of asbestos exposure occup environ med bray f ferlay j soerjomataram i siegel rl torre la jemal a global cancer statistics globocan estimates of incidence and mortality worldwide for cancers in countries ca cancer j clin doi103322caac21492 manzini vde p recchia l cafferata m et al malignant peritoneal mesothelioma a multicenter study on cases ann oncol doi101093annoncmdp shih ca ho sp tsay fw lai kh hsu pi diffuse malignant peritoneal mesothelioma kaohsiung j med sci doi101016jkjms201305003 0c' | Colon_Cancer |
overexpression of epithelial cell adhesion molecule epcam has been associated with chemotherapeutic resistanceleads to aggressive tumor behavior and results in an adverse clinical outcome the molecular mechanism by whichepcam enrichment is linked to therapeutic resistance via nrf2 a key regulator of antioxidant genes is unknown wehave investigated the link between epcam and the nrf2 pathway in light of therapeutic resistance using head andneck squamous cell carcinoma hnscc patient tumor samples and cell lines we report that epcam was highlyexpressed in nrf2positive and hpvnegative hnscc cells in addition cisplatinresistant tumor cells consisted of ahigher proportion of epcamhigh cells compared to the cisplatin sensitive counterpart epcamhigh populationsexhibited resistance to cisplatin a higher efï¬ciency in colony formation sphere growth and invasion capacity anddemonstrated reduced reactive oxygen species ros activity furthermore nrf2 expression was significantly higher inepcamhigh populations mechanistically expression of nrf2 and its target genes were most prominently observed inepcamhigh populations silencing of epcam expression resulted in the attenuation of expressions of nrf2 and sod1concomitant with a reduction of sox2 expression on the other hand silencing of nrf2 expression rendered epcamhighpopulations sensitive to cisplatin treatment accompanied by the inhibition of colony formation sphere formation andinvasion efï¬ciency and increased ros activity the molecular mechanistic link between epcam expression andactivation of nrf2 was found to be a concerted interaction of interleukin6 il6 and p62 silencing of p62 expressionin epcamhigh populations resulted in the attenuation of nrf2 pathway activation suggesting that nrf2 pathwayactivation promoted resistance to cisplatin in epcamhigh populations we propose that therapeutic targeting the nrf2epcam axis might be an excellent approach to modulate stress resistance and thereby survival of hnscc patientsenriched in epcamhigh populationscorrespondence syed s islam drsyedsislamgmailcom1department of biochemistry and molecular biology university of chittagongchittagong bangladesh2department of pathology mcgill university montreal qc canadafull list of author information is available at the end of the edited by b zhivotovskyintroductionhead and neck squamous cell carcinoma hnsccaffects more than patients per year12 resistanceto chemotherapeutic drugs limits the overall treatment 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 0coutcome in hnscc patients3 response to chemotherapeutic drugs is partly mediated by the keap1nrf2 signaling system4 nrf2nfe2l2 nuclear factor erythroid2like is a key transcription factor which in the normalbasal state functions as cytoprotective response to oxidative and electrophilic stress under oxidative stressstate nrf2 dissociates from cytoplasmic inhibitor keap1kelch like echassociated protein translocate into thenucleus and activates nrf2 transcriptional genes andprotects cells against oxidative stress mediates detoxiï¬cation and participates in atpdependent drug efï¬ux5abnormalities of keap1nrf2 pathwaylead to amechanism of oncogenesis and chemo and radioresistance in a variety of cancers including hnscc4inhibition of nrf2 expression by sirna augmentedcarboplatininduced tumor growth inhibition in a xenograft mouse model6 recent studies have indicated thatkeap1nrf2 pathway is engaged in sustaining csc cancerstem celllike properties in cancers and causes resistanceto therapeutic agentscscs exhibit enhanced selfrenewal properties lead todisease recurrence and most importantly exhibit thestrongest therapeutic resistance within the tumor cellspopulation7 elevated expression of nrf2 target genescontribute to therapeutic resistance and facilitate survivalof cscs10 several cell surface markers such as cd44cd133 cd24 cd49f and aldh have been proposed forthe detection and isolation of cscs from tumors1112many studies also emphasized the potential use of epithelial cell adhesion molecule epcam as a marker ofcscs due to its ubiquitous overexpression in tumors13epcam was originally identiï¬ed as a novel tumorspeciï¬ccell surface antigen and overexpressed in a large numberof cancers14 and involved in cell migration proliferation and differentiation18 due to its wide expressionepcam may be a potential target for molecular intervention for therapeutically resistant tumors and requiresfurther investigationa recentstudy reported that nrf2 knockdowninhibits the selfrenewal capacity of glioma stemcells19 furthermore nrf2 signaling is activated inspheroids in breast and colon cancer cells where highnrf2 activity in spheroids has correlated with therapeutic resistance20 however it is unknown how thenrf2 pathway and epcam interact and play roles inthe development of chemotherapeutic resistance inview of the importance of epcam and nrf2 signalingin the development of chemoresistance and the limited understanding of the link between epcam andthe nrf2 pathway we investigated the potential role ofnrf2 signaling in cscs with special emphasis onepcamenriched cells that leads to chemotherapeuticresistanceofï¬cial of the cell death differentiation associationresultscancer stem cell markers are upregulated in hpvnegativeand nrf2 overexpressing hnscc tumorsgiven the role of nrf2 signaling in chemotherapeuticresistance and csc survival2122 we ï¬rst explored theexpression of several prominent csc markers in hnsccusing cases from tcga dataset we used normalizedmrna zscores and compared several csc markers withinnrf2high and nrf2low tumors statistically significantdifferences were obtained for all csc markers comparingthe nrf2high and nrf2low groups with the most significant relationship found in epcam p fig 1asince hpv human papillomavirus has emerged as anovel risk factor for hnsccs23 we therefore comparednrf2 expression in hpvpositive and hpvnegative patientgroups from our own archived tumor samples no significant differences were noted between the hpv groupsand nrf2 expression p fig 1b a significantexpression difference was noted in epcam cd49f andstemness factor sox2 p p p fig 1cin hpvnegative versus hpvpositive groups in additioncd44 p cd49fp epcam p and sox2 p showed significantly higherexpressions in the nrf2high group fig 1d csc markersincluding sox2 were significantly increased in the tumortissue compared with matched normal tissues fig 1eepcam is expressed in cisplatin resistant cells and epcaminhibition sensitizes cells to cisplatin and inhibits hnscccell proliferationtumors cisplatin resistantnext we evaluated epcam transcript levels from agroup of cisplatin resistant n and sensitive n hnscc patienttumors showed relatively high expression of epcamcompared with cisplatin sensitive tumors fig2a this ï¬nding led us to test the cisplatin resistancein vitro for which we established a line of cisplatinresistant fadu cells termed as fadures we established acisplatinresistant fadures cells by maintaining parentalfadu cells in a series of cisplatin concentrations for weeks before these cells were stably grown in μmcisplatin as shown in supplementary fig s1a fadurescells exhibited higher resistance to cisplatin treatmentcompared to parental fadu cells we then treated faducells μm of cisplatin for days and analyzed the epcamexpression by western blot fadu cells maintained in μm of cisplatin showed higher epcam expression incontrast to untreated parental cells supplementary figs1b to assess the role of epcam in cisplatin resistanceuntreated patient tumor cells scc15 and fadu cellswere transfected with siepcam and siscramble for hwashed and followed by cisplatin treatment for an additional h and assessed for cell viability supplementary 0cnoman et al cell death and disease page of fig cancer stem cell markers cscs are upregulated in hpvnegativenrf2 overexpressing hnscc tumors a cd44 cd133 epcam andcd49f were compared between nrf2high and low groups using the tcga dataset ratios were calculated by dividing the mrna expression of thenrf2high group by that of the nrf2low group b nrf2 expression was compared between hpv and hpv group using our own dataset n ratio was calculated by dividing the expression intensity of the hpv group by that of the hpv group c cscs expression comparedbetween hpv and hpv group ratio was calculated by dividing the expression intensity of the hpv group by that of the hpv group dexpression of cscs was compared between nrf2high and nrf2low group ratios were calculated by dividing the expression intensity of the nrf2high group by that of the nrf2low group e cancer stem markers were compared between hnscc and matched normal tissues ratio was calculatedby dividing the mrna expression of the tumor sample by that of the matched normal samples in all cases whiskers indicate the maximum and theminimum values pvalues were calculated using students t testfig s2 whereas parental cells were found to be somewhat resistantto cisplatin treatment knockdown ofepcam with siepcam enhanced the sensitivity to cisplatin treatment fig 2b to examine the resistancefurther cells were treated with different concentrations ofcisplatin and determined the ec50 fig 2c furthermoresilencing epcam significantly reduced epcam transcriptlevel and inhibited cell proliferation fig 2d echemotherapeutic resistance is associated with increasednrf2 transcriptional activity and epcam overexpressionit was reported that inhibition of nrf2 reverses theresistance to cisplatin of hnscc cells22 to further assessthe role of nrf2 in chemotherapeutic resistance wecompared nrf2 target genes in cisplatin sensitive n and resistant n hnscc patients tumor cells by realtime quantitative polymerase chain reaction qrtpcrthe unsupervised heat map analysis showed that nrf2target genes sod2 slc3a1 akrc1 gclc ho1nqo1 and sod1 were highly upregulated in thecisplatinresistant tumor cells compared to the cisplatinsensitive tumor counterparts fig 3a suggesting thatcisplatin treatment potentially plays a significant role innrf2 pathway activation to establish the link betweennrf2 and epcam in resistance freshly isolated cisplatinresistant n and sensitive n patient tumor cellswere subjected to ï¬ow cytometry analysis and quantiï¬edthe epcam expression approximately epcampositive cell population was found in cisplatin resistanttumors while onlycella epcampositiveofï¬cial of the cell death differentiation association 0cnoman et al cell death and disease page of fig epcam is expressed in cisplatin resistant cells and epcam inhibition sensitizes hnscc cells to cisplatin and inhibits hnscc cellproliferation a expression of epcam mrna in hnscc patients cisplatin resistant and sensitive tumor cells b doseresponse and cell viability ofhnscc patient tumor cells top panel scc15 middle panel and fadu bottom panel cells cell viability of siepcam and scrambled sirnatransfected cells were monitored following exposures of cells to different concentrations of cisplatin c ec50 of cisplatin in parental siscrambled andsiepcam transfected patient tumor cells top panel scc15 middle panel and fadu bottom panel cells the ec50 differences between siscrambleand siepcam cells were compared d relative epcam mrna expression in hnscc patient tumor cells top panel scc15 middle panel and fadubottom panel cells following transfection of cells by siscrambled and siepcam pvalues were calculated using students t test e cell proliferationwas determined following transfection of cells by siscrambled and siepcam siscrambled and siepcam cell growth was compared on day nsdenote not significant p p p population was detectable in cisplatin sensitive tumorsfig 3b based on these epcam cell fractions in resistantand sensitive groups we hereafter termed these twopopulations epcamhigh and epcamlow immunostainingfor epcam in cisplatin resistance n tissues showedenhanced expression of epcam fig 3c fluorescenceactivated cell sorting facs sorted epcamhigh cellofï¬cial of the cell death differentiation associationfraction was highly resistant to cisplatin compared to theepcamlow cell fraction fig 3d etumorsresistantfunctionallyto cisplatin showedenhanced expression of epcam coupled with theincreased level of sox2 and abcg5 fig 3f indicatingenrichment of epcam coincident with stemlike anddrug resistant features in cells we then analyzed nrf2 0cnoman et al cell death and disease page of fig chemotherapeutic resistance is associated with increased nrf2 transcriptional activity and epcam overexpression a heat map ofhierarchically clustering based on the expression of nrf2 pathway target genes reveals distinct expressions in cisplatin resistant and sensitive patienttumor cells significantly upregulated gene expression intensity marked as red and downregulated genes are marked as blue b hnscc patient tumorcells were isolated from treatment resistant and sensitive patients and epcam positive cells identiï¬ed by ï¬ow cytometry c immunoï¬uorescenceimages of epcam expression were captured from the cultured cisplatin resistant and sensitive hnscc patient tumor cells scale bar µm d cellviability determination in parental epcamhigh and epcamlow cells after treating the cells with different cisplatin concentrations e apoptotic celldetermination in epcamhigh and epcamlow cells after cisplatin µm treatment f epcam sox2 and abcg5 protein levels were determined fromcisplatin resistant and sensitive patient tumor cells by western blotting g transcript levels of epcam and nrf2 in hnscc patient tumor cells assessedby qrtpcr values represents ±sd for three independent experiments hi transcript levels of epcam and nrf2 in scc15 and fadu cells assessed byqrtpcr values represents ±sd for three independent experiments j nrf2 and sod1 protein expression in cisplatintreated scc15 and fadu cellsand epcam transcript levels by qrtpcr and found thatboth transcripts were increased in cisplatinresistanttumor cells fig 3g suggesting that resistance to cisplatin is due in part to an increased level of nrf2 transcriptional activity and epcam overexpression toconï¬rm this ï¬nding in cell lines scc15 and fadu cellswere treated with cisplatin μm for days and wereassessed for the level of nrf2 and epcam transcriptscisplatin treatment significantly increased the nrf2 andepcam expression levels fig 3hj immunoblot analysis conï¬rmed that both nrf2 and sod1 expression werehigher in cisplatin treated cells fig 3jnrf2 pathway is predominantly activated in epcamhighcells and epcam knockdown inactivates the nrf2arepathwaytumorto explore if the nrf2 pathway is exclusively activatedin epcamhigh cells freshly isolated cisplatinresistant andsensitive patientcells were facs sortedepcamhigh cells were predominantly detected in thecisplatinresistant cell fraction compared to sensitive cellsfig 4a cells were treated either with cisplatin or vehiclefor days and analyzed by ï¬ow cytometry the resultscorroborated the results obtained in patient tumor cellsfig 4a next we cultured cisplatin treated fadu cells inofï¬cial of the cell death differentiation association 0cnoman et al cell death and disease page of fig nrf2 pathway is predominantly activated in epcamhigh cells and epcam knockdown inactivates the nrf2are pathway a epcamhighand epcamlow cells were determined in hnscc patient tumor cells scc15 and fadu cells using ï¬ow cytometry b total cellular protein levels ofepcam nrf2 and sod1 were determined in epcamhigh and epcamlow cells by western blot analysis c nrf2 transcript levels in epcamhigh andepcamlow cells p compared to epcamlow group d sod1 nqo1 and akrc1 transcript levels in epcamhigh and epcamlowcells p compared to epcamlow group e fadu cells were transfected with siepcam and scrambled sirna and epcam nrf2 sod1 and sox2 transcript levelswere determined by qrtpcr analysis in epcamhigh cells f g protein levels of epcam nrf2 sod1 and sox2 were determined by western blotanalysis in fadu and scc15 hnscc cells after silencing epcam in epcamhigh cells h a luciferase assay was used to detect reporter gene activity fromares i immunostaining of hnscc cells stained with epcam green nrf2 red and dapi blue after epcam knockdown scale bar μmgrowth supplemented csc medium for days facssorted for epcamhigh and epcamlow cells were recultured in csc medium for an additional daysepcamhigh cells overexpressed epcam nrf2 and sod1proteins and nrf2 transcripts fig 4b c in additionepcamhigh cells overexpressed sod1 nqo1 andakrc1 transcripts fig 4d these results indicated thatthe nrf2 signaling pathway is exclusively activated in theepcamhigh cells to determine whether the elevated nrf2in epcamhigh cells is epcam dependent welevelsilenced epcam expression by siepcam and observedthat epcam nrf2 sod1 and sox2 proteins and transcripts were attenuated in epcamhigh cells fig 4egthese observations prompted us to hypothesize thatepcam might regulate the expression of antioxidantfactors via the nrf2are antioxidant response elementsofï¬cial of the cell death differentiation association 0cnoman et al cell death and disease page of fig nrf2 inhibition in epcamhigh cells sensitizes cells to cisplatin treatment coupled with the abrogation of production of reactiveoxygen species ros a epcamhigh and epcamlow cells were determined in hnscc patient tumor cisplatinresistant and untreated tumor cellsusing ï¬ow cytometry legend 1untreated and 2cisplatin resistant patient tumor cells b protein levels of nrf2 and sod1 were measured insinrf2 silenced and siscrambled epcamhigh cells by western blot c epcam protein was measured in sinrf2 silenced and siscramble cells by westernblot d sox2 and abcg5 protein levels were determined in sinrf2 silenced and siscrambled epcamhigh cells by western blot e cell viability wasanalyzed after incubation of cisplatin for h in sinrf2 and siscrambled epcamhigh cells e f ros activity was measured from e patient tumor cellsand f fadu cells facs sorted epcamhigh and epcamlow cells after cisplatin or sinrf2rna treatment values represent ±sd from triplicate sampledwells p compared with untreated groupspathway this hypothesis was tested by transfection offadu and scc15 cells with an are luciferase reporterhnscc cells with or without epcam knockdown weretransiently transfected with an are luciferase reporterplasmid at h post transfection the cells were assayedfor luciferase activity epcam knockdown decreased theluciferase reporter activity with a comparable decreasedstaining intensity in epcam and nrf2 fig 4h i theseresults suggest that the inhibitory effects of epcamknockdown on cell growth and cisplatin resistance correlates with the degree of nrf2 activation in csclikeepcamhigh cellsnrf2 inhibition in epcamhigh cells sensitizes cells tocisplatin treatment coupled with the abrogation ofproduction of reactive oxygen speciesan increasing number of reports suggest that cisplatinmediated csc enrichment and resulting resistance substantially limits the positive outcome of the disease24furthermore in a group of hnscc patient tumors highexpression of epcam has been reported to correlate withtherapeutic resistance2526 to explore the possible functionallink between chemotherapeutic resistance andepcam we ï¬rst sorted epcamhigh cells by ï¬ow cytometry from cisplatin and vehicletreated fadu cells andfound that higher percentage of epcamhigh cells vs fig 5a in cisplatin treated cells knockdown ofnrf2 in epcamhigh cells attenuated the expression ofnrf2 and nrf2 target gene sod1 proteins fig 5b concomitant with the attenuation in expression of epcamsox2 and abcg5 fig 5c d additionally nrf2 silencingin epcamhigh cells showed a significant increased sensitivity to cisplatin treatment fig 5evarious antioxidant enzymes are induced by nrf2pathway activation that reduce the intracellular ros levelresulting in cells becoming drug resistant27 hence wespeculated that chemotherapeutic resistance might likelybe due to the reduction of ros in epcamhigh cells toaddress this possibility we used two approaches to analyze mitochondrial ros generation first we sortedepcamhigh and epcamlow cells from treatment naivepatient tumor cells and fadu cells treated cells withofï¬cial of the cell death differentiation association 0cnoman et al cell death and disease page of cisplatin and measured the mitochondrial ros using aï¬uorescent indicator ros activity was decreased at and μm cisplatin concentrations in epcamhigh cellswhile increased in epcamlowcells fig 5f suggesting thattherapeutic resistance was partly caused by reducing rossecondly we knocked down nrf2 by sinrf2rna incisplatintreated fadu cells and measured the ros levelros levels steadily increased in both epcamhigh andepcamlow cells fig 5gnrf2 inhibition eliminates colonyforming capacity spheregrowth and invasion capacity in epcamhigh cellswe hypothesize that cells overexpressing epcam mayacquire higher colony forming capacity increased spheregrowth and invasion capacity to test this fadu andscc15 cells were grown in growth factor supplementedcsc medium for days to allow epcam enrichmentfacs sorted and quantiï¬ed for the percent of epcamhighand epcamlow cells sorted cells were evaluated for thedegree of colonyforming capacity sphere formation andinvasiveness epcamhigh populations are highly efï¬cientin forming colonies sphere growth and invasive capacitycompared to epcamlow cells fig 6ac knockdown ofnrf2 in epcamhigh cells demonstrated reduced colonyformation sphere growth and invasive capacity as compared to scramble sirna treated cells fig 6dfinterleukin6 and p62 are involved in the activation of thenrf2 pathway and resistance to cell death in epcamhighcellsaccumulating evidence indicates that both interleukin il6 and the nrf2mediated antioxidant pathwaycontribute to chemotherapeutic resistance in oral squamous cell carcinoma2829 to conï¬rm the role of il6 inthe activation of nrf2 in epcamhigh cells we assessed il mrna transcripts from a group of hnscc patienttumors treated either with chemotherapy cisplatin n doxorubicin n or chemoradiotherapy crt n or tumors obtained after debulking surgery n without treatment il6 mrna was increased in thechemotherapy and chemoradiotherapy tumors comparedwith matched adjacent normal and surgery alone tumorfig 7a to determine the effects ofil6 on theexpression of nrf2 fadu cells were treated with eithercisplatin μm or il6 pgml alone or in a combination of cisplatin and il6 and assessed for nrf2expression by immunoï¬uorescence labeling a detectableincrease in nrf2 expression in the cytoplasm and nucleuswas observed in the cisplatintreated cells fig 7baddition of il6 significantly increased the cytoplasmicand nuclear nrf2 expression fig 7b western blot analysis showed that il6 treatment activated expression ofnrf2 in cisplatin treated cells fig 7c no changes inkeap1 mrna and protein expression levels wereofï¬cial of the cell death differentiation associationobserved fig 7d next we determined whether il6plays role in preventing or reducing ros activity undercisplatin and il6 treatment conditions we found thattreatment with il6 alone reduces ros generation whilecells treated with cisplatin and il6 in combination further reduces the level of ros fig 7e tocilizumab is ahumanized antihuman il6 receptor monoclonal antibody which has been shown to controls resistance toradiation by suppressing oxidative stress via nrf2 pathway28 cisplatintreated cells undergoing il6 and tocilizumab ngml treatment were analyzed by westernblot for the expressions of sod1 and nrf2 il6 alonetreatment enhanced sod1 expression via the nrf2 pathway while tocilizumab inhibited the expression fig 7fin addition il6 treatment significantly reduced the rosproduction while tocilizumab inhibited fig 7g suggesting that il6 is likely involved in the activation of nrf2and plays a role in therapeutic resistance by reducing rosactivityto analyze the possible involvement of p62 in nrf2activation in the chemotherapeutic resistant epcamhighcells p62 protein was analyzed by western blotting p62protein in the epcamhigh cells was increased concomitant with an increase in microtubuleassociatedprotein 1a1b light chainii lc3b fig 7h it appearslikely that the increase in p62 is directly related toepcam expression fig 7h knockdown of epcamdiminished p62 expression suggesting a correlationbetween epcam and p62 fig 7i accordingly epcamsilencing in fadu cells depleted the growth of spheresfig 7i silencing of p62 by p62sirna revealed theinhibition of nrf2 p62 and sod1 fig 7j furthermorep62 knockdown also diminished the efï¬ciency of spheregrowth fig 7j interestingly keap1 expression levelincreased following p62mediated silencing fig 7j theexpression of epcam remained unchanged after p62mediated silencing suggesting epcammediated p62upregulation in these cells fig 7j k the expression levelof lc3b was also reduced during p62mediated silencingfig 7j k silencing of p62 further caused the reductionin expression of nrf2 target genes sod2 ho1 andakrc1 fig 7l all together these results suggested thatnrf2 activation in epcamhigh csclike cells were associated with the increased levels of il6 and p62 inhnscc cellsdiscussionin this study we have shown the role of the nrf2pathway activation because the cellular response to electrophilic agents is partially mediated by this pathway andlikely plays a significant role in therapeutic resistancethrough activation of nrf2 enrichment of cscs andlowering of ros activity we report that increased nrf2activity is associated with the enrichment of cscs and 0cnoman et al cell death and disease page of fig nrf2 inhibition eliminates colony forming capacity sphere growth and invasion capacity in epcamhigh cells a colonyforming assaywas carried out and quantiï¬ed from sorted epcamhigh and epcamlow cell populations b sphereforming efï¬ciency was determined and quantiï¬edusing sphere formation assay from epcamhigh and epcamlow cell populations c invasive potential of epcamhigh and epcamlow cells wasdetermined and quantiï¬ed by transwell invasion assay pvalues were calculated using students t test p p p dfnumbers of soft agar colonies formed d sphere formation e and invasion capacity f were quantiï¬ed in sinrf2rna and siscramble epcamhighcells scale bar μm values represent mean ± sd from three independent experiments p p p pvalues werecalculated using students t testdemonstrated a previously unknown link betweenepcam and the nrf2 pathway a leading cause of chemotherapeutic resistancerecent studies have highlighted the association betweenthe nrf2 pathway and cscs for examplein neuralstemprogenitor cells nrf2 overexpression modulatedofï¬cial of the cell death differentiation association 0cnoman et al cell death and disease page of fig il6 interleukin6 and p62 are involved in the activation of the nrf2 pathway and cell death resistance in epcamhigh cells a realtime pcr analysis of il6 expression in hnscc tumor tissues from matched adjacent normal n untreated surgery only n cisplatintreated n doxorubicin n and chemoradiotherapy n treated tumor tissues transcripts levels were normalized to betaactin b theimmunoï¬uorescence images of cytoplasmic and nuclear nrf2 in fadu cells after 5day cisplatin treatment with or without pgml il6 legend cytoplasmic and 2nuclear nrf2 scale bar μm c nrf2 protein levels after 5day posttreatment with cisplatin or combination of cisplatin and pgml of il6 analyzed by western blotting d keap1 mrna and protein expression in fadu cells 5day posttreatment with cisplatin alone orwith pgml of il6 e ros level was determined in fadu and scc15 cells after treating cells with cisplatin il6 and combination of il6 andcisplatin f after h cisplatin treatment with vehicle or pgml il6 or combination of il6 and ngml tocilizumab cell lysates were subjectedto western blotting and the levels of sod1 and nrf2 proteins were determined g after h cisplatin treatment with vehicle or pgml il6 or il6with ngml tocilizumab the ros production was analyzed h p62 and lc3b were measured in epcamlow and epcamhigh fadu cells by westernblotting i p62 protein was determined in epcamlow and epcamhigh fadu cells following scrambled sirna and epcamsirna transfectionquantiï¬cation and representative images of spheres formed by siscrambled and epcamsirna transfected epcamhigh cells are presented scale bar μm values represent three separate experiments p compared with siscramble group j epcamhigh cells were transfected withscrambled or p62sirna and protein levels of nrf2 p62 keap1 sod1 epcam and lc3b were assessed quantiï¬cation analysis and representativeimages of spheres formed by siscrambled and p62sirna transfected epcamhigh cells are presented scale bar μm values represent threeseparate experiments p compared with sicontrol group k l transcript levels of epcam lc3b sod1 ho1 and akrc1 were determined insiscrambled and p62sirna transfected cells by qrtpcr values represent mean ± sd from three separate experiments p p valuescompared with sicontrol groupneurosphere formation efï¬ciency as well as neural differentiation30 in addition nrf2 knockdown in primaryhuman glioblastoma cells decreased the selfrenewalcapacity of glioma stem cells31 as additional evidencecscschemotherapies and are considered alternative causes ofconventionalare highlyresistanttotumor relapse and aggressiveness cd44 cd133 cd24and aldh activity are frequently used for the detectionand isolation of cscs from tumor tissues and manycancer cell lines epcam has evolved as a potential cscmarker due to its involvement in cell signaling migrationmetastasis and therapeutic resistance the link betweenofï¬cial of the cell death differentiation association 0cnoman et al cell death and disease page of in the clinicalepcam and acquisition of csclike properties is supported by epcam inhibition activation of wntbetacatenin signaling enriched the epcam cell populationwhereas rna interferencebased blockage of epcamattenuated csc activities in cancer cells32 these reportshighlight a critical role for epcam in the development ofcsclike featuressetting epcamexpression is associated with an unfavorable prognosis inbreast cancer33 furthermore low ros levels are correlated with the maintenance of a subpopulation of drugresistant cscs within tumors34 since the mechanisticinsights into the functions of epcam have only beenrecently explored the relationship between epcam andan association with regulation of the nrf2 pathway hasnever been described moreover thus far no studies haveexplored the association between the nrf2 pathway andepcam expression in the context of csclike featuresand drug resistance as a molecular mechanism of differential antioxidant capacity and stress resistance ofcscs we identiï¬ed a direct association between epcamand nrf2 signaling with respect to drug resistance andenrichment of csclike features in hnscc cellsseveral noteworthy ï¬ndings have emerged from ourstudy first epcam was highly expressed in hpvnegative tumors nrf2positive tumors were highly enriched in a epcam cell population and epcam was highlyexpressed in hnscc tumors compared to normalcounterparts these observations suggest a direct association between epcam and the nrf2 pathway in concordance we found that nrf2 and its target genes weresignificantly upregulated in the cisplatinresistant hnscctumors compared to cisplatin sensitive tumors thefunctional implication is that nrf2 activation led to theinduction of stemness and drug resistance features byoverexpressing sox2 and abcg5 proteins in epcamhighcell population while knockdown of epcam by sirnaattenuated the expression of nrf2 sod1 and sox2secon | Colon_Cancer |
" triple negative breast cancer tnbc remains recalcitrant to most targeted therapy approaches however recent clinical studies suggest that inducing tumor damage can render tnbc responsive to immunotherapy we therefore tested a strategy for immune sensitization of murine tnbc 4t1 tumors through combination of focused ultrasound fus thermal ablation and a chemotherapy gemcitabine gem known to attenuate myeloid derived suppressor cells mdscsmethods we applied a sparse scan thermally ablative fus regimen at the tumor site in combination with systemically administered gem we used flow cytometry analysis to investigate the roles of monotherapy and combinatorial therapy in mediating local and systemic immunity we also tested this combination in rag1 mice or t cell depleted wild type mice to determine the essentiality of adaptive immunity further we layered programmed cell death protein pd1 blockade onto this combination to evaluate its impact on tumor outgrowth and survivalresults the immune modulatory effect of fus monotherapy was insufficient to promote a robust t cell response against 4t1 consistent with the dominant mdsc driven immunosuppression evident in this model the combination of fusgem significantly constrained primary tnbc tumor outgrowth and extended overall survival of mice tumor control correlated with increased circulating antigen experienced t cells and was entirely dependent on t cell mediated immunity the ability of fusgem to control primary tumor outgrowth was moderately enhanced by either neoadjuvant or adjuvant treatment with anti pd1 thermally ablative fus in combination with gem restricts primary tumor outgrowth improves survival and enhances immunogenicity in a murine metastatic tnbc model this treatment strategy promises a novel option for potentiating the role of fus in immunotherapy of metastatic tnbc and is worthy of future clinical evaluationtrial registration numbers nct03237572 and nct04116320 metastatic breast cancer brca particularly the triple negative breast cancer tnbc phenotype is resistant to most chemical and molecularly targeted therapeutic approaches interestingly tnbc is often infiltrated with immune cells and the presence of these cells has been shown to have a favorable prognosis in patients treated with neoadjuvant chemotherapy1 early studies in the use of immunotherapies targeting the pd1programmed death ligand pd l1 checkpoint inhibitory axis showed some efficacy2 in tnbc compared with other brca subtypes which are generally recalcitrant to checkpoint blockade activity in the tnbc subtype may be related to the relatively high immune infiltration and correlated with the higher mutational burden observed in tnbc greater immunotherapy efficacy in tnbc has been recently observed with the use of antibodies targeting the pd1pd l1 checkpoint inhibitory axis in combination with nab paclitaxel5 this outcome suggests that inducing tumor damage augments antitumor immunity either by promoting antigen availability or disrupting the immunosuppressive tumor microenvironment tme found in tnbcamong the potential networks in tnbc that could constrain the activity of antitumor immunity is the presence of immunosuppressive myeloid cell subsets these have the capacity to impair adaptive immunity and promote tumor growth and metastasis among these cell types myeloid derived suppressor cells mdscs prevail as a heterogeneous population of immature myeloid cells which serve the eponymous role of suppressing the antitumor immune response limiting both t cell activation and effector functions6 increased levels of this cell type have been demonstrated in tumor tissues of patients with primary brca while those with metastatic disease bear the highest abundance of circulating mdscs8 studies have sheybani a0nd et a0al j immunother cancer 20208e001008 101136jitc2020001008 0copen access shown that approaches that either stimulate myeloid cells with inflammatory mediators or eliminate mdsc can improve antitumor immunity9to this end the central premise put forth in this study is that focused ultrasound fusa safe noninvasive and nonionizing strategy for localized acoustic energy deposition into tissuescan synergize with immunotherapy in a murine model of metastatic tnbc fus is capable of rapidly heating tumors to thermally ablative temperatures its extracorporeal application obviates the need for catheterization injection or implantation fus can be targeted with millimeter precision under mri or ultrasound guidance thereby allowing for thermal damage and destruction of tumor tissue without compromising healthy intervening or peripheral tissues the bioeffects of fus hold distinct implications for tumor antigenicity immune cell activation and trafficking13 thermally active fus regimes have elicited antitumor immune responses in implantable models of melanoma15 pancreatic16 prostate17 colon20 kidney21 and brca23 pertaining to the challenge of myeloid cell immunosuppression in tnbc thermally ablative fus has been shown to induce the expression of heat shock proteins24 and proinflammatory cytokines including interleukin il12 interferonÎ ifnÎ and tumor necrosis factorα tnfα from a variety of cancer cell lines and after in vivo treatment of tumors26 whether the ability of fus to induce these inflammatory mediators is sufficient to overcome myeloid suppression in the context of brca is currently under debate with some studies showing activation of antigen presenting cells and t cell recruitment in patients with brca treated with thermally ablative fus28 while others show that additional innate stimuli are needed to support antitumor immunity23 notably some studies have suggested that a sparse scan thermal ablation regimen more effectively recruits and activates dendritic cells dcs and antitumor immunity than total thermal ablation perhaps by limiting thermal denaturation of tumor antigens and innate stimuli31based on the improved myeloid cell maturation that occurs with sparse scan regimens we herein tested the ability of a sparse scan partial thermal ablation fus regimen as a monotherapy to promote antitumor immunity in an aggressive syngeneic model of metastatic murine tnbc with extensive granulocytic mdsc involvement that is recalcitrant to anti pd1 while some activity is evident with the partial ablation approach significantly greater control was achieved by targeting mdsc inhibition in combination with thermally ablative fus this control was completely dependent on the adaptive immune responsemoreover we demonstrate that layering anti pd1 immune checkpoint blockade onto this combinatorial regimen moderately improves tumor growth restriction these data suggest that in disease settings where myeloid allied approaches to attenuate myeloid immunosuppression may be employed to reveal the full immunotherapeutic immunosuppression predominates potential of thermally ablative fus once immunosuppressive myeloid cells are accounted for fus treatment can promote adaptive immunity that in turn potentiates immune checkpoint blockademethodscell line maintenance4t1 and e0771 cell lines were maintained in rpmi l glut or dulbeccos modified eagles medium dmem gl d glucose l glutamine respectively supplemented with fetal bovine serum fbs at °c and co2 thawed cells were cultured for up to three passages and maintained in logarithmic growth phase for all experiments cells tested negative for mycoplasmaeight week old to week old female balbc or c57bl6 mice were obtained from nci charles river nci crl or the jackson laboratory female balbc rag1 mice were obtained from the jackson laboratory 4t1 or e0771 cells were subcutaneously implanted into the right flank of mice mice were housed on a hour12 hour lightdark cycle and supplied food ad libitum tumor outgrowth was monitored via digital caliper measurements tumor volume was calculated as follows volume lengthwidth22 approximately days 4t1 or days e0771 following tumor implantation mice were randomized into groups in a manner that ensured matching mean starting tumor volume across experimental groupsin vivo ultrasoundguided fus partial thermal ablationmice were treated with fus either days 4t1 cohorts or days e0771 postimplantation on treatment day mice were anesthetized with intraperitoneal injection of ketamine mgkg zoetis and dexdomitor mgkg pfizer in sterilized saline mouse flanks were shaved and depilated following which ultrasound guided fus thermal ablation was performed using one of the two systems system and treatment details are provided in online supplementary materials and methods mice that did not receive fus treatment consistently underwent anesthesia and depilation of the flank additionally these mice underwent a sham treatment consisting of exposure to the °c degassed water bath exposure for min following sham or fus treatment all mice were moved to a heating pad and given antisedan for anesthesia reversal and recoverygemcitabine therapygemcitabine gem mgmouse in µl volume mylan diluted in saline and filter sterilized through a µm syringe filter was administered intraperitoneally once a week on the day of fus treatment following which administration was repeated for an additional weeks administration of gem doses was based on existing literature demonstrating the use of gem for inhibition of mdscs in 4t112 the initial dose of gem was administered immediately prior to sham or fus treatment sheybani a0nd et a0al j immunother cancer 20208e001008 101136jitc2020001008 0cmice that did not receive gem received an intraperitoneal injection of vehicle treatment µl of sterile saline at the time points specifiedpd1 blockade therapyfor checkpoint inhibitor therapy the rat anti mouse pd1 antibody αpd1 rmp114 diluted in sterilized saline was administered intraperitoneally every days for a total of five doses µg per mouse treatment was initiated on day early αpd1 or day delayed αpd1t cell depletionst cell depletion antibodiesanti cd8 clone bio x cell and anti cd4 gk15 clone bio x cellwere diluted in sterilized saline and administered intraperitoneally every to days starting at day days post fus for a total of seven doses µg of each antibody for a total µg per mouseimmunohistochemistryon day sham or fus exposed tumors were excised and fixed in neutral buffered formalin sigma fixed tumors were paraffin embedded sectioned and stained for hematoxylin and eosin digital images of stained slides were acquired using the vectra automated quantitative pathology imaging system akoya biosciences whole slide screening and image capture were subsequently performed using phenochart akoya biosciencesflow cytometrymice were bled at days and via tail vein and samples were rbc lysed hybri max sigma and stained for flow cytometry analysis at days post tumor implantation tissues were obtained from euthanized tumor bearing animals for immune response assessment in order to gain resolution into tissue resident versus vascular immune cell populations mice were injected intravenously with rat anti mouse cd45 fitc clone f11 bd biosciences min prior to euthanasia 4t1 tumors spleens cardiac blood axillary and brachial tumor draining lymph nodes tumor dlns pooled and nondraining inguinal lymph nodes were harvested processed and stained for flow cytometry analysis additional details are provided in online supplementary materials and methodssamples were acquired on an attune nxt flow cytometer thermofisher scientific and data were analyzed with flowjo treestar or fcs express de novo software a representative gating strategy for granulocytic myeloid derived suppressor cell g mdsc and cd44 t cells is provided in online supplementary figure statistical analysisall statistical analyses were performed in graphpad prism graphpad software a detailed description of statistical methods for each experiment is provided in the corresponding figure legendopen accessanimal study approvalall animal work was performed under a protocol approved by the animal care and use committee at the university of virginia and conformed to the national institutes of health guidelines for the use of animals in researchresultspartial thermal ablation of established tnbc tumors promotes peripheral dc activation but has limited impact on the presence of t cells and other myeloid cell subsetsto achieve partial thermal ablation of 4t1 tumors we used an ultrasound guided fus system equipped with a single element therapeutic transducer driven at mhz figure 1a online supplementary figure a grid of sonications was overlaid on the ultrasound visible tumor and ablated in a raster pattern under b mode ultrasound guidance figure 1bc the exceptionally small focus of this system rendered a low ablation fraction of total tumor volume immediately following ablation tumors displayed evidence of coagulative necrosis in the ablated zone with surrounding periablative margins figure 1d one week following fus partial thermal ablation tumors and secondary lymphoid organs were excised for immunological characterization by flow cytometry figure 1b fus partial thermal ablation of 4t1 tumors conferred a significant increase fold in the absolute number of cd11c hi dcs within the axillary tumor draining lymph node adln of mice figure 1e while this was accompanied by a nearly threefold elevation in the absolute number of cd86 dcs within the adln figure 1f the percentage of dcs expressing cd86 did not change figure 1g increased numbers of dcsand cd86 dcs in particularsuggest fus is promoting the maturation or trafficking of these cells in the dlns where they could encounter and activate t cells however this did not translate to tumor growth restriction data not shown we also did not observe significant differences in the absolute number of activated t cells in 4t1 tumors figure 1h or dlns data not shown following fus exposure suggesting limitations in the ability of fus activated dc to further drive an antitumor t cell responseimmune profiling by flow cytometry revealed that irrespective of fus exposure of the intratumoral cd45 immune cell population is comprised of cd11b myeloid cells figure 1i similarly approximately of the circulating immune cell population in 4t1 tumor bearing mice is comprised of myeloid cells a striking fold elevation in circulating myeloid burden compared with naive mice online supplementary figure notably ly6g granulocytic myeloid derived suppressor cells g mdscs significantly dominated the immune cell repertoire within 4t1 tumors relative to other myeloid including f480 macrophages ly6c cell subsets monocytic myeloid derived suppressor cells m mdscs and cd11c hi dcs figure 1j fus partial thermal ablation did not significantly alter the absolute number per sheybani a0nd et a0al j immunother cancer 20208e001008 101136jitc2020001008 0copen access figure partial thermal ablation of established tnbc tumors promotes peripheral dc activation but has limited impact on the presence of t cells and other myeloid cell subsets a design overview of a custom ultrasound guided fus system consisting of a mhz single element transducer orthogonally co registered to an mhz linear ultrasound imaging array the tumor bearing flank of each anesthetized mouse was acoustically coupled to ultrasound transducers via degassed water bath maintained at °c sham mice were similarly positioned but did not undergo sonications b schematic illustration of fus partial thermal ablation scheme and study layout for evaluation of immune sequelae in 4t1 tumor bearing mice a grid of sonications was applied in a raster pattern onto the b mode ultrasound visible tumor in total two planes of sonication spaced mm apart were applied to each tumor grid points were spaced mm apart within a single plane one week following thermal ablation tumors and secondary lymphoid organs were excised for sham n6 or fus treated n5 mice and processed for flow cytometry c representative b mode ultrasound images of ectopic 4t1 tumors either before top or during bottom fus exposure sonication grid depicting targets red points is superimposed on b mode image during treatment subsequent to thermal ablation hyperechoic signatures yellow arrow are occasionally observed d representative he staining of either sham 4t1 tumors or those resected immediately following fus partial thermal ablation zoomed insets depict the transition from necrotic to intact tumor tissue within the periablative zone scale bars400 µm and µm on left and right inset respectively e absolute number of cd11c hi dcs in the axillary tumor draining lymph node adln of 4t1 tumor bearing mice p00136 vs sham f absolute number of cd86 cd11c hi dcs in the adln p00063 vs sham g percentage of cd86 subset out of total cd11c hi dcs within adln h absolute number of intratumoral cd44 cd8 and cd44 cd4 t cells and regulatory t cells tregs per gram tumor i percentage of cd11b myeloid cells out of total cd45 immune cells across tumor spleen adln inguinal dln idln and nontumor draining axillary and inguinal lns ndlns p005 vs all other groups irrespective of fus exposure specifically tumor vs spleen p00226 tumor spleen vs all other organs p00001 j absolute number of intratumoral myeloid cells cd11c hi dcs f480 macrophages ly6c monocytic myeloid derived suppressor cells m mdscs ly6g granulocytic myeloid derived suppressor cells g mdscs per gram 4t1 tumor p00001 vs all other cell types irrespective of fus exposure all data represented as mean±sem significance assessed by unpaired t test fh or two way analysis of variance followed by tukey multiple comparison correction ik nsnot significant dcs dendritic cells fus focused ultrasound hifu high intensityfocused ultrasoundsheybani a0nd et a0al j immunother cancer 20208e001008 101136jitc2020001008 0cgram tumor of these myeloid cell subsets these observations led us to formulate the hypothesis that widespread immunosuppressive mechanisms associated with the 4t1 tme must be addressed in order to facilitate the t cell response to fusfus partial thermal ablation in combination with gem constrains primary tnbc tumor outgrowth and extends overall survivalour observation of the overwhelming mdsc burden following 4t1 tumor implantation warranted implementation of an allied therapeutic strategy in order to counter this immunosuppressive barrier to this end we tested a combinatorial paradigm incorporating gem a myelosuppressive chemotherapy demonstrated to inhibit mdscs transiently in the 4t1 model without consequence to t cell phenotype or function12to evaluate the efficacy of fus and gem in combination we used a preclinical ultrasound guided fus system to achieve partial thermal ablation of established 4t1 tumors 14d after tumor implantation average tumor volume of mm3 in combination with the single session of fus thermal ablation we initiated gem therapy mgmouse which was then readministered weekly for a total of three gem doses figure 2a combinatorial therapy synergized to produce significant constraint of 4t1 tumor outgrowth compared with sham and monotherapy groups figure 2bcby termination of treatments at day 4t1 tumors exposed to fusgem combination saw nearly and reductions in average volume compared with sham or gem exposed tumors respectively figure 2b two dimensional tumor projections at day postimplantation saw a nearly fold reduction in area from sham to combinatorial therapy setting figure 2de in a fraction of mice treated with fusgem we observed complete regression of 4t1 tumors although transient figure 2c tumor outgrowth eventually rebounded after termination of treatments 4t1 tumor bearing mice receiving fusgem treatment additionally saw the greatest extension in overall survival with and increases in median survival time compared with sham and gem groups respectively hrs and for fusgem relative to sham and gem groups respectively figure 2f we additionally observed that fusgem significantly constrained outgrowth in a separate c57bl6 metastatic mammary carcinoma model e0771 online supplementary figure to further the clinical relevancy of these findings we applied this combinatorial strategy with the research grade analog of a clinical ultrasound guided fus system theraclion echopulse that is already ce marked for applications in breast fibroadenoma thyroidparathyroid gland and varicose vein ablation and currently in use for multiple clinical trials leveraging fus thermal ablation in combination with cancer immunotherapy we observed that partial thermal ablation using the theraclion visualization and treatment unit mhz in combination open accesswith gem controlled 4t1 tumor outgrowth to a degree comparable with that observed with the custom in house system online supplementary figure these findings lend credence to the notion that the impact of combining gem with fus may be conserved across partial thermal ablation regimens moreover they demonstrate that the efficacy of fus partial thermal ablation in combination with gem can be recapitulated on a system with a larger focus and in line image guidance that is currently in use clinicallycombination of fus partial thermal ablation with gem increases the levels of circulating t cellslymphocytesin particular cd8 and cd4 t cellsplay an important role in responding to tumor antigen and generating a durable antitumor response based on the extended protective effect observed in mice treated with fusgem flow cytometry analysis was performed to evaluate the contribution of t cells in generating systemic and local tumor control we sampled the circulating immune cell repertoire in 4t1 tumor bearing mice via serial tail bleeds days and prior to readministration of gem and a terminal cardiac bleed at the time of spleen harvest day figure 3a combinatorial therapy significantly elevated absolute number of cd8 and cd4 t cells in the circulation at days and figure 3bc and ef moreover a trend threefold to fivefold increase in circulating t cells was noted in the fus group relative to sham figure 3bc and ef from days to systemic cd44 expressing antigen experienced t cell populations both cd8 and cd4 saw a steady significant increase after combinatorial therapy figure 3d and g a similar modest trend was noted for the fus monotherapy group relative to sham and gem figure 3d and g these changes were concordant with a decrease in circulating myeloid cd11b cells in gem recipient groups demonstrating the ability of gem to partially alleviate circulating myeloid burden figure 3hsplenomegaly is a common signature that arises in parallel with the leukemoid reaction to 4t1 tumors that is the expansion of immunosuppressive myeloid cells during tumor progression we observed that combinatorial therapy most significantly reverses splenomegaly online supplementary figure 6ab consistent with this observation immunological characterization of spleens revealed a significant decrease in cd11b myeloid cellsa reduction in fusgem spleens relative to sham or monotherapy figure 3i while there appeared to be a trend toward more cd11b cells in the monotherapy groups compared with the sham this difference was not significant and there was no difference between these groups in terms of absolute cd11b cell numbers within the spleen data not shown the decrease in myeloid cells in the combination treatment group was accompanied by a significant corresponding elevation in lymphocytes in the spleen following fusgem treatment relative to these sham and gem groups combination therapy elevated splenic cd8 t lymphocytes by fold and sheybani a0nd et a0al j immunother cancer 20208e001008 101136jitc2020001008 0copen access figure combination of focused ultrasound fus partial thermal ablation with gemcitabine gem constrains primary triple negative breast cancer outgrowth and extends overall survival a overview of experimental design for evaluation combination of fus with serial gem treatment in murine mammary carcinoma b average 4t1 tumor outgrowth in sham n7 fus monotherapy n5 gem monotherapy n10 and combinatorial fusgem therapy groups n10 data are represented up to select time points corresponding with mouse dropout due to humane endpoints all data represented as mean±sem significance assessed on outgrowth up to day by repeated measures mixed effects model implementing restricted maximum likelihood method followed by tukey multiple comparison correction p005 vs all other groups specifically sham vs fusgem p00001 fus vs fusgem p00001 shamgem vs fusgem p00026 c 4t1 tumor outgrowth from individual mice in sham fus shamgem or fusgem groups data represent outgrowth from initiation of treatments at day up to removal of mouse from study for meeting a humane endpoint d representative images of 4t1 tumors excised at day scale bar1 cm e quantification of 2d tumor areas from images in previous panel f kaplan meier curve depicting overall survival of sham treatment n9 fus monotherapy n6 gem monotherapy n10 and combinatorial fusgem therapy n10 recipient mice significance assessed by log rank mantel cox test p005 vs all other groups specifically sham vs fus p02154 sham vs fusgem p00001 sham vs shamgem p00050 fus vs fusgem p00021 fus vs shamgem p00312 fusgem vs shamgem p00041sheybani a0nd et a0al j immunother cancer 20208e001008 101136jitc2020001008 0copen accessfigure combination of focused ultrasound fus partial thermal ablation with gemcitabine gem increases the levels of circulating t cells a overview of experimental design to understand the impact of fus andor gem treatment on circulating immune cells bc absolute number of circulating cd8 t cells at day b and day c d percentage of circulating cd8 t cells expressing cd44 from days to ef absolute number of circulating cd4 t cells at day e and day f g percentage of circulating cd4 t cells expressing cd44 from days to h percentage of cd11b myeloid cells out of total cd45 immune cell in circulation from days to ik percentage of myeloid cells i cd8 t cells j and cd4 t cells k out of total cd452 immune cells all data represented as mean±sem all data representative of sham n6 fus monotherapy n4 gem monotherapy n9 and combinatorial fusgem therapy n6 groups significance assessed by analysis of variance followed by tukey multiple comparison correction for b c e f or fishers least significant difference lsd without multiple comparisons correction for ik significance for d g and h assessed by repeated measures mixed effects model implementing restricted maximum likelihood method followed by fishers lsd without multiple comparisons correction p005 vs all other groups unless otherwise indicated p001 p0001 vs groups indicatedsheybani a0nd et a0al j immunother cancer 20208e001008 101136jitc2020001008 0copen access fold figure 3j and cd4 t lymphocytes by fold and fold figure 3k these elevations were accompanied by a modest increase in percentage of foxp3 regulatory t cells tregs online supplementary figure 6e additionally increases in percentage of nk and b cells were noted twofold to fivefold online supplementary figure 6cd these findings indicate that combinatorial therapy with fusgem promotes a systemic lymphocyte response that is discrete from the effects of either intervention alone which may account for reduced mortality associated with pulmonary metastasescombinatorial fusgem therapy does not promote robust local antitumor t cell responsesgiven the robust systemic immune signatures within the blood and spleen following fusgem we assayed 4t1 tumors at a time point within the window of tumor growth restriction and subsequent to termination of treatments ie day to interrogate whether tumor control correlates with an increase in the effector functions of the intratumoral t cell response figure 4a approximately hours prior to euthanasia mice received intravenous brefeldin a injection to inhibit cytokine secretion for subsequent intracellular cytokine staining by flow cytometry immune characterization of tumors at days postimplantationthat is days subsequent to final gem administrationrevealed no significant changes in absolute number of antigen experienced cd44 cd8 or cd4 t lymphocytes figure 4bc moreover the polyfunctionality of these t cells as denoted by ifnÎ and granzyme b expression was not significantly altered figure 4de however intratumoral functional changes were noted in the myeloid compartment gem monotherapy modestly increased il 12p40 production by dcs fold but this was not conserved in the combinatorial therapy group figure 4f moreover while fus monotherapy generated a trend in elevated tnfα production by intratumoral g mdscs gem recipient groups saw a significant increase threefold relative to sham figure 4g these findings indicate that changes in the myeloid compartment in response to monotherapy and combination therapy may contribute to tumor control but are unlikely to drive the protective response entirely interestingly intratumoral t cell representation correlates poorly with circulating lymphocytes suggesting a transitory immune response that either cannot be fully characterized at this time point or is hampered by additional modes of immunosuppressionprotection conferred by combination of fus and gem is dependent on adaptive immunitysince our findings revealed no obvious advantage or function of adaptive immunity in the local tme we next investigated the overarching role of the adaptive immune system in protection offered by combinatorial therapy with fusgem to this end we utilized an rag1 model that is deficient in t and b cells to address the hypothesis that mature t andor b cells play a role in the observed response wild type wt or rag1 mice bearing 4t1 tumors were randomized into groups in a manner that preserved similarity in average initial tumor volumes mice were subsequently treated with either gem monotherapy or the combination of fusgem the tumor growth inhibition offered by fusgem was entirely lost in rag1 mice relative to their wt counterparts with average 4t1 tumor volume in rag1 mice being over fivefold higher than that of wt mice on termination of treatments figure 5a of note despite a trend toward loss of protection in rag1 mice tumor outgrowth in response to gem monotherapy did not significantly stratify between wt and rag1 settings figure 5a we also observed a complete loss of fusgem mediated survival benefit over gem monotherapy in the rag1 setting figure 5b while these results demonstrate that an intact adaptive immune response is required for both the overall survival benefit and restriction of primary tumor offered by fusgem therapy they do not delineate the relative roles of t andor b cellsthus to address the hypothesis that the protective effect of fusgem is specifically dependent on cd4 and cd8 t cells we depleted these populations via serial coinjections of cd8 depleting and cd4 depleting antibodies in 4t1 tumor bearing wt mice on a fusgem figure 5c depletions were maintained between day and day and flow cytometry analysis of circulating immune cells at day confirmed that the target t cell populations were effectively depleted in all mice online supplementary figure consistent with the tumor escape observ | 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 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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 |
alcoholic liver disease ald is a chronic alcoholinduced disorder of the liver for which there are few effectivetherapies for severe forms of ald and for those who do not achieve alcohol abstinence in this study we used asystematic drugrepositioning bioinformatics approach querying a large compendium of geneexpression proï¬les toidentify candidate us food and drug administration fdaapproved drugs to treat ald one of the top compoundspredicted to be therapeutic for ald by our approach was dimethyl fumarate dmf an nuclear factor erythroid related factor nrf2 inducer we experimentally validated dmf in liver cells and in vivo our work demonstrates thatdmf is able to significantly upregulate the nrf2 protein level increase nrf2 phosphorylation and promote nrf2nuclear localization in liver cells dmf also reduced the reactive oxygen species ros level lipid peroxidation andferroptosis furthermore dmf treatment could prevent ethanolinduced liver injury in ald mice our results provideevidence that dmf might serve as a therapeutic option for ald in humans and support the use of computationalrepositioning to discover therapeutic options for aldintroductionoxidative stress is implicated in the development ofdiverse liver disorders such as alcoholic liver diseaseald12 ald encompasses a variety of chronic liverdiseasesincluding liver steatosis fatty liver hepatitiscombined with ammation ï¬brosis cirrhosis andultimately hepatocellular carcinoma hcc3 althoughalcohol abstinence is effective for patients with mild aldsteatosis there are few effective therapies for severeforms of ald and for those who do not achieve alcoholabstinence corticosteroid is the only treatment option toimprove the shortterm survival of severe alcoholiccorrespondence yongheng chen yonghenc163com orting liu liuting818126com1department of oncology nhc key laboratory of cancer proteomics statelocal joint engineering laboratory for anticancer drugs national center feriatrics clinical research xiangya hospital central south university changsha hunan china2department of gastroenterology xiangya hospital central south university changsha hunan chinathese authors contributed equally ye zhang shuang zhaoedited by m agostinihepatitis ah patients4 however many of these patientsdo not respond to this treatment and experience severeadverse effects such as infection5 therefore there is anurgent need to develop novel targeted therapeutics totreat severe forms of ald or patients who fail to achievealcohol abstinence the computational repositioning offood and drug administration fdaapproved drugs is apromising and efï¬cient avenue for discovering new uses6given the high costs possible side effects high failurerate and long testing periods for developing new medicines an fdaapproved compound was known to begenerally safe in humans and available for clinical use7 itis possible to identify safe drugs with potentialforrepurposing in other conditions by using computationalstrategies which can eliminate the need for a phase isafety trial and expedite phase ii efï¬cacy trials analysis ofinteractions between genes and fdaapproved drugsallow the pursuit of new indications for treating diseaseswith no fdaapproved pharmacotherapiesrecent advancements in computing and the dramaticexpansion of available highthroughput datasets have 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 0cenabled the development of drug repurposing to identifynovel treatment options for ald thus in this study weaimed to identify a new therapeutic option with potential forrepositioning in ald we used a systematic computationalapproach based on both public geneexpression patterns inald and the interactions between genes and fdaapproved drugs interestingly we identiï¬ed nuclear factorerythroid 2related factor nrf2 as a novel therapeutictarget in ald8 nrf2 is a basic leucine zipper bziptranscription factor that regulates the expression of certainproteins which protect cells against oxidative stress underunstressed conditions nrf2 is kept in the cytoplasm bykelch likeechassociated protein keap1 and cullin3upon oxidative stress nrf2 is phosphorylated at ser40 andreleases from keap1 then translocates into the nucleus inthe nucleus nrf2 forms a heterodimer with one of thesmall maf proteins maff mafg and mafk binds tothe antioxidant response element are in the promoterregions of many antioxidative enzymes and regulates thetranscription of these enzymes such as glutamatecysteineligase catalytic gclc and heme oxygenase1 ho1more surprisingly we found that the fdaapproved nrf2inducer9 dimethyl fumarate dmf which has not previously been described to have a therapeutic associationwith ald was determined to have a strong therapeuticpotential for repositioning in ald we evaluated the efï¬cacy of dmf for ald in liver cells and in vivo using anethanolinduced mouse model concordant with our computational prediction the experimental results demonstratethat dmf is able to significantly ameliorate ethanolinducedliver injury compared to untreated groupsresultscomputational repositioning of fdaapproved drugs foraldto identify efï¬cient therapeutic strategies for patientswith liver diseases we downloaded drug datasets thatcontain both clinical application and animal test fromgene expression omnibus wwwncbinlmnihgovgeogse accession number gse28619 and then weused a bioinformatics approach to testthe drugrepositioning potential of fdaapproved drugs for aldfrom this approach we computed the activity score ofcandidate drugs and compared geneexpression proï¬les inresponse to these drugs in ald then we annotated theknown gene targets of the topscoring candidates andqueried fdaapproved drugbank using gene targets as aninput which displayed an output of a list of chemicalcompounds notably ald cells are known to abnormallyexpress molecules in the antioxidant response pathwaythus we aimed to study one of the ï¬ve topscored candidate genes nrf2 among nrf2compound interactionsthe main use of dmf is previously tested with some success in multiple sclerosis patients with relapsing formsofï¬cial of the cell death differentiation associationfocused on thesuggesting that dmf used in the clinic may affect the aldgeneexpression signature this analysis led us to focus ondrugs targeting molecules fig 1a the majority of knownphysiologic or pharmacological nrf2 inducers are electrophilic molecules that covalently modify by oxidation oralkylation cysteine residues presentin the thiolrichkeap1 protein10 dmf is one of the known nrf2 inducers which has been tested for the treatment of multiplesclerosis and approved in for its drug bioavailabilityand efï¬cacy11 currently mmf has been used to develop asecond generation of nrf2 inducers as prodrugs12therefore wefumarateregulationmechanism of nrf2 in liver disorders the generation oftoxic metabolites by ethanol such as lipidperoxidationproducts contributes to the pathogenesis of alcoholic liverinjury fumarates prevent ros accumulation via the nrf2pathway in liver cells therefore we used an ald mousemodel six mice a group and hepatic ï¬brosis rat modelnine rats a group to examine the role of fumarates in vivohepatic lipid accumulation was distinctively increased inethanolfed rats in order to address the role of dmf inhepatic lipid accumulation we administered ald micewith dmf at mgkgday or mgkgday for daysin order to address the role of dmf in hepatic ï¬brosis weadministered hepatic ï¬brosis rats with dmf at mgkgday or mgkgday for weeks dmf ameliorated thehepatic steatosis induced by ethanol as observed in liversections stained with hematoxylin and eosin he fig 1band supplementary fig s1a at the same time the highlycrosslinked collagen fraction increased significantly during ethanolinduced ï¬brosis progression while collagendeposition was partly reduced under dmf treatment fig1c and supplementary fig s1b to substantiate theï¬nding that dmf increases the activity of nrf2 pathwayto inhibit ald we collected liver sections from normaland ald mice and checked nrf2 and gclc proteinlevels in the mouse model we performed immunohistochemistry ihc and western blotting for nrf2 andgclc results revealed that dmf treatment significantlyincreased nrf2 and gclc protein levels in ald mouseliver when compared to the matched control groups fig1df and supplementary fig s1c ddmf and mmf activate the nrf2 pathway in liver cellsregulator ofnrf2 is an essentialthe antioxidantresponse pathway which promotes the expression of various genes in response to oxidative stress1314 fumaratesprotect neurons and astrocytes against ros damage15 todetermine whether dmf or mmf regulates the nrf2protein level in liver cells we cultured hepg2 and lo2cells under the treatment of μm dmf and mmf fordifferent lengths of time and found that both dmf andmmf increased the protein level of nrf2 in a timedependent manner fig 2a and supplementary fig s2a 0czhang et al cell death and disease page of fig see legend on next pageofï¬cial of the cell death differentiation association 0czhang et al cell death and disease page of see ï¬gure on previous pagefig computational repositioning of food and drug administration fdaapproved drugs for alcoholic liver disease ald a schematicrepresentation of the bioinformatics workï¬ow for the repositioning approach used to identify potential candidate drugs and genes for the treatmentof ald b dimethyl fumarate dmf prevents ethanolinduced hepatic steatosis mice were fed with the control diet or ethanol diet containing vv ethanol respectively followed by treatment with mgkg dmf or mgkg dmf by oral gavage for days tissue sections from the mouseliver were prepared for hematoxylin and eosin he staining scale bars are μm c dmf decreases ethanolinduced hepatic ï¬brosis mice were fedas in b tissue sections from the mouse liver were prepared for collagen staining scale bars are μm d e dmf increases endogenous nrf2 andgclc to activate the nrf2 signaling pathway in the mouse liver immunohistochemical staining of nrf2 and gclc proteins in mouse liver tissuesliver tissue sections from different groups were stained immunohistochemically with antinrf2 antibody d or antigclc antibody e as indicateddata shown are from one mouse from each group scale bars are μm f nrf2 and gclc in mouse liver sections were compared against actb bywestern blotting the statistical analysis of all samples is shownfurther results revealed that the nrf2 protein level wasupregulated with increased dmf and mmf concentrationsfig 2b and supplementary fig s2b phosphorylationserine40 is required for nrf2 activation1617 to conï¬rmthe activation of nrf2 we treated hepg2 or lo2 cellswith dmf and mmf respectively as indicated thendetermined the level of phosphorylated nrf2 protein bywestern blotting results showed that dmf and mmftreatment significantly increased the phosphorylation levelof nrf2 when we adjusted the sample loading to keep thenrf2 level constant fig 2c and supplementary fig s2cindicating that nrf2 was activatedin addition wechecked the protein levels of nrf2regulated genes15 ourdata showed that dmf and mmf treatment promoted theexpression of gclc and ho1 protein levels fig 2a bmoreover nrf2 knockdown dramatically decreasedgclc and ho1 protein upon either normal condition orfumarates treatment fig 2d and supplementary fig s2dcollectively our results demonstrate that fumarates activate the nrf2 pathway in liver cellsonce phosphorylated nrf2 can translocate into thenucleus and activate transcription of various detoxiï¬cation and antioxidant enzymes upon exposure to stresses18to examine whether fumarates regulated nrf2 nuclearlocalization in liver cells we treated hepg2 or lo2 cellswith dmf and mmf at different concentrations for hfig 2e then cells were lysed and subjected to cytosolicand nuclear fraction extraction we found that dmf fig2e left pannel and mmf fig 2e right pannel promotednrf2 nuclear accumulation in a dosedependent mannermoreover we performed immunoï¬uorescence in livercells confocal microscopy data showed that nrf2expression and nuclear localization were enhanced inhepg2 cells upon dmf and mmf treatment fig 2ftaken together our data provide evidence that fumaratesactivate nrf2 and promote its translocation from cytoplasm to the nucleusdmf and mmf reduce the ros level by activating nrf2 inliver cellsthe relative levels of gsh and gssg are associatedwith various disease aging and cell signaling events19ofï¬cial of the cell death differentiation associationto illustrate the potency offumarates as antioxidantagents we performed the reaction to convert total glutathione and the oxidized form gssg to the reducedform gsh then we measured both total glutathioneand gssg in the luminescent reaction scheme with thegsh probe the results showed that dmf and mmfinduced a dosedependent increase of intracellular gshfig 3a b doxorubicin dox an effective anticanceragent can induce the generation of ros which then leadsto oxidative damage of cellular and mitochondrial membranes2223 27dichloroï¬uorescin diacetate dcfhdais a speciï¬c indicator of ros formation24 and has beenused widely as a ï¬uorescence probe in cells2526 confocalmicroscopy data revealed that ros were accumulated inhepg2 cells with the presence of dox while dmf andmmf blocked the doxinduced accumulation of rosfig 3c and supplementary fig s3a then we performedsirna transfection in hepg2 cells to knock down nrf2and observed a significant increase of ros upon doxtreatment even in the presence of dmf and mmf fig3d and supplementary fig s3b moreover we usedmitotracker® red cmxros kit an agent which can bepassively transported through the cell membrane anddirectly gathered on the active mitochondria to test theeffect of fumarates on the mitochondrial ros level wefound a significant reduction of h2o2 or ethanolinducedmitochondrial ros under fumarates treatment fig 3eand supplementary fig s3cthese results suggest aresistant effect of fumarates in response to ros by activating the nrf2 pathwaydmf and mmf reduce rosinduced lipid peroxidation andferroptosis in liver cellsrecent studies showed accumulation of ros can lead tolipid peroxidation and ferroptosis27 therefore we speculated that fumarates regulate rosinduced ferroptosis toexamine ferroptosis in dox or ethanoltreated cells weexamined the levels of hepatic malondialdehyde mdaand nadpnadph content2829 consistent with rosinduced ferroptosis we found that dox or ethanoltreatment significantly increased lipid peroxidation fig4a b and decreased nadph content fig 4c we 0czhang et al cell death and disease page of fig see legend on next pageofï¬cial of the cell death differentiation association 0czhang et al cell death and disease page of see ï¬gure on previous pagefig dimethyl fumarate dmf and mmf activate the nrf2 pathway in liver cells a dmf or mmf treatment increases endogenous nrf2gclc and ho1 protein level in a timedependent manner hepg2 or lo2 cells were either untreated or treated with μm dmf or mmf for differentlengths of time followed by being lysed and subjected to western blotting with the indicated antibodies b dmf or mmf treatment increasesendogenous nrf2 gclc and ho1 protein level in a dosedependent manner hepg2 or lo2 cells were either untreated or treated with dmf ormmf at the indicated concentrations for h actb is shown as a loading control c dmf or mmf increases the nrf2 s40 phosphorylation levelhepg2 or lo2 cells were treated as in b analyzed by western blotting with nrf2 phospho s40 antibody and normalized against nrf2 proteinthe sample loading was adjusted to keep the nrf2 level constant d nrf2 knockdown decreases gclc and ho1 protein levels under normal orfumarates condition hepg2 or lo2 cells were transfected with sinrf2 or negative control nrf2 gclc and ho1 protein levels were determined bywestern blotting e dmf or mmf promotes nrf2 nuclear accumulation after treated with μm dmf left panel or mmf right pannel for hhepg2 or lo2 cells were subjected to cytosolic and nuclear fractionation and nrf2 protein levels were determined by western blotting histone3h3 and αtubulin were used as nuclear and cytoplasmic markers respectively while actb was used as a wholecell lysate maker f hepg2 cells weretreated with dmso μm dmf or μm mmf for h as indicated then paraformaldehyde ï¬xed blocked and processed for immunoï¬uorescencewith dapi blue or antibody against nrf2 green nrf2 staining is shown on the left and the merged nrf2 and dapi on the right bar μm relativenrf2 ï¬uorescence intensity was calculated using imagej software the ratio was quantiï¬ed mean values were calculated from the individualdistributions in ten cells per conditionobserved a decrease of mda levels and a restoration ofnadph when we added fumarates into liver cells pretreated with dox or ethanol fig 4ac more evidencewas obtained when we detected the protein level of gpx4an important ferroptosis regulator which can inhibit cellmembrane phospholipid peroxidation results showedthat compared with dmso treatment gxp4 was substantially decreased under ethanolstimulated conditionindicating a promoting role of ethanol in liver lipid peroxidation and ferroptosis however we observed arestoration of the gxp4 protein level when we addedferrostatin1 an inhibitor of ferroptosis into hepg2 andlo2 cells pretreated with ethanol fig 4d and supplementary fig s4a a similar result was detected in mouseliver primary cells ethanol treatment lead to a significantdecrease offerrostatin1restored gpx4 protein pretreated with ethanol fig 4eand supplementary fig s4b in addition we treated livercells with erastin an inducer of ferroptosis which playsthe opposite role to ferrostatin1 in ferroptosis and foundfumarates led to an accumulation of gxp4 and nrf2protein even in the presence of ethanol or erastin fig 4ef we also detected lipid peroxidation with c11bodipy undecanoic acid by measuring the ï¬uorescenceintensity in red color consistent with our previousresults an increase of ros production was observedunderthe treatment of ethanol and erastin whileferrostatin1 or fumarates can inhibit lipid peroxidationinduced by ethanol supplementary fig s4c suggestinga preventive effect of fumarates in rosinduced lipidperoxidation and ferroptosisendogenous gpx4 whiledmf inhibits ethanolinduced lipid peroxidation andferroptosis in vivothese results strongly suggest that dmf prevents rosinduced liver injury and ferroptosis via activating thenrf2 pathway we therefore studied the role of dmf inrosinduced ferroptosis in mice hepatocytes treated withofï¬cial of the cell death differentiation associationethanol or not compared to the untreated group andferrostatin1 treated group groups treated by ferroptosisinducer erastin and ethanol had smaller ruptured mitochondria fig 5a these cellular morphological featuresare characteristic of ferroptosis however dmf ameliorated the ferroptosis induced by ethanol as observed bytransmission electron microscopymore evidence was obtained when we performed western blotting and ihc compared with the normal micethe protein levels of 4hne which indicated an increasedlipidperoxidationinduced ferroptosis were higher in aldmouse livers while the gpx4 protein level was lower incontrast dmf treatment could block lipidperoxidationinduced ferroptosis by decreasing the protein levels of4hne and increasing the protein levels of gpx4 in vivofig 5bf these data further validate fumarates as inhibitors of the lipidperoxidationinduced ferroptosisdiscussionusing a computational repositioning of existing drugsbased on the publicly available geneexpression data todiscover therapies for ald we inferred that the nrf2inducer dmf could serve as a therapeutic option for aldand performed experimental validations which demonstrated the efï¬cacy of dmf in ameliorating ald in livercells and in the mouse model the precise mechanism ofaction for dmf is unknown but it is known to activatethe nrf2 antioxidant pathway although dmf has notpreviously been suggested as a therapy for ald previousstudy has shown that nrf2 prevents alcoholinducedfulminant liver injury30 in this study we found thatfumarates activate the nrf2 signaling pathway promoting nrf2 phosphorylation and nuclear localization inliver cells nrf2 further activates the transcription ofgenes encoding various detoxiï¬cation and antioxidantenzymes in response to rosoxidative stress is implicated in the development ofdiverse liver disorders such as ald nonalcoholic fatty 0czhang et al cell death and disease page of fig see legend on next pageofï¬cial of the cell death differentiation association 0czhang et al cell death and disease page of see ï¬gure on previous pagefig dimethyl fumarate dmf and mmf reduce the ros level by activating nrf2 in liver cells a b dmf or mmf enhances cellular redoxpotential by increasing gsh level hepg2 cells were treated with or without different concentrations of dmf a or mmf b for h and thenassessed for cellular gsh and gssg levels denotes p ns denotes no signiï¬cance error bars represent mean ± sd for triplicate experiments cfumarates block dox or ethanolinduced ros accumulation hepg2 cells were pretreated with dox or ethanol for h followed by treatment with μm dmf or mmf for another h as indicated cells were loaded with dcfhda μm and incubated for min at °c in the darkfluorescence images were acquired by a confocal microscope bar μm d nrf2 knockdown accumulates ros damage in liver cells either with orwithout fumarates hepg2 cells were transfected with sinrf2 and treated as in c fluorescence images were obtained e fumarates block h2o2 orethanolinduced mitochondrial ros accumulation lo2 cells were pretreated with h2o2 or ethanol for h followed by the treatment with μmdmf or mmf for another h as indicated cells were incubated with mitotracker® red cmxros red at °c in the dark images were acquired byï¬uorescence microscope bar μmliver disease nafld and hcc2 elevated cellularstresses which are induced by alcohol hepatic viruses ordrugs play a vital role in the initiation and progression ofmultiple liver pathologies31 certain stressed conditions can cause the accumulation of cellular rosuncontrolled production of ros results in oxidativestress on tissues and cells and causes lipid peroxidation34the nrf2 antioxidant pathway is a highly conservedsignal transduction pathway that allows cells tissues andans to survive under oxidative stress conditions35 ourstudy showed that fumarates activate the nrf2 signalingpathway reduce the cellular ros level and protect livercells from ethanolinduced oxidative injuryferroptosis is an iron and rosdependent form of celldeath which is characterized by the accumulation of lipidlevels3637 ros accumulationhydroperoxides to lethalcould directly react with unsaturated fatty acids whichmay lead to a destruction of the mitochondrial membrane a massive release of substances promoting apoptosisand increased ferroptosis dysregulation offerroptosis has been implicated in various pathologicalprocesses including cancer neurodegenerative diseasesacute renal failure druginduced hepatotoxicity ischemiareperfusion injury and tcell immunity3839 our studyshowed that fumarates upregulate the protein level ofgpx4 a gshdependent enzyme that reduces lipidhydroperoxides while decrease lipid peroxidation andferroptosis and thus ameliorate ethanolinduced liverinjury in the ald mouse model fig in addition theseï¬ndings support that fumarates could also be effective inother ferroptosisassociated diseasesin recent years drug repurposing has gained more andmore attention for accelerating drug development40given the high costs possible side effects high failure rateand long testing periods for developing new medicines7drug repurposing provides an attractive approach to meetthe need for improved diseases treatment for exampledisulï¬ram an old alcoholaversion drug has emerged as acandidate for treating highrisk breast cancer7 hippeastrine hydrobromide hh which has been used to preventavian uenza h5n1 has become a promising drug forinhibiting zika virus zikvinfection41 topiramate aofï¬cial of the cell death differentiation associationsafe and effective drug for treating neurological diseasesis capable of ameliorating ammatory bowel disease42in this study we demonstrate that computational repositioning of fdaapproved drugs by analyzing publicgeneexpression data can be used to infer drug therapiesfor ald and offer experimental evidence that the nrf2inducer dmf is capable of ameliorating disease pathophysiology in the ald mouse model dmf was alreadyestablished as a safe and effective drug for treating multiple sclerosis43 additional clinical investigation will beneeded to test whether dmf could beneï¬t patients suffering from aldmaterials and methodscell culture and treatmentcell culture was performed as previously described44hepg2 or lo2 cells were cultured in dmemhigh glucose medium hyclone sh3002201 or rpmi mediummodiï¬ed hyclone sh3080901 supplemented with fetal bovine serum gibco penicillin andstreptomycin gibco at °c in a humidiï¬edatmosphere containing co2 for fumarate treatmentcells were ï¬rst cultured in the medium which containedfetal bovine serum then dmf sigmaaldrich and mmf sigmaaldrich of different concentrations were added into the medium the treatmentsto increase cell oxidative stress and ferroptosis wereperformed by adding ethanolsigmaaldrich e7023 mm doxorubicindox solarbio d8740 μm anderasitin selleck s7242 μm to the culture medium for hthen we treated liver cells with fumarates orferrostatin1 sigmaaldric sml0583 μm for another h all the concentrations are ï¬nal concentrations in theculture mediumwestern blottingwestern blotting was performed as previously mentioned4546 hepg2 or lo2 cells were lysed in ripa lysisbufferbeyotime p0013b containing protease andphosphatase inhibitors cell debris was removed by centrifugation while celllysates were boiled for minand centrifuged at °c before loading on or 0czhang et al cell death and disease page of fig see legend on next pageofï¬cial of the cell death differentiation association 0czhang et al cell death and disease page of see ï¬gure on previous pagefig dimethyl fumarate dmf and mmf reduce rosinduced lipid peroxidation and ferroptosis in liver cells a b fumarates obviouslyreverse dox or ethanolinduced lipid peroxidation hepg2 a and lo2 b cells were pretreated with μm dox mm ethanol or μm erastinfor h followed by μm ferrostatin1 or μm fumarates for h thereafter cells were lysed and subjected to lipid peroxidation malondialdehydemda assay c fumarates reverse dox or ethanolinduced ferroptosis lo2 cells were pretreated with μm dox mm ethanol or μm erastinfor h followed by μm ferrostatin1 or μm fumarates for h thereafter cells were lysed and subjected to nadpnadph assay denotesp denotes p and ns denotes no signiï¬cance error bars represent mean ± sd for triplicate experiments df fumarates block lipidperoxidation and ferroptosis in liver cells hepg2 cells lo2 cells d f or mouse liver primary cells e were pretreated with mm ethanol or μmerastin for h as indicated followed by μm ferrostatin1 or μm fumarates for another h thereafter cells were lysed and subjected to westernblotting for nrf2 and gxp4 with actb as loading control the statistical analysis of all samples is shown fsdspage gels then proteins were transferred ontopvdf membranes merck millipore ltd ipvh00010 forwestern blotting analysis the primary antibodies tophosphors40 nrf2 abcam ab76026 workingdilution nrf2 proteintech 163961ap working dilution gclc proteintech 126011ap working dilution ho1 proteintech 107011ap working dilution αtubulin proteintech 660311lg working dilution gxp4 abcam ab125066 working dilution histone3 proteintech 1ap working dilution actbβactin proteintech 205361ap working dilution werecommercially obtainedrna interferenceknocking down of nrf2 was performed by rnainterference following the manufacturers instructions forlipofectamine rnaimax reagent invitrogen the knockdown efï¬ciency was determined by westernblotting synthetic sirna oligo nucleotides were obtainedcommercially from genepharma co ltd list of effectivesequences is as follows sinrf21 ²gguugagacuaccaugguutt3²sinrf22 ²ccagaacacucaguggaautt3²sinrf23 ²gccuguaaguccuggucautt3²negative control ²uucuccgaacgugucacgutt3²cytoplasmic and nuclear extractsfor nrf2 nuclear translocation experiments cells werecultured in the medium which contained fetal bovineserum then dmf and mmf of different concentrationswere added into the medium for h in all 10cmdiameter plates of hepg2 and lo2 cells were lysed andcytosolic and nuclear fractions were separated followingthe protocol provided by the nuclear and cytoplasmicextraction kit manufacturer active motif inc the nuclear pellets were washed three times with phosphate buffered saline containing freshly added proteaseand phosphatase inhibitors the cytosolic supernatantwas centrifuged to remove any nuclear contamination andtransferred to a new tube both the cytosolic and nuclearfractions were boiled separately in sds sample buffer andanalyzed by western blotofï¬cial of the cell death differentiation associationgsh analysishepg2 cells were plated into white and ï¬atbottom well plates and cultured for h at °c then we treatedcells with dmso or fumarates and incubated for another h for ï¬uorescent gsh assay we ï¬rst washed cells withhanks balanced salt solution solarbio h1045500 thendetermined the levels of reduced and oxidized gsh bygshgssg assay kit promega v6611 according to themanufacturers protocol totalrelative luminescenceunits rlu are graphed as means ± sd denotes p ns denotes no signiï¬cance graphed data represents oneof three experimental repeatsmeasurement of cell lipid peroxidation and nadpnadphassayin thefumarates erasitin μm orliver cells were plated into 60mm dishes and culturedfor h at °c the treatments to increase cell lipidperoxidation were performed by adding ethanol mmdoxorubicindox μm and erasitin μm to theculture medium for h then we treated liver cells with orwithoutferrostatin1 μm for another h all the concentrations are ï¬nalconcentrationslipidperoxidation assay and nadpnadph assay we ï¬rstwashed cells with °c precooled phosphate bufferedsaline then determined the levels of cell lipid peroxidation by mda assay kit beyotime s0131 and nadpnadph quantitation colorimetric kit biovision k347according to the manufacturers protocol the totalhepatic mda content and nadpnadph levels aregraphed as means ± sd graphed data represent one ofthree experimental repeatsculture medium forimmunoï¬uorescence staininghepg2 cells were plated into glass bottom cell culturedishes nest and pretreated with or withoutdox for h followed by addition of dmf and mmf intothe medium thereafter cells were ï¬rst ï¬xed with paraformaldehyde biosharp then permeabilized in triton x100 amresco blocked by bovine serum albumin amresco in pbs buffersigmaaldrich p5368 and lastly incubated with theindicated primary nrf2 antibody working dilution 0czhang et al cell death and disease page of fig see legend on next pageofï¬cial of the cell death differentiation association 0czhang et al cell death and disease page of see ï¬gure on previous pagefig dimethyl fumarate dmf inhibits ethanolinduced lipid peroxidation and ferroptosis in vivo a dmf prevents ethanolinducedferroptosis mice were fed as indicated on the ï¬nal day morning the mice were given alcohol liquid gkg or maltodextrin control by gavageand sacriï¬ced after h in addition ferrostatin1 mgkg and erastin mgkg were provided min before | Colon_Cancer |
" autism spectrum disorder asd is a developmental disorder and the effective pharmacologicaltreatments for the core autistic symptoms are currently limited increasing evidence particularly that from clinicalstudies on asd patients suggests a functional link between the gut microbiota and the development of asdhowever the mechanisms linking the gut microbiota with brain dysfunctions gutbrain axis in asd have not yet beenfull elucidated due to its genetic mutations and downregulated expression in patients with asd ephb6 which alsoplays important roles in gut homeostasis is generally considered a candidate gene for asd nonetheless the role andmechanism of ephb6 in regulating the gut microbiota and the development of asd are unclearresults here we found that the deletion of ephb6 induced autismlike behavior and disturbed the gut microbiota inmice more importantly transplantation of the fecal microbiota from ephb6deficient mice resulted in autismlikebehavior in antibiotictreated c57bl6j mice and transplantation of the fecal microbiota from wildtype miceameliorated the autismlike behavior in ephb6deficient mice at the metabolic level the disturbed gut microbiota inephb6deficient mice led to vitamin b6 and dopamine defects at the cellular level the excitationinhibition eibalance in the medial prefrontal cortex was regulated by gut microbiotamediated vitamin b6 in ephb6deficient mices our study uncovers a key role for the gut microbiota in the regulation of autismlike social behavior byvitamin b6 dopamine and the ei balance in ephb6deficient mice and these findings suggest new strategies forunderstanding and treating asdkeywords gut microbiota asd ephb6 vitamin b6 dopamine ei balance correspondence tgaosmueducn lijming3sysueducn ying li and zhengyi luo contributed equally to this work2state key laboratory of an failure research key laboratory of mentalhealth of the ministry of education guangdonghong kongmacao greaterbay area center for brain science and braininspired intelligenceguangdong province key laboratory of psychiatric disorders collaborativeinnovation center for brain science department of neurobiology school ofbasic medical sciences southern medical university guangzhou peoples republic of china1department of pathology sun yatsen memorial hospital sun yatsenuniversity guangzhou peoples republic of 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 0cli microbiome page of autism spectrum disorder asd which affects approximately of the population around the worldis mainly characterized by impaired social interactionand communication and restricted and repetitive behavior although early behavioral and educationalinterferences have shown effective ameliorative roleson autistic symptoms of asd patients the effectivepharmacological therapies for the treatment of coreautistic symptoms remain limited [ ]thataccumulating evidence showsthe gutbrainmicrobiota axis plays a key role in regulating homeostasis of the human body gut microanisms reportedlyparticipate in many neuropsychiatric disorders suchas anxiety disorders depression and epilepsy in most asd patients changes in gut microanismsand serious gastrointestinal problems have been observed [] interestingly several studies have foundthat the gut microbiota play important role in modulating the asdlike phenotypes of mice [] aclinical study showed that microbiota transfer therapycan improve gastrointestinal problems and autisticsymptoms in asd patients aged to years and thisbenefit can last for years [ ] these studiessuggest that the gutbrainmicrobiota axis might havea significantimpact on the development of asdhowever the contribution of the gut microbiota tothe dysregulation of brain function has not been fullyelucidatedephb6 which belongs to the eph family of receptortyrosine kinases is located on chromosome 7q in twostage genome research on susceptibility lociinautism found transcripts mapped to the chromosome 7qregion that are associated with a predisposition to autincluding ephb6 more recent studies haveismsuggested that ephb6 is a candidate asdassociatedgene [] and recent genomic studies have foundthat ephb6 is mutated in some asd patients [ ]most importantly transcriptome analyses have shownthat ephb6 is downregulated in asd patients [ ]although ephb6 plays an important role in regulatingeph receptor signaling networks t cellfunctionsintestinal epithelium and epithelialdevelopment ofhomeostasis []the role and mechanisms ofephb6 involved in regulation of the gut microbiota andasd remain unclearin our study we found that ephb6 is functionallyassociated with asd and regulates autismlike socialbehavior by gut microbiotamediated vitamin b6 anddopamine moreestablished thefunctionallink between dysregulated gut microbiotaand excitationinhibition ei imbalance in the medial prefrontal cortex mpfc a key gutbrain functional axis in ephb6deficient miceimportantly weresultsthe deletion of ephb6 led to autismlike behavior andgut microbial disturbance in micealthough ephb6 has been identified as a candidate geneassociated with asd whether and how ephb6 functions inasd remain unclear to address these unanswered questions we established ephb6knockoutko mice andfound that ephb6 was deleted in different tissues includingthe colon brain lung and spleen in these mice comparedwith ephb6 wildtype wt mice additional file figure s1cd however the brain and body weights thebody length and the daily dietary consumption were similarbetween the two groups of mice despite the deletion ofephb6 additional file figure s1ehthan the wt mice fig 1bpatients with asd often display repetitive stereotypedbehavior and social deficits interestingly we found thatthe ko mice spent more time selfgrooming than thewt mice fig 1a in the marble burying test the komice buried similar marbles as the wt mice additionalfile figure s1j and in the social partition test the komice spent less time sniffing at the partition regardlessof whether a familiar or novel mouse was placed in thecagein the threechambered social approach task both the wt and komice spent similar lengths of time in bilateral chambersduring the first 10min trial which indicated that the experimental environment was normal fig 1c howeverthe ko mice spent a similar length of time in chamberswith an unfamiliar mouse or inanimate object fig 1dand also showed less preference for the social mousestranger over the object than the wt mice fig 1fgif a novel social partner stranger was placed in theempty wire cage the ko mice still spent a similar lengthof time in the two chambers fig 1e and showed lesspreference for the novel mouse over the familiar mousethan the wt mice fig 1h these results sufficientlyconfirmed that the ko mice exhibited abnormal socialinteraction olfactory cues have generally been consideredto be of the utmost importance in communication amongmice [ ] in the olfactory habituationdishabituationtest repeated presentation of cotton swabs saturated withthe same odor resulted in increasingly decreased lengthsof time spent sniffing at cotton swabs and the presentation of cotton swabs saturated with a new odor increasedthe time spent sniffing these findings were obtained withboth the wt and ko mice however the ko miceshowed less interest in cotton swabs saturated with socialodor than the wt mice fig 1i these results indicatedthat the ko mice exhibited communication deficits eventhough their ability to discriminate and habituate differentodors was normalasd is often accompanied by other mental diseasessuch as hyperactivity anxiety and intellectual disabilityin the open field test the ko mice showed the same 0cli microbiome page of fig see legend on next page 0cli microbiome page of see figure on previous pagefig the deletion of ephb6 led to autismlike behavior and gut microbial disturbance in mice a the 8weekold male ko mice spent more time selfgrooming than wt mice n mice for each group b in social partition test ko mice spent less time sniffing the partition than wt mice n mice respectively ch in threechambered social approach task time spent in chambers during different 10min trials ce trajectory diagram duringthe second 10min trial f were showed ko mice showed less preference for the social mouse over the object g and less preference for the novelsocial mouse over the familiar social mouse h than wt mice n mice respectively i in olfactory habituationdishabituation test ko mice spentless time sniffing social odors than wt mice n mice for each group j in elevated plus maze test ko mice spent less time in open arm and moretime in closed arm than wt mice n mice respectively k the intestinal permeability of 8weekold wt and ko mice was detected using fitcdextran n mice for each group l the mrna expressions of tight junction molecules were detected in colon of 8weekold wt and ko mice n mice respectively m the mrna expressions of cytokines were detected in colon of 8weekold wt and ko mice n mice respectively nr 16srrna gene sequencing of gut microbiota of 8weekold wt and ko mice the species richness n and diversity o of gut microbiota were similarwhile the microbial composition p was different between the two groups relative abundance of different bacteria in phylum level was showed qat genus level the relative abundance of mucispirillumn was decreased in ko mice r n mice for each group data shown are mean ± sem ormedian ± iqr twotailed unpaired students t test a ce gh jm mannwhitney test no q r mixed design anova with genotype asindependent factor and stimulitrials as repeatedmeasure factor b i anosim analysis p p p p wt ephb6 mice koephb6 mice fitc fluorescein isothiocyanate statistical values are presented in additional file table s2locomotor activities and spent almost the same time inthe center area as the wt mice additional file figures1kl in the elevatedplusmaze testthe ko micespent less time in the open arm and more time in theclosed arm than the wt mice fig 1j which impliedthat the ko mice displayed anxietylike behavior in themorris water mazethe ko mice exhibited normalspatial learning and memory similarly to the wt miceadditional file figure s1mo collectively the resultsshowed that the deletion of ephb6 in mice resulted inautismlike behavior including stereotyped behavior andsocial deficits accompanied by anxietylike behavior butdid not result in any evidence of intellectual disabilityephephrin signaling reportedly modulates gut epithelial development and homeostasis and it is also generallyaccepted that many asd patients present gastrointestinal gi symptoms [ ] and a changed gut microbiota composition we then questioned whether komice would suffer from gi problems measurement ofthe intestinal permeability by fluorescein isothiocyanatefitcdextran revealed that the intestinal permeabilityof ko mice was significantly increased compared withthat of the wt mice fig 1k accordingly the mrnaexpression of cldn4 a tight junction molecule in thecolon of ko mice was lower than that in the colon ofwt mice fig 1l in addition we found that the colonof the ko mice presented substantially increased mrnaexpression of il1β a proinflammatory factor and decreasedexpression of il6 which exerts an antiinflammatory effectcompared with that of the wt mice fig 1m the gi problems in the ko mice were not accompanied by morphological changes in the distal ileum proximal colon liver orlung additional file figure s1pthe integrity of the intestinal mucosa is important formaintaining the balance of the ecological environmentin the animals gut we then detected the fecal microbialpopulations of mice by 16s rrna gene sequencing nodifferences in the microbial species richness and diversitywere found between the two groups fig 1n o notablya principal coordinates analysis of the braycurtis distanceshowed that the fecal microbiota of the ko mice clustereddifferently from that of the wt mice fig 1p which indicated that the gut microbial composition differed betweenthe two groups at the phylum level the differencesbetween the two groups were caused by a decreased abundance of deferribacteres in the fecal microbiota of the komice fig 1q at the genus level mucispirillum which isa genus belonging to the phylum deferribacteres wasdecreased in the fecal microbiota of the ko mice fig 1rin general our results indicated that the deletion of ephb6in mice resulted in increased intestinal permeability andchanges in the gut microbial compositionmany studies have indicated that gi problems and thebehavioral abnormalities associated with asd always appearin parallel in patients we thus questioned which of thesesymptoms appears first in the ko mice and found that themicrobial species richness and diversity did not differ between the 34weekold wt and ko mice additional file figure s2ab the principal coordinates analysis revealedthat the gut microbiota of 4weekold ko mice clustered differently from that of 4weekold wt mice additional file figure s2d whereas the gut microbiota of 3weekold komice clustered similarly to that of 3weekold wt miceadditional file figure s2c in addition 4weekold butnot 3weekold ko mice showed increased selfgroomingand decreased interest in social odors compared with sameaged wt mice additional file figure s2eh these resultsfurther implied a possible relationship between the abnormalbehavior and gut microbial dysbiosis in mice with deletion ofephb6transplantation of the fecal microbiota from ephb6deficient mice caused autismlike behavior in spf c57bl6j miceasd is generally considered a neurodevelopmental disorder postnatal developmental disorder can also causeautism in patients and the postnatal mutation ofnrxn1 in neurons leads to autismlike behavior in mice 0cli microbiome page of additionally the gut microbiota of asd patientscould induce autismlike behavior in mice thereforeto study the relationship between gut microbial dysbiosisand autismlike behavior in mice with deletion of ephb6we gavaged the fecal microbiota from 8weekold malewt or ko mice to 3weekold spf male c57bl6j micefor week fig 2a three weeks after the gavage offecal microbiota the gut microbial composition of spfc57bl6j mice treated with the fecal microbiota fromthe ko mice differed from that of spf c57bl6j micetreated with the fecal microbiota from the wt micefig 2bd more interestingly c57bl6j mice that weregastrically perfused with the fecal microbiota from theko mice displayed increased selfgrooming fig 2e andpartially decreased social behavior fig 2fi comparedwith the control mice the two groups of mice showedsimilar behaviors in the open field test and elevated plusmaze test additional file figure s3ad furthermorewe orally gavaged the suspending solution offecalmicrobiota from the wt or ko mice to antibioticpretreated spf male c57bl6j mice after pretreatmentwith antibiotics for days 3weekold spf male c57bl6j mice was gavaged orally with the fecal microbiota of8weekold male wt or ko mice for days fig 2japproximately weeks after fecal microbial colonizationwe similarly found that the gut microbiota of spf c57bl6j mice treated with the fecal microbiota from the ko miceclustered differently from that of the control mice fig 2km we subsequently found that c57bl6j mice that weregastrically perfused with the fecal bacteria from the komice showed increased selfgrooming fig 2n and partiallydecreased social behavior fig 2or additionally the twogroups of mice showed similar behaviors in the openfield test and elevatedplusmaze test additional file figure s3eh moreover the fecal microbiota from weekold but not 3weekold ko mice induced increasedselfgrooming and partial social deficits in 3weekold spfc57bl6j micecompared with c57bl6j micegavaged with fecal microbiota from sameaged wtmice additional file figure s4ah collectively thefecal microbiota from ephb6deficient mice caused increased selfgrooming and partially impaired socialbehavior in c57bl6j micewe subsequently questioned whether the gut microbiota continue to play a role in autismlike behavior inadult mice first we orally gavaged a mixture of antibiotics to 6weekold male spf c57bl6j mice for weekand found that this antibiotic treatment greatly disruptedthe gut microbiota and induced decreased selfgroomingand partial social deficits in young adult c57bl6j miceadditional file figure s5ai these results indicatedthat the gut microbiota was related to autismlike behavioreven in adult mice and that different gut microbiota compositions likely contributed to different behaviors such asselfgrooming and social behavior we then gavaged thefecal microbiota from 8weekold male wt or ko micedirectly to 6weekold spf male c57bl6j mice for week and found that the fecal microbiota from ko micealso induced a disturbed gut microbiota increased selfgrooming and partial social deficits in adult c57bl6jmice fig 3ai unexpectedly we also found that metabolites of the gut microbiota from the ko mice inducedpartial social deficits in c57bl6j mice fig 3jm thegut microbiota without metabolites from the ko mice stillcaused partial social deficits in c57bl6j mice additionalfile figure s5jmoverall our results indicated that the gut microbiotaplays an important role in autismlike behavior even inadult micetransplantation of the fecal microbiota from wildtypemice ameliorated autismlike behavior in adult ephb6deficient miceno previous study has focused on the effectiveness ofmicrobiota transplantation in adult asd patients wesubsequently orally gavaged the fecal microbiota from weekold male wt mice to 8weekold ko mice for week a week later we found that the gut microbiota ofthe ko mice gavaged with the fecal microbiota of thewt mice clustered differently from that of the ko micegavaged with sterile pbs fig 4b the phylumlevelanalysis revealed that the relative abundance of deferribacteres was increased in the ko mice gavaged with thefecal microbiota of the wt mice fig 4c at the genuslevel mucispirillum which is a genus belonging to thephylum deferribacteres was also increased in the komice treated with the fecal microbiota of the wt micefig 4da functional analysis revealed that the ko mice exhibited increased social behavior fig 4fi after gavage withthe fecal microbiota from the wt mice and a decreasedtendency of selfgrooming fig 4e these results indicated that gut microbial dysbiosis was responsible forautismlike behavior in mice with deletion of ephb6gut microbiotamediated vitamin b6 homeostasisregulated social behavior in ephb6deficient micebecause the abnormal behaviors were likely due tobrainrelated problems we attempted to determine howthe gut microbiota affected the brain and subsequentlycaused autismlike behavior in ephb6deficient micefirst we attempted to identify the key region of thebrain affected by the dysregulated gut microbiota inmice with deletion of ephb6 and that was responsiblefor the resulting autismlike behavior studies on asdpatients or mouse models have shown that the hippocampus cerebellum and mpfc are implicated in asd[ ] after processed with a threechambered social 0cli microbiome page of fig see legend on next page 0cli microbiome page of see figure on previous pagefig transplantation of the fecal microbiota from ephb6deficient mice caused autismlike behavior in 3weekold spf c57bl6j mice aischematic of the fecal microbiota transplantation a the 3weekold spf male c57bl6j mice were orally gavaged with the fecal microbiotafrom 8weekold male wt or ko mice each contained eight healthy mice from at least three cages for week after weeks the fecalmicrobiota of the treated c57bl6j mice were sequenced bd eight treated c57bl6j mice of each group were selected randomly from at leastthree cages and selfgrooming test e olfactory habituationdishabituation test f threechambered social approach task gi open field testand elevated plus maze test were conducted with an interval of at least days ei n mice respectively jr schematic of the fecalmicrobiota transplantation j the 3weekold spf male c57bl6j mice were orally gavaged with antibiotics ampicillin vancomycin neomycinmetronidazole for days and then orally gavaged with the fecal microbiota from 8weekold male wt or ko mice each contained eight healthymice from at least three cages for another days after days the fecal microbiota of the treated c57bl6j mice were sequenced km sixtreated c57bl6j mice of each group were selected randomly from at least two cages and selfgrooming test n olfactory habituationdishabituation test o threechambered social approach task pr open field test and elevated plus maze test were conducted with an intervalof at least days nr n mice respectively data shown are mean ± sem or median ± iqr twotailed unpaired students t test e h i nq r mannwhitney test b c k l mixed design anova with genotype as independent factor and stimulitrials as repeatedmeasure factor f oanosim analysis d m p wt col or ko col colonized with the fecal microbiota from ephb6 or ephb6 mice abx pretreated withantibiotics ampicillin vancomycin neomycin metronidazole statistical values are presented in additional file table s2approach task the protein expression of cfos in thempfc of the ko mice was significantly higher than thatin the mpfc of the wt mice additional file figures6ac asd is generally considered to be caused by anincreased ratio of synaptic excitation and inhibition andasd children exhibit elevations in the restingstateneuronal activity therefore whether the mpfc ismodulated by the gut microbiota of the ko mice needsto be further investigated because the mpfc tissue wastoo small for some experiments we used pfc tissuefrom mice in our subsequent studythe first question we asked was whether the bacteriacould directly modulate the mpfc undoubtedly we didnot detect any bacterial dna or colonies in the pfctissues of the wt or ko mice additional file figures6de because metabolites of the gut microbiota fromthe ko mice also induced social deficits in c57bl6jmice we hypothesized that some substances that hadbeen affected by gut microbial dysbiosis caused socialdeficits in the ko miceto identify the significantly changed metabolites wedetected the metabolites in the target tissue that is thepfc of the ko mice using nontargeted metabolomicsstrategies surprisinglythe metabolites in the pfcshowed significant differences between the two groupsof mice as demonstrated by orthogonal partialleastsquares discriminant analysis fig 5a a kegg pathwayanalysis identified four pathways that were significantlyenriched in the differentially changed metabolites andthese included the vitamin b6 metabolism pathway dueto the decreased relative abundances of pyridoxaminepm and pyridoxal ²phosphate plp in the pfc ofthe ko mice fig 5bdvitamin b6 in the body is mainly derived from dietand gut bacteria synthesis and is then absorbed in theintestine we then detected the levels of vitamin b6 inthe feces blood and pfc of mice and found that theephb6deficient mice presented increased fecal levels ofpyridoxine pn decreased plasma levels of pm andplp and decreased levels of plp in the pfc fig 5ekone week after gavage the ko mice gavaged with thefecal microbiota from the wt mice exhibited decreasedlevels of pn in feces and tended to show increased levelsof pm in plasma and increased levels of plp in plasmaand the pfc compared with the ko mice gavaged withsterile pbs fig 5ek these results indicated that thegut microbiota regulated the level of vitamin b6 in thefeces blood and pfc of mice probably by regulatingthe absorption of vitamin b6 in intestinewe subsequently supplied vitamin b6 to the ko mice toclarify its effect on autismlike behavior however intragastric supplementation with vitamin b6 did not amelioratethe social deficits in the ko mice additional file figures7ac one hour after the intraperitoneal injection of mgplp the ko mice presented higher levels of plp in plasmafig 6b and increased social behavior fig 6df comparedwith the control mice no changes in selfgroomingfig 6c and social novelty fig 6g were detected inthe ko mice after the injection of plp additionallythe intraperitoneal injection of or mg of plp exerted noeffect on the social behavior of c57bl6j mice fig 6hjmoreover after being fed without vitamin b6 for weeksc57bl6j mice presented lower plasma plp levels anddecreased social behavior fig 6kn conclusively ourresults proved the existence of a relationship between gutmicrobiotamediated defects of vitamin b6 and socialdeficits in ephb6deficient micegut microbiotamediated vitamin b6 homeostasisregulated dopamine in the pfc of ephb6deficient micevitamin b6 as a cofactor has been implicated in morethan biochemical reactions in cellsincluding thebiosynthesis and catabolism of amino acids and neurotransmitters as the most important active substances in the brain we first detected neurotransmittersin the pfc of mice by highperformance liquid chromatography hplc and found similar levels of glutamategammaaminobutyric acid gaba glycine aspartic 0cli microbiome page of fig fecal microbiota transplantation from ephb6deficient mice partially induced social deficits in 6weekold spf c57bl6j mice aischematic of the fecal microbiota transplantation a the 6weekold spf male c57bl6j mice were orally gavaged with fecal microbiota from weekold male wt or ko mice for week after week the fecal microbiota of the treated c57bl6j mice were sequenced bd n mice foreach group and selfgrooming test e olfactory habituationdishabituation test f threechambered social approach test gi were conductedwith an interval of at least days ei n mice respectively jm fecal metabolites from 8weekold male wt and ko mice were orallygavaged to 6weekold spf male c57bl6j mice for week j after week olfactory habituationdishabituation test k and threechamberedsocial approach task l m were conducted with an interval of at least days n mice respectively data shown are mean ± sem ormedian ± iqr twotailed unpaired students t test e h i m mannwhitney test b c mixed design anova with genotype as independentfactor and stimulitrials as repeatedmeasure factor f k anosim analysis d p p wt col or ko col colonized with fecalmicrobiota or fecal metabolites from ephb6 mice or ephb6 mice statistical values are presented in additional file table s2acid serine and glutamine in the wt and ko micegavaged with sterile pbs or the fecal microbiota fromthe wt mice fig 7a b interestingly the pfc of theko mice exhibited decreased dopamine levels and increased 5hydroxytryptamine 5ht levels than that ofthe wt mice fig 7c treatment with the fecal microbiota from the wt mice increased the level of dopaminebut did not affect the level of 5ht in the pfc of theko mice compared with the levels found in the komice gavaged with sterile pbs the levels of noradrenaline epinephrine and dihydroxyphenyl acetic aciddopac did not differ among the three groups of micefig 7c more excitingly the level of dopamine in thepfc of spf c57bl6j mice gavaged with the fecalmicrobiota from the ko mice was lower than that in thepfc of c57bl6j mice gavaged with the fecal microbiotafrom the wt mice additionalfile figure s8aeadditionally the intraperitonealinjection of plp increased the level of dopamine in the pfc of the komice fig 7d and vitamin b6 deficiency decreased thelevel of dopamine in the pfc of spf c57bl6j micefig 7e brieflyindicated that gutthese results 0cli microbiome page of fig transplantation of the fecal microbiota from wildtype mice ameliorated autismlike behavior in adult ephb6deficient mice a schematic ofthe the fecal microbiota transplantation the 8weekold male wt and ko mice were orally gavaged with the fecal microbiota from 8weekoldmale wt mice eight healthy mice from at least three cages or sterile pbs for week after week the fecal microbiota of the treated wt andko mice were sequenced bd and behavioral tests were conducted with an interval of at least days ei bd 16s rrna gene sequencing ofthe fecal microbiota from mice principal coordinates analysis of braycurtis distance b the relative abundance of deferribacteres c at phylumlevel and the relative abundance of mucispirillum d at genus level were showed at phylum level the range of on x axis was used for therelative abundance of p_bacteroidetes p_firmicutes and p_proteobacteria and the range of on x axis was used for other bacteria n mice respectively ei selfgrooming test e olfactory habituationdishabituation test f and threechambered social approach task giwere performed n mice respectively data shown are mean ± sem or median ± iqr oneway anova e h i kruskalwallis testc d mixed design anova with genotype as independent factor and stimulitrials as repeatedmeasure factor f anosim analysis b p p p wt ephb6 mice ko ephb6 mice fmt fecal microbiota transplantation pbs phosphatebuffered saline statisticalvalues are presented in additional file table s2microbiotamediated vitamin b6 homeostasisaffect the level of dopamine in the pfc of micecouldto determine whether the decrease in dopamine contributed to the autismlike behavior of ephb6deficientmice and considering the fast metabolism of dopaminein the brain we injected agonists of dopamine receptorsinto the mpfc of mice the deletion of ephb6 had noeffect on the mrna expression of dopamine receptorsor tyrosine hydroxylase th in the mpfc or ventraltegmental area vta fig 7f we then injected anagonist of dopamine d1 receptor d1r skf38393or dopamine d2 receptor d2r quinpirole into thempfc of mice the results showed that the ko miceexhibited increased social behavior fig 7gj afterinjection with skf38393 compared with the ko miceinjected with artificial cerebrospinalfluid acsfhowever no differences were found between c57bl6j mice injected with acsf and c57bl6j mice 0cli microbiome page of fig gut microbiota regulated vitamin b6 in ephb6deficient mice ad in nontargeted metabolomics analysis the metabolites in pfc of weekold male wt and ko mice were differently clustered by orthogonal partial least squares discriminant analysis a the enriched keggpathways associated with differential metabolites b the relative abundance of pyridoxamine pm c and pyridoxal ²phosphate plp d wereshowed n mice respectively ek the fecal microbiota from 8weekold wt mice or pbs were gavaged to 8weekold wt or ko mice for week e one week later the level of pm f plp g and pyridoxine pn h in feces of mice were detected n mice respectively thelevel of pm and plp in plasma i j n mice respectively and level of plp in pfc k n mice respectively of mice were alsodetected data shown are mean ± sem r bd oneway anova fk p p p wt ephb6 mice ko ephb6mice fmt fecal microbiota transplantation pbs phosphatebuffered saline pfc prefrontal cortex pm pyridoxamine plp pyridoxal ²phosphate pnpyridoxine statistical values are presented in additional file table s2injected with skf38393 fig 7kmin contrastquinpirole did not increase social behavior in the komice additionalfile figure s8fh and the d1rantagonistinc57bl6j mice fig 7nq in short these results indicated that dysregulated gut microbiota and vitaminb6 defect led to autismlike behavior via the d1rmediated pathway in ephb6deficient miceinduced decreased social behaviut microbiota regulated the ei balance in the mpfc ofephb6deficient miceit is generally thought that d1rs modulate ga | Colon_Cancer |
" appendectomy for acute appendicitis aa is considered one of the most common emergencysurgeries however emergency appendectomy accompanied with complex lesions such as extensive abscessformation is not recommended in most cases therefore nonoperative management followed by intervalappendectomy ia for aa has been tried herein we present three aa cases with specific etiology that underwentinterval appendectomycase presentation case a 68yearold man was diagnosed aa with intestinal malrotation and intraabdominalabscesses he initially treated with conservative therapy and underwent laparoscopic ia after detailed preoperativeexaminationcase a 22yearold man had been under treatment for pancolitistype ulcerative colitis uc also bothered byrightlower abdominal pain several times a year the appendix always appeared swollen on every ct taken duringsymptoms he underwent laparoscopic ia pathological finding revealed typical uc histological features in theresected appendix after the surgery he never suffered from terrible right lower abdominal paincase a 69yearold woman complaining a right lower abdominal pain had undergone ct examination whichrevealed aa with appendiceal mass irregular wall thickness of the cecum and mediastinal and paraaortic lymphnode swelling the operation was carried out after conservative therapy the pathological diagnosis revealed brafmutated colorectal carcinoma she had received systematic chemotherapy after the surgery and all metastaticlesions have completely disappeared interval appendectomy provided us with much clearer anatomical information and precise therapeuticstrategies avoiding technical and general operative complications and also induced fast recovery and short lengthof hospital stay interval appendectomy is a reasonable procedure and could be recommended in case of aa withsome different etiologykeywords interval appendectomy malrotation ulcerative colitis appendiceal carcinoma correspondence kasagisurg2medkyushuuacjp1department of surgery national hospital anization fukuoka higashimedical center koga 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 httpcreativecommonslicensesby40 0ckasagi surgical case reports page of appendectomy for aa is considered the most commonemergency surgeries and prompt appendectomy haslong been a standard treatment for aa [] currentlylaparoscopic appendectomy la becomesthe firsttherapeutic choice for aa la is a safe and effectiveprocedure for the treatment of simple appendicitis andthis approach is superior to open appendectomy oa interms of postoperative wound infections analgesia requirement length of hospital stay return to work andoverall recovery [ ]immediate appendectomy is technically demandingwith distorted anatomy adhesive loops of bowel anddifficulty to close the appendiceal stump because of theinflamed tissues then early la for aa with suchconditions may be converted to oa ileocecal resectionor right hemicolectomy therefore nonoperative management followed by iafor aa has been tried in many hospitals initially aamay be managed in an elective nonsurgical manner including intravenous antibiotics and selective percutaneous drainage and then carrying out operation mostlyla [] this management has been performed especially in pediatric patients however the validity ofia is still controversial in adult patients [ ]herein we present three cases of aa with specific etiology who eventually underwent interval appendectomydiscussing beneficial effects of interval appendectomy foraa with different etiologycase presentationcase a 68yearold man complaining a leftlower abdominalpain had visited our hospital ct revealed the cecum located in the left lower side of the abdomen and a swollen blindend structure with intraabdominal abscessesfig 1a finally he was diagnosed as aa with intestinalmalrotation he received conservative treatment withfig a ct findings the cecum was located in the left lower side of the abdomen the swollen appendix was surrounded by intraabdominalabscesses arrow heads b the clinical course of this patient he was discharged at day and underwent an interval appendectomy at day cthe gastrografin enema findings the cecum arrow located in leftlower side of the abdomen d the trocar placement of the intervalappendectomy note the mirror image trocar placement against an ordinal laparoscopic appendectomy e intraoperative findings iliocecalstructures were located in the left lower abdomen 0ckasagi surgical case reports page of intravenous antibiotics which ameliorated his symptomand inflammatory findings fig 1b he left the hospitalat day afterwards we carried out detailed preoperative examination on his outpatient visit the gastrografinenema confirmed that the cecum was located in the leftlower side revealing the presence of intestinal malrotation fig 1c we underwent a laparoscopic ia using amirrorimage trocar placement fig 1d the iliocecalstructures were found at the left side of the abdomenfig 1e he was discharged from the hospital at thepostoperative day case a 22yearold man had been under the treatment forpancolitistype ulcerative colitis uc fig 2a b and hasbeen also bothered by rightlower abdominal pain several times a year the appendix always appeared swollenon every ct taken during symptoms fig 2c on everyonset of symptoms he received intravenous followed byoral antibiotics which always ameliorated his symptomsendoscopy examination revealed that the appendicealorifice was almost normal fig 2d both uc and aamight cause his right lower abdominal pain he wastreated with conservative treatment at first he underwent a laparoscopic ia in order to obtain the accuratediagnosis there were no operative complications andhe left the hospital days after the operation thepathologicalfindings showed not only aa featuresinflammatory cell infiltration but also histological characteristics typical of uc crypt distortion in the resectedappendix fig 2ecase a 69yearold woman complaining a right lower abdominal pain undergone ct examination which revealedruptured aa with appendiceal mass and irregular wallthickness of the cecum fig 3a which made us suspectcolon cancer moreoverthere were mediastinal andparaaortic lymph node swelling suspecting malignantlymphoma or lymph node metastases serum soluble il2r cea and ca199 were uml normal range ngml normal range and uml normal range respectively and those serumfindings made us suspect malignant lymphoma or epithelial neoplasm conservative therapy with intravenousantibiotics for aa was started seven days after the initiation of conservative therapy her general condition andinflammatory signs were significantly improved fig 3bshe underwent ileocolectomy ie extended appendectomy because there were strong suspicions of malignancy for making a pathological diagnosis she left thehospital days after the operation without any complications fig 3b the pathological features concludedpoorly differentiated adenocarcinoma with peripherallymph node metastases fig 3c d the genotype analyses revealed rightside colorectal carcinoma with braffig a b colonoscopic findings continuous ulcerative lesions and erythema were observed c ct fingings ct revealed a swollen appendix onevery symptomatic course arrow heads d colonoscopic findings the appendiceal orifice was almost normal arrow heads e pathologicalexamination the pathological finding revealed inflammatory cells infiltration and crypt distortion in the resected appendix 0ckasagi surgical case reports page of fig a ct findings ct revealed acute appendicitis arrow heads with abdominal abscess and irregular wall thickness of the cecum circle bthe clinical course of this patient she underwent interval operation at day and left the hospital at postoperative day c a macrofinding ofthe resected an the arrow heads indicate the appendix the oval indicates the tumor lesion d pathological examination pathological featuresrevealed poorly differentiated adenocarcinomamutation and liver metastasis lesion appeared later shereceived chemotherapy bevacizumab 5fufolinateoxaliplatin cpt11 months later either mediastinaland paraaortic lymph node swelling and all metastaticlesions had completely disappeared she was kept on acomplete response cr at the final visit year and months after the operationdiscussionthere are still controversies over the efficacy of ia foracute appendicitis [ ] we presented here three interesting cases of aa who underwent ia which eventually proved to be a very effective therapeutic choice sofar there have been case reports with a single case regarding the efficacy of ia the current manuscript contains three cases with different unique etiologies wecan contrast one case with another in order to understand how the managements are different some reportssuggested that the recurrence rate of aa during thewaiting time for ia is and the complication rateof surgery for recurrent aa is as high as however especially in a phlegmon or appendiceal massia may have some advantages for example providing adefinite diagnosis ruling out any underlying malignancyand avoiding unwanted injury to the surrounding tissue[ ] and also the advantage of ia is to performthe operation at a time when the peritonitis has resolvedpotentially resulting in fewer intraoperative andor postoperative complications there are some analysesabout costeffectiveness of ia [ ] however costbenefits of ia remain controversial ia requires onemore additional admissionone for conservative treatment and one for surgical therapy ia might requiremuch more medical resources nevertheless ia providesa lot of benefits accurate preoperative informationavoiding technical and general operative complicationsand improves patients qol fast recovery and shortlength of postoperative hospital stay particularly forcases with some characteristic etiology like we presentedin the current manuscriptinterval operation also 0ckasagi surgical case reports page of provides us with precise therapeutic strategies that leadto well results and prognosis therefore we advocateinterval operation for aa with unique etiologiesin case aa with appendiceal mass was initially treatedwith conservative therapy which enabled further examinations for intestinal malrotation the anatomical information was extremely useful making the operation safe andavoiding technical complications ia seemed to induce hisfast recovery and short length of hospital stay those results seemed to be one of the greatest benefits of iarecent several reports have shown a significant negative association between appendectomy and uc []however the majority of the studies deal with the history of appendectomy before the development of uc[ ] there are a few published or unpublished dataabout the course of uc when appendectomy is performed after uc diagnosis those reports suggested thatthe disease seems to become milder after appendectomy[ ] case underwent ia electively after which henever suffered from terrible right lower abdominal painalthough there were no direct objective testimonies tothe symptomatic improvements such as blood tests andpathological findings in this case ia seemed to ameliorate his uc conditionthere have been considerable studies on appendicealadenocarcinoma in a recent report about primary appendiceal carcinomas with an average age of years old were t3t4 tumors and of them had lymph nodemetastasis more than were poorly differentiated stageiv disease represented of the cases and 5year overall survival was for all stages [ ] such information reinforces the indication of any surgical interventionappendectomyileocolectomy required to treat appendiceal inflammatory mass after conservative treatment in patients with metastatic disease they often have peritoneuminvolvement patients who were submitted to surgicalcytoreduction had a median recurrencefree survival of years and an overall survival of years was achievedwhen patients could undergo a complete cytoreductionunfortunately complete cytoreduction was achieved onlyin of the patients in case t3 tumor was accompanied with peripheral and distant lymph nodes liver metastasis we had undergone a complete cytoreduction afterthe conservative therapy for aa and were able to obtainthe accurate diagnosis without any postoperative complications furthermore in spite of the poor prognosis withbraf gene mutation those therapeutic strategies enabledthe induction of precise early systematic chemotherapyresulting in a well prognosisthe ideal interval is thought to be approximately months needless to say appendectomy can be performed easily once the inflammation abates the interval ofcase was ideal in this regard however the intervals ofcase and case were short in case he had repeatedlysuffered abdominal pains caused by appendicitis whichwere always alleviated by a short course of antibioticsthere were no abscesses around the appendix when anappendectomy may be performed easily such short intervalcan be acceptable in case there were strong suspicionsof malignancy and the appendix was perforated thereforewe performed the ileocolectomy as soon as the inflammation around the appendix somewhat abated in order topromptly start appropriate therapy against malignancyalthough the beneficial role of ia for aa is still controversial there are some advantages for selected aa caseswith specific etiology we present representative three aacases with intestinal malrotation inflammatory bowel disease and colorectal malignancy in these cases interval appendectomy provided us with much clearer anatomicalinformation and precise therapeutic strategies avoidingtechnical and general operative complications moreoverinterval appendectomy also induced postoperative fast recovery and short length of hospital stayinterval appendectomy is a reasonable procedure andcould be recommended in case of acute appendicitis suspiciously having some different etiologyabbreviationsaa acute appendicitis ia interval appendectomy uc ulcerative colitisla laparoscopic appendectomy oa open appendectomy cr complete responseacknowledgementsnone declaredauthors contributionsyk and hu wrote the manuscript yk and yn performed the surgery and etand sk participated in the surgery yk kn ta yn mi and hu participated inthe study design and coordination kt developed histological staining anddiagnosed findings hu and yk performed total anization of writing themanuscript all authors read and approved the final manuscriptfundingthis manuscript was not funded externallyavailability of data and materialsdata sharing is not applicable to this as no datasets were generatedor analyzed during the current studyethics approval and consent to participatethe present study was conducted in accordance with the ethical standardsof our institutionconsent for publicationconsent was obtained from the patient and patients family for thepublication of this case report and accompanying imagescompeting interestswe have no competing interestsauthor details1department of surgery national hospital anization fukuoka higashimedical center koga japan 2department of pathology nationalhospital anization fukuoka higashi medical center koga japan 0ckasagi surgical case reports page of received june accepted august referencesquartey b interval appendectomy in adults a necessary evil j emergtrauma shock ditillo mf dziura jd rabinovici r is it safe to delay appendectomy inadults with acute appendicitis ann surg udgiri n curras e kella vk nagpal k cosgrove j appendicitis is it anemergency am surg frazee rc roberts jw symmonds re snyder sk hendricks jc smith rw a prospective randomized trial comparing open versus laparoscopicappendectomy ann surg guller u hervey s purves h muhlbaier lh peterson ed eubanks s laparoscopic versus open appendectomy outcomes comparison based ona large administrative database ann surg ahmed i deakin d parsons sl appendix mass do we know how to treatit ann r coll surg engl oliak d yamini d udani vm lewis rj arnell t vargas h initialnonoperative management for periappendiceal abscess dis colon rectumskoubokristensen e hvid i the appendiceal mass results of conservativemanagement ann surg hoffmann j lindhard a jensen he appendix mass conservativemanagement without interval appendectomy am j surg adalla sa appendiceal mass interval appendicectomy should not be therule br j clin pract lai hw loong cc chiu jh chau gy wu cw lui wy intervalappendectomy after conservative treatment of an appendiceal mass worldj surg vons c barry c maitre s pautrat k leconte m costaglioli b amoxicillin plus clavulanic acid versus appendicectomy for treatment ofacute uncomplicated appendicitis an open label noninferiorityrandomized controlled trial lancet hori t machimoto t kadokawa y hata t ito t kato s laparoscopicappendectomy for acute appendicitis how to discourage surgeons usinginadequate therapy world j gastroenterol corfield l interval appendicectomy after appendiceal mass or abscess inadults what is best practice surg today andersson re petzold mg nonsurgical treatment of appendiceal abscess orphlegmon a systematic review and metaanalysis ann surg lare s raminder n brandon b richard n interval appendectomy findingthe breaking point for costeffectiveness j am coll surg hung wl che cl chew ww wing yl watchful waiting versus intervalappendectomy for patients who recovered from acute appendicitis with tumorformation a costeffectiveness analysis j chin med assoc gent ae hellier md grace rh swarbrick et coggon d inflammatorybowel disease and domestic hygiene in infancy lancet rutgeerts p dhaens g hiele m geboes k vantrappen g appendectomyprotects against ulcerative colitis gastroenterology duggan ae usmani i neal kr logan rf appendicectomy childhoodhygiene helicobacter pylori status and risk of inflammatory bowel diseasea case control study gut russel mg stockbrugger rw is appendectomy a causative factor inulcerative colitis eur j gastroenterol hepatol ekbom a appendicectomy and childhood hygiene different sides of thesame coin gut benedix f primary appendiceal carcinoma epidemiology surgery andsurvival results of a german multicenter study ejso nitecki ss the natural history of surgically treated primary adenocarcinomaof the appendix ann surg lieu ch systemic chemotherapy and surgical cytoreduction for poorlydifferentiated and signet ring cell adenocarcinomas of the appendix annoncol darwazeh g cunningham sc kowdley gc a systematic review ofperforated appendicitis and phlegmon interval appendectomy or waitandsee am surg publishers notespringer nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations 0c" | 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 |
" medical practice variation in caesarean section rates is the most studied type of practice variation inthe field of obstetrics and gynaecology this has not resulted in increased homogeneity of treatment betweengeographic areas or healthcare providers our study aim was to evaluate whether current study designs on medicalpractice variation of caesarean section rates were optimized to identify the unwarranted share of practice variationand could contribute to the reduction of unwarranted practice variation by meeting criteria for audit and feedbackmethods we searched pubmed embase ebscocinahl and wileycochrane library from inception to march24th studies that compared the rate of caesarean sections between individuals institutions or geographicareas were included study design was assessed on selection procedure of study population data source casemixcorrection patient preference aggregation level of analysis maternal and neonatal outcome and determinantsprofessional and anizational characteristicsresults a total of studies were included most studies measured the caesarean section rate in the entirestudy population instead of using a sample national databases were most often used as information source casemix correction was performed in studies the robson classification was used in of thestudies following its endorsement by the who in the most common levels of aggregation were hospitallevel and grouped hospitals eg private versus public the percentage of studies that assessed therelationship between variation in caesarean section rates and maternal outcome was neonatal outcome determinants professional and anizational characteristics and patient preference s study designs of practice variation in caesarean sections varied considerably raising questions abouttheir appropriateness studies focused on measuring practice variation rather than contributing to the reduction ofunwarranted practice variation future studies should correct for differences in patient characteristics casemix andpatient preference to identify unwarranted practice variation practice variation studies could be used for audit andfeedback if results are presented at lower levels of aggregation and appeal to intrinsic motivation of physicians forexample by including the health effects on mother and childkeywords caesarean section medical practice variation study design characteristics correspondence mdhvinkvunl mdhvinkgmailcom1department health sciences faculty of science talma institute vrijeuniversiteit de boelelaan hv amsterdam the netherlands2department of obstetrics and gynaecology university medical centergroningen groningen 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 0cvink bmc pregnancy and childbirth page of the caesarean section has been the most performed surgical procedure worldwide for many decades it hasbeen extensively studied both to optimize treatment and to understand why deviations from optimal treatment occur this longterm popularity has not resulted in evidence based homogeneoustreatmentbetween geographic areas or healthcare providers [ ]the magnitude of the variation has raised questionsabout regional differences in quality of healthcare especially in countries with similar resources moreover years of study on practice variation shows no trend ofincreasing regional l one worldwide conformity variation in medical practice can be divided in warranted and unwarranted practice variation variationis warranted when it results from variation in need forexample due to varying rates of extreme premature deliveries extremely preterm deliveries are centralized atinstitutions with the highest expertise of neonatal careas it yields the most optimal outcome these institutions may deviate from the national average as theyserve a highrisk populationmedical practice variation is unwarranted if it cannotbe explained by patient characteristics or patient preference [ ] to identify unwarranted practice variationstudies should compare study groups that are comparable in terms of relevant patient characteristics or makethem comparable through careful casemix correction patient preference is important when both modesof delivery vaginal delivery and caesarean section arean acceptable option variation of caesarean sectionrates is desirable to allow for differences in patient preference across healthcare providers and random or unmeasured differences in need of having a caesareansection when a reported rate deviates more from an acceptable range differences may less likely be attributableto differences in patient preference as both over andundertreatment of caesarean sections harms mother andchild it is therefore likely that quality of healthcare formother and child can be improved by reducing unwarranted practice variation of caesarean sections sufficientevidence on risks and benefits of caesarean sections mayhelp to reduce variation higher quality evidence will result in better guidance on the optimal caesarean sectionrate for specific obstetric conditions subsequentlyuptodate clinical guidelines and clinical support systems may facilitate clinicians to implement recent evidence finally shared decision making should beincorporated in daily clinical practice to empower patients to decide what suits them best improving the process of generating evidence implementing clinical guidelines and incorporating shareddecision making requires healthcare professionals tochange their clinical behavior which is complex auditand feedback is a nonclinical intervention that supportschange of clinical behavior literature shows thathealthcare professionals may be encouraged with information relevant to them one example is performancefeedback on a low level of aggregation ie group or individual level another is to tap into the intrinsic motivation to do well for patients evidence shows thatunnecessary caesarean sections cause an increase in maternal death rates and may affect infant health negatively monitoring and reporting mother and child healthmay motivate change to reduce unnecessary caesareansections audit and feedback has been put forwardas a way through which research can contribute to thereduction of practice variation but it is unclearwhether current research designs of studies on medicalpractice variation of caesarean section rates can be usedfor this purposemedical practice variation in caesarean section rateshas been extensively studied studies started as earlyas and the interest for the topic has remainedstrong however unwarranted medical practicevariation of caesarean section rates has not been reduced the objective of this scoping review is to evaluatewhether studies on medical practice variation of caesarean section rates have used an optimal study design toidentify unwarranted practice variation and when identified can also be used for audit and feedback to contributereduction of unwarranted practicevariationto themethodsto evaluate the characteristics of all caesarean sectionvariation studies we opted for a scoping review wefollowed the prisma statement the researchprotocol was not publishedsearch strategywe searched studies that compared caesarean sectionrates between individual healthcare professionals hospitals groups of hospitals or geographic areas a comprehensive search was performed in collaboration with amedical librarian in the bibliographic databases pubmedembase ebscocinahl and wileycochrane libraryfrom inception up to march 24th the followingterms were used including synonyms and closely relatedwords as index terms or freetext words practice patterns caesarean section the freetext term caesarean section was only used in titles the meshtermpractice patterns includes several descriptions of practice variation the search strategy was performed without date orsearchlanguage restriction the full 0cvink bmc pregnancy and childbirth page of strategies for all databases can be found in additional filestudy selectionall studies that reported on any variation in caesareansection rates between individual healthcare professionals hospitals groups of hospitals or geographicareas were included we included any type of study designie crosssectional study designs and both prospective and retrospective longitudinal studiesexclusion criteriawe excluded studies that were not published in englishand studies that did not publish data on variation of caesarean section rates between healthcare professionalshospitals groups of hospitals or geographic areasprocess of study identification and selectiontitles and abstracts were downloaded and entered inendnote version x81 duplicates were removed tworesearchers mv and pdb screened titles and abstractsthe researchers independently decided whether to include the for full text screening disagreementwas resolved by consensus if no consensus could beachieved a third researcher made the decision evdhfull text screening was performed by two researcherswho decided independently whether to include the or not mv and pdb in case of disagreement thethird researcher evdh decided whether the metthe predefined inclusion criteria data on the designcharacteristics of the studies were extracted by one researcher mv these data were randomly crosscheckedby a second researcher pdbdata extractionto describe the variation of caesarean section rates wescored the minimum and maximum caesarean sectionrate when caesarean section rates of multiple yearswere reported the rate of the most recent year was usedas an indicator for the risk of selection bias we scoredthe selection of study population we differentiated between the use of a study sample or the entire studypopulation to estimate the caesarean section rate theuse of a study sample was considered as a high risk ofselection bias if the study lacked a description of thesampling frame the assessment of the caesarean sectionrate of the entire study population was considered as alow risk of selection bias to indicate the risk of information bias we differentiated between the use of electronic patientepf a national database orquestionnaires the use of epf and databases were considered as low risk of bias as the information was collected by attending healthcare professionalsfilesidentification of unwarranted practice variationto identify whether studies distinguished between warranted and unwarranted practice variation we scoredwhether casemix correction was performed and if patient preference was taken into accountno restriction was imposed on the method of casemix correction examples of casemix corrections include calculating an adjusted or expected caesarean section rate reporting stratified odds ratios by patientcharacteristics or using logistic regression to adjust forpatient characteristics we extracted which variableswere used for casemix correction and whether the robson classification was used the latter is the system proposed by the world health anisation who andthe international federation of obstetrics and gynaecology figo to classify caesarean section casemix no restriction was imposed on how patient preferencewas measured measuring patient preference requiresadditional data collection this could be unfeasible forlarge cohort studies unless a truly random sample of sufficient size is used we assessed whether all studies tookpatient preference into account registered the cohortsize and noted how patient preference was measured ifpatient preference was measured we assessed whether asample was used and whether it was randomusefulness for audit and feedbackto evaluate whether the studies were able to providehealthcare professionals feedback on their clinical behavior in order to reduce unwarranted practice variationwe extracted the aggregation level of analysis that wasused and differentiated betweenindividual physiciangroup of physicians hospital hospital category regionor country similarly we scored whether maternal andneonatal outcomes were measured as outcome reporting informs healthcare professionals on their clinicalperformance we extracted all reported variablesin addition we extracted several explanatory factorsthat might contribute to unwarranted practice variationincluding anizational characteristics ie profitstatusor teachingstatus of the hospital and physician characteristics ie physician gender and age furthermore wescored whether studies analysed financial consequencesof unwarranted practice variation of caesarean sectionratesresultsthe process of study identification and selection is schematically presented according to the prisma statementin fig a total of records were identified from pubmed from embase from cochraneand from cinahl after duplication we screened abstracts for eligibility in total s were 0cvink bmc pregnancy and childbirth page of fig prisma flowchartfig average of the reported minimum and maximum caesarean section rate per year is not presented in fig as only studies areonly included until 24th of march 0cvink bmc pregnancy and childbirth page of selected for fulltext screening we excluded studiesthe reasons for exclusion are presented in fig intotal studies met the inclusion criteria and were included the included studies and their design characteristics and reported variation in caesarean section ratesare listed in additional file the included studies were published between and the cohorts that were studied varied between and more than million women most studies analyzed variation of caesarean section rates in the unitedstates studies followed by brazil studies andaustralia studies the number of studies per country are shown in additional file a wide variation incaesarean section rates is reported some subsaharanregions perform caesarean sections in less than ofthe deliveries by contrast the reported caesarean section rate of some municipalities in brazil reached the variation of caesarean section rates did not decrease over time figure shows the average reportedminimum and maximum caesarean section rates peryear the outlier in is one study that reported variation between four hospitals in rio de janeiro in oneprivate hospital more than of the women deliveredby a caesarean section in a similar situation occurred only two studies were included both reportingon variation in indiain studies a sample of the study populationwas used to estimate the caesarean section rate the majority of the studies studies measured themode of delivery of the entire study population bothmethods were used in eight studies and the selection frame was unclear in nine studies an exampleof a study in which both methods were used was theanalysis of variation in caesarean section rates betweencountries some country estimates were based on a sample of their population while others were based on registries ofin the samplebasedstudies studies defined a selection frame designed to select a representative samplethe entire populationthe majority of studies used data from registries suchas national databases studies or electronicpatient files studies questionnaires were usedin studies eg the demographic and healthsurvey dhs that was used in studies suchquestionnaires were sent to a sample of households inorder to collect information on live births of the pastyears in studies multiple data sources wereused or the data source was not describedidentification of unwarranted practice variationcasemix correction was performed in studies the variables that were used for casemix correction areshown in additional file baseline patient characteristics were observed in studies some studies didnot describe patient characteristics per cohort but didperform a correction for maternal or neonatal characteristics many different maternal and obstetric variables were used as baseline characteristic or for casemix correction age parity gestational age birthweight and maternal education level are the characteristics that were used most often morethan half of the variables were only used in one or twostudiesto reduce this heterogeneity and to increase the quality of casemix correction the who in recommended to use the robson classification as the standardfor casemix correction for studies on caesarean sectionrates in the period out of studiesused the robson classification four of these studies did perform additional casemix correction byusing specific patient characteristics the who notesthat the robson classification should especially be usedwhen comparing caesarean section rates between healthcare facilities or within healthcare facilities over time in the period studies compared caesarean section rates between healthcare providers individuallevel andhospital category of which used the robson classification within the same period of the studiesthat compared caesarean section rates between geographic areas used the robson classification thepercentage of studies that corrected for casemix did notchange over time figure shows the number of studiesthat corrected for casemix by yearlevel group of physicians hospitalthe effect of casemix correction is shown in fig of the studies that corrected for casemix studies calculated an adjusted caesarean sectionrate figure shows these rates per study categorizedper aggregation level the remaining studies calculated an expected caesarean section rate reportedstratified odds ratios by patient characteristics or usedlogistic regression to adjust for patient characteristicsat provider level individual physician group of physicians and hospital level of the studies and at geographic level regional or national of the studiescorrected for casemix figure shows that at the provider level casemix correction had a substantial impacton the provider caesarean section rate at the geographic level the impact of casemix correction wascomparatively smallsix studies took patient preference into accountthese studies did not assess variation of caesarean section rates for a specific obstetric condition for instancepatients with a history of caesarean section in which acaesarean section and vaginal delivery are both an acceptable option all studies measured patientpreference by questionnairesthat were handed tomothers that gave birth between one day and years 0cvink bmc pregnancy and childbirth page of fig number of studies per year with and without casemix correction is not presented in fig as only studies are only included until24th of march rates was least often studied at the level of the individualphysician and group of physicians no clear timetrend was observed in the choice of aggregation level ortrend in observed variation based on the level of aggregation the largest variation in caesarean section rates is reported on both the lowest ie individual level and thehighest ie international level of aggregationneonatal outcomes were captured in andmaternal outcomes in of the studies all variables that were used to measure these outcomes arelisted in additional file many different variables wereused to measure neonatal and maternal outcomes neonatal mortality apgar score maternal mortalityandhaemorrhage are outcomes that were measured mostoften half of the outcome variables were used in justone single study table shows the numbers of studiesper aggregation level that took neonatal and maternaloutcome into account neonatal and maternal outcomeswere most often reported if variation of caesarean nicu admissionprior to the survey patient preference was assessed byposing a variety of questions on the reason of the caesarean section and perceived influence on decision makingno studies reported the quality of decision making in of the studies a sample of the study populationwas used to measure patient preference samples variedbetween and women and three studies used apredefined sampling frameusefulness for audit and feedbacktable shows the study characteristics by aggregation levelthe majority of the studies used one aggregationlevel a minority used two and only three studies used three aggregation levels healthcare providersindividual physicians group of physicians hospitals or hospital categories were compared in studies andgeographic areas national or regional in studies hospitals and grouped hospitals eg private vspublic hospitals were most often used as aggregation levelof analysis medical practice variation of caesarean sectionfig the effect of casemix correction on different aggregation levels 0cvink bmc pregnancy and childbirth page of table study characteristics by aggregation levelnumber of studiesaverage cohort sizemedian cohort sizeentire population measured instead of samplereported variation in caesarean section rate average ofstudiesreported variation in caesarean section rate median ofstudiescasemix correctionmedical outcomeindividuallevelgroup ofphysicians hospitallevelhospitalcategoryregionnational sections was studied atphysiciansthe level ofthe individualdeterminants and financial consequencesa limited number of studies explored determinants toexplain medical practice variation of caesarean sectionrates in an additional analysis hospital characteristics orphysician characteristics were used in studies to explain differences in caesarean section rates thevariables that were used are listed in additional file financial consequences of unwarranted variation of caesarean section rates were calculated in six studiesdiscussionalmost four decades have past and studies werepublished following opits first report on variation ofcaesarean section rates between geographic areas inaustralia clearly the issue raised by the first studies hasnot lost its sense of urgency among researchers nor forthe funders of research because the magnitude of unwarranted variation was considered problematic andremained stable over time while previous reviewsnarrowed their focus on measuring variation betweengeographic areas [ ] or studied the difference between public and private hospitals the focus of thisreview was on the presence of study characteristics thatmay help to reduce unwarranted variation of caesareansection ratesstrengths and limitationsa strength of our review was the systematic search andselection procedure which allowed us to identify almost all studies on medical practice variation that compared caesarean section rates this resulted in a largenumber of included studies a second strength is thehigh level of detail of our analysis the selection of theindividual variables that were used for casemix correction outcomes or determinants enabled us to present anindepth overview of study characteristicsseverallimitations should be addressed first weaimed to describe study characteristics of all relevantpublished studies ie irrespective of the quality in theenglish language and therefore did not perform a qualityassessment of the included studies second we were unable to retrieve all publications that were selected forfulltext screening in order to limit the number of missing s we contacted the authors of missing sby email or through researchgate however this wasnot successful for of the studiesinterpretationfirst we appraised whether studies were designed to distinguish between warranted and unwarranted practicevariation by performing casemix correction and analysing patient preference casemix correction is an essential aspect of the quality of studies on practice variationbecause without casemix correction it remains unclearwhat share of the variation is attributable to health differences between populations and thus to what extentthe variation is warranted our results show that only of the studies that compared caesarean section ratesbetween healthcare providers performed casemix correction casemix correction was performed by calculating adjusted rates expected rates stratified odds ratiosor logistic regression analysis over time we observed noimprovement in the performance of casemix correctionpatient preference another important aspect to identify unwarranted practice variation was only measured insix studies without the assessment of patient preference it remains unclear whether practice variation canbe explained by differences in the outcome of shared decision making this is especially important when bothmodes of delivery vaginal delivery and caesarean section are an acceptable option patients with a historyof caesarean section breech or twin delivery are examples in which information on patient preference is necessary to differentiate between warranted and unwarrantedmedical practice variation [] our results show 0cvink bmc pregnancy and childbirth page of that none of studies that assessed variation in these specific obstetric situations patient preference was analysedto improve the identification of unwarranted practicevariation future studies should not only measure patientpreference but should focus on the implementation ofshared decision making recent literature shows thatseveral shared decision making measures are availablewhich could be included in the study design of medicalpractice variation studies to improve comparability both within healthcare facilities and between them robson proposed what latercame to be known as the robson classification systemfor assessing monitoring and comparing caesarean section rates in the who and the figo jointly endorsed this classification as the international standardfor casemix correction [ ] our data show that following the publication of this guideline by the who in the robson classification was only used in of all studies comparing providers and in studies comparing regions literature shows that casemixcorrection can be improved even more if additional patient characteristics are considered only of thestudies that were published between and andused the robson classification did perform additionalcasemix correction ie age adjusted caesarean sectionrates within robson groups studies that performedcasemix correction with or without robson classification used a wide variation of maternal and obstetriccharacteristics to identify unwarranted practice variation we advise to at least use the robson classificationand to standardise variables for additional casemix correction a delphi procedure can help obstetricians andmidwives to reach consensus on which variables to use the necessity to which casemix correction is neededdepends on the level of aggregation the lower the levelof aggregation the more case mix correction contributesto a valid description of clinical performance health care providers often operate in a network andtreat a subset of the entire population that subset ismore likely to differ between providers as they get morespecialized and the casemix differences may justify differences in caesarean section rates this requiresthat studies aimed at lower levels of aggregation placemore emphasis on casemix correction reporting standards and appropriate small number statistics once casemix has been appropriately controlled forfuture studies should aim to decompose regional unwarranted variation into lower levels of aggregation thisdecomposition allows regional variation to be attributedto health care providers however disaggregation ofcontributions to lower levels of aggregation is not without major risk on its own as groups of physicians getsmaller their clientbase may diverge more from theregional averagefor example due to specializationreporting differences or chance within providers eghospitals further disaggregation to the physician levelcould help identify individual contributions while thelower number of observations may harm both validityand reliability they may also provide a stronger stimulusfor change if the information is interpreted intelligentlyand presented in a motivating environmentstimulus for change might be further enhanced whenoutcome reporting becomes integrated in future studydesigns healthcare professionals are intrinsically motivated to deliver the best healthcare for their patients reporting outcomes in casemix corrected feedbackinformation directly stimulates the intrinsic motivationto improve outcome for patients if it becomes visiblethat increased caesarean section rates do not yield improved maternal and neonatal outcomes professionalsmight be encouraged to adapt clinical behavior our results show that the wide variation in outcome variablesdemands consensus and standardization studies will become more comparable and better interpretable when astandard set of outcomes is used for maternal infectiousmorbidity outcomes after caesarean delivery a core outcome set cos is developed we encourage to develop a cos for neonatal outcomes after caesareansectionforty years of research on caesarean section rates havebeen unable to reduce unwarranted practice variationour study shows that most studies do not meet the criteria to identify unwarranted practice variation and cannot be used for audit and feedback to contribute to thereduction of unwarranted practice variation future studies should correct for differences in patient characteristics and patient preference present results at low levelsof aggregation and appeal to intrinsic motivation by including the health effects on mother and childsupplementary informationsupplementary information accompanies this paper at httpsdoi101186s12884020031693additional file search strategies additional file contains the searchstrategies that were used for the databases pubmed embase ebscocinahl and wileycochrane libraryadditional file list of included studies additional file contains a listof all studies that were included in this scoping review per study theresults are summarized we used the following definitions for theindependent variables used in the table year year of publication author first author of study included title title of study included study period years from which caesarean section rates were reported inexample if a researcher performed a questionnaire survey in andincluded deliveries from y prior to the survey we reported study period caesarean section rate unadjusted caesarean section rate ofmost recent year reported cohort size the cohort size from which thecaesarean section rate is calculated if caesareans section rates from 0cvink bmc pregnancy and childbirth page of multiple years were reported we noted specifically the cohort size of thecohort that was used to calculate the most recent caesarean section rate data source data source that was used by the authors to calculate thereported caesarean section rate it is reported as other if data source isunknown or multiple data sources are used casemix correction thestudy reported an adjusted caesarean section rate expected caesareansection rate reported stratified odds ratios by patient characteristics orused logistic regression to adjust for patient characteristics yn aggregation level aggregation level of analysis outcome outcomes maternal or neonatal were noted yn determinants anisational orphysician characteristics were used to explain reported difference in caesarean section rates between healthcare professionals hospitals groupsof hospitals or geographic areas ynadditional file studies per country additional file describes thenumber of studies on medical practice variation of caesarean sectionrates that were conducted in each count | 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 |
"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 |
"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 |
"purpose squamous cell carcinomas and adenocarcinomas are the most common types of cervical cancercompared to squamous cell carcinomas adenocarcinomas are more common in younger women and have apoorer prognosis yet so far no useful biomarkers have been developed for these two types of cancer in thefollowing study we examined the combination of cytokeratin p63 p40 and muc5ac for distinguishingsquamous cell carcinoma scc from adenocarcinoma of the cervix aecmaterials and methods a total of scc and aec were collected immunohistochemical analyses wereconducted to determine the expression of ck56 p63 p40 ck7 and muc5ac one pathologist who was blinded tothe patients clinical and pathological data interpreted the staining resultsresults muc5ac and ck7 were detected in and of aec cases compared to and of scccases p the specificity of muc5ac was higher than that of ck7 in aec p the sensitivity of muc5accombined with p40 or p63 was similar to that of ck7 but the specificity was slightly higher than that of ck7 inaec moreover the expression of muc5ac was correlated with the degree of tumor differentiation inadenocarcinomas p and was not related to the prognosis of cervical adenocarcinoma and subtypess muc5ac may be useful as a biomarker for differential diagnoses between squamous carcinoma andadenocarcinoma of the cervixkeywords cervical adenocarcinoma cervical squamous cell carcinoma muc5ac ck7 correspondence xiaofangzhangsdueducn hailing li and xiaotong jing contributed equally to this work2department of pathology school of basic medical science shandonguniversity jinan shandong p r china5department of pathology school of basic medical science shandonguniversity jinan shandong p r 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 0cli diagnostic pathology page of thelastintroductioncervical cancer is the fourth most common carcinomain women responsible for of cancerrelateddeaths worldwide [ ] squamous carcinoma is themost common type of cervical carcinoma followed byadenocarcinoma nevertheless overthreedecades a significant increase in adenocarcinoma caseshas been observed in many developed countries especiallyin younger women papsmear screening also knownas pap test is still considered the main screening methodfor cervical cancer especially for squamous carcinoma compared to squamous carcinoma the adenocarcinomaof the cervix is more common in younger women and hasa poorer prognosis therapeutic approaches includechemoradiotherapy ccrt which has been proven tobe effective for squamous carcinoma of the cervix but notfor adenocarcinoma of the cervix due to its highchemo and radioresistance therefore differentiatingadenocarcinoma from squamous carcinoma is importantin order to provide patients with most suitable therapyp63 p40 and cytokeratin 56ck56 are the mostcommon panel ofimmunochemical markers for thediagnosis of squamous carcinoma p63 and ck56are traditional markers that indicate squamous differentiation in primary lung neoplasms most squamouscarcinomas and large cell carcinomas are positive forck56 warth found that the probability of acorrect sqcc diagnosis using ck56 is p63 a transcriptional regulator has a crucial role in thedevelopment and differentiation of stratified squamousepithelium it is usually strongly expressed in the basalkeratinocytes [] vosmik analyzed patientswith cervical squamous cell carcinoma and found that had positive expression of p63 p40 is a new specific marker for distinguishing squamouscarcinomas from adenocarcinoma whose specificity isabout in lung carcinomas however the positiveexpression of ck56 p63 and p40 are only found in a fewadenocarcinomas [ ] kriegsmann suggested theuse of either ck56 or p40 over p63 in the routine diagnostic setting ck7 is expressed in many ductal andglandular epithelial cells mainly gallbladder hepatic ductsand pancreatic ducts in tissues of the female genital tractovary endometrium fallopian tube and cervix and in thebreast lung and urinary tract tissues in the normalcervical tissue and adenocarcinoma ck7 staining wasobserved in the columnar cells of endocervical glandshashiguchi found the different rates of ck7 in patientswith cervical intraepithelial neoplasia and those with invasivecarcinomas vs [ ] thus far no efficientmarkers have been developed for distinguishing squamouscell carcinoma and adenocarcinoma in the endocervixmucins are a family of large glycoproteins expressedon the epithelial cell surfaces including ducts of lacrimalglands in the eye salivary glands the lining of the respiratory gastrointestinal urothelial and reproductive tracts muc5ac belongs to gelforming mucins multiple histological studies have highlighted that muc5acis expressed in the conjunctiva middle ear nasopharynxlungs gallbladder and stomach under normal conditionswhere it provides protection to corresponding epithelialsurfaces from different factors some research hasshown that muc5ac may be a potential biomarker inpancreatic cancer tissues dimaio found thatanterior gradient homolog and muc5ac are usefulpositive markers of adenocarcinoma in the setting ofabsent or diminished p63 and cytokeratin staining inesophageal carcinoma it is also expressed in theendocervix yamanoi found that muc5ac waslargely expressed in typical legh atypical legh gasmda and gasnonmda thus we speculated thatmuc5ac could be expressed in other adenocarcinomasand might be used for the differential diagnosis of adenocarcinoma and squamous carcinoma the aim of thisstudy was to examine the combination of cytokeratin p63 p40 and muc5ac for distinguishing squamous cellcarcinoma scc from the adenocarcinoma in the cervixaecmaterials and methodstissue sampleswe analyzed poorly to moderately differentiated cervical squamous carcinoma scc and adenocarcinomasof endocervix aec all tissues were collected from thedepartment of human pathology of qilu hospitalshandong university china from to specimenswere retrieved from the pathology files of the departmentof pathology at the same hospital after collection all specimens were fixed in buffered formalin hematoxylin eosin he stains were available for review paraffin blockswere used for immunohistochemical staining all the slideswere reviewed by two experienced pathologistshistopathological and clinical variables including agetumor size differentiationinfiltrate depth and lymphnode metastasis were summarized in table followupinformation was available in aec with the followuptime ranging from to months mean monthsimmunohistochemistryfour to five micronthick paraffin sections of the cases were dewaxed rehydrated in graded alcohols andprocessed using the pv9000 detection kit zsbio commerce store beijing china briefly antigen retrieval wasperformed in a microwave oven for min in mm trisedta buffer mm tris base mm edta solution tween ph endogenous peroxidase activitywas blocked with a h2o2methanol solution for min slides were then incubated in normal goat 0cli diagnostic pathology page of table comparison of clinicopathological features between cervical squamous cell carcinoma and cervical adenocarcinomasquamous cell carcinomasn adenocarcinoman Ï p valueage¤ size cm ¥ unknowndifferentiationpoormoderatewellunknown infiltrate depth of mesenchyme¤ unknownlymph node metastasisnoyesunknown serum for min to prevent nonspecific binding sampleswere then incubated overnight at °c with a primary antibody phosphate buffered saline pbs was used instead ofthe first antibody as a negative control consequentlysamples were incubated with reagent atroomtemperatureroomfor min and reagent attemperature for min finally the tissues were stainedwith diaminobenzidine dab the antibodies used in thisstudy are listed in table scoring methodstaining results were interpreted by one pathologist whowas blinded to the patients clinical and pathologicaldata for ck56 ck7 and muc5ac more than oftumor cells with a membrane or cytoplasmic brownyellow granules were considered positive for p63 andp40 the positive standard was that more than oftumor cells have brownyellow granules in the nucleusstatistical analysisstatistical analysis was performed with spss softwareversion spss inc chicago ii usa chisquareor fishers exacttests were used when comparingfrequencies between two groups probability values lessthan were considered statistically significantresultsthe expression of ck56 p63 p40 ck7 and muc5ac inscc and aecihc for the five proteins was performed on humanprimary cervical cancersincluding scc and aec as shown in fig and fig muc5ac ck56and ck7 were mainly expressed in the cell membranetable immunohistochemical antibodiesantibodymuc5acnozm0395ck56ck7p40p63zm0313zm0071zm0472zm0406vendorzsbio commerce store beijing chinazsbio commerce store beijing chinazsbio commerce store beijing chinazsbio commerce store beijing chinazsbio commerce store beijing chinadiluationready to useready to useready to useready to useready to use 0cli diagnostic pathology page of fig the expression of ck56 p63 p40 ck7 and muc5ac in a case of poordifferentiated squamous cell carcinoma by ihc a he b ck56positive staining c p63 positive staining d p40 positive staining e ck7 positive staining f muc5ac negative staining Ãfig the expression of ck56 p63 p40 ck7 and muc5ac in case of poordifferentiated adenocarcinoma invasive stratified mucinproducingcarcinoma ismile by ihc a he b ck56 negative staining c p63 negative staining d p40 negative staining e ck7 negative staining f muc5acpositive staining à 0cli diagnostic pathology page of and cytoplasm while p40 and p63 were mainly locatedin the nucleussignificant effect on the prognosis of cervical adenocarcinoma patients p as shown in fig tumorin thecells ofwe found that muc5ac exhibited prominent immucervical aecnoreactivitymuc5ac and ck7 were detected in and of aec cases compared to and of scc casesbesides for aec the specificity of muc5ac was muchhigher than that of ck7 p moreover the sensitivity of ck56 p40 and p63 was and respectively and the specificity was and respectively in aec table through the combined detection of p40 or p63 wecompared muc5ac and ck7 again we found that thesensitivity and specificity of muc5ac in aec combinedwith p40 or p63 were and respectively and respectively the sensitivity and specificity of ck7 combined with p40 or p63 were and and respectively table thesensitivity of muc5ac combined with p40 or p63 wassimilar to that of ck7 while the specificity was slightlyhigher than that of ck7expression of muc5ac and ck7 in cervicaladenocarcinoma subtypeswe further detected the expression of muc5ac insubtypes of aec table among cases of usualtype cervical adenocarcinoma cases were muc5acpositive and cases were ck7 positive and there wasno statistical difference p in cases of mucinous adenocarcinoma nosthe expression rate ofmuc5ac and ck7 were both moreover out of cases of gastric mucinous adenocarcinomaexpressed muc5ac and of them were ck7 positivep the positive rate of the muc5ac in mucinouscarcinoma intestinal type villous tubular adenocarcinomaendometrioid adenocarcinoma clear cell carcinoma serouscarcinoma adenosquamous carcinoma and invasive stratified mucinproducing carcinoma ismile was and respectively the expressionrate of muc5ac had no statistical difference among thesesubtypes all p correlation between muc5ac expression andclinicopathological characteristics in cervicaladenocarcinomathis study further analyzed the relationship between theexpression of muc5ac and clinicopathological featuresin cervical adenocarcinoma table the expression oftumormuc5ac was correlated with the degree ofdifferentiation p a lower degree oftumordifferentiation was associated with a lower expressionrate of muc5ac there was no significant correlationbetween the expression of muc5ac protein and agetumor size depth of myometrialinvasion and lymphnode metastasis all p kaplan meier analysisrevealed that the expression of muc5ac protein had nodiscussion and sidentification of previously unutilized sensitive biomarkers is still a priority for improved differential diagnosis of cervical aec and scc at present ck56 p63p40 and ck7 are the main biomarkers for differentiatingcervical adenocarcinoma from squamous cell carcinomack56 is a kind of high molecular weight basal cellkeratin 58kda and 56kda which is mainly expressed inthe basal cells of squamous epithelium and ductal epithelium and some squamous epithelial germinal layercells myoepithelial cells and mesothelial cells butpoorly expressed in glandular epithelial cells someresearch results showed that ck56 has high sensitivityand specificity in the diagnosis ofsquamous celltable sensitivity and specificity of muc5acãck56ãck7ãp40ãp63 in cervical squamous cell carcinoma and adenocarcinomamarkerssensitivityspecificitysquamous cell carcinomasn adenocarcinoman muc5acck7ck56p40p63ck56and p40ck56and p63muc5acand p40muc5acand p63ck7and p40ck7and p63 0cli diagnostic pathology page of table the correlation of muc5ac and the clinical variants inthe cervical adenocarcinomathe expression of muc5acpositivenegativeÏ valuep valueage¤ v size cm ¥ differentiationpoorwellmoderateinfiltrate depthof mesenchyme¤ lymph nodemetastasisnoyescarcinoma [] in contrast other studies showedhigh sensitivity but low specificity when diagnosing thistype of tumor p63 is a member of the p53 family a classical tumorsuppressor gene family it is located on chromosome3q27 filho showed good sensitivity whendetecting squamous cell carcinoma with a positive rateof contrary kaufmann suggested thatp63 could also be expressed in a small number of adenocarcinoma basal cell carcinoma and transitional epithelial carcinoma moreover p63 can also be used as amarker of myoepithelial cells and prostate basal cellstherefore p63 lacks absolute specificity for squamousdifferentiationp40 is a subtype of p63 protein expressed in squamousepithelial cells including epidermis and hair folliclesurothelial cells myoepithelial cells ofthe mammarygland sweat gland and salivary gland and basal cells ofthe prostate which are highly specific in labeling squamous epithelium bishop showed that in cases of squamous cell carcinoma of the lung and cases of adenocarcinoma of the lung the sensitivity andspecificity of p63 were and respectivelythe sensitivity and specificity of p40 in the diagnosis ofsquamous cell carcinoma of the lung were and respectively therefore p40 is considered as ahighly specific and sensitive tumor biomarker of squamous epithelial origin in this study we used immunohistochemistry to detectck56 p63 and p40 in cervical squamous cell carcinomaand adenocarcinoma the sensitivity of ck56 p40 andp63 was and respectively and thespecificity was and respectivelymoreover the specificity of ck56 is slightly lower thanthat of p40 and p63 we also found that a combinationof ck56 with p40 or p63 slightly decreased the sensitivity and and increased the specificity and which in turn increased the accuracy of diagnosing squamous cell carcinomack7 is a kind of low molecular weight keratin mainlyexpressed in glandular epithelium and transitional epithelial cells of most normal tissues many studieshave found that ck7 is not only expressed in adenocarcinoma but also in squamous intraepithelial neoplasiacervical squamous cell carcinoma lung squamous cellcarcinoma and esophageal squamous cell carcinomalee found a positive expression of ck7 in fig survival analysis of muc5ac expression in cervical adenocarcinoma 0cli diagnostic pathology page of table expression of muc5ac and ck7 in differentadenocarcinoma subtypessubtypesmuc5acusual typeÏ valueck7p valuepositivenegativemucinous adenocarcinoma nospositivenegativegastric typepositivenegativeintestinal typepositivenegativevillous tubular adenocarcinomapositivenegativeendometrioid adenocarcinomapositivenegativeclear cell carcinomapositivenegativeserous carcinomapositivenegativeismilepositivenegativeadenosquamous carcinomapositivenegativeismile invasive stratified mucinproducing carcinoma cases with scc and cases withciniii furthermore yamada found that ck7expression in esophageal squamous cell carcinoma butalso in iiiaiib stage esophageal squamous cell carcinoma suggest poor tumor differentiation and thus canbe used as an independent prognostic factor ourstudy showed that the positive rate of ck7 was incervical poorly differentiated squamous cell carcinomawhich further suggested that ck7 is not an ideal markerfor differentiation between squamous cell carcinoma andadenocarcinomamucin is a high molecular weight glycosylated proteinsecreted by epithelial cells in the respiratory tractgastrointestinal tract and urogenital tract which has animportant role in the protection of epithelium cell adhesion signal transduction immune activation and inhibition at present at least mucins have been found inthe female reproductive system riethdorf and albarracin used immunohistochemistrymethods to detect the expression of muc5ac in different female reproductive system malignant tumors theyfound that muc5ac was highly expressed in cervicaladenocarcinoma and poorly expressed inendometrial adenocarcinoma all of themwere expressed in the primary ovarian mucinous tumor but not in colon adenocarcinoma therefore they concluded that muc5ac could beused as an effective marker to distinguish the origin ofpelvic tumors and distinguish primary ovarian tumorsand colorectal metastasis as well as endometrial adenocarcinoma from cervical metastasis [ ] in thisstudy we found positive expression of muc5ac in cases of cervical adenocarcinoma and in cases of squamous carcinoma which wasconsistent with riethdorfs study the sensitivity ofmuc5ac and ck7 to cervical adenocarcinoma was and respectively but the specificity ofmuc5ac was much higher than that of ck7 through the joint detection of p40 or p63 wecompared muc5ac and ck7 again and found that thesensitivity and specificity of muc5ac combined withp40 or p63 were and respectively and respectively the sensitivity and specificity ofck7 combined with p40 or p63 were and and respectively these results showed thatthe sensitivity of muc5ac combined with p40 or p63was similar to that of ck7 butthe specificity wasslightly higher than that of ck7 therefore muc5ac issuperior to ck7 in the diagnosis of cervical adenocarcinoma and squamous cell carcinomabesides we preliminarily detected the expression ofmuc5ac in different types of cervical adenocarcinomaand found no significant difference these data suggestedthat muc5ac has no diagnostic significance in the classification of cervical adenocarcinoma at the same time weanalyzed the relationship between the expression ofmuc5ac and the prognosis of cervical adenocarcinomaand the result revealed that muc5ac was not related tothe prognosis of cervical adenocarcinomaoverall our observations strongly suggest that muc5acmay be useful as a biomarker for differential diagnosesbetween squamous carcinoma and adenocarcinomaabbreviationsscc squamous cell carcinoma aec adenocarcinoma of the cervixck cytokeratin he hematoxylin eosin pbs phosphate buffered salinedab diaminobenzidine ismile invasive stratified mucinproducingcarcinoma 0cli diagnostic pathology page of authors contributionsxiaofang zhang designed the study and drafted the manuscript hailing liand xiaotong jing analyzed the data and carried out theimmunohistochemistry jie yu and tingguo zhang read the pathologicalsections jinan liu collected the clinical data and carried our followupshiming chen made the slides the authors read and approved the finalmanuscript downey p cummins r moran m gulmann c if it's not ck56 positive ttf negative it's not a squamous cell carcinoma of lung apmis warth a muley t herpel e meister m herth fj schirmacher p weichert whoffmann h schnabel pa largescale comparative analyses ofimmunomarkers for diagnostic subtyping of nonsmallcell lung cancerbiopsies histopathology fundingthis work was supported by the national natural science foundation ofchina no and technology development foundation of yantaino ws017availability of data and materialsnot applicableethics approval and consent to participateall tissue samples from patients were collected and protocols wereperformed according to the procedures approved by the research ethicscommittee of shandong medical university all patients provided informedconsentcompeting intereststhe authors declare that they have no competing interestsauthor details1department of pathology weifang traditional chinese hospital weifangshandong p r china 2department of pathology school of basic medicalscience shandong university jinan shandong p r china 3department ofpathology the fourth hospital of jinan the third affiliated hospital ofshandong first medical university jinan shandong p r china 4departmentof oncology yuhuangding hospital yantai shandong p r china5department of pathology school of basic medical science shandonguniversity jinan shandong p r chinareceived july accepted august referenceskurman rj carcangiu ml herrington cs who classification of tumours offemale reproductive ans4th ed lyon iarc press takeuchi s biology and treatment of cervical adenocarcinoma chin jcancer res young rh clement pb endocervical adenocarcinoma and its variants theirmorphology and differential diagnosis histopathology forouzanfar mh foreman kj delossantos am lozano r lopez ad murraycj naghavi m breast and cervical cancer in countries between and a systematic analysis lancet galic v herzog tj lewin sn neugut ai burke wm lu ys hershman dlwright jd prognostic significance of adenocarcinoma histology in womenwith cervical cancer gynecol oncol favero g pierobon j genta ml araujo mp miglino g del cpdm deandrade ch fukushima jt baracat ec carvalho jp laparoscopicextrafascial hysterectomy completion surgery after primary chemoradiationin patients with locally advanced cervical cancer technical aspects andoperative outcomes int j gynecol cancer rose pg java jj whitney cw stehman fb lanciano r thomas gm locallyadvanced adenocarcinoma and adenosquamous carcinomas of the cervixcompared to squamous cell carcinomas of the cervix in gynecologiconcology group trials of cisplatinbased chemoradiation gynecol oncol ma y fan m dai l kang x liu y sun y xiong h liang z yan w chen kexpression of p63 and ck56 in earlystage lung squamous cell carcinoma isnot only an early diagnostic indicator but also correlates with a goodprognosis thorac cancer kaufmann o fietze e mengs j dietel m value of p63 and cytokeratin as immunohistochemical markers for the differential diagnosis of poorlydifferentiated and undifferentiated carcinomas am j clin pathol barbieri ce pietenpol ja p63 and epithelial biology exp cell res senoo m pinto f crum cp mckeon f p63 is essential for the proliferativepotential of stem cells in stratified epithelia cell pozzi s zambelli f merico d pavesi g robert a maltere p gidrol xmantovani r vigano ma transcriptional network of p63 in humankeratinocytes plos one 200943e5008 vosmik m laco j sirak i beranek m hovorkova e vosmikova h drastikovam hodek m zoul z odrazka k prognostic significance of humanpapillomavirus hpv status and expression of selected markers her2neuegfr vegf cd34 p63 p53 and ki67mib1 on outcome after chemoradiotherapy in patients with squamous cell carcinoma of uterine cervixpathol oncol res nobre ar albergaria a schmitt f p40 a p63 isoform useful for lung cancerdiagnosis a review of the physiological and pathological role of p63 actacytol stolnicu s hoang l hankobauer o barsan i terinte c pesci a avielronens kiyokawa t alvaradocabrero i oliva e and others cervicaladenosquamous carcinoma detailed analysis of morphologyimmunohistochemical profile and clinical outcomes in cases modpathol toyoshima m momono y makino h kudo t oka n sakurada j suzuki hkodama h yoshinaga k cytokeratin 7positivecytokeratin 20negative cecaladenocarcinoma metastatic to the uterine cervix a case report world jsurg oncol hashiguchi m masuda m kai k nakao y kawaguchi a yokoyama maishima s decreased cytokeratin expression correlates with theprogression of cervical squamous cell carcinoma and poor patientoutcomes j obstet gynaecol res lee h lee h cho yk cytokeratin7 and cytokeratin19 expression in highgrade cervical intraepithelial neoplasm and squamous cell carcinoma andtheir possible association in cervical carcinogenesis diagn pathol krishn sr ganguly k kaur s batra sk ramifications of secreted mucinmuc5ac in malignant journey a holistic view carcinogenesis thornton dj rousseau k mcguckin ma structure and function ofthe polymeric mucins in airways mucus annu rev physiol rose mc voynow ja respiratory tract mucin genes and mucinglycoproteins in health and disease physiol rev balmaña m duran a gomes c llop e lópezmartos r ortiz mr barrabés sreis ca peracaula r analysis of sialyllewis x on muc5ac and muc1mucins in pancreatic cancer tissues int j biol macromol dimaio ma kwok s montgomery kd lowe aw pai rkimmunohistochemical panel for distinguishing esophageal adenocarcinomafrom squamous cell carcinoma a combination of p63 cytokeratin muc5ac and anterior gradient homolog allows optimal subtyping humpathol yamanoi k ishii k tsukamoto m asaka s nakayama j gastric gland mucinspecific oglycan expression decreases as tumor cells progress from lobularendocervical gland hyperplasia to cervical mucinous carcinoma gastrictype virchows arch reisfilho js simpson pt martins a preto a gartner f schmitt fcdistribution of p63 cytokeratins and cytokeratin in normal and neoplastic human tissue samples using tarp4 multitumor tissuemicroarray virchows arch yamada a sasaki h aoyagi k sano m fujii s daiko h nishimura myoshida t chiba t ochiai a expression of cytokeratin predicts survival instage iiiaiib squamous cell carcinoma of the esophagus oncol rep baker ac eltoum i curry ro stockard cr manne u grizzle we chhieng dmucinous expression in benign and neoplastic glandular lesions of theuterine cervix arch pathol lab med 0cli diagnostic pathology page of riethdorf l o'connell jt riethdorf s cviko a crum cp differentialexpression of muc2 and muc5ac in benign and malignant glandularlesions of the cervix uteri virchows arch albarracin ct jafri j montag ag hart j kuan sf differential expression ofmuc2 and muc5ac mucin genes in primary ovarian and metastatic coloniccarcinoma hum pathol publishers notespringer nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations 0c" | Colon_Cancer |
"eph receptors and the corresponding eph receptorinteracting ephrin ligands jointly constitute a critical cellsignaling network that has multiple functions the tyrosine kinase epha2 which belongs to the family of ephreceptors is highly produced in tumor tissues while found at relatively low levels in most normal adult tissuesindicating its potential application in cancer treatment after years of investigation a large amount of dataregarding epha2 functions have been compiled meanwhile several compounds targeting epha2 have beenevaluated and tested in clinical studies albeit with limited clinical success the present review briefly describes thecontribution of epha2ephrin a1 signaling axis to carcinogenesis in addition the roles of epha2 in resistance tomoleculartargeted agents were examined in particular we focused on epha2s potential as a target for cancertreatment to provide insights into the application of epha2 targeting in anticancer strategies overall epha2represents a potential target for treating malignant tumorskeywords epha2 receptor ephrin a1 cancer therapy targetintroductionephrin receptors eph represent the most importantclass of receptor tyrosine kinases rtks epha1 thefirstly described eph receptor was identified in livercancer cells while screening for rtks in nowadays there are eph receptors and relatedligands ephrins eph receptor signaling contributesto multiple biological events mostly causing cellcellrepulsion or adhesion therefore eph receptors and thecorresponding ligands have essential functions in tissuepatterning neuronal targeting and blood vessel development in the embryo [ ] meanwhile eph proteins arefound in high levels in multiple malignancies with suchoverexpression significantly contributing to carcinogenesis eph receptors are single transmembrane proteins withnterminal and intracellular domains withextra correspondence zqxiao2001hotmailcom sumin27126com2research center of carcinogenesis and targeted therapy xiangya hospitalcentral south university changsha hunan china3thoracic surgery department hunan cancer hospital and the affiliatedcancer hospital of xiangya school of medicine central south universitychangsha hunan chinafull list of author information is available at the end of the ligandbinding and intrinsic enzymatic activities respectively [ ] eph receptors are grouped into a and bcategories according to their extracellular domainswhich determine the binding affinity for ligands ephreceptorinteracting proteins or ephrins [ ] nineepha and five ephb receptors are found in humans the ligands for eph receptors ephrins are anchored tothe cell membrane they also comprise two subcategoriesincluding ephrin a ephrin a15 and ephrin bephrin b13 [ ]to modulatory processessome eph receptors especially epha2 attract increasing attention because of demonstrated or hypothesizedcontributionscontrollingcarcinogenesis and tumor progression fig thepresent manuscript reviewed the clinical associationsand biological and cellular consequences of epha2overexpression in cancer potential opportunities fortherapeutic intervention based on epha2 targeting areparticularly discussedepha2ephrin a1 signalingthe epha2 receptor is a 130kda transmembrane glycoprotein with amino acids the epha2 gene in 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 0cxiao of hematology oncology page of fig historical development and breakthroughs in targeting epha2 in cancerhumans is found on chromosome 1p36 its initial detection occurred in while screening a hela cell cdnalibrary comprising degenerate oligonucleotides engineered to interact with highly conserved domains oftyrosine kinases epha2 was originally termedepithelial cell kinase eck since it was detected in mostepithelial cellsepha2 interacts with any of the eight different ephrinafamily ligands with overt preference to ephrin a1 ephrin a1 represents a gpianchored proteincontaining amino acids apparent molecular weight kda the human ephrin a1 gene is located on1q21q22 this tnfα earlyinducible gene product wasfirstly described in human umbilical vein endothelialcells huvecs three decades ago and shown tobind epha in ephrin a1s expression patternin cancer seems to differ from that of epha2 with attenuation in a variety of aggressive tumors particularlythose overexpressing epha2 under normal conditions epha2 interacts with ephrina1 on the neighboring cell and induce diverse signalingnetworks following celltocell contact as membraneproteins ephrins are engaged in both forward termedephrinepha2 forward and reverse called epha2ephrinreverse signaling from ephrin ligands to epha2 and viceversa this is also known as ephrinepha2 bidirectionalsignaling [ ] forward signaling is often cellrepulsive and promotes epha2 oligomerization andtherefore enhancing kinase activityphosphorylationthe immediate biologicalconsequences of epha2phosphorylation include decreased cellextracellularmatrix ecm attachment ephrin a1associated epha2induction inhibits focal adhesion kinase fak extracellularand aktphosphorylation to regulate motility viability and proliferation in multiple malignant cell lines [ ] whereasreverse signaling is more likely to be adhesive and isgenerally considered as kinaseindependent due to lacking enzyme activity in ephrin a1 however the reverseregulated protein kinaseserksignaling by ephrin a1 is largely poorly understood inaddition epha2 possesses ligandindependent kinase activity in cultured cancer cells which might partially explain its malignant effects in the nonphosphorylatedstate [ ] actually epha2ephrin a1 interaction orepha2 ligandindependent kinase activity likely functions through multiple factors acting jointly eg celltype and the microenvironment altogether the epha2ephrin a1 signaling regulates multiple cellular processesproliferation survival migration morphology celltocell repulsion and adhesion in embryonic developmentangiogenesis and tumorigenesis fig epha2 in cancerdifferent from the majority of eph kinases that aremostly synthesized during the developmental processepha2 is mainly restricted to proliferating epithelialcells in adults epha2 expression in the adult occurs in normal tissues only when they have highlyproliferating epithelial cells where its importanceand function are not well understood however anaccumulating body ofsuggests humanepha2 is abundantly expressed in diverse cancerssuch as prostate lung esophageal colorectal cervical ovarian and breast and skin cancers epha2 is upregulated at thegene and protein levelstissuespecimens and established cancer cell lines [ ] inparticular most elevated epha2 expression is consistently detected in cells with highest malignancy in addition epha2 expression has associations withpoor prognosis elevated metastatic potential and reduced survival of tumor patients [ ] moreoverepha2 is nota biomarker of malignantcharacter but also an active participant in malignantprogression [ ] consequently epha2s expression patterns and functional relevance in malignanciesmake this protein an attractive therapeutic target incancerin human tumorevidencesimply 0cxiao of hematology oncology page of fig expression and biological pathways linked with epha2 the interaction of cellmembranebound epha2 with ephrin a1 induces forward orreverse signals in the corresponding cells under normal conditions cellcell contacts allow epha2 to interact with ephrin a1 which inducesepha2 phosphorylation and activates its downstream signaling tyrosine phosphorylation of epha2 promotes the generation of a complex withccbl subsequently induces epha2 degradation this leads to suppression of ecm attachment cell proliferation cell migration and angiogenesisin the malignant state loss of cellcell contacts induces receptorligand interaction and degradation of epha2 in addition tyrosinephosphorylation of epha2 could be rapidly reversed by the phosphatase lmwptp further leading to the overexpression and accumulation ofunphosphorylated form of epha2 this leads to promotion of ecm attachment cell proliferation cell migration and angiogenesisthere is considerable interest in the mechanisms thatgovern epha2 expression and in understanding howthese mechanisms are subverted in cancer emergingevidence links high epha2 protein amounts with epha2regulation at the mrna level as well as protein stabilityalthough the precise mechanisms governing epha2 upregulation in cancer remain largely undefined [ ]epha2 mrna is tightly regulated to date a fewsomatic mutations of epha2 have been reported [] in addition epha2 amplification detected in only alow percentage of cases in pancreatic cancer samples the epha2 promoter comprises dnadamageresponsive p53binding sites and this receptoris upregulated by ultravioletray uv treatment epha2 is overexpressed in rastransformed cells andtransgenic mice overexpressing ras suggesting epha2as a direct transcriptional target of rat sarcoma rasrapidly accelerated fibrosarcoma raferk signaling epha2 gene expression is also reduced by multiplestimuli such as signaling by the cmyc and estrogen receptor these observations are intriguing given thatepha2 consistently shows highest expression in breasttumor cells with most pronounced aggressiveness andno expression of estrogen receptor erα [ ] thusit is tempting to speculate that epha2 overexpression inbreast cancer might be linked to the loss of hormone dependence that frequently arises in advanced stages of thediseasedecreased ligandmediated receptorinternalizationand degradation consequently enhancing protein stability might help increase epha2 amounts in malignantcells an interesting consequence of epha2 stimulationby ligand or antibodyis epha2 phosphorylationinternalization and degradation [] after liganddependent induction epha2 aggregation occurs at thecelltyrosine phosphorylationsurfacefollowed by 0cxiao of hematology oncology page of promoting the generation of a complex with ccblwhich is internalized into early endosomes for subsequent epha2 degradation studies have shown thatccbl overexpression decreases the levels of the epha2protein likely by enhancing protein degradation tyrosine phosphorylation of epha2 could also be rapidly reversed by lowmolecularweight protein phosphataselmwptp a phosphatase binding to and dephosphorylating epha2 increased lmwptp expressionfunctions to reduce epha2 phosphotyrosine contentcontributing to elevated epha2 levels in cancer cellsdespite epha2 overexpression in cancer phosphorylatedepha2 is found in lower amounts in cancer cells incomparison with nontransformed epithelial cells unlike many other receptor tyrosine kinases the enzymatic activity of epha2 does not depend on ligand interaction or receptor autophosphorylation [ ] it isconsidered that deficient celltocell contact commonlyfound in malignant cells and insufficient levels of ephrina1 on cancer cells reduce epha2 phosphorylation targeting epha2 in canceroverexpression and aggressive features of epha2 intumor cells and relatively low expression in most normaladult tissues make this protein a potential therapeutictarget in cancer the epha2ephrin a1 system could betargeted for cancer treatment at least via two mechanisms first epha2s oncogenic features could be inhibited eg decreasing epha2 expression promotingepha2 degradation and blocking endogenous epha2activation alternatively the epha2 receptor could beemployed to deliver therapeutics exogenous drugs orendogenous immune cells to cancer cells and associatedvessels therapies targeting epha2 in cancer are shownin table and fig inhibiting epha2 expressiongiven the positive association of epha2 overexpressionwith aggressive clinical and pathological features in human cancers investigators have examined the potentialof downregulating epha2 in preclinical models shortinterfering rnas sirnas for gene knockdown constitute a great tool for protein function assessment genediscovery and drug development [ ] and have beenapplied to silence epha2 in human cancer cells forexamplein pancreatic adenocarcinomaderived cellssequencespecific sirna targeting epha2 suppressesepha2 expression retarding tumor growth in a nudemouse xenograft model in addition treatment withepha2specific sirna significantly reduces malignancyin glioma nonsmall cell lung cancer nsclc and breast cancer cells however despite the greatsuccess in in vitro knockdown in vivo sirna delivery ischallengingand] asefficient[aresultbiocompatible delivery systems for systemic sirna administration have been evaluated for instance epharna the 12dioleoylsnglycero3phosphatidylcholinedopc nanoliposomal epha2targeted therapeutic hasbeen developed in the nude mouse model administered ovarian tumors intraperitoneally epharna wasshown to be taken up by the tumor reducing epha2levels in the animals h following single treatment this finding indicates that treatment with epharna reduces tumor growth in the ovarian cancer mousexenograft model in addition both signal dosing andmultidosing of epharna have an excellent safety profile in many mammalian species including nonhumanprimates promoting epha2 degradationartificial ligands or antibodies interacting with epha2could suppress signaling by promoting internalizationand degradationsoluble ephrin a1 and ephrin a1fcplasma membranebound ephrins and soluble ephrinswith artificial clusteringdimerization associated withantibodies targeting coohterminal epitope tags orfusion to immunoglobulin g igg fc potently promoteepha2 phosphorylation and degradation featuresincludingephrin a1 has been demonstrated to be present at lowlevels and to possess tumor suppressing propertiesdependent on celltocell contact in a variety of tumors[ ] transfection with fulllength human ephrina1 into glioblastoma multiforme gbm cells exhibits adramatic suppression of epha2 and inhibits multiplemalignantimpaired anchorageindependent growth proliferation and migration of great interest ephrin a1 was shown to be released asa soluble monomeric entity by gbm and breast cancercells this soluble ephrin a1 could function in ainduce epha2 internalization andparacrine mannerdownregulation elicitsubstantial alterations of cellmorphology and inhibit cell migration in treated gbmcellsin a juxtacrine interactionindependent manner treatment with ephrin a1conditioned mediaabolishes the phosphorylation of erk induced by emptyvectorconditioned media which might contain growthfactors moreover treatment with a fusion protein ofmonomeric ephrin a1 mea1 also induced phosphorylation and degradation of in human breast cancer cells thus ephrin a1associated tumor suppressionmight result from epha2 downregulation as well asdirect signaling through epha2in addition to soluble ephrin a1 ephrin a1fcobtained by fusing recombinant ephrin a1 to humanigg fc for dimerization shows ephrinlike features andinduces epha2 phosphorylation treatment with 0cxiao of hematology oncology page of table summary of epha2 targeted therapies against cancermechanism method orcompoundcancer typeexact effects on epha2effects in vitrodecrease epha2 expressionepharnaovarian cancerdecrease in vivo epha2 expressionpromote epha2 degradationsoluble ephrin a1 and ephrin a1fcephrin a1glioblastoma multiformeinduce epha2 internalization anddownregulationinhibit cell migrationmonomericephrin a1ephrin a1fcephrin a1fcbreast cancerinduce epha2 phosphorylation anddegradationpancreatic cancerinduce epha2 degradationinhibit cell motility and invasiongastric cancerinduce epha2 phosphorylation anddegradationinhibit cell growthepha2 monoclonal antibodyea12breast cancerinduce epha2 phosphorylation anddegradationinhibit cell growth disruptangiogenesisea2 andb233breast cancerinduce epha2 phosphorylation anddegradationinhibit tumor growth in vivoeffectsin vivoinhibittumrowthinhibittumrowthinhibittumrowthref d2 scfvlymphomaprevent epha2ephrin interactionshm16melanomaantibody internalizationds8895abreast cancer and gastriccancerinhibit epha2 phosphorylationinhibit cell proliferation induceapoptosisinhibit cell migration and invasion ds8895abreast cancer and gastriccancer3f23mbreast ovarian nonsmallcell lung cancerinduce epha2 phosphorylationkill tumor cells in vitroblock endogenous epha2 activationinhibit ephephrin interactionsepha2fcpancreaticinhibit epha2 phosphorylationinhibit angiogenesislithocholicacidprostate and coloncancerinhibit epha2 phosphorylationinhibit cell rounding retractioninhibittumrowthinhibittumrowthinhibittumrowthunipr126prostate cancerinhibit epha2 phosphorylationunipr126prostate cancerinhibit epha2 phosphorylationunipr129prostate cancerunipr1331prostate cancerinhibit epha2 phosphorylation andblock kinase domain enzymatic activityblock epha2 phosphorylation andactivationinhibit cell rounding retractioninhibit cell rounding disrupt angiogenesisdisrupt angiogenesischolanicacidgw406476d10prostate cancerinhibit epha2 phosphorylationinhibit cell retractionprostate cancerprostate cancerinhibit epha2 phosphorylationinhibit epha2 phosphorylationinhibit cell retractioninhibit kinase activity of epha2dasatinibmelanomainhibit epha2 phosphorylation andkinase activityinhibit cell migration and invasion 0cxiao of hematology oncology page of table summary of epha2 targeted therapies against cancer continuedmechanism method orcompounddasatinibexact effects on epha2pancreatic cancercancer typeinhibit epha2 phosphorylation andkinase activityeffects in vitroinhibit cell growthinhibit cell survivalglioblastomanonsmall cell lungcancercandidate4aalwii41alwii41inhibit epha2 phosphorylationinhibit cell survivallung cancerinhibit cell survival proliferationmigration increased apoptosisepha2 as drug delivery targetpeptideantibodydrug conjugatesephrin a1pe38qqrglioblastoma multiforme decrease epha2 expressionmedi547prostate cancerinduce epha2 phosphorylation anddegradationinhibit cell survivalinhibit cell survivalmedi547endometrial cancerinduce epha2 internalization anddegradationinhibit cell survival induceapoptosismedi547ovarian cancerinduce epha2 degradationinhibit cell survival andproliferation induce apoptosisantibodydirected nanotherapeuticsytplmm310melanomabreast prostate gastricand esophageal cancerepha2based immunotherapydcvaccinecolon cancer murinedcvaccinecolon cancer murineand melanoma humancar t cellsglioblastomadecrease epha2 expressioncar t cellsgliomacar t cellslung cancercar t cellsesophageal squamouscell carcinomainhibit cell survivalinhibit cell survivalinhibit cell survivaleffectsin vivoinhibittumrowthinhibittumrowthinhibittumrowthinhibittumrowthinhibittumrowthinhibittumrowthinhibittumrowthinhibittumrowthinhibittumrowthinhibittumrowthinhibittumrowthref inreducedresultedamountsephrin a1fcofmembraneassociated epha2 and inhibited cellularmotility and invasion in pancreatic ductal adenocarcinoma cells in addition proteasomal degradation wasdemonstrated to play a critical role in ephrin a1fcassociated epha2 catabolism as the proteasome suppressor mg132 markedlyephrin a1fcinhibitsrelated epha2 degradation likewise ephrin a1fc increases epha2 phosphorylation decreases epha2 protein expression and inhibits growth in gastric cancercells dimeric ephrin a1fc suppresses rasmitogenactivated protein kinase mapk signaling toreduce growth factorassociated erk phosphorylation[] 0cxiao of hematology oncology page of fig targeting epha2 in cancer epha2s expression patterns and functional relevance in malignancies make this protein an attractivetherapeutic target in cancer accordingly epha2 overexpression has been targeted with several approaches such as decrease epha2 expressionpromote epha2 degradation block endogenous epha2 activation epha2 as drug delivery target epha2based immunotherapy and epha2based combination therapeuticsepha2 monoclonal antibodythe large extracellular domain of epha2 provides anantigen that is frequently upregulated on tumor cells[ ] in addition ligand stimulation is sufficient toinduce epha2 degradation these evidences suggest thatepha2 could elicit a particularly attractive monoclonalantibody and antibodies that mimic the actions ofephrin a1 would be expected to function similarly as theligandstudies have shown that several agonist monoclonalantibodies raised against epha2 induce its internalizationand degradation suppressing its malignant features forexample kinch isolated antibodies from miceafterimmunization with the pcdna3ecdepha2fcexpression plasmid and identified ea12 that dosedependently elevated phosphotyrosine amounts in epha2these authors demonstrated that ea12 inhibits morethan of soft agarformed colonies in breast cancercells compared with vehicletreated controls these findingsthe growthsuppressive effects ofepha2specific antibodies correlate with their capabilityof stimulating epha2 autophosphorylation and degradation coffman two demonstrated thatindicate thatantibodiesincluding ea2 and b233 promote epha2phosphorylation and degradation in cancer cells the antibody ea2 mgkg administered ip was shown tosignificantly decrease breast and lung cancer cell growthin vivo relative to the matched isotype controls igg11a7 goldgur isolated and characterized theantiepha2 singlechain antibody d2 scfv which washighly specific to epha2 and blocked ligand interaction incos7 cells indeed treatment with d2 scfv inducedapoptosis and reduced cell proliferation in the lymphomacell line in addition sakamoto showed that oneof the epha2 mabs produced shm16 interacts with anepha2 epitope differing from that affecting ephrin a1binding to epha2 shm16 was clearly internalized in cellsand inhibited malignant features in melanoma cells however shm16 showed no effects on ephrin a1 interactionwith epha2 on the cell surface while recognizing a different epha2 epitope shm16 was shown to be clearly internalized by a375 cellsantibodydependent cellular cytotoxicity adcc killscells via perforingranzyme trail and fasl adcc also affects adaptive tumor immunity and its enhancementtumorremarkablycouldalterthe 0cxiao of hematology oncology page of generatedmicroenvironment [ ] ds8895a a newly developed humanized antiepha2 mab afucosylated foradcc enhancement wasby mouseimmunization with recombinant human epha2 and further humanized as human igg1 treatment withds8895a of epha2positive breast and gastric cancercells was shown to partially inhibit ephrin a1associatedepha2 phosphorylation in agreement treatment withds8895a inhibits tumor growth in epha2positive human breast and gastric cancer xenografts in mice another epha2 effectorenhanced agonist monoclonalantibody that exhibits adcc activity is 3f23m 3f23m was obtained by fusing the mouse parental antibody b233 and the humanized antibody 3f2 3f23madministration dosedependently increased epha2 phosphorylation in the breast cancer cellline which wassimilar to that of the parental antibodies 3f2wt andb233 3f23m significantly inhibited ovarian breastand lung cancer cell lines which were cocultured withperipheral blood monocytes from a healthy donor however 3f23m was minimally toxic in the absence of nkcells on the other hand interaction with nks was increased by 250fold for 3f23m in comparison with3f2wt with improved affinity to fcγriiia modifyingthe fc portion of the epha2 antibody resulted in enhanced interaction with fcγriiia administration of3f23m significantly induced tumor growth inhibition ina breast cancer xenograft orthotopic model comparedwith the isotype control antibody and phosphate buffersaline pbs groupsblocking epha2 activationcompounds binding to epha2 or ephrin a1 couldsuppress signaling by direct antagonist effectstopeptidesandalternativesinhibiting ephephrin interactionssmall molecules that block epha2 could representefficientantibodiesrecently small molecules disrupting the ephephrincomplex have been described with most exertingpharmacological activitiesthethereby acting asligandbinding domain of epha2common proteinprotein interaction ppiinhibitorsthe ephrin binding site in eph receptors allow highaffinity binding of small molecules [ ]through targeting ofit was hypothesized that soluble receptors repressepha signaling by suppressing the interactions of endogenous ephrins with epha receptors epha2fc represents a soluble protein chimera involving the fusion ofepha2s extracellular domain with human igg1 fcpreventing interactions of several ephrin a ligands withendogenous receptors and potently inhibiting ephareceptor activation in cultured cells by interacting withephrin a1 epha2fc could induce ephrininitiatedreverse signaling treatment with epha2fc was shownto dosedependently inhibit epha2 receptor phosphorylation and activity in addition epha2fc strongly inhibited angiogenesis and microvessel growth in vitro as wellas growth in pancreatic tumor xenografts furthermore soluble epha2fc was demonstrated to inhibitendothelial cell migration upon t1 mouse mammaryadenocarcinomaangiogenesisin vitro moreover epha2fc inhibited t1 tumrowth in vivo and reduced tumor vascular density andgrowth while increasing cell apoptosiscellinducedtumorlithocholic acid lca 3a5b3hydroxycholan24oicacid a secondary bile acid produced by prokaryotic transformation of chenodeoxycholic acidis considered anepha2 antagonist lca interacts with the nuclear receptor farnesoid x receptor fxr and the gproteincoupledreceptor gproteincoupled bile acid receptor gpbar1also called tgr5 under physiological conditions molecular modeling investigations revealed thatlca mimics ephrin a1 in interacting with epha2 via insertion of its cyclopenta[a]perhydrophenanthrene scaffoldinto the hydrophobic epha2 receptor ligandbindingchannel generating a salt bridge involving arg103 anessential amino acid in ephrin a1 recognition lcawas shown to competitively and reversibly inhibit epha2ephrin a1 binding ki μm without reducing epha2skinase activity further functional assays revealed thatlca inhibits epha2 autophosphorylation and blocksephrin a1related prostate cancer cell cytotoxicitythe specificity of lac in antagonizing eph receptor hasbeen demonstrated with no detected effects on otherrtks including egfr vascular endothelial growth factorreceptor vegfr insulinlike growth factor receptorigf1r and the insulin receptor however lca is alsoconsidered to interact with epha and ephb receptors indicating an interaction with the highly conserved region ofeph receptor family members thus lca has beenused as a prototype for designing or identifying other ppisamino acid conjugates of lca were shown to effectivelydisrupt epha2 binding to ephrin a1 and to suppressepha2 phosphorylation in intact cells thereby bluntingmalignancy unipr126n3ahydroxy5bcholan24oylltryptophan a novel antagonist derived from lcainhibits epha2 phosphorylation and angiogenesis in cultured cells in the low micromolar range unipr126was shown to disrupt the epha2ephrin a1 complex andto inhibit epha2 phosphorylation in prostate cancer cellsat a level 6fold higher pic50 unipr129 n3ahydroxy5bcholan24oyllbhomotryptophanthe lhomotrp conjugate of lca another newly developedppi based on the in silico model of the epha2unipr126complex also disrupts epha2ephrin a1 interactionic50 nm ki nmin agreementunipr129 was shown to inhibit ephrin a1fcassociated 0cxiao of hematology oncology page of prostate cancer cell cytotoxicity and angiogenesis in vitroin addition both unipr129 and unipr126 reduce polygon formation but unipr129 ic50 μm was 4foldmore potent than unipr126 ic50 μm ic50values in inhibiting ephrin a1related epha2 phosphorylation were and μm respectively for unipr129 andunipr126 furthermore unipr126 showed cytotoxicityin huvecs increasing lactic dehydrogenase ldh release unlike unipr129 comparing efficacy for prostatecancer cell retraction unipr129 and unipr126 had similar strengths and were much more potent compared withlca likewise a series of ltrp derivatives of lca havebeen synthesized and a compound defined as compound was identified as the most potent antagonist disruptingepha2 binding to ephrin a1 this compound blocking epha2 phosphorylation ic50 μm was times more efficient compared with lca ic50 μmin inhibiting prostate cancer cells treatment with compound significantly reduced the percentage of retractedcells stimulated by ephrin a1fc in addition unipr1331n3bhydroxyd5cholen24oylltryptophan wasidentified as the first orally bioavailable small moleculeantagonizing the ephephrin system unipr1331was obtained by conjugating ltryptophan with the parentcompound 3βhydroxyd5cholenic acid which serves asbioisostere analogues of lca the activity of unipr1331in blunting epha2 binding to ephrin a1 pic50 was ten times increased compared with that of the parent3βhydroxyd5cholenic acid pic50 and barelystronger than lca pic50 administration ofunipr1331 was shown to inhibit gbm growth and to extend the time to progression in a subcutaneous xenograftmodel through inhibition of angiogenesis [ ] cholanic acid 5bcholan24oic acid is another moleculecompetitively inhibiting epha2 binding to ephrin a1 withincreased potency compared with lca cholanic acidhas a specific and reversible interaction with epha2sligandbinding domain blocking epha2 phosphorylationand prostate cancer cell cytotoxicity in contrast to lcapromiscuous binding cholanic acid is more selective forepha receptors cholanic acid inhibits eph receptor phosphorylation at noncytotoxic levels it inhibits epha2 activation by ephrins ic50 μm more effectivelycompared with lca ic50 μm in additioncholanic acid suppresses epha2 phosphorylation viadirect binding to the epha2 kinase domain rather thaninhibiting epha2 kinase activitybesides lca and its analogues small molecules thatinterfere with the epha2ephrin a1 system comprise thefollowing i the fxr agonist gw4064 a stilbenecarboxylic acid dosedependently disrupts the epha2ephrin a1 complex ic50 μminhibits epha2phosphorylation ic50 μm and blocks epha2activation in prostate cancer cells ii the disalicylicacidfuranyl derivative 76d10 ²²1e4e3oxopenta14diene15diylbisfuran52diylbis2hydroxybenzoic acid inhibits ephrin interaction with epha2reducing epha2 phosphorylation stimulated by ephrina1 fc and inhibiting epha2mediated cell retraction inprostate cancer cells inhibiting kinase activity of epha2the successful development of specific rtk inhibitors hasprompted subsequent efforts for identifying comparabletargets unlike other anticancer approaches targeted therapies are relatively less toxic multiple small moleculeepha2 inhibitors interacting with the intracellular kinasedomain have been describedckitdasatinib bms354825 represents an oral kinaseinhibitor simultaneously targeting breakpoint clusterregionabelson bcrablplateletderivedgrowth factor receptor pdgfr and sfks [ ]its anticancer features have been demonstrated in earlyand latephase clinical studies of chronic myelogenousleukemia cml a variety of studies have demonstratedthat dasatinib directly reduces epha2 phosphorylationand kinase activity [ ] however the promiscuous targeting profile of dasatinib makes data interpretation ambiguous dasatinib has also been recently usedas a lead structure for developing epha2inhibitors withameliorated targeting profiles the novel epha2 inhibitor candidate 4a based on dasatinib was shown tofeature an ameliorated selectivit | 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 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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 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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 |
four to nine percent of the sequences transcription are long noncoding rnas lncrnas inmammalian genomes canzio ji lncrna was regarded as the noise ofgenome transcription and did not have biological functions at ï¬rst however an increasing numberof studies have reported that lncrna is widely robinson involved in chromosomeedited bylei dengcentral south university chinareviewed byhao linuniversity of electronic science andtechnology of china chinainner mongolia university chinajuan wangcorrespondencenan dudunan05aliyuncomganfeng xiexiegfaliyuncom these authors share ï¬rst authorshipspecialty sectionthis was submitted tomolecular medicinea section of the frontiers in cell and developmentalbiologyreceived june accepted june published august citationliu z zhang y han x li c yang xgao j xie g and du n identifying cancerrelated lncrnasbased on a convolutional neuralnetwork front cell dev biol 103389fcell202000637frontiers in cell and developmental biology wwwfrontiersinaugust volume 0cliu a method to identify cancerrelated lncrnasgenomicimprintingchromatin modiï¬cationsilencingtranscriptional activationinterference andnuclear transport cheng 2018a recently it has beenproven to be associated with many kinds of cancerstranscriptionalthe secondary structure spliced form and subcellularlocalization of most lncrnas are conserved karner which is very important for lncrna to execute functionshowever compared to the functions of micrornas mirnasand proteins the function oflncrna is more diï¬cult todetermine according to the position of lncrna in the genomerelative to proteincoding genes it can be divided into ï¬ve typessense antisense bidirectional intronic and intergenicmany researchers have found lncrnas play an important rolein cancers avgeris cheng 2018b zhao and neurodegenerative diseases peng and zhao as other biological molecules zhang t bai cheng 2019a liang although manyresearchers have veriï¬ed many associations between lncrnasand cancers by biological experiments compared with ourknowledge about diseaserelated genes we still do not knowenough about diseaserelated lncrnas considering the timeand money cost of ï¬nding diseaserelated lncrnas more andmore researchers tend to use computational methods to identifydiseaserelated lncrnas these methods could be divided intothree categories machine learning methods network methodsand other methodsmachine learning methods build models based on thesimilarities of diseases orlncrnas and their biologicalcharacteristics cheng cheng 2019b zeng zou lan developed thelncrnadisease association prediction ldap which is amethod based on bagging support vector machine svm toidentify lncrnadisease associations they used similarities oflncrnas and diseases as the features yu developedcollaborative ï¬ltering naive bayesian classiï¬er cfnbc based onnaive bayesian they integrated mirnalncrna associationsmirnadisease associations and lncrnadisease associationsto infer more lncrnadisease associations considering thediscriminative contributions of the similarity association andinteraction relationships among lncrnas disease and mirnasxuan 2019a developed a dual convolutional neuralnetwork cnn with attention mechanisms to predict diseaserelated lncrnasnetwork methods are the most common way to identifyassociations between diseases and lncrnas nowadays gu yu zhang j kuang wang l liu thiskind of method would build one or multiple networks toinfer new information wang l built a lncrnamirnadisease interactive network and used their novel methodldlmd to predict associations between lncrnas and diseasessumathipala used a multilevel network topologywhich includes lncrnaprotein proteinprotein interactionproteindisease relationship to use network diï¬usion algorithmto predict diseaserelated lncrnas the graph convolutionalnetwork gcn and cnn were used on a lncrnamirnadisease network by xuan 2019b deng builtlncrna similarity network disease similarity network mirnasimilarity network and their associations then they calculatedthe metapath and feature vector for each lncrnadisease pair inthe heterogeneous information networkother methods may borrow the feature extraction methodor similarity conjecture of network methods but the core ofthis method is matrix decomposition or matrix completionlu developed the geometric matrix completionlncrnadisease association gmclda which is a methodbased on geometric matrix completion they calculated diseasesimilarity based on disease ontology do and calculatedthe gaussian interaction proï¬le kernel similarity for lncrnasthen they inferred diseaserelated lncrnas based on theassociation patterns among functionally similar lncrnas andsimilar diseases wang y proposed a weightedmatrix factorization to capture the interintraassociationsbetween diï¬erent types of nodes then they approximated thelncrnadisease association matrix using the optimized matricesand weights to predict diseaserelated lncrnas localityconstrained linear coding label propagation latent dirichletallocation llclplda was developed by xie firstly localconstraint features of lncrnas and diseases wereextracted by localityconstrained linear coding llc thenthey predicted diseaserelated lncrnas by label propagationlp strategyhowever previous methods did not consider the regulatingtarget gene expression of lncrna which is an important functionof lncrna and plays an important role in associations betweenlncrnas and diseases in addition deep learning methods arean important tool and have shown their power in bioinformaticschen lv wei wu zhao 2019abc therefore in this paper we used thisinformation as features of lncrna in addition the expressionof lncrna in diï¬erent tissues were also used as the featuresof lncrna then the deep belief network dbn was used toencode and the cnn was used to classifymethodsfeature extractiontissue expression speciï¬city of long noncodingrnacompared with proteincoding geneslncrna shows strongtissue speciï¬city the speciï¬city of lncrnas in diï¬erent kindsof tissues and cell types has been proven by many biologicalexperiments the diï¬erent expression also plays an importantrole in essential cellular processes sasaki testedthe expression of lncrnas in diï¬erent tissues and found lncrnas exhibited tissuespeciï¬c expression and oflncrnas were only expressed in one discrete tissue thereforethe expression of lncrnas in diï¬erent tissues were used asthe featureswe obtained the expression of lncrnas in diï¬erenttissues which included adipose adrenal breast colon heartkidney liver lung lymph node ovary placenta prostate testisand thyroidtherefore the dimension of each lncrnas expression featureis frontiers in cell and developmental biology wwwfrontiersinaugust volume 0cliu a method to identify cancerrelated lncrnastherefore the dimension of each lncrnas target gene featureis deep belief networkthe dbn can eï¬ectively learn complex dependencies betweenvariables zhao 2019d the dbn contains many layers ofhidden variables which can eï¬ectively learn the internal featurerepresentation of the data and can also be used as an eï¬ectivenonlinear dimensionality reduction methodwhen the observable variables are known the joint posteriorprobabilities of the hidden variables are no longer independentof each other so it is diï¬cult to accurately estimate the posteriorprobabilities of all hidden variables the posterior probability ofearly dbn is generally approximated by monte carlo methodbut its eï¬ciency is relatively low which makes its parameterlearning diï¬cult in order to eï¬ectively train the dbn weconvert the sigmoid belief network of each layer to a restrictedboltzmann machine rbm the advantage of this is that theposterior probabilities of the hidden variables are independentof each other which makes it easy to sample in this way thedbn can be regarded as being stacked from top to bottom bymultiple rbms and the hidden layer of the lth rbm is used asthe observable layer of the l 1th rbm further the dbn canbe trained quickly by layerbylayer training that is starting fromthe bottom layer and training only one layer at a time until thelast layer the speciï¬c layerbylayer training process is to trainthe rbm of each layer in turn from bottom to top assuming wehave trained the rbm in the ï¬rst l1 layer we can calculate theconditional probability of the bottomup hidden variablesphihi Ï bi wihiwhere bi is the bias of ith layer of rbm wi is the connectionweight hi is the ith layer of rbmthe process of training dbn is as followsfigure the number of target genes for each long noncoding rnalncrnafigure the distribution of the number of target genes lncrna longnoncoding rnareversetarget gene of long noncoding rnaquantitativechainreaction qrtpcr and western blot were used to testthe diï¬erentexpression genes after knocking down oroverexpressing lncrnastranscriptasepolymerasewe obtained target genes of lncrna from lncrna2targetinput train dataset Ëvn learning rate λjiang as we can see in figure there are kinds of lncrnasone lncrna has more than target genes then we drawthe distribution of the number of target genes correspondingto lncrnaasshown in figure most ofthe target genes arecorresponding to less than ï¬ve lncrnas therefore if we usedthem to be the features of lncrnas the features would be sparsetherefore we only select the most common target genes to bethe features the genes which are corresponding to more thanï¬ve lncrnas were selected as the features of lncrnas there are kinds of genes then we need to encode these genesf [g1 g2 · · · g45]where g1 denotes the ï¬rst gene of these genes and f denotesthe feature of lncrna for each lncrna if g1 is the target geneof it then g1 otherwise g1 output weight matrix wl bias al and blfor l 1linitialization wi al bi sample from train dataset Ëh0for i lsample hi based on phi Ëhiendset hi1as the train sample to train lth layer ofrbmendsince the dimension of expression feature and target genefeature are diï¬erent we should reduce the dimension of targetgene feature and make it the same as the expression featurestherefore in this paper two layers of rbm were used to builda dbn modelthe number of nodes oftheand respectively sigmoid function was used astwo layers was thefrontiers in cell and developmental biology wwwfrontiersinaugust volume 0cliu activation functionÏ x extherefore the dimension of ï¬nal features is f cid20 g1 g2 · · · g13e1 e2 · · · e13 cid21a method to identify cancerrelated lncrnasconvolutional neural networkthe power of cnn in dealing with bioinformatic problems hasbeen proven by many researchers we selected cnn as theclassiï¬er based on two reasons the dimension of features is which can be regarded as an image the outstandingperformance of cnn in image classiï¬cationthere are ï¬ve layers in our cnn model the structure of cnnis shown as table where g1 g2 · · · g13 denotes target gene feature after dbnand e1 e2 · · · e13 denotes the expression of lncrnas in diï¬erent tissuestable the structure of convolutional neural network cnnlayersparameterconvolutional layerpooling layerconvolutional layerpooling layerfully connected layeroutputfilter kernel size activation function tanhpool size activation function tanhfilter kernel size activation function tanhpool size activation function tanhunits activation function tanhunits activation function sigmoidwork framefigure shows the work frame of our method dbncnnthere are three steps of our methods firstly we should extractfeatures of lncrnas there are two parts of features expressionfeature and target gene feature then dbn was used to encodethe target gene feature after encoding the two kinds of featureswere combined together finally cnn was used to classifyresultsdata descriptionthe known associations between lncrna and diseases wereobtained from lncrnadisease database bao wetotally obtained kinds of cancerrelated lncrnas the numberof their corresponding lncrnas is shown as figure as shown in figure peoples understanding of cancerrelated lncrnas varies widely we have known more than lncrnas for some cancers but few lncrnas are known for somecancers to better build our model we only selected cancerswhich have more than related lncrnas therefore kindsof cancers were selectedfigure work frame of deep belief network dbnconvolutional neural network cnn lncrna long noncoding rnafrontiers in cell and developmental biology wwwfrontiersinaugust volume 0cliu a method to identify cancerrelated lncrnasfigure the number of long noncoding rnas lncrnas for each cancertable the performance of deep belief network dbnconvolutional neuralnetwork cnn in cancerscancerarea undercurve aucarea under precisioncurve auprcervical cancerbreast cancercolorectal cancerstomach cancerurinary bladder cancerlung cancerovarian cancerthyroid cancerprostate cancerliver cancerpancreatic cancerovarian epithelial cancergallbladder cancerendometrial cancercolon canceresophageal cancerthetargetgenes oflncrnas were obtained fromlncrna2target database we have discussed about this insection target gene of long noncoding rnafigure the receiver operating characteristic roc curves of the threemethods dbn deep belief network cnn convolutional neural network pcaprincipal component analysisfigure the area under the precisionrecall curve aupr of the threemethods dbn deep belief network cnn convolutional neural network pcaprincipal component analysisthe expression oftissues wasobtained from noncodev5 zhao we only usedhuman datalncrnas in diï¬erentthe performance of deep beliefnetworkconvolutional neural networkwe did 10cross validation on each cancer area under the curveauc cheng dao zhang and areaunder the precisionrecall curve aupr were used to evaluatethe performance of dbncnn the results are shown in table as we can see in table the performance of dbncnn isquite diï¬erent in diï¬erent cancers this may be caused by thediï¬erent sample sizes the average auc is and aupr is comparison experimentsto verify the superior of dbncnn we compared it with similarmethods since the main function of dbn is to reduce dimensionprincipal component analysis pca has the same functiontherefore instead of using dbn to encode we used pca thistime and cnn was used to classify the features after pca we callthis method pcacnn in addition we also used the deep neuralnetwork dnn to replace cnn so this comparison method wascalled dbndnnwe used these three methods to test on cancers andsummarized the results to get a ï¬nal auc and aupr for eachmethod the receiver operating characteristic roc curves areshown in figure as shown in figure the blue curve denotes the results ofdbncnn the red and black curves denote pcacnn anddbndnn respectively as we can see dbncnn performedbest among these three methods the auc of dbncnn is which is better than and for pcacnn anddbndnn respectivelyfrontiers in cell and developmental biology wwwfrontiersinaugust volume 0cliu a method to identify cancerrelated lncrnasas shown in figure the aupr of dbncnn is the highestwith the least standard errorcase studyliu found down syndrome cell adhesion molecule antisense rna dscamas1 is associated with breast cancerby constructing two suppression subtracted cdna librariesmartensuzunova reported the associationbetween h19 and bladder cancer they also pointed out that h19could be the biomarker of bladder cancershi measured the expression level of lncrnasloc554202 in breast cancer tissues and found that loc554202was signiï¬cantly increased compared with normal control andassociated with advanced pathologic stage and tumor sizeconclusionsincreasing evidence has shown the relationship between lncrnasand cancers lncrnas could be the biomarkers to help diagnosecancer and also help researchers understand the mechanismof cancers compared with peoples knowledge of diseaserelated protein coding genes we knew few about diseaserelated lncrnas however the biological experiments for ï¬ndingdiseaserelated lncrnas are timeconsuming and expensivetherefore in this paper we proposed a novel method foridentifying cancerrelated lncrnas we called this methoddbncnn which is a fusion of dbn and cnn two kindsof features were used based on the biological background sincelncrnas have tissuespeciï¬c expression and the expression ofcancer tissues is diï¬erent from normal tissues the expressionoftissues could provide importantin diï¬erentlncrnasreferencesavgeris m tsilimantou a levis p k tokas t sideris d c stravodimosk loss of gas5 tumour suppressor lncrna an independentmolecular cancer biomarker for shortterm relapse and progression in bladdercancer patients br j cancer 101038s4141601803206bai y dai x ye t zhang p yan x gong x plncrnadba repository of plant lncrnas and lncrnarbp protein interactions currbioinform bao z yang z huang z zhou y cui q and dong d lncrnadisease an updated database of long noncoding rnaassociateddiseases nucleic acids res d1034d1037 101093nargky905canzio d nwakeze c l horta a rajkumar s m coï¬ey e l duï¬y ee antisense lncrna transcription mediates dna demethylationto drive stochastic protocadherin α promoter choice cell 653e15 101016jcell201903008chen x shi w and deng l prediction of disease comorbidity usinghetesim scores based on multiple heterogeneous networks curr gene ther cheng l computational and biological methods for gene therapy currgene ther cheng l hu y sun j zhou m and jiang q 2018a dincrna afor exploring diseasecomprehensive webbased bioinformaticsassociations 101093bioinformaticsbty002ncrna functionbioinformaticstoolkitandcheng l jiang y ju h sun j peng j zhou m 2018busingcrossontologyinfacrontsimilaritiescalculatingtermtheirexecutelncrnasinformation for us to identify cancerrelated lncrnas inadditionregulation function byinteracting with their target genes therefore the target genesof lncrnas can also be the features of lncrnas to encode thefeatures dbn was used to reduce the dimension finally cnnwas used to identify real cancerrelated lncrnas based on theï¬nal featureto verify the eï¬ectiveness of our method we compareddbncnn with pcacnn and dbndnn since pca canalso reduce the dimension of features and dnn can also doclassiï¬cation the results showed that dbncnn performedbest finally case studies have been done to verify the accuracy ofour results we found potential lncrnas for kinds of cancerswhich can be a kind of guidance for researchers ï¬nding novelcancerrelated lncrnasdata availability statementthe datasets presented in this study can be found in onlinerepositoryrepositoriesrepositories theandnumbersbethesupplementary materialaccessionnamesfoundcantheofinauthor contributionsnd and gx designed the research zl performed the researchand wrote the manuscript yz and xh acquired the dataand reviewed and edited the manuscript cl xy and jganalyzed the data all authors reviewed the manuscript andprovided commentsinformation ï¬ow by a random walk bmc genomics 19suppl 101186s1286401743386cheng l yang h zhao h pei x shi h sun j 2019a metsigdisa manually curated resource for the metabolic signatures of diseases briefbioinform 101093bibbbx103cheng l zhao h wang p zhou w luo m li t 2019bcomputational methods for identifying similar diseases molecular therapynucleic acids 101016jomtn201909019dao f y lv h zulï¬qar h yang h su w gao h acomputational platform to identify origins of replication sites in eukaryotesbrief bioinform 101093bibbbaa017 [epub ahead of print]deng l li w and zhang j ldah2v exploring metapaths acrossmultiple networks for lncrnadisease association prediction ieeeacmtransac comput biol bioinform 101109tcbb20192946257 [epubahead of print]gu c liao b li x cai l li z li k global network randomwalk for predicting potential human lncrnadisease associations sci rep 101038s4159801712763zjij tangj xia kjandjiang rtumorigenesis microenvironment currbioinformlncrna injiang q wang j wu x ma r zhang t jin s lncrna2targeta database for diï¬erentially expressed genes after lncrna knockdown oroverexpression nucleic acids res d193d196 101093nargku1173karner h webb ch carmona s liu y lin b erhard m functional conservation of lncrna jpx despite sequence and structuraldivergence j mol biol 101016jjmb201909002frontiers in cell and developmental biology wwwfrontiersinaugust volume 0cliu a method to identify cancerrelated lncrnaskuang l zhao h wang l xuan z and pei t a novel approachbased on point cut set to predict associations of diseases and lncrnas currbioinform lan w li m zhao k liu j wu fx pan y ldap a webserver for lncrnadisease association prediction bioinformatics 101093bioinformaticsbtw639liang c changlu q he z tongze f and xue z gutmdisorder acomprehensive database for dysbiosis of the gut microbiota in disorders andinterventions nucleic acids res liu d rudland p sibson d and barraclough r identiï¬cation ofmrnas diï¬erentiallyexpressed between benign and malignant breast tumourcells br j cancer 101038sjbjc6600456liu x hong z liu j lin y alfonso rp zou q computational methods for identifying the critical nodes in biologicalnetworks brief bioinform 101093bibbbz011lu c yang m li m li y wu f and wang j predicting humanlncrnadisease associations based on geometric matrix completion ieee jbiomed health inform 101109jbhi20192958389 [epub ahead of print] protein function predictionto deep learning proteomics 19e1900119lv z b ao c y and zou qfrom traditionalclassiï¬er 101002pmic201900119martensuzunova e s böttcher r croce c m jenster g visakorpi t andcalin g a long noncoding rna in prostate bladder and kidneycancer eur urol 101016jeururo201312003peng j and zhao t reduction in tom1 expression exacerbatesalzheimers disease proc natl acad sci usa 101073pnas1917589117robinson e k covarrubias s and carpenter s the how and why oflncrna function an innate immune perspective biochim biophys acta generegul mech 101016jbbagrm2019194419sasaki y t sano mideue t kin t asai k and hirose t identiï¬cation and characterization of human noncoding rnas withtissuespeciï¬c expression biochem biophys res commun 101016jbbrc200704034sumathipala m maiorino e weiss s t and sharma ashi y lu j zhou j tan x he y ding j long noncodingrna loc554202 regulates proliferation and migration in breast cancer cellsbiochem biophys res commun 101016jbbrc201402144network diï¬usion approach to predictlncrna disease associationsusing multitype biological networks lion front physiol 103389fphys201900888wang l xuan z zhou s kuang l and pei t a novel modelassociations based on the lncrnafor predicting lncrnadiseasemirnadisease interactive network curr bioinformwang y yu g wang j fu g guo m and domeniconi c weightedmatrix factorization on multirelational data for lncrnadisease associationprediction methods 101016jymeth201906015wei l su r wang b li x zou q and gao x integrationof deep feature representations and handcrafted featuresto improvethe prediction of n 6methyladenosine sites neurocomputing 101016jneucom201804082wu b zhang h lin l wang h gao y zhao l a similarity searching system for biological phenotype images using deepconvolutional encoderdecoder architecture curr bioinform xie g huang s luo y ma l lin z and sun y llclplda a novelmodel for predicting lncrnadisease associations mol genet genomics 101007s00438019015908xuan p cao y zhang t kong r and zhang z2019a dualconvolutional neural networks with attention mechanisms based methodfor predicting diseaserelated lncrna genes front genet 103389fgene201900416xuan p pan s zhang t liu y and sun h 2019b graph convolutionalnetwork and convolutional neural network based method for predictinglncrnadisease associations cells 103390cells8091012yu g fu g lu c ren y and wang j brwlda birandomwalks for predicting lncrnadisease associations oncotarget 1018632oncotarget19588yu j xuan z feng x zou q and wang l a novel collaborativeï¬ltering model for lncrnadisease association prediction based on the naïvebayesian classiï¬er bmc bioinform 101186s1285901929850zeng x x wang w deng g s bing j x and zou q prediction ofpotential diseaseassociated micrornas by using neural networks mol thernucleic acids 101016jomtn201904010zhangand deng lj zhang z chen zintegratinglncrnadisease associationieeeacm transac comput biol bioinform multiple heterogeneous networks for novelinference 101109tcbb20172701379zhang t tan p wang l jin n li y zhang l rnalocate aresource for rna subcellular localizations nucleic acids res d135d138 101093nargkw728zhang z m tan j x wang f dao f y zhang z y and linh early diagnosis of hepatocellular carcinoma using machinelearning method front bioeng biotechnol 103389fbioe2020zhao t cheng l zang t and hu y 2019a peptidemajor histocompatibilitycomplex class i binding prediction based on deep learning with novel featurefront genet 103389fgene201901191and cheng lidentifyingalzheimers diseaserelated proteins by lrrgd bmc bioinform 101186s1285901931247zhao t hu y zang t2019bzhao t hu y zang t and cheng l mrtfb regulates the expressionof nomo1 in colon proc natl acad sci usa 101073pnas2000499117zhao t hu y zang t and wang y 2019c integrate gwas eqtland mqtl data to identify alzheimers diseaserelated genes front genet 103389fgene201901021zhao t wang d hu y zhang n zang t and wang y 2019d identifyingalzheimers diseaserelated mirna based on semiclustering curr gene ther zhao y li h fang s kang y wu w hao y noncode an informative and valuable data source of long noncoding rnas nucleicacids res d203d208 101093nargkv1252zou q xing p wei l and liu b gene2vec gene subsequenceembedding for prediction of mammalian n6methyladenosine sites frommrna rna 101261rna069112118conï¬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 liu zhang han li yang gao xie and du this is an openaccess distributed under the terms of the creative commons attribution license ccby the use distribution or reproduction in other forums is permitted providedthe original authors and the copyright owners are credited and that the originalpublication in this is cited in accordance with accepted academic practiceno use distribution or reproduction is permitted which does not comply with thesetermsfrontiers in cell and developmental biology wwwfrontiersinaugust volume 0c' | Colon_Cancer |
objectives paired box protein8 pax8 immunohistochemical expression can be used as a diagnostic marker for epithelial cells tumors this study aimed at investigating the immunohistochemical expression of pax8 among sudanese females diagnosed with cervical endometrial and ovarian cancers between december and may by studying their formalinfixed paraffin embedded blocksresults sixty patients diagnosed with female reproductive tract cancers were included who aged ± years range cervix was the most common cancer site in patients regarding cancer stage there was and of the study population had stage 3b and 2b respectively the histopathological diagnosis included and poorly moderately and well differentiated cervical squamous cell carcinoma scc as well as and endometrial adenocarcinoma metastatic adenocarcinoma endocervical adenocarcinoma and ovarian mucinous cyst adenocarcinoma respectively pax8 was positively expressed in endometrial adenocarcinoma endocervical adenocarcinoma and ovarian mucinous cyst adenocarcinoma poorly and moderately differentiated scc all patients diagnosed with well differentiated scc and metastatic adenocarcinoma showed no expression of pax8 a statistically significant was seen for pax8 expression and the different histopathological diagnosis p value keywords female reproductive cancer paired box protein8 immunohistochemical expressionintroductionpaired box protein8 pax8 is a member of the family paired box proteins paxs [ ] pax8 consists of amino acids with a molecular weight of approximately kilo dalton and its molecular properties are located on chromosome 2q13 [] pax8 is a transcription factor that regulates ans development during the embryonic period as well as to maintain normal cellular functions in some cells after birth [ ] during the embryonic period pax8 also plays a significant role correspondence nouh_saadoutlookcom alfarrabi college for science and technology khartoum sudanfull list of author information is available at the end of the in the development of genital ans derived from the mesonephric and the m¼llerian ducts [] in a previous experiment the deletion of the pax8 gene resulted in dysfunctional uterus absence of the endometrium and the vaginal opening also resulted in poor development of the myometrial tissue several studies have described the immunohistochemical utility of pax8 as a diagnostic marker for epithelial cells neoplasms of many glands and ans such as thyroid thymus and kidney as well as some female reproductive tract tumors [ ]in a healthy female reproductive tract pax8 shown to be overexpressed in the epithelial cells of the endocervix and the endometrium [] pax8 was found to be expressed among endometrioid carcinomas transitional 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 0cali a0et a0al bmc res notes page of undifferentiated cell carcinomas and the metastatic carcinomas at a range of and [ ] whereas for the ovarian carcinomas pax8 was under expressed considering that few studies have investigated the immunohistochemical expression of pax8 in carcinomas of the endometrium and uterine cervix in the different parts of the world but none from sudan yet [ ] this study aimed at investigating the immunohistochemical expression of pax8 among sudanese females diagnosed with cervical endometrial and ovarian carcinomasmain textmaterials and a0methodsstudy design and a0population characteristicsthis is a descriptive retrospective hospital based study conducted at different histopathology laboratories during the period from december till may in khartoum state sudan we retrieved archived formalin fixed paraffin embedded blocks previously collected from female patients with cervical endometrial or ovarian carcinomas the retrieved formalin fixed paraffin blocks represent all the female population admitted at the hospitals for reproductive malignancies diagnosis the participants demographic data was collected including age place of residence the clinical data including site of cancer cancer grade and the histopathological diagnosis were also collectedsections preparation for a0immunohistochemistry stainingtwo sections were cut using rotary microtome leica germany from each histopathological block then one slide was stained by hematoxylin and eosin staining technique the other slide was mounted onto 3aminopropyltriethoxysilane coated slides for immunohistochemistry to retrieve pax8 tissues antigen we treated the sections with citrate buffer at ° a0c for a0min in a waterbath then the tissue sections were rinsed first in distilled water and later with tris buffer saline tbs this was followed by treatment with peroxidase block hydrogen peroxide in methyl alcohol for a0min to quench endogenous peroxidase activity the slides were then placed in a humid chamber then the slides were drained and rinsed in two successive changes of tris buffer wash buffer for a0 min each nonspecific proteinprotein interactions were blocked by incubating and treating the tissue sections in a humid chamber with the power block casein in phosphate buffered saline for a0 min then the remaining solution was drained from the slides the sections were then incubated in the primary antibody pax8 antipax8 rabbit antihuman monoclonal antibody ab189249 abcam united kingdom at room temperature in the humid chamber according to the manufacture instructionsobserving the yellowishbrown or brown appearance of the nucleus was considered a positive result for the pax8 for the negative control we omitted the incubation with the primary antibody step instead we incubated the section in the phosphate buffer saline pbsresults interpretationsfor the interpretation of the results we depended on the intensity as well as the number of the cells that expressed the marker and the expression was graded into categories negative no staining less than of the cells were expressing the marker of the cells were expressing the marker more than of the cells were expressing the marker more than of the cells were expressing the marker the slides were interpreted and validated by two expert pathologists blindly of each other results photomicrographs were taken using olympus sp350 camera olympus imaging america inc usastatistical analysisthe statistical analysis of the results was done using ibm spss statistics vs the chisquared test was performed to compare the frequencies of categorical variables statistical significance level was defined as p value at confidence intervalresultscharacteristics of a0the a0study participantsthe study included patients diagnosed with female genital tract cancer patients aged ± a0years range a0years patients were grouped into age groups those aged a0 years constituted half of the study participants the remaining were and patients distributed across the remaining age groups of a0 years a0 years and a0 years respectively according to patients place of residence patients were originating from the four regions of sudan most of the patients were from western part of sudan followed by from the central part of sudanregarding the site of cancer the cervix was the most commonly involved patients there were and endometrial and ovarian cancer respectively based on the international federation of gynecology and obstetrics figo cancer grading the majority of the study population was diagnosed with stage 3b and 2b cancer and of the patients respectively the were and stage 4b 3a 2a 1b and 4a respectively 0cali a0et a0al bmc res notes page of no statistically significant association between figo staging and age group was found p value histologically there were squamous cell carcinoma scc all of which were cervical cancers and adenocarcinoma scc and adenocarcinoma were further classified into poorly differentiated scc moderately differentiated scc and well differentiated scc endometrium adenocarcinoma metastatic adenocarcinoma endocervical adenocarcinoma and ovarian mucinous cyst adenocarcinomabased on age groups age group showed no statistically significant relationship with either patients place of residence cancer site cancer histological type figo staging and cancer histopathological type table a0immunohistochemical expression of a0paxthe immunohistochemical expression of pax8 was shown as a yellowishbrown or brown staining of the nucleus fig a0 based on site of cancer all endometrium carcinoma showed positive expression of pax8 with p value there were only patients who had positive expression of pax8 including adenocarcinoma and scc a statistically significant difference was noted for the pax8 staining and cancer type with p value the analysis of pax8 staining results based on the histopathological diagnosis showed that all patients who were diagnosed with well differentiated scc and metastatic adenocarcinoma had negative results for the pax8 expression while of the endometrium adenocarcinoma were found positive for the pax8 expression a statistically significan was t seen for pax8 expression and the different histopathological diagnosis p value table a0table classification of a0participants demographic and a0clinical diagnosis based on a0age groupage group no total no p value a0years a0years a0years a0yearsresidence of patient central sudan east sudan west sudan north sudansite of cancer cervix endometrium ovarycancer histological type scc adenocarcinomafigo staging stage stage 2a stage 2b stage 3a stage 3b stage 4a stage 4bhistopathological cancer grades well differentiated scc poorly differentiated scc moderately differentiated scc endometrium adenocarcinoma endocervical adenocarcinoma metastatic adenocarcinoma ovarian mucinous cyst adenocarcinomascc squamous cell carcinoma 0cali a0et a0al bmc res notes page of fig immunohistochemical expression of pax8 among the different histopathological cancer types and grades the immunohistochemical expression of pax8 is shown as a yellowishbrown or brown staining of the nucleus a well differentiated scc negative b metastatic adenocarcinoma negative c poorly differentiated scc positive d moderately differentiated scc positive e endometrium adenocarcinoma positive f ovarian mucinous cystadenocarcinoma positive g endocervical adenocarcinoma positive and h endometroid adenocarcinoma positive 0cali a0et a0al bmc res notes page of table association of a0clinical diagnosis and a0the a0immunohistochemical expression of a0pax8pax results no total no p valuepositivenegativecancer histological type scc adenocarcinomacancer site cervix endometrium ovaryfigo staging stage stage 2a stage 2b stage 3a stage 3b stage 4a stage 4bcancer histopathological grading well differentiated scc poorly differentiated scc moderately differentiated scc endometrium adenocarcinoma endocervical adenocarcinoma metastatic adenocarcinoma ovarian mucinous cyst adenocarcinomascc squamous cell carcinoma discussionprevious studies on the immunohistochemical expression of pax8 in the normal female reproductive tract showed that pax8 was expressed in the endometrial endocervical and ovarian epithelial cells as well as in nonciliated epithelial cells of the fallopian tubes [ ] this study investigated the immunohistochemical expression of pax8 in sudanese patients who were diagnosed with female reproductive tract cancers patients on the 5th decade of life were constituting half of the study participants with no statistically significant association between age group and the type of cancer however previous studies had suggested other risk factors which could contribute in the development of certain gynecological cancer [ ]regarding the place of residence the majority of patients coming from western sudan this result is in contrary with a previous study in sudan conducted by saeed et a0al in which they showed that the percentage of patients suffering from different types of cancers residing in central and northern sudan were higher compared to the other regions in sudan nevertheless these findings could suggest the involvement of environmental risk factors however the limited study samples size is insufficient to support this suggestion therefore further research with a larger samples size investigating the potential environmental risk factors is essential for strategic prevention and protection measuresthe reported number of female patients with cervical cancer was high compared to ovarian and endometrium cancer similar results were seen previously among sudanese females also the high frequency of stages 3b and 2b compared to the other stages were comparable to previous study conducted in sudan this similarity underscores a delayed response among sudanese females in seeking healthcare and urge the need for health promotion and education to encourage young sudanese females for the early signs detection and seeking healthcare as early as possible for a better treatmentregarding the classification based on the histopathological diagnosis most of the female diagnosed with scc this result was also similar to previous study investigated the prevalence of the different gynecologic cancer in sudan however the expression of pax8 among the studied samples was relatively low compared to previous studies [ ] this could be attributed 0cali a0et a0al bmc res notes page of to the site of cancer development while agrees with another study where pax8 was expressed only in patient interestingly a high frequency of pax8 expression was noted among females diagnosed with endometrium cancer compared to scc this finding is in contrary with a previous report where pax8 was expressed among only of the studied samples also the result was strongly in accordance with other studies [ ] besides that the lack of pax8 expression among those who were diagnosed with well differentiated scc and metastatic adenocarcinoma could play a significant role in either gynecologic cancer differentiation or in detection of endometrium adenocarcinoma progression to metastatic adenocarcinoma [ ]conclusionalthough pax8 showed a significant expression among adenocarcinomas lesions and negative expression was noted among those with well differentiated scc and metastatic adenocarcinoma pax8 might not be beneficial when used alone as a diagnostic marker for the tumors that occur in the female reproductive tractlimitations¢ the small sample size investigated in this study reduced the ability of using the expression of pax8 as a diagnostic marker therefore a largescale study is needed and it should include other types of malignant tumors encountered in the female reproductive systemacknowledgementsthe authors would like to acknowledge the medical staff for their interest and cooperation during the study and thanks to all who participated in completing this studyauthors contributionseta nsm and ees provided conceptual framework for the study guidance for interpretation of the data and performed data analysis eta ees irs lah and amm performed laboratory work nsm ees msm aay and aa performed the statistical analysis nsm msm ees and aa participated in the manuscript preparation revision and coordination all authors read and approved the final manuscriptfundingnot applicableavailability 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 participateethical approval was obtained from the research ethics committee of the faculty of medical laboratory sciences university of khartoum sudan ethical approval no fmlsrec002042 all participant approved to participate by signing an informed consentconsent for publicationnot applicablecompeting interestsno competing interests to discloseauthor details department of histopathology and cytology faculty of medical laboratory sciences university of khartoum khartoum sudan department of histopathology and cytology faculty of medical laboratory sciences national university khartoum sudan alfarrabi college for science and technology khartoum sudan faculty of medicine sinnar university sennar sudan molecular biology department faculty of medical laboratory sciences nile university khartoum sudan faculty of dentistry ibn sina university khartoum sudan department of neurology mayo clinic jacksonville fl usa department of radiology mayo clinic jacksonville fl usa institute of endemic diseases university of khartoum khartoum sudan mycetoma research center university of khartoum khartoum sudan faculty of medicine nile university khartoum sudan received july accepted august references gruss p walther c pax in development cell mansouri a hallonet m gruss p pax genes and their roles in cell differentiation and development curr opin cell biol macchia pe lapi p krude h pirro mt missero c chiovato l souabni a baserga m tassi v pinchera a pax8 mutations associated with congenital hypothyroidism caused by thyroid dysgenesis nat genet vilain c rydlewski c duprez l heinrichs c abramowicz m malvaux p renneboog bt parma j costagliola s vassart g autosomal dominant transmission of congenital thyroid hypoplasia due to lossoffunction mutation of pax8 j clin endocrinol metab park s vk c genetics of congenital hypothyroidism j med genet dahl e koseki h balling r pax genes and anogenesis bioessays lang d powell sk plummer rs young kp ruggeri ba pax genes roles in development pathophysiology and cancer biochem pharmacol stoykova a gruss p roles of paxgenes in developing and adult brain as suggested by expression patterns j neurosci mittag j winterhager e bauer k grummer r congenital hypothyroid female pax8deficient mice are infertile despite thyroid hormone replacement therapy endocrinology bouchard m de caprona d busslinger m xu p fritzsch b pax2 and pax8 cooperate in mouse inner ear morphogenesis and innervation bmc dev biol mittag j winterhager e bauer k grummer rje congenital hypothyroid female pax8deficient mice are infertile despite thyroid hormone replacement therapy endocrinolog laury ar perets r piao h krane jf barletta ja french c chirieac lr lis r loda m hornick jl a comprehensive analysis of pax8 expression in human epithelial tumors am j surg pathol wong s hong w hui p buza n comprehensive analysis of pax8 expression in epithelial malignancies of the uterine cervix int j gynecol pathol ozcan a shen ss hamilton c anjana k coffey d krishnan b truong ld pax expression in nonneoplastic tissues primary tumors and metastatic tumors a comprehensive immunohistochemical study mod pathol bowen nj logani s dickerson eb kapa lb akhtar m benigno bb mcdonald jf emerging roles for pax8 in ovarian cancer and endosalpingeal development gynecol oncol 0cali a0et a0al bmc res notes page of ozcan a liles n coffey d shen ss truong ld pax2 and pax8 expression in primary and metastatic m¼llerian epithelial tumors a comprehensive comparison am j surg pathol distinguishing ovarian mucinous neoplasms from colonic and appendiceal mucinous neoplasm bmc res notes nesrin r kilic d risk factors for cervical cancer results from a hospital ozcan a liles n coffey d shen ss truong ldjtajosp pax2 and pax8 based casecontrol study int j hematol oncol expression in primary and metastatic m¼llerian epithelial tumors a comprehensive comparison am j surg pathol nonaka d tang y chiriboga l rivera m ghossein r diagnostic utility of thyroid transcription factors pax8 and ttf2 foxe1 in thyroid epithelial neoplasms mod pathol tacha d zhou d cheng l expression of pax8 in normal and neoplastic tissues a comprehensive immunohistochemical study appl immunohistochem mol morphol bowen nj logani s dickerson eb kapa lb akhtar m benigno bb mcdonald jfjgo emerging roles for pax8 in ovarian cancer and endosalpingeal development gynecol oncol k¶bel m kalloger se boyd n mckinney s mehl e palmer c leung s bowen nj ionescu dn rajput a ovarian carcinoma subtypes are different diseases implications for biomarker studies plos medicine 2008512e232 nonaka d chiriboga l soslow ra expression of pax8 as a useful marker in distinguishing ovarian carcinomas from mammary carcinomas am j surg pathol tong gx devaraj k hamelebena d yu wm turk a chen x wright jd greenebaum e pax8 a marker for carcinoma of m¼llerian origin in serous effusions diagn cytopathol laury ar perets r piao h krane jf barletta ja french c chirieac lr lis r loda m hornick jljtajosp a comprehensive analysis of pax8 expression in human epithelial tumors am j surg pathol tong gx devaraj k hamelebena d yu wm turk a chen x wright jd greenebaum ejdc pax8 a marker for carcinoma of m¼llerian origin in serous effusions diagn cytopathol chu pg chung l weiss lm lau sk determining the site of origin of mucinous adenocarcinoma an immunohistochemical study of cases am j surg pathol brunner ah riss p heinze g meltzow e brustmann h immunoexpression of pax in endometrial cancer relation to highgrade carcinoma and p53 int j gynecol pathol ozcan a shen ss hamilton c anjana k coffey d krishnan b truong ldjmp pax expression in nonneoplastic tissues primary tumors and metastatic tumors a comprehensive immunohistochemical study mod pathol aldaoud n erashdi m alkhatib s abdo n almohtaseb a graboskibauer a the utility of pax8 and satb2 immunohistochemical stains in saeed me cao j fadul b kadioglu o khalid he yassin z mustafa sm saeed e efferth t a fiveyear survey of cancer prevalence in sudan anticancer res saeed me cao j fadul b kadioglu o khalid he yassin z mustafa sm saeed e efferth tjar a fiveyear survey of cancer prevalence in sudan anticancer res mohamed keh ashmeig aaa cervical cancer our experience in sudan philadelphia aacr elhasan lme bansal d osman of enan k abd farag eab prevalence of human papillomavirus type in sudanese women diagnosed with cervical carcinoma j cancer res ther tacha d zhou d cheng ljai morphology m expression of pax8 in normal and neoplastic tissues a comprehensive immunohistochemical study appl immunohistochem mol morphol ord³±ez ng value of pax immunostaining in tumor diagnosis a review and update adv anat pathol gailey mp bellizzi am immunohistochemistry for the novel markers glypican pax8 and p40 δnp63 in squamous cell and urothelial carcinoma am j clin pathol yemelyanova a gown am holmes bj ronnett bm vang r pax8 expression in uterine adenocarcinomas and mesonephric proliferations int j gynecol pathol liang l zheng w liu j liang sx assessment of the utility of pax8 immunohistochemical stain in diagnosing endocervical glandular lesions arch pathol lab med wong s hong w hui p buza njijogp comprehensive analysis of pax8 expression in epithelial malignancies of the uterine cervix int j gynecol pathol de andrade dap da silva vd de macedo mg de lima ma de andrade vm andrade cemc schmidt rl reis rm dos reis r squamous differentiation portends poor prognosis in low and intermediaterisk endometrioid endometrial cancer plos one 20191410e0220086publishers 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 |
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 |
tenascinc tnc is an extracellular matrix ecm glycoprotein that plays an important rolein cell proliferation migration and tumour invasion in various cancers tnc is one of themain protein overexpressed in breast cancer indicating a role for this ecm molecule in cancer pathology in this study we have evaluated the tnc lossofffunction in breast cancercells in our approach we used dsrna sharing sequence homology with tnc mrna calledatnrna we present the data showing the effects of atnrna in mdamb231 cells bothin monolayer and threedimensional culture cells treated with atnrna were analyzed forphenotypic alterations in proliferation migration adhesion cell cycle multicaspase activation and the involvement in epithelial to mesenchymal transition emt processes as complementary analysis the oncogenomic portals were used to assess the clinical implication oftnc expression on breast cancer patients survival showing the tnc overexpression associated with a poor survival outcome our approach applied first in brain tumors and then inbreast cancer cell lines reveals that atnrna significantly diminishes the cell proliferationmigration and additionally reverses the mesenchymal cells phenotype to the epithelial onethus tnc could be considered as the universal target in different types of tumors wheretnc overexpression is associated with poor prognosisa1111111111a1111111111a1111111111a1111111111a1111111111open accesscitation wawrzyniak d grabowska m gÅodowiczp kuczyÅski k kuczyÅska b fedorukwyszomirska a downregulation oftenascinc inhibits breast cancer cells developmentby cell growth migration and adhesionimpairment one e0237889 101371 pone0237889editor lucia r languino thomas jeffersonuniversity united statesreceived may accepted august published august copyright wawrzyniak this is anopen access distributed under the terms ofthe creative 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 supportingintroductioninformation filesfunding this work was supported by the ministryof science and higher education of the republic ofpoland by know program kk was supported byncbr program powr03020000i03216competing interests the authors have declaredthat no competing interests existthe tumor microenvironment is composed of the surrounding stromal cells such as endothelialcells in blood vessels immune cells fibroblasts and the extracellular matrix ecm [ ] during carcinogenesis is often perturbed and deregulated while during embryonic development isstrictly controlled to maintain homeostasis in tumors the composition of the ecm differsfrom that of normal tissue and enables new interactions that affect the function of cancer cellsand are critical in modulating invasion associated with cell migration and growth the tumorassociated ecm presents several tumorassociated antigens that are generally more abundant one 101371 pone0237889 august one 0cdownregulation of tenascinc inhibits breast cancer cells developmentand possibly more stable than those of the cell surface [] consequently these proteins represent possible valuable targets for tumor imaging and therapy [ ] ecm proteins such as fibronectin fn and tenascin have isoforms that are expressed in a tissue specific manner generatedby alternative splicing of their primary transcripts one of the most consistent isoform changesin the ecm of many tumors is the upregulation of the glycoprotein tenascinc tnc tncalongside tenascinx tnx tenascinr tnr and tenascinw tnn are members of wellconserved among vertebrates tenascin family tn [] numerous isoforms of tnc can beproduced through alternative splicing of nine fibronectin type iii regions between repeats and at the premrna level there is a considerable amount of literature on the contribution of different splicingdependent tnc domains in specific biological functions changes in thetnc isoforms expression pattern have been then described in a number of malignancies andtheir nature appears to be tumortype specific recent studies have demonstrated that somesplice variants are specific to diseased tissues [] in breast tissues expression of two tncvariants one containing domain d and the other both b and d was found to be associated withinvasive phenotype tnc promotes cell migration angiogenesis inhibit focal contact formation and also act as a cell survival factor [] its importance was found in the development and progression of different types of neoplasm including colon and breast cancerfibrosarcoma lung cancer melanoma squamous cell carcinoma bladder cancer and prostaticadenocarcinoma [ ] tnc is also highly expressed in highgrade gliomas which correlatesas well with the invasiveness of glioma cells [] in the brain it is important for the development of neural stem cells [ ] and moreover is suspected to be a potential marker for glioblastoma multiforme gbm stem cells gsc previously we have shown that tnc is overexpressed in gbm and can be a good target inrnai approach with 164nt long dsrna complementary to the mrna of tnc which wecalled atnrna we conducted the experimental therapy for gbm patients the discoverythat tnc presents a dominant epitope in glioblastoma prompted us to investigate the potentialof atnrna to block the tnc expression and its effect on the growth of human breast cancers where tnc overexpression was also established and linked with the highest malignancyinvasion capability and metastasis ability this view is supported by mock who showedthat gbm patients with antibodies against the egflike repeats of tnc antibody targetvcedgftgpdcae have a significantly better prognosis than other patients thus weassumed that in the light of the satisfactory results of brain tumors experimental therapy breastcancer could be the next possible object of interest to establish the atnrna approachhere we demonstrate that atnrna approach can be successfully used in breast cancercells impairing the basic hallmarks of tumor cells with the performed analysis of proliferation migration rate multicaspases induction pathway cell cycle analysis spheroids viabilityand the involvement of tnc in emt induction we have then interrogated the impact of tncon breast cancer growth showing its potency to be also the promising therapeutic target inbreast cancer treatmentresultsoncogenomic in silico analysis reveals the tnc correlation with poorsurvival of breastcancer patientsto look deeper into the tnc function we performed the analysis of genomewide breast cancerdata with available oncogenomic portals such as gepia the human protein atlas cbioportaland ppisurv based on the status of three important receptors conventionally used for breastcancer subtyping ie estrogen receptor er progesterone receptor pr and human epithelialreceptor her2 breast cancer is classified as luminal a luminal b her2 positive and triple one 101371 pone0237889 august one 0cdownregulation of tenascinc inhibits breast cancer cells developmentnegative basallike triplenegative and her2overexpressing breast cancer yields a poorpatient prognosis because of a high incidence of metastases disease progression and resistanceto current chemotherapy regimens we first compared the expression level of tenascincin subtypes of breast cancer using gepia program fig a in s1 file mrna level of tncwere higher in triplenegative and her2 subtypes compared to the luminal a and luminal bsubtypes which have a better prognosis for patient survival therefore we chose mdamb231cells as a model for in vitro experiments because it is the most invasive cell line from breast cancer models mdamb231 cell genome clusters with the basal subtype of breast cancer sincethe cells also lack the growth factor receptor her2 they represent a good model of triplenegative breast cancer what is important adams showed that only invasive cell linessuch as mdamb231 or mdamb468 express tenascinc whereas the tumor cell lines witha low invasive capacity mcf7 and t47d do notas a next step we compared the expression levels of tenascin genes tnc tnn tnrtnxb in invasive breast cancer using gepia program fig 1a mrna level of tnc washighly expressed in breast cancer tissue brca interestingly expression levels of tnn andtnxb were significantly lower in breast cancer tissues fig 1a there was no significant difference in tnr gene expression between breast cancer and nontumor tissueswe also examined the expression of tenascin proteins in normal and malignant tissues byquerying data from the human protein atlas tnc in most cases and partly tnn wereexpressed at medium levels whereas tnr was not detected fig 1b and 1c tnc and tnrlevels were undetectable in samples from normal breast adipocytes glandular and myoepithelial cells taken together our results demonstrate that mrna and protein levels of tnc is relatively higher in invasive breast cancer tissues than those in normal tissuesthe cbioportal analysis enabled to look for the mutations in the tnc gene it appeared thattnc gene mutations measured for breast cancer patients are present as somatic mutation only in cases since these mutations seem to be irrelevant for breast cancer wedid not perform any further analysiswith ppisurv portal we looked through the transcriptomic data to correlate the tncexpression with the different clinical parameters such as survival or prognosis of the canceras the initial step of analysis we performed the alignment of tnc and other proteins from thetenascin family such as tenascinx tnx looking for the homology between these two proteinsat the top of that we made the analysis of the homology between the tnc and tnx with the relation to atnrna sequence the alignment of these two ecm proteins shows that they shareonly limited number of nucleotides query cover and for short and long transcriptionalvariants in the protein nterminus region respectively the sequence alignment analysis clearlyshow the atnrna matching exclusively to the tnc sequence identity fig 2appisurv analysis based on the kaplanmeier statistics showed very clearly the strong correlation with tnc expression and patients survival the high expression level has a greatimpact on the shorter survival for the patients thus suggesting also that tnc can be also considered as the prognostic factor for breast cancers p fig 2b at the same time weanalyzed also the available data for the tnx the results showed an inverse correlation oftnxb mrna expression and survival p fig 2c analysis was carried out on thegroup of patient n for tnc and n for tnxbatnrna mediates the downregulation of tnc mrna and proteinexpression in breast cancer cellsto achieve downregulation of tnc expression in breast cancer cell line transfection withvarious concentration of atnrna and nm was performed h after one 101371 pone0237889 august one 0cdownregulation of tenascinc inhibits breast cancer cells developmentfig tenascin is highly expressed in breast invasive carcinoma tcgabrca a messenger rna levels of tnc tenascinctnn tenascinw tnr tenascinr tnxb tenascinx genes in specimens from patients with invasive carcinoma of the breast one 101371 pone0237889 august one 0cdownregulation of tenascinc inhibits breast cancer cells developmentvs nontumor samples rna sequencing data were retrieved from the database of tcga and analysed using the gepia geneexpression profiling interactive analysis online web server httpgepiacancerpkucn the red boxes represent cancer specimensgrey boxes represent healthy breast specimens significance value � p b summary of tenascin expression patterns in breastcancer tissues and healthy breast determined by immunohistochemical staining data were retrieved from the human protein atlasdatabase case numbers of invasive breast cancer are shown na not available nd not detectable in healthy breast column means that tnc and tnr are not detected in nontumor samples results in normal breast are based on immunohistochemical stainingof a single sample c representative images of immunohistochemical staining for tnc tnn and tnr in breast healthy tissue andinvasive breast carcinoma specimens the images shown here are of the tissue sections from tissue microarray arrays tmas stainedwith appropriate antibodies tnccab004592 tnncab010907 tnrcab022343 all the images were taken at à magnification101371 pone0237889g001transfection the expression level of tnc was examined by qrtpcr analyses significantdownregulation of tnc mrna expression was observed compared to control treated withscrambled rna the level of tnc was decreased from at a concentration of nmatnrna up to for cells treated with nm atnrna in comparison to the controlp fig 3afig the oncogenomic analysis of the survival association with tenascinc and tenascinx in breast cancer asequence alignment of tenascinc versus tenascinx with their relation to atnrna the sequence alignment analysisclearly show the atnrna matching exclusively to the tnc sequence identity relation of tnc b and tnxbc gene expression to survival of breast cancer patients survival analysis performed with the use of a dataset breastcancer geo gse7390 and gse3494 deposited in and tools available from the ppisurv web portal tnxbl longtranscription variant tnxbs short transcription variant101371 pone0237889g002 one 101371 pone0237889 august one 0cdownregulation of tenascinc inhibits breast cancer cells developmentfig expression level of tnc and immune response genes after atnrna treatment in mdamb231 cell line a relative expression level of theexpression of tnc oas1 oas3 rig1 ifi16 and tlr3 established by qrtpcr relative expression was calculated using theδδcp method statisticalevaluation of atnrna versus scrambled sirnas ccontrol cells was performed using oneway anova followed by tukeys posthoc test effect of poly ic μgml on immune response genes oas1 oas3 rig1 ifi16 tlr3 in the figure presented as purple bars the results for hprtnormalized expressionof mrna are expressed as fold change of target gene expression relative to the control without poly ic treatment which is defined as b the proteinexpression levels of tnc and hprt c western blot analysis reveals efficient tnc silencing in mdamb231 cells with atnrna compared to cells treatedwith sirnas ccontrol the data represents the means ± sd from independent experiments significance value � p �� p ��� p 101371 pone0237889g003the qrtpcr analysis was also supported by direct analysis of the protein expression levelwe have observed already a decrease in tnc protein expression upon 50nm atnrnatreatment the highest concentration 100nm used led to the dramatic drop of the protein one 101371 pone0237889 august one 0cdownregulation of tenascinc inhibits breast cancer cells developmentexpression measured as the of the decrease fig 3b and 3c these observations were fullyconsistent with relative tnc expression level measured by qrtpcrinterferon response to atnrnato establish interferon induction in breast cancer mdamb231 cultured cells we looked forinterferon stimulated genes isg including oas1 oas3 rig1 tlr3 and ifi16 genes theanalysis was carried out with the qrtpcr fig 3a changes after atnrna measured byqrtpcr were not significant as shown basically for all of the genes in the concentrationrange of nm in parallel a synthetic form of dsrna polyipolyc poly ic wastransfected as a positive control poly ic has been used extensively as a tlr3 ligand to induceantiviral response [] we showed that transfection with poly ic μgml efficientlyinduced the expression of immune response genes oas1 oas3 rig1 ifi16 tlr3 inmdamb231 cells this enhancement in mrna expression was 7fold higher in poly ictreated cells than in untreated cells fig 3a purple barstnc knockdown inhibits cells proliferation and leads to the changes inmigration rate and adhesion potential of breast cancer cellsin order to investigate the involvement of tnc on breast cancer cells proliferation mdamb cell line was treated with atnrna and the realtime cell proliferation assay was performed the cells ability to proliferate was measured for h we have noticed time and concentrationdependent decrease in proliferation rate the most effective concentration ofatnrna was nm with decrease from after and h respectively fig 4anoteworthy nm and nm of atnrna was already sufficient concentration for the efficient inhibition of breast cancer cells proliferation the dosedependent effect of atnrna inmdamb231 proliferation potential resulted in standard sigmoidal doseresponses with ic50of ± nm after h ± nm after h and ± nm after h of treatmentfig 4bto get more insight into the downregulation of tnc expression by atnrna on themobility of breast cancer cells realtime measurements of migration was carried out wefound that downregulation of tnc expression by atnrna significantly impaired the cellmigration in breast cancer cell lines fig 4c the results were quantitatively assessed during h of experiment and showed that mdamb231 cells transfected with atnrna had thelowest motility beginning from h post transfection it was established that atnrnadelayed the migration of mdamb231 cells by ± h ± h ± h and ± h with and 100nm concentration respectively notably the most effective concentration which affected the migration potential of the cells was 10nm when compared to the control the observed delay was ± hsince tnc is implicated also in cellmatrix attachment we further looked at the adhesionability of atnrna treated cells the cells were conducted to realtime adhesion assay withxcelligence system compared with the controls tnc knockdown resulted in increased cellsadhesion on average fig 4dtnc promotes apoptosis and is involved in cell cycle regulation in breastcancer cellsto determine additionally the effect of atnrna on the cell cycle progression themdamb231 cells were treated with different concentrations of atnrna for h andthe cell cycle analysis was assessed by muse1 cell analyzer cells transfected with atnrna one 101371 pone0237889 august one 0cdownregulation of tenascinc inhibits breast cancer cells developmentfig activity of atnrna in proliferation migration and adhesion proliferation of breast cancer in culture was monitored in realtime using xcelligencesystem a impedance was recorded every min but to improve the clarity of the graphs only every fourth readout was plotted data show the mean ± sd ofthree independent measurements b dosedependent effects of atnrna on proliferation was evaluated using nonlinear regression by fitting experimentalvalues to sigmoidal bellshaped equation c migration of mdamb231 cancer cells was studied using xcelligence system serumdepleted cells weretransfected with increasing concentrations of atnrna from to nm or scrambled sirnas ccontrol impedance ci values of each experimentalcondition was recorded over time plotted against time fitted to fourparameter logistic nonlinear regression model and et50 was calculated for each atnrnaconcentration to generate doseresponse curves et50 value was normalized to the data obtained for untreated cells and plotted as normalized half maximaleffective time et50 of cell migration against atnrna concentrations d adhesion of mdamb231 cell line was observed in realtime using xcelligencesystem graph shows the final impendence values minus the initial values for the respective samples differences between ci values for atnrna treated andcontrol cells were statistically evaluated using oneway anova followed by tukeys posthoc test symbols above the bars significance value ��� p compared to cells treated with scrambled sirnas ccontrol101371 pone0237889g004showed the cell cycle distribution with the concentrationdependent decrease in cell numberin g0g1 phase increased in s phase and unchanged in g2m phase compared to the cellstransfected with unspecific control rna fig 5a atnrna impacted the cell cycle by almostdoubling the cells sphase fraction from to for the highest atnrna concentrationthus resulted with the cells arrest in s phase the cell cycle analysis proved the nontoxic effectof atnrna since we did not observe the increase in g1 population s phase arrest persistsfollowing up to h of atnrna treatment fig 5bthus to examine whether atnrnainduced apoptosis would be associated with the caspases activation the expression levels and activity of caspases such as caspase1 and in the atnrnatreated mdamb231 cells were assessed using muse1 cell one 101371 pone0237889 august one 0cdownregulation of tenascinc inhibits breast cancer cells developmentfig effect of atnrna on breast cancer cell cycle phase analysis mdamb231 cells were transiently transfected with increasing concentrations ofatnrna from to nm or scrambled sirna ccontrol for a and h b the cells were then fixed and added with propidium iodide pirnase a staining solution and analyzed in muse1 cell analyzer using modfit lttm software a percentage of cell distribution in each cell cycle phase wassummarized and shown the cell cycle distribution profile image is shown as a representative result of three independent experiments101371 pone0237889g005analyzer as shown in fig 6a breast cancer cells treated with atnrna exhibited enhancedmulticaspase activity in a concentrationdependent manner the multicaspase activity was ± ± ± and ± respectively at and nm atnrna concentration compared with the control fig 6b thus we observed almost5fold increase of the population of the apoptotic cells with the lowest atnrna concentration whereas almost 12fold with the highest oneatnrna has an impact on spheroids integrityto visualize the involvement of tnc in tumor formation the 3d culture model was appliedsince 3d cell culture models mimic better the in vivo behavior of cells in tumour tissues andare excellent surrogates to predict tumorigenic potential in vivo the ability to spheroid maintenance of breast cancer cells was assessed after atnrna treatment we have observed that one 101371 pone0237889 august one 0cdownregulation of tenascinc inhibits breast cancer cells developmentfig effect of atnrna on multiple caspase activation caspase1 and in mdamb231 cell linebreast cancer cells were transiently transfected with increasing concentrations of atnrna from to nm orscrambled sirnas ccontrol for h the transfected cells were then incubated with muse1 multicaspase reagent followedby analysis of the percentage of cell population in live caspase caspasedead and dead in muse1 cell analyzer a thepercentage of live caspase caspasedead and dead cells profile image is shown as a representative result from one of threeindependent experiments b the graphical representation of percentage of live and exhibiting caspase activity cellpopulation transfected with atnrna and scrambled sirnas statistical evaluation of atnrna versus scrambled cells one 101371 pone0237889 august one 0cdownregulation of tenascinc inhibits breast cancer cells developmenttreated with scrambled sirnas was performed using oneway anova followed by tukeys posthoc test significance value��� p compared to scrambled control ccontrol error bars represent sd101371 pone0237889g006downregulation of tnc led to the disintegration of the spontaneously forming spheroids ofmdamb231 the clearly visible effect on the spheroid viability was observed even with thelowest atnrna concentration the increased concentrations of dsrna had a great impacton spheroids integrity resulting in structure disintegration at the highest concentration of nm fig 7a the atnrna application influenced the spheroid volume and shape displaying the total shrinking of the compact structure into the small fragments with the highestatnrna concentration fig 7ato have a better insight into the mdamb231 spheroids structure and the atnrnaimpact on their viability the confocal microscopy imaging was assessed the analysis of fluorescent labelling with greenfluorescent calceinam of living and dead cells within the spheroid with the livedead viabilitycytotoxicity kit was carried out as revealed by the image ofthe untreated mdamb231 cells compact multicellular spheroids were obtained fluorescence images revealed the overall morphology of the mdamb231 spheroids fig 7b thecell density in the core of the untreated spheroid was high and no dead cells were identifiedthe similar pictures were obtained for the control furthermore all conditions with differentatnrna concentrations resulted with losing the spheroid density increased dimensionsand appearing a higher population of dead cells it is worthy of note that the spheroids treatedwith increasing atnrna concentrations did not display a smooth contour following h oftreatment and subsequently their round shape was markedly altered by the treatments afterthe treatment with 50nm concentrations the spheroids showed the strong overrepresentationof dead cells fig 7b thus the 100nm concentration of atnrna was most likely too highfor the cells viability and we were not able to keep the spheroids in shape that would allow forthe imagingtnc is involved in emt processesas the consequence of these finding we analyzed the expression level of proteins involved inemt processes we took into account two main emt markers ecadherin and vimentinwestern blot analysis shows the significant increase of ecadherin level followed by the dropof the expression of vimentin protein fig 8a these observations were concentrationdependent showing the highest efficacy for atnrna at the concentration of 100nm for ecadherin expression similarly for vimentin we have observed the highest decrease of expressionupon atnrna transfection at 100nm concentration fig 8bdiscussiontnc is the main ecm protein of various tumors and its overexpression is repeatedlyobserved in breast cancer cells both in vitro and in vivo indicating a role for this extracellularmatrix glycoprotein in neoplastic pathology moreover its high expression correlates withworsened patient survival prognosis in several cancer types in breast cancers severalstudies demonstrate that high expression of tnc is not only an indicator of poor prognosisbut also correlates with metastasis to distinct ans such as lymph nodes liver and lung [] tnc plays a substantial role in emt that is believed to be a key mechanism in cancer progression whereby cancer cells acquire more aggressive behavior [ ] in human breast cancer specimens tnc is coexpressed with the mesenchymal marker vimentin themechanistic role of tnc in the process of emt remains poorly defined however studies one 101371 pone0237889 august one 0cdownregulation of tenascinc inhibits breast cancer cells developmentfig effects of atnrna on viability and spheroid structure in mdamb231 cells a monolayer cultures weretransfected with indicated amounts of atnrna oligonucleotides and after hrs cellular spheroids of mdamb cells were generated from cells in perfecta3d1 96well hanging drop plate and cultured for up to hoursscale bars μm scrambled sirnas cscr b the viability of the atnrna transfected cells within spheroidsusing livedead cell imaging kit left and middle panels present live cells green and dead cells red respectively one 101371 pone0237889 august one 0cdownregulation of tenascinc inhibits breast cancer cells developmentthe right panels show merge of two fluorescent images scrambled sirnas cscr concentrations ofatnrna used for transfection fluorescence images were taken using leica tcs sp5 confocal laser scanningmicroscope and plan apo à na oilimmersion objective scale bars μm101371 pone0237889g007suggest that tnc can induce an emt like phenotype in mcf7 breast cancer cells via theαvβ6 and αvβ1 integrins [ ] many studies on various cancer tissues have demonstrateddownregulation of epithelial markers including ecadherin plakoglobin and cytokeratin aswell as the upregulation of mesenchymal markers such as ncadherin and vimentin andexpression of emt transcription factors snai and twist since these changes towardsmesenchymal phenotype could correlate with invasiveness metastatic potential and poorpatients outcome we have investigated the effect of tnc knockdown on the expression levelsof emt markers our results show that down regulation of tnc reverses the malignant phenotype of the cancer cells as the experimental result we observed the downregulation of mesenchymal markervimentin followed by the upregulation of epithelial markerecadherinthis indicates atnrna as a potential therapeutic agent which could switch the mesenchymal phenotype of breast cancer cells to the epithelial one inhibiting the ability to metastasisand invasion additionally it has been also shown that tnc as the ecm component plays alsofig the effect of tnc downregulation on emt process of mdamb231 cells a the protein expression levelsof ecadherin vimentin and gapdh b western blot analysis of atnrna effects on the emt process revealed asignificant increase in ecadherin level followed by the drop of the expression of vimentin protein the data representsthe means ± sd from independent experiments101371 pone0237889g008 one 101371 pone0237889 august one 0cdownregulation of tenascinc inhibits breast cancer cells developmenta role in cell to cell or cellmatrix attachment most probably inhibiting the cells migration inapproach with atnrna it seems that tnc in breast cancer cell line plays an antiadhesiverole which would affect the cell migration and invasion ability in addition to emt processesthus tnc downregulation seems to enhances the adhesiveness of cancer cells showing thedirect involvement of tnc in cell adhesive properties targeting the tnc in potential therapymight be also highly beneficial since it has been already established that tnc maintain a stemcell niche in the brain tumors thus could promote the tumor cell invasion therefore its overexpression largely contributes to radiochemotherapy resistance and tumor recurrence infact it has been shown that targeting gbm invasion increases tumor sensitivity to temozolomide tnc also promotes stromal events such as the angiogenic switch and the formationof more but leaky blood vessels involving wnt signaling and inhibition of dickkopf1 dkk1in a neuroendocrine tumor model we observed significant atnrnamediated downregulation of tnc was in concordance with the observed changes in proliferation and migration rates the results show thatthe atnrna transfected cells lose their ability to migrate thus showing the involvement oftnc in breast cancer invasiveness our data indicate also that the downregulation of tncexpression in mdamb231 cancer cell lines inhibits proliferation along with induction of celldeath we were able to determine the tnc impact on the apoptosis by measuring level of bothcaspases initiating intracellular events caspase2 and effector caspases3 and orend demonstrated that tnc causes cdk2 inactivation and blocks cell cycle progression from g1 phase to s phase of anchoragedependent fibroblasts by interfering withfibronectinsyndecan4 interactions for the proliferation of anchoragedependent cellsattachment to the ecm is required detachment of fibroblasts by a pure tenascinc substratum results in g1phase arrest by inactivation of the cdk2 complex in a ckidependent manner in contrast to fibroblasts proliferation of most tumor cells is stimulated by tnc this shows that the effect of tnc on proliferation is cell typespecific and suggests that in cancer cells the cdk2 complex is not repressed in the presence of tnc in leukemia breast carcinoma and glioma were found subpopulations of stemlike cells that support tumor growth in such cells adhesion on the tnc substratum may override the g0g1 and gls cellcycle checkpoints which may explain the increased proliferation rate the g1s transitionis enforced by cyclin ecdk2 activation via syndecan4 related signalling it has been shownthat tnc in tumor cells binds to the syndecan4 binding site in fibronectin thereby blockingsyndecan4 ligation releasing the tumor cells from the suppressive effect of fibronectin ontheir proliferation this would a | Colon_Cancer |
" oral administration is the most common way to deliver drugs to the systemic circulation or targetans orally administered drugs are absorbed in the intestine and metabolized in the intestine and liver in theearly stages of drug development it is important to predict firstpass metabolism accurately to select candidatedrugs with high bioavailability the caco2 cell line derived from colorectal cancer is widely used as an intestinalmodel to assess drug membrane permeability however because the expression of major drugmetabolizingenzymes such as cytochrome p450 cyp is extremely low in caco2 cells it is difficult to predict intestinalmetabolism which is a significant factor in predicting oral drug bioavailability previously we constructed a mouseartificial chromosome vector carrying the cyp cyp2c9 cyp2c19 cyp2d6 and cyp3a4 and p450 oxidoreductasepor 4cypsmac genes and increased cyp expression and metabolic activity in hepg2 cells via transfer of thisvectorresults in the current study to improve the caco2 cell assay model by taking metabolism into account weattempted to increase cyp expression by transferring the 4cypsmac into caco2 cells the caco2 cells carryingthe 4cypsmac showed higher cyp mrna expression and activity in addition high metabolic activity availabilityfor permeation test and the potential to assess drugdrug interactions were confirmeds the established caco2 cells with the 4cypsmac are expected to enable more accurate prediction ofthe absorption and metabolism in the human intestine than parental caco2 cells the mammalian artificialchromosome vector system would provide useful models for drug developmentkeywords mammalian artificial chromosome chromosome transfer cytochrome p450 intestinal metabolismcaco2 cell correspondence kazukitottoriuacjp1division of genome and cellular functions department of molecular andcellular biology school of life science faculty of medicine tottoriuniversity nishicho yonago tottori japan2chromosome engineering research center cerc tottori university nishicho yonago tottori 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 0cohta bmc biotechnology page of bioavailability is an important area of concern in drugdevelopment poor oral bioavailability has led to drugwithdrawal oral drug bioavailability is often limited bymetabolizing enzymes and efflux transporters in the gut the caco2 cell line derived from human colon carcinoma is a commonly used model for estimating the intestinal absorption of new drug candidates althoughcaco2 cells express a variety of efflux and uptake transporters they have an absence or low levels of cytochrome p450 cyp isoforms such as cyp3a4 andcyp2c that are typically expressed in the human intestinal epithelium therefore caco2 cells are of limited use in evaluating the role of metabolism inintestinal absorption after oral administration to predict the intestinal absorption of drugs more accurately itis necessary to modify caco2 cells to increase their expression of cyp isoformssome studies reported that cyp3amediated metabolism in caco2 cells was enhanced by transfection withboth cyp3a4 and cyp oxidoreductase por [ ] treatment with 1α25dihydroxyvitamin d3 [ ] or the combination of transfection with cyp3a4 and treatment withboth sodium butyrate and 12otetradecanoylphorbol13acetate in contrast few studies aimed at enhancingmultiple cyp isoforms in caco2 cells have been performed honkakoski and his collaborators created caco2cell lines expressing nuclear receptors pregnane x receptor and constitutive androstane receptor [] thesenuclear receptors upregulated the expression of somecyp isoforms in caco2 cells but cyp activities remainedvery low in the absence of 1α25dihydroxyvitamin d3therefore a new approach is needed to introduce multiple cyp isoforms in caco2 cellsmammalian artificial chromosome ac vectors derived from native chromosomes have several advantagesover conventional vectors acs segregate freelyfrom host chromosomes through a set of cell divisionsand are adapted to carry multiple target genes with a desired copy number and mbsized genomic regions withendogenous regulatory elements furthermore acs carrying genes of interest can be transferred into varioustarget celllines via microcellmediated chromosometransfer mmct considering these advantages acshave been used to generate several model cells for pharmacokinetic and toxicokinetic studies previously a lack of cyp3a4 expression in the caco2cell line was addressed through the introduction of exogenous cyp3a4 and por which is a coenzyme ofcyps via a human artificial chromosome hac vectorderived from human chromosome [ ] the haccarrying cyp3a4 and por genes conferred sufficientcyp3a activity to parental caco2 cells to be useful forpredicting the intestinal extraction ratio in humansrecently a mouse artificial chromosome mac vectorconstructed from native mouse chromosome wasused to increase the activity of multiple cyps in hepg2cells which are a liver cancer cell line typically exhibiting low cyp activity in this study four cyp genescyp3a4 cyp2c9 cyp2c19 cyp2d6 and a pene were loaded on the mac 4cypsmac and transferred to hepg2 cells tchepg2 to make the cellsmore suitable as a model to evaluate drugdrug interactions ddis and hepatotoxicity in the initial screeningof candidate drugs the expression and activity ofcyps in tchepg2 were comparable to those in humanhepatocytes and this expression was sustained after along culture period because of the stability of the macin human cells regarding the assessment of ddisthe activity of cyps in tchepg2 was reduced in a concentration and timedependent manner by specific inhibitors which reflects the conditions in primary humanhepatocytes furthermore metabolic toxicity of aflatoxinb1 which is converted to its active metabolite viacyp3a4 and exerts hepatotoxicity through dna damage was clearly recapitulated in tchepg2 cells rather than parental hepg2 cells this study suggestedthat tchepg2 can provide a useful model to assess notonly hepatic metabolism but also cypmediated hepatotoxicity during the early stages of drug development andthe system using the mac can improve the existingcellbased modelin the current study we aimed to utilize previouslyconstructed 4cypsmac to generate a novel caco2 cellline with increased activity of multiple major cyps the4cypsmac was transferred to caco2 cells via mmctto establish caco2 cells carrying the 4cypsmac andthe caco2 4cypsmac cells were examined to determine whether they exhibited sufficient cyp activity foruse in initial drug screeningresultsmmct and analyses of acquired clonescho cells carrying a mac vector with cyp2c9cyp2c19 cyp2d6 cyp3a4 por and gfp genes wereprepared 4cypsmac fig 1a using cho cells asdonor cells and caco2 cells as recipient cells weattempted to generate caco2 cells carrying the 4cypsmac via mmct fig 1a after selection four drugresistant gfppositive clones were obtained caco24cypsmac fig 1b to examine whether the cypand por genes were introduced into the obtainedclones genomic pcr analyses were performed chocells with the 4cypsmac and caco2 cells were usedas positive and negative controls respectively consequently a band of the desired size was observed for eachprimer set in the candidate clones fig 1c next achromosome specimen was prepared from the acquired 0cohta bmc biotechnology page of fig introduction of the 4cypsmac into caco2 cells a transfer of the 4cypsmac into caco2 cells the structure of the mac carrying fourcyps and por is shown at the top a cag promoter was placed upstream of each gene a schematic view of the transfer of the 4cypsmac tocaco2 cells is shown at the bottom the 4cypsmac was transferred from cho cells to caco2 cells through the mmct method b an image ofgfp fluorescence in parental caco2 cells and caco2 4cypsmac cells the gfp fluorescence indicates the presence of the 4cypsmac in thecaco2 cells the white bars indicate a distance of μm c results of genomic pcr analyses to detect four cyp and por transgenes on the macin caco2 cells donor cho cells and caco2 cells are positive and negative controls respectively d images of fish analyses of caco2 cellscarrying the 4cypsmac red and green signals indicate the mac and transgenes respectively the arrow shows the 4cypsmac and the insetshows an enlarged image of the 4cypsmacclones and fish analysis was performed to check thekaryotype fish analysis revealed that a single copy of the4cypsmac existed in the candidate clones fig 1drtqpcr analysis was performed to examine themrna expression level of the introduced cyps and porin the obtained clones compared with that in parentalcaco2 cells the gene expression level was particularlyhigh in the caco2 4cypsmac and caco2 4cypsmac clones fig among the introduced genespor expression was only slightly enhanced in theseclones basal expression of por in parental caco2 cells ishigh as observed in a previous study caco2 4cypsmac showed high expression of the majority of genescompared with caco2 4cypsmac the caco2 cellline consists of a heterogeneous population of cells therefore difference of the gene expression levels between obtained clones may partly depend on the population into which the 4cypsmac has been introducedalthough the other two clones obtained by mmct stillshowed higher expression levels than the parental caco2cells the level of increase was moderate therefore we selected caco2 4cypsmac and caco2 4cypsmac clones for further analyses to evaluate the availability asan improved model system these results suggest that wesuccessfully transferred the 4cypsmac to caco2 cellsand the genes on the mac were highly expressedmonolayer formation of caco2 4cypsmac cellswe seeded caco2 4cypsmac and caco2 4cypsmac cells at a concentration of à cellswell 0cohta bmc biotechnology page of fig gene expression analyses of caco2 cells with the 4cypsmac the expression levels of the four cyps and por in caco2 cells with the4cypsmac the relative expression levels of the four cyps and por genes of the parental caco2 cells and acquired clones were analyzedthrough rtqpcr gapdh was used for normalization mean ± se n in a millicell 24well cell culture insert plate the caco 4cypsmac cells spread across the entire membrane and formed a cell layer while the caco2 4cypsmac cells did not spread and there were gaps in thecell layer fig 3a caco2 4cypsmac appeared toaggregate and form multiple layers rather than spreadand form a single layer it was reported that multilayeredareas appeared in the cell population for late passagecells the teer value was measured using amillicellers fig 3b the teer value is an index oftight junction formation and when the value is almostconstant it is considered that a cell layer has formedwith the exception of the caco2 4cypsmac clonethe caco2 cells and caco2 4cypsmac cellsshowed an increase in teer value untilit plateauedafter d the caco2 cells and caco2 4cypsmac showed almost equivalent teer values because monolayer formation is essential for the permeation test thesubsequenttests were performed using the caco24cypmac cellsculture timedependent change in gene expressiontotal rna was extracted from caco2 cells andcaco2 4cypsmac cells on the 4th 11th and22nd days after seeding we compared the expressionlevel of each gene on each day and confirmed thatthe expression levels of the four cyps and por increased in both the caco2 cells and caco2 4cypsmac cells fig 3c the expression levels of allgenes analyzed were significantly higher in the caco24cypsmac cells on the 22nd day than those inparental caco2 cells the gene expression levelincreased depending on the culture time and the geneexpression levels of the caco2 4cypsmac cellsestablished in the current study were higher thanthose of parental caco2 cells with the exceptions ofcyp3a4 and cyp2d6 the expression levels in parental caco2 cells were higher in all cases until day the parental caco2 cell line appears to have higherpotential to enhance the expression of cyp2c9 andcyp2c19 during differentiation 0cohta bmc biotechnology page of fig monolayer formation assay a images of bright and gfp fluorescence from cells seeded on the membrane of a millicell24 plate in caco24cypsmac cells did not spread throughout the membrane and did not form a cell layer but in caco2 4cypsmac there were no gapsbetween cells and they formed a cell layer the white bars indicate a distance of μm b transepithelial electrical resistance teer values ofcaco2 cells caco2 4cypsmac and caco2 4cypsmac cells c culture timedependent change in gene expression the relative expressionlevel was evaluated in caco2 and caco2 4cypsmac mean ± se n the expression levels in caco2 and caco2 4cypsmac at day were compared with those in humanadult intestine additional file figure s1 in caco24cypsmac the expression levels of cyp2c9 andcyp2c19 were comparable and that of cyp2d6 washigher than in human adult intestine although cyp3a4expression was significantly enhanced in caco2 4cypsmac compared with that in parental caco2 cellsthe expression was stilllower than in human adultintestinecyp metabolic activity measurementa p450glo assay with each specific substrate wasemployed to measure the metabolic activity of cyps inthe caco2 4cypsmac cells which had high geneexpression levels as confirmed through rtqpcr analysis the activities of all four cyps were higher in thecaco2 4cypsmac clone than in caco2 cellsfig 4a the resultsthe introducedindicate that4cypsmac expressed functional cyps and increasedthe total activity of each cyp in the caco2 cells theenhancements in the rates of metabolic activity ofcyp2c9 cyp2c19 and cyp2d6 were generally correlated with those of mrna expression however therewas a gap between the enhancement of the rate ofcyp3a4 mrna expression and that of metabolic activity this may have been because the parental caco2 originally had extremely low expression of cyp3a4mdz permeability testa permeation test was conducted using midazolammdz a cyp3a substrate to examine whether the cellsreflected the behavior of small intestinal epithelial cellsin terms of mdz permeation the penetration test wasperformed on day after cell seeding when the teervalue plateaued we measured the amount of ²ohmdz in each of the donor side apical recipient sidebasal and intracellularly the amounts of ²oh mdz 0cohta bmc biotechnology page of fig activity of each cyp in the caco2 4cypsmac cells a the metabolic activity of each cyp in caco2 4cypsmac cells the relative activityfor each cyp was measured by comparing the parental caco2 cells and the caco2 4cypsmac mean ± se n b permeability test usingmdz the permeability test was performed d after seeding caco2 cells and caco2 4cypsmac whereby μm mdz was added to theapical side and after min the apical intracellular and basal supernatants were collected the ²oh mdz in the supernatant was measuredthrough lcmsms c cyp3a4 inhibition test ketoconazole an inhibitor of cyp3a4 was added to the caco2 4cypsmac and incubated for h a luminescent substrate was measured to detect cyp3a4 activity with different concentrations of ketoconazolein alllayers of the caco2 4cypsmac cells werehigher than in those of caco2 cells fig 4b moreoverer was calculated using eq and the results were and for caco2 and caco2 4cypsmac respectively er indicates the rate of metabolism during cellpermeation cyp3a4 was scarcely expressed in parentalcaco2 cells so the er value was extremely low howevercaco2 4cypsmac cells showed an er of which was higher than in the caco2 cells and mdz wasmetabolized by cyp3a4 when passing through the cellsinhibition testto determine the availability of the established clone forthe assessment of ddis we added ketoconazole an inhibitor of cyp3a4 to the caco2 4cypsmac cells andexamined whether the metabolic activity was reduced ketoconazole at and μm was addedand cells were incubated at °c for h followed by themeasurement of metabolic activity the metabolic activityof cyp3a4 decreased as the inhibitor concentration increased fig 4c the activity of cyp3a4 in caco2 4cypsmac appeared to be sufficient for the inhibition test compared with that in parental caco2 cells in addition to thepermeation test for cyp3a4 inhibition of cyp3a4s function by ketoconazole in caco2 4cypsmac cells was alsoconfirmed this suggests that the established cells could beused for ddi testing of drugs that are substrates ofcyp3a4 therefore the caco2 4cypsmac cells moreaccurately reflect the behavior of cyp3a4 substrates in human epithelial cells than parental caco2 cellsdiscussionin the current study we introduced four cyps and porinto caco2 cells to increase their drug metabolic 0cohta bmc biotechnology page of abilities which are typically low this study was intendedto establish a better human cell model to more preciselyevaluate the behavior of drugs in the small intestine the4cypsmac was successfully introduced into the caco2cells and successfully increased cyp activityin contrastin this studythe gene expression and activity of cyps in tchepg2 carrying the 4cypsmac are either comparableto or higher than those in primary human hepatocytes to the other cypscyp3a4 mrna expression was still low in caco2 carrying the 4cypsmac compared with the level in human adult intestine despite the significant enhancementof mrna expression regarding the genes on the4cypsmac each is present as a single copy becausethe nature of gene regulation is supposed to differ between hepg2 and caco2 changing copy number of thecyp3a4 gene may further optimize the expression profile of caco2 cells to that of human intestinethe established caco2 4cypsmac cells with particularly high gene expression showed high activity in all cypsin the future we will conduct metabolic tests inhibitiontests and permeation tests using drugs that are substratesfor other cyps and investigate whether the caco2 4cypsmac cells reflect the behavior of drugs in small intestinal epithelial cells the cyp expression level in the humansmall intestine is reported to be approximately forcyp3a4 approximately for cyp2c9 approximately for cyp2c19 and approximately for cyp2d6 it will be necessary to evaluate whether the proportion ofcyp expression in the caco2 4cypsmac is close tothat of the human small intestineif cyp metabolic capacity is guaranteed in the established clones the established cell line can be used asnew human small intestine model cells in recent yearssmall intestine model cells prepared from induced pluripotent stem cells have been reported but such cells areconsidered difficult to use for screening large quantitiesof drug candidate compounds however caco2cells are easy to handle therefore it is possible to usecypmodified caco2 cells to test large numbers of candidate compounds as a first screeningwako osaka japan supplemented with fetal bovine serum fbs and μgml g418 parental caco2cells atcc® htb37¢ atcc manassas va usawere maintained in dulbeccos modified eagles mediumdmem wako supplemented with fbs memnonessential amino acids gibco thermo fisher scientific waltham ma usa m hepes gibco mmsodium pyruvate gibco mm glutamax gibcoand penicillinstreptomycin wako caco2 cells withthe 4cypsmac were maintained in the above mediumsupplemented with μgml g418 these cells werecultured at °c in co2microcellmediated chromosome transfertransfer of 4cypsmac from cho cells to caco2 cellswas performed using a standard procedure brieflydonor cho cells were cultured in f12 medium supplemented with fbs and μgml colcemid after h microcells were isolated through centrifugation withdmem containing cytochalasin b and filtration thenmicrocells suspended in phytohemagglutinin p phapdmem were poured onto caco2 cells in a 6cm dishand incubated for min the cells were treated withpolyethylene glycol peg solution g of peg1000 ml of dmem ml of dimethyl sulfoxide for minfollowed by washing with dmem after h of recoveryculture cells were seeded in a 24well collagencoatedplate corning ny usa and maintained with selectionmedium h after seeding thereafter the medium waschanged twice a week to obtain drugresistant clonesbecause the mac carries a gfp gene gfppositiveclones were selected from the drugresistant clonesgenomic pcr analyseswe extracted genomic dna from cell lines using a genomic dna extraction kit with dnasefree rnase gentra systems minneapolis mn usa the primers forthe genomic pcr are listed in additional file tables1 they amplified each gene region on the 4cypsmac we used kod fx takara otsu japan in accordance with the manufacturers instructionsthe mammalian artificial chromosome vector systemwould provide useful models for drug development theestablished caco2 cells with the 4cypsmac are expected to more accurately predict absorption and metabolism in the human intestine than parental caco2 cellsmethodscell culturechinese hamster ovary cho cells jcrb0218 jcrbcell bank nibiohn osaka japan carrying the 4cypsmac were maintained in hams f12 nutrient mixturefish analysestrypsinized cells were incubated for min in m kcland fixed with methanol and acetic acid and thenslides were prepared using standard methods fish analyses were performed using the fixed metaphase of each cellhybrid using digoxigeninlabeled roche germany dna[mouse cot1 dna invitrogen carlsbad ca usa] andbiotinlabeled dna [pac 4cypspor] essentially as described previously chromosomal dna was counterstained using dapi sigmaaldrich st louis mo usaimages were captured using an axioimagerz2 fluorescencemicroscope carl zeiss germany 0cohta bmc biotechnology page of rtqpcrwe extracted mrna using the rneasy mini kit qiagen germany and synthesized firststrand cdna usingthe high capacity cdna reverse transcription kit applied biosystems foster city ca usa the primersare listed in additional file table s1 for rtqpcranalysis tb green premix ex taq takara was usedand relative mrna expression was evaluated throughthe δδct method gapdh was used for normalizationculture timedependent expression level change of fourcypscaco2 cells and caco2 4cypsmac were seededin a 6cm dish at a concentration of à cellswell cells were lysed using trizol invitrogen causa on days and after seeding and rnawas extracted and purified using an rneasy mini kitqiagen thereafter cdna synthesis was performedusing the highcapacity cdna reverse transcriptionkit applied biosystemsactivity test of the four cypswe tested the metabolic activity ofthe four cypsusing the p450glo¢ assay promega madison wiusa the luminogenic substrates used for the testwere luciferinipa cyp3a4 luciferinme egecyp2d6 luciferinh cyp2c9 and luciferinhege cyp2c19 cells wereseeded in 48wellcollagencoated plates corning at a density of à cellswell after h the medium was changedand h later we added transport medium tm containing substrate tm was prepared using hanks balanced salt solution hbss with mm nahco3 mm glucose and mm hepes which was adjusted to ph after incubation we added detectionreagent and measured the luminescence using an infiniteandcyp2c19 required h of incubation while cyp2d6and cyp3a4 required h after the measurementthe cells were washed with pbs dissolved in lysisbuffer and diluted fivefold the same amount of celltiter glo promega was added to μl of the lysateand luminescence measurement was performed tonormalize data to the number of viable cellswako cyp2c9f500 platereadermidazolam mdz permeability testthe obtained clones were assessed in an mdz permeation test each cell was seeded on a 24well cell cultureinsert plate millipore billerica ma usa at a concentration of à cellswell the medium was changedonce a week after seeding and every d after the secondweek transepithelial electrical resistance teer wasmeasured using millicellers millipore before mediumexchange the test was performed d after seedingfor the test tm ph prepared by adding mmnahco3 mm glucose and mm hepes tohbss at ph was used the donor side solutionwas prepared by dissolving μm mdz in tm ph the acceptor side solution was prepared by adding fbs to tm ph on the test day themedium was removed from the culture insert seededwith the cells and the cells were rinsed twice withtm ph tm ph and tm ph wereadded to the apical and basal chambers respectivelyand cells were incubated at °c for min the testwas started by adding the donor side solution to thedonor side chamber and the acceptor side solution tothe acceptor side chamber thirty minutes after thestart of the test the solution in the donor side andacceptor side chambers was collected moreover tomeasure the amount of mdz and ²hydroxy mdz²oh mdz in the cells after the test the cultureinsert was quickly rinsed three times with icecoldtm ph the membrane was cut using a cutterand μl of icecold tm ph was added cellswere detached from the membrane through sonicationand a cell suspension was used as a sample thesesamples were deproteinized and stored at °cuntil measurementlcmsms was used for the measurement of mdzand ²oh mdz in the samples qtrap5500 sciexframingham ma usa and a prominence uflc system shimadzu kyoto japan were combined for measurement the hplc conditions and msms conditionsare shown in additional file table s2 quantitativeanalysis was performed in multiple reaction monitoringmode mass transitions mz were formdz for ²oh mdz and for ²oh mdz d4 data were analyzed usinganalyst software sciexequation formula to calculate extraction ratio erer ¼metabolite donorþreceiverþintracellularpþparent receiverþintracellularððþ þ pmetabolite donorþreceiverþintracellularðþtokyo chemicalinhibition testketoconazoleindustry tokyojapan was used as an inhibitor against cyp3a4 andchanges in metabolic activity were measured using ap450glo assay with luciferinipa cells were seededin a 48well collagencoated plate at à cellswell and the medium was changed after d thenext daythe medium was collected cells werewashed twice with pbs and then μl of tm ph containing ketoconazole tokyo chemical industry at and μm was added toeach set of three wells tm was adjusted to ph byadding mm nahco3 mm glucose and mm 0cohta bmc biotechnology page of received april accepted august referencesbenet l wu c hebert m wacher v intestinal drug metabolism andantitransport processes a potential paradigm shift in oral drug delivery jcontrol release xie f ding x zhang qy an update on the role of intestinal cytochromep450 enzymes in drug disposition acta pharm sin b takenaka t kazuki k harada n kuze j chiba m iwao t matsunaga t abes oshimura m kazuki y development of caco2 cells coexpressingcyp3a4 and nadphcytochrome p450 reductase using a human artificialchromosome for the prediction of intestinal extraction ratio of cyp3a4substrates drug metab pharmacokinet hu m li y davitt cm huang sm thummel k penman bw crespi cltransport and metabolic characterization of caco2 cells expressing cyp3a4and cyp3a4 plus oxidoreductase pharm res schmiedlinren p thummel ke fisher jm paine mf lown ks watkins pbexpression of enzymatically active cyp3a4 by caco2 cells grown onextracellular matrixcoated permeable supports in the presence of1alpha25dihydroxyvitamin d3 mol pharmacol fan j liu s du y morrison j shipman r pang ks upregulation oftransporters and enzymes by the vitamin d receptor ligands 1alpha25dihydroxyvitamin d3 and vitamin d analogs in the caco2 cell monolayer jpharmacol exp ther cummins cl mangravite lm benet lz characterizing the expression ofcyp3a4 and efflux transporters pgp mrp1 and mrp2 in cyp3a4transfected caco2 cells after induction with sodium butyrate and thephorbol ester 12otetradecanoylphorbol13acetate pharm res korjamo t honkakoski p toppinen mr niva s reinisalo m palmgren jjmonkkonen j absorption properties and pglycoprotein activity of modifiedcaco2 cell lines eur j pharm sci korjamo t monkkonen j uusitalo j turpeinen m pelkonen o honkakoskip metabolic and efflux properties of caco2 cells stably transfected withnuclear receptors pharm res kublbeck j hakkarainen jj petsalo a vellonen ks tolonen a reponen pforsberg mm honkakoski p genetically modified caco2 cells withimproved cytochrome p450 metabolic capacity j pharm sci mes to hbss the cells were preincubated for h at °c and 1000fold diluted cyp3a4 substrate wasadded one hour later μl of the supernatant wascollected from the well mixed with μl of detectionreagent and the luminescence value was measuredusing an infinite f500 plate reader wakosupplementary informationsupplementary information accompanies this paper at httpsdoi101186s12896020006378additional file figure s1 comparison of gene expression betweenhuman small intestine and day culture of caco2 and caco2 4cypsmac table s1 primer sequences for genomic pcr and rtqpcrtable s2 lcmsms analysis conditions mdz ²oh mdzabbreviationscyp cytochrome p450 ac artificial chromosome mmct microcellmediated chromosome transfer por p450 oxidoreductase hac humanartificial chromosome mac mouse artificial chromosome ddi drugdruginteraction cho chinese hamster ovary mdz midazolamteer transepithelial electrical resistance er extraction ratioacknowledgmentswe thank satoru iwado at tottori university for technical assistance with theexperiments we also thank dr hiroyuki kugoh dr masaharu hiratsuka drhiroyuki satofuka and dr takahito ohira at tottori university for criticaldiscussions this research was partly performed at the tottori bio frontiermanaged by tottori prefecture we thank edanz group wwwedanzeditingcomac for editing a draft of this manuscriptauthors contributionsall authors conceived and designed the experiments yo and kka performedthe experiments yo sa kko and yk wrote the paper mo and yksupervised the study all authors read and approved the final manuscriptfundingthis work was supported in part by the basis for supporting innovative drugdiscovery and life science research binds from the japan agency formedical research and development amed under grant numberjp18am0301009 ykavailability of data and materialsthe data and materials used andor analyzed during the current study areavailable from the corresponding author on reasonable requestethics approval and consent to participatenot applicableconsent for publicationnot applicablecompeting interestsdr mitsuo oshimura is ceo and a shareholder of trans chromosomics incdr satoshi abe is a member of trans chromosomics inc and the otherauthors declare no conflict of interestauthor details1division of genome and cellular functions department of molecular andcellular biology school of life science faculty of medicine tottoriuniversity nishicho yonago tottori japan 2chromosomeengineering research center cerc tottori university nishicho yonagotottori japan 3trans chromosomics inc nishicho yonagotottori japan 4laboratory of biopharmaceutics meijipharmaceutical university noshio kiyose tokyo japan oshimura m uno n kazuki y katoh m inoue t a pathway fromchromosome transfer to engineering resulting in human and mouseartificial chromosomes for a variety of applications to biomedicalchallenges chromosom res satoh d abe s kobayashi k nakajima y oshimura m kazuki y human andmouse artificial chromosome technologies for studies of pharmacokineticsand toxicokinetics drug metab pharmacokinet kazuki y hoshiya h takiguchi m abe s iida y osaki m katoh m hiratsukam shirayoshi y hiramatsu k ueno e kajitani n yoshino t kazuki k ishiharac takehara s tsuji s ejima f toyoda a saka | 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 |
"purpose squamous cell carcinomas and adenocarcinomas are the most common types of cervical cancercompared to squamous cell carcinomas adenocarcinomas are more common in younger women and have apoorer prognosis yet so far no useful biomarkers have been developed for these two types of cancer in thefollowing study we examined the combination of cytokeratin p63 p40 and muc5ac for distinguishingsquamous cell carcinoma scc from adenocarcinoma of the cervix aecmaterials and methods a total of scc and aec were collected immunohistochemical analyses wereconducted to determine the expression of ck56 p63 p40 ck7 and muc5ac one pathologist who was blinded tothe patients clinical and pathological data interpreted the staining resultsresults muc5ac and ck7 were detected in and of aec cases compared to and of scccases p the specificity of muc5ac was higher than that of ck7 in aec p the sensitivity of muc5accombined with p40 or p63 was similar to that of ck7 but the specificity was slightly higher than that of ck7 inaec moreover the expression of muc5ac was correlated with the degree of tumor differentiation inadenocarcinomas p and was not related to the prognosis of cervical adenocarcinoma and subtypess muc5ac may be useful as a biomarker for differential diagnoses between squamous carcinoma andadenocarcinoma of the cervixkeywords cervical adenocarcinoma cervical squamous cell carcinoma muc5ac ck7 correspondence xiaofangzhangsdueducn hailing li and xiaotong jing contributed equally to this work2department of pathology school of basic medical science shandonguniversity jinan shandong p r china5department of pathology school of basic medical science shandonguniversity jinan shandong p r 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 0cli diagnostic pathology page of thelastintroductioncervical cancer is the fourth most common carcinomain women responsible for of cancerrelateddeaths worldwide [ ] squamous carcinoma is themost common type of cervical carcinoma followed byadenocarcinoma nevertheless overthreedecades a significant increase in adenocarcinoma caseshas been observed in many developed countries especiallyin younger women papsmear screening also knownas pap test is still considered the main screening methodfor cervical cancer especially for squamous carcinoma compared to squamous carcinoma the adenocarcinomaof the cervix is more common in younger women and hasa poorer prognosis therapeutic approaches includechemoradiotherapy ccrt which has been proven tobe effective for squamous carcinoma of the cervix but notfor adenocarcinoma of the cervix due to its highchemo and radioresistance therefore differentiatingadenocarcinoma from squamous carcinoma is importantin order to provide patients with most suitable therapyp63 p40 and cytokeratin 56ck56 are the mostcommon panel ofimmunochemical markers for thediagnosis of squamous carcinoma p63 and ck56are traditional markers that indicate squamous differentiation in primary lung neoplasms most squamouscarcinomas and large cell carcinomas are positive forck56 warth found that the probability of acorrect sqcc diagnosis using ck56 is p63 a transcriptional regulator has a crucial role in thedevelopment and differentiation of stratified squamousepithelium it is usually strongly expressed in the basalkeratinocytes [] vosmik analyzed patientswith cervical squamous cell carcinoma and found that had positive expression of p63 p40 is a new specific marker for distinguishing squamouscarcinomas from adenocarcinoma whose specificity isabout in lung carcinomas however the positiveexpression of ck56 p63 and p40 are only found in a fewadenocarcinomas [ ] kriegsmann suggested theuse of either ck56 or p40 over p63 in the routine diagnostic setting ck7 is expressed in many ductal andglandular epithelial cells mainly gallbladder hepatic ductsand pancreatic ducts in tissues of the female genital tractovary endometrium fallopian tube and cervix and in thebreast lung and urinary tract tissues in the normalcervical tissue and adenocarcinoma ck7 staining wasobserved in the columnar cells of endocervical glandshashiguchi found the different rates of ck7 in patientswith cervical intraepithelial neoplasia and those with invasivecarcinomas vs [ ] thus far no efficientmarkers have been developed for distinguishing squamouscell carcinoma and adenocarcinoma in the endocervixmucins are a family of large glycoproteins expressedon the epithelial cell surfaces including ducts of lacrimalglands in the eye salivary glands the lining of the respiratory gastrointestinal urothelial and reproductive tracts muc5ac belongs to gelforming mucins multiple histological studies have highlighted that muc5acis expressed in the conjunctiva middle ear nasopharynxlungs gallbladder and stomach under normal conditionswhere it provides protection to corresponding epithelialsurfaces from different factors some research hasshown that muc5ac may be a potential biomarker inpancreatic cancer tissues dimaio found thatanterior gradient homolog and muc5ac are usefulpositive markers of adenocarcinoma in the setting ofabsent or diminished p63 and cytokeratin staining inesophageal carcinoma it is also expressed in theendocervix yamanoi found that muc5ac waslargely expressed in typical legh atypical legh gasmda and gasnonmda thus we speculated thatmuc5ac could be expressed in other adenocarcinomasand might be used for the differential diagnosis of adenocarcinoma and squamous carcinoma the aim of thisstudy was to examine the combination of cytokeratin p63 p40 and muc5ac for distinguishing squamous cellcarcinoma scc from the adenocarcinoma in the cervixaecmaterials and methodstissue sampleswe analyzed poorly to moderately differentiated cervical squamous carcinoma scc and adenocarcinomasof endocervix aec all tissues were collected from thedepartment of human pathology of qilu hospitalshandong university china from to specimenswere retrieved from the pathology files of the departmentof pathology at the same hospital after collection all specimens were fixed in buffered formalin hematoxylin eosin he stains were available for review paraffin blockswere used for immunohistochemical staining all the slideswere reviewed by two experienced pathologistshistopathological and clinical variables including agetumor size differentiationinfiltrate depth and lymphnode metastasis were summarized in table followupinformation was available in aec with the followuptime ranging from to months mean monthsimmunohistochemistryfour to five micronthick paraffin sections of the cases were dewaxed rehydrated in graded alcohols andprocessed using the pv9000 detection kit zsbio commerce store beijing china briefly antigen retrieval wasperformed in a microwave oven for min in mm trisedta buffer mm tris base mm edta solution tween ph endogenous peroxidase activitywas blocked with a h2o2methanol solution for min slides were then incubated in normal goat 0cli diagnostic pathology page of table comparison of clinicopathological features between cervical squamous cell carcinoma and cervical adenocarcinomasquamous cell carcinomasn adenocarcinoman Ï p valueage¤ size cm ¥ unknowndifferentiationpoormoderatewellunknown infiltrate depth of mesenchyme¤ unknownlymph node metastasisnoyesunknown serum for min to prevent nonspecific binding sampleswere then incubated overnight at °c with a primary antibody phosphate buffered saline pbs was used instead ofthe first antibody as a negative control consequentlysamples were incubated with reagent atroomtemperatureroomfor min and reagent attemperature for min finally the tissues were stainedwith diaminobenzidine dab the antibodies used in thisstudy are listed in table scoring methodstaining results were interpreted by one pathologist whowas blinded to the patients clinical and pathologicaldata for ck56 ck7 and muc5ac more than oftumor cells with a membrane or cytoplasmic brownyellow granules were considered positive for p63 andp40 the positive standard was that more than oftumor cells have brownyellow granules in the nucleusstatistical analysisstatistical analysis was performed with spss softwareversion spss inc chicago ii usa chisquareor fishers exacttests were used when comparingfrequencies between two groups probability values lessthan were considered statistically significantresultsthe expression of ck56 p63 p40 ck7 and muc5ac inscc and aecihc for the five proteins was performed on humanprimary cervical cancersincluding scc and aec as shown in fig and fig muc5ac ck56and ck7 were mainly expressed in the cell membranetable immunohistochemical antibodiesantibodymuc5acnozm0395ck56ck7p40p63zm0313zm0071zm0472zm0406vendorzsbio commerce store beijing chinazsbio commerce store beijing chinazsbio commerce store beijing chinazsbio commerce store beijing chinazsbio commerce store beijing chinadiluationready to useready to useready to useready to useready to use 0cli diagnostic pathology page of fig the expression of ck56 p63 p40 ck7 and muc5ac in a case of poordifferentiated squamous cell carcinoma by ihc a he b ck56positive staining c p63 positive staining d p40 positive staining e ck7 positive staining f muc5ac negative staining Ãfig the expression of ck56 p63 p40 ck7 and muc5ac in case of poordifferentiated adenocarcinoma invasive stratified mucinproducingcarcinoma ismile by ihc a he b ck56 negative staining c p63 negative staining d p40 negative staining e ck7 negative staining f muc5acpositive staining à 0cli diagnostic pathology page of and cytoplasm while p40 and p63 were mainly locatedin the nucleussignificant effect on the prognosis of cervical adenocarcinoma patients p as shown in fig tumorin thecells ofwe found that muc5ac exhibited prominent immucervical aecnoreactivitymuc5ac and ck7 were detected in and of aec cases compared to and of scc casesbesides for aec the specificity of muc5ac was muchhigher than that of ck7 p moreover the sensitivity of ck56 p40 and p63 was and respectively and the specificity was and respectively in aec table through the combined detection of p40 or p63 wecompared muc5ac and ck7 again we found that thesensitivity and specificity of muc5ac in aec combinedwith p40 or p63 were and respectively and respectively the sensitivity and specificity of ck7 combined with p40 or p63 were and and respectively table thesensitivity of muc5ac combined with p40 or p63 wassimilar to that of ck7 while the specificity was slightlyhigher than that of ck7expression of muc5ac and ck7 in cervicaladenocarcinoma subtypeswe further detected the expression of muc5ac insubtypes of aec table among cases of usualtype cervical adenocarcinoma cases were muc5acpositive and cases were ck7 positive and there wasno statistical difference p in cases of mucinous adenocarcinoma nosthe expression rate ofmuc5ac and ck7 were both moreover out of cases of gastric mucinous adenocarcinomaexpressed muc5ac and of them were ck7 positivep the positive rate of the muc5ac in mucinouscarcinoma intestinal type villous tubular adenocarcinomaendometrioid adenocarcinoma clear cell carcinoma serouscarcinoma adenosquamous carcinoma and invasive stratified mucinproducing carcinoma ismile was and respectively the expressionrate of muc5ac had no statistical difference among thesesubtypes all p correlation between muc5ac expression andclinicopathological characteristics in cervicaladenocarcinomathis study further analyzed the relationship between theexpression of muc5ac and clinicopathological featuresin cervical adenocarcinoma table the expression oftumormuc5ac was correlated with the degree ofdifferentiation p a lower degree oftumordifferentiation was associated with a lower expressionrate of muc5ac there was no significant correlationbetween the expression of muc5ac protein and agetumor size depth of myometrialinvasion and lymphnode metastasis all p kaplan meier analysisrevealed that the expression of muc5ac protein had nodiscussion and sidentification of previously unutilized sensitive biomarkers is still a priority for improved differential diagnosis of cervical aec and scc at present ck56 p63p40 and ck7 are the main biomarkers for differentiatingcervical adenocarcinoma from squamous cell carcinomack56 is a kind of high molecular weight basal cellkeratin 58kda and 56kda which is mainly expressed inthe basal cells of squamous epithelium and ductal epithelium and some squamous epithelial germinal layercells myoepithelial cells and mesothelial cells butpoorly expressed in glandular epithelial cells someresearch results showed that ck56 has high sensitivityand specificity in the diagnosis ofsquamous celltable sensitivity and specificity of muc5acãck56ãck7ãp40ãp63 in cervical squamous cell carcinoma and adenocarcinomamarkerssensitivityspecificitysquamous cell carcinomasn adenocarcinoman muc5acck7ck56p40p63ck56and p40ck56and p63muc5acand p40muc5acand p63ck7and p40ck7and p63 0cli diagnostic pathology page of table the correlation of muc5ac and the clinical variants inthe cervical adenocarcinomathe expression of muc5acpositivenegativeÏ valuep valueage¤ v size cm ¥ differentiationpoorwellmoderateinfiltrate depthof mesenchyme¤ lymph nodemetastasisnoyescarcinoma [] in contrast other studies showedhigh sensitivity but low specificity when diagnosing thistype of tumor p63 is a member of the p53 family a classical tumorsuppressor gene family it is located on chromosome3q27 filho showed good sensitivity whendetecting squamous cell carcinoma with a positive rateof contrary kaufmann suggested thatp63 could also be expressed in a small number of adenocarcinoma basal cell carcinoma and transitional epithelial carcinoma moreover p63 can also be used as amarker of myoepithelial cells and prostate basal cellstherefore p63 lacks absolute specificity for squamousdifferentiationp40 is a subtype of p63 protein expressed in squamousepithelial cells including epidermis and hair folliclesurothelial cells myoepithelial cells ofthe mammarygland sweat gland and salivary gland and basal cells ofthe prostate which are highly specific in labeling squamous epithelium bishop showed that in cases of squamous cell carcinoma of the lung and cases of adenocarcinoma of the lung the sensitivity andspecificity of p63 were and respectivelythe sensitivity and specificity of p40 in the diagnosis ofsquamous cell carcinoma of the lung were and respectively therefore p40 is considered as ahighly specific and sensitive tumor biomarker of squamous epithelial origin in this study we used immunohistochemistry to detectck56 p63 and p40 in cervical squamous cell carcinomaand adenocarcinoma the sensitivity of ck56 p40 andp63 was and respectively and thespecificity was and respectivelymoreover the specificity of ck56 is slightly lower thanthat of p40 and p63 we also found that a combinationof ck56 with p40 or p63 slightly decreased the sensitivity and and increased the specificity and which in turn increased the accuracy of diagnosing squamous cell carcinomack7 is a kind of low molecular weight keratin mainlyexpressed in glandular epithelium and transitional epithelial cells of most normal tissues many studieshave found that ck7 is not only expressed in adenocarcinoma but also in squamous intraepithelial neoplasiacervical squamous cell carcinoma lung squamous cellcarcinoma and esophageal squamous cell carcinomalee found a positive expression of ck7 in fig survival analysis of muc5ac expression in cervical adenocarcinoma 0cli diagnostic pathology page of table expression of muc5ac and ck7 in differentadenocarcinoma subtypessubtypesmuc5acusual typeÏ valueck7p valuepositivenegativemucinous adenocarcinoma nospositivenegativegastric typepositivenegativeintestinal typepositivenegativevillous tubular adenocarcinomapositivenegativeendometrioid adenocarcinomapositivenegativeclear cell carcinomapositivenegativeserous carcinomapositivenegativeismilepositivenegativeadenosquamous carcinomapositivenegativeismile invasive stratified mucinproducing carcinoma cases with scc and cases withciniii furthermore yamada found that ck7expression in esophageal squamous cell carcinoma butalso in iiiaiib stage esophageal squamous cell carcinoma suggest poor tumor differentiation and thus canbe used as an independent prognostic factor ourstudy showed that the positive rate of ck7 was incervical poorly differentiated squamous cell carcinomawhich further suggested that ck7 is not an ideal markerfor differentiation between squamous cell carcinoma andadenocarcinomamucin is a high molecular weight glycosylated proteinsecreted by epithelial cells in the respiratory tractgastrointestinal tract and urogenital tract which has animportant role in the protection of epithelium cell adhesion signal transduction immune activation and inhibition at present at least mucins have been found inthe female reproductive system riethdorf and albarracin used immunohistochemistrymethods to detect the expression of muc5ac in different female reproductive system malignant tumors theyfound that muc5ac was highly expressed in cervicaladenocarcinoma and poorly expressed inendometrial adenocarcinoma all of themwere expressed in the primary ovarian mucinous tumor but not in colon adenocarcinoma therefore they concluded that muc5ac could beused as an effective marker to distinguish the origin ofpelvic tumors and distinguish primary ovarian tumorsand colorectal metastasis as well as endometrial adenocarcinoma from cervical metastasis [ ] in thisstudy we found positive expression of muc5ac in cases of cervical adenocarcinoma and in cases of squamous carcinoma which wasconsistent with riethdorfs study the sensitivity ofmuc5ac and ck7 to cervical adenocarcinoma was and respectively but the specificity ofmuc5ac was much higher than that of ck7 through the joint detection of p40 or p63 wecompared muc5ac and ck7 again and found that thesensitivity and specificity of muc5ac combined withp40 or p63 were and respectively and respectively the sensitivity and specificity ofck7 combined with p40 or p63 were and and respectively these results showed thatthe sensitivity of muc5ac combined with p40 or p63was similar to that of ck7 butthe specificity wasslightly higher than that of ck7 therefore muc5ac issuperior to ck7 in the diagnosis of cervical adenocarcinoma and squamous cell carcinomabesides we preliminarily detected the expression ofmuc5ac in different types of cervical adenocarcinomaand found no significant difference these data suggestedthat muc5ac has no diagnostic significance in the classification of cervical adenocarcinoma at the same time weanalyzed the relationship between the expression ofmuc5ac and the prognosis of cervical adenocarcinomaand the result revealed that muc5ac was not related tothe prognosis of cervical adenocarcinomaoverall our observations strongly suggest that muc5acmay be useful as a biomarker for differential diagnosesbetween squamous carcinoma and adenocarcinomaabbreviationsscc squamous cell carcinoma aec adenocarcinoma of the cervixck cytokeratin he hematoxylin eosin pbs phosphate buffered salinedab diaminobenzidine ismile invasive stratified mucinproducingcarcinoma 0cli diagnostic pathology page of authors contributionsxiaofang zhang designed the study and drafted the manuscript hailing liand xiaotong jing analyzed the data and carried out theimmunohistochemistry jie yu and tingguo zhang read the pathologicalsections jinan liu collected the clinical data and carried our followupshiming chen made the slides the authors read and approved the finalmanuscript downey p cummins r moran m gulmann c if it's not ck56 positive ttf negative it's not a squamous cell carcinoma of lung apmis warth a muley t herpel e meister m herth fj schirmacher p weichert whoffmann h schnabel pa largescale comparative analyses ofimmunomarkers for diagnostic subtyping of nonsmallcell lung cancerbiopsies histopathology fundingthis work was supported by the national natural science foundation ofchina no and technology development foundation of yantaino ws017availability of data and materialsnot applicableethics approval and consent to participateall tissue samples from patients were collected and protocols wereperformed according to the procedures approved by the research ethicscommittee of shandong medical university all patients provided informedconsentcompeting intereststhe authors declare that they have no competing interestsauthor details1department of pathology weifang traditional chinese hospital weifangshandong p r china 2department of pathology school of basic medicalscience shandong university jinan shandong p r china 3department ofpathology the fourth hospital of jinan the third affiliated hospital ofshandong first medical university jinan shandong p r china 4departmentof oncology yuhuangding hospital yantai shandong p r china5department of pathology school of basic medical science shandonguniversity jinan shandong p r chinareceived july accepted august referenceskurman rj carcangiu ml herrington cs who classification of tumours offemale reproductive ans4th ed lyon iarc press takeuchi s biology and treatment of cervical adenocarcinoma chin jcancer res young rh clement pb endocervical adenocarcinoma and its variants theirmorphology and differential diagnosis histopathology forouzanfar mh foreman kj delossantos am lozano r lopez ad murraycj naghavi m breast and cervical cancer in countries between and a systematic analysis lancet galic v herzog tj lewin sn neugut ai burke wm lu ys hershman dlwright jd prognostic significance of adenocarcinoma histology in womenwith cervical cancer gynecol oncol favero g pierobon j genta ml araujo mp miglino g del cpdm deandrade ch fukushima jt baracat ec carvalho jp laparoscopicextrafascial hysterectomy completion surgery after primary chemoradiationin patients with locally advanced cervical cancer technical aspects andoperative outcomes int j gynecol cancer rose pg java jj whitney cw stehman fb lanciano r thomas gm locallyadvanced adenocarcinoma and adenosquamous carcinomas of the cervixcompared to squamous cell carcinomas of the cervix in gynecologiconcology group trials of cisplatinbased chemoradiation gynecol oncol ma y fan m dai l kang x liu y sun y xiong h liang z yan w chen kexpression of p63 and ck56 in earlystage lung squamous cell carcinoma isnot only an early diagnostic indicator but also correlates with a goodprognosis thorac cancer kaufmann o fietze e mengs j dietel m value of p63 and cytokeratin as immunohistochemical markers for the differential diagnosis of poorlydifferentiated and undifferentiated carcinomas am j clin pathol barbieri ce pietenpol ja p63 and epithelial biology exp cell res senoo m pinto f crum cp mckeon f p63 is essential for the proliferativepotential of stem cells in stratified epithelia cell pozzi s zambelli f merico d pavesi g robert a maltere p gidrol xmantovani r vigano ma transcriptional network of p63 in humankeratinocytes plos one 200943e5008 vosmik m laco j sirak i beranek m hovorkova e vosmikova h drastikovam hodek m zoul z odrazka k prognostic significance of humanpapillomavirus hpv status and expression of selected markers her2neuegfr vegf cd34 p63 p53 and ki67mib1 on outcome after chemoradiotherapy in patients with squamous cell carcinoma of uterine cervixpathol oncol res nobre ar albergaria a schmitt f p40 a p63 isoform useful for lung cancerdiagnosis a review of the physiological and pathological role of p63 actacytol stolnicu s hoang l hankobauer o barsan i terinte c pesci a avielronens kiyokawa t alvaradocabrero i oliva e and others cervicaladenosquamous carcinoma detailed analysis of morphologyimmunohistochemical profile and clinical outcomes in cases modpathol toyoshima m momono y makino h kudo t oka n sakurada j suzuki hkodama h yoshinaga k cytokeratin 7positivecytokeratin 20negative cecaladenocarcinoma metastatic to the uterine cervix a case report world jsurg oncol hashiguchi m masuda m kai k nakao y kawaguchi a yokoyama maishima s decreased cytokeratin expression correlates with theprogression of cervical squamous cell carcinoma and poor patientoutcomes j obstet gynaecol res lee h lee h cho yk cytokeratin7 and cytokeratin19 expression in highgrade cervical intraepithelial neoplasm and squamous cell carcinoma andtheir possible association in cervical carcinogenesis diagn pathol krishn sr ganguly k kaur s batra sk ramifications of secreted mucinmuc5ac in malignant journey a holistic view carcinogenesis thornton dj rousseau k mcguckin ma structure and function ofthe polymeric mucins in airways mucus annu rev physiol rose mc voynow ja respiratory tract mucin genes and mucinglycoproteins in health and disease physiol rev balmaña m duran a gomes c llop e lópezmartos r ortiz mr barrabés sreis ca peracaula r analysis of sialyllewis x on muc5ac and muc1mucins in pancreatic cancer tissues int j biol macromol dimaio ma kwok s montgomery kd lowe aw pai rkimmunohistochemical panel for distinguishing esophageal adenocarcinomafrom squamous cell carcinoma a combination of p63 cytokeratin muc5ac and anterior gradient homolog allows optimal subtyping humpathol yamanoi k ishii k tsukamoto m asaka s nakayama j gastric gland mucinspecific oglycan expression decreases as tumor cells progress from lobularendocervical gland hyperplasia to cervical mucinous carcinoma gastrictype virchows arch reisfilho js simpson pt martins a preto a gartner f schmitt fcdistribution of p63 cytokeratins and cytokeratin in normal and neoplastic human tissue samples using tarp4 multitumor tissuemicroarray virchows arch yamada a sasaki h aoyagi k sano m fujii s daiko h nishimura myoshida t chiba t ochiai a expression of cytokeratin predicts survival instage iiiaiib squamous cell carcinoma of the esophagus oncol rep baker ac eltoum i curry ro stockard cr manne u grizzle we chhieng dmucinous expression in benign and neoplastic glandular lesions of theuterine cervix arch pathol lab med 0cli diagnostic pathology page of riethdorf l o'connell jt riethdorf s cviko a crum cp differentialexpression of muc2 and muc5ac in benign and malignant glandularlesions of the cervix uteri virchows arch albarracin ct jafri j montag ag hart j kuan sf differential expression ofmuc2 and muc5ac mucin genes in primary ovarian and metastatic coloniccarcinoma hum pathol publishers notespringer nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations 0c" | Colon_Cancer |
" national economies are increasingly facing the challenge of having to finance the prevention andtreatment of human diseases and of having to compensate for the resulting loss of economic production physicalinactivity is demonstrably closely related to the risk of developing certain disease group physical inactivity results indirect and indirect burdens that the present study intends to quantify in hungary for the period between and methods based on the data of the hungarian public finances this study determines the direct and indirect costsincurred by hungary due to illnesses and through the par method it quantifies the financial burden of physicalinactivity incurred by the hungarian treasuryresults the total financial burden of illnesses in hungary showed a decreasing tendency from to eventhough the year saw an increase in costs compared to similarly while total public expenditure on illnessesassociated with physical inactivity increased by when compared to the total amount attributable to medicalconditions stemming from physical inactivity still showed a decrease of billion huf in the overall period the biggesteconomic burden is posed by cardiovascular diseases hypertension and type diabetess the increase in the economic burden associated with physical inactivity can be attributed to thecombined effect of two factors changes in total expenditure on specific disease groups which showed an increase inthe period under review and changes in the physical activity levels of the hungarian population which showed animprovement over the period under review initiatives in hungary aimed at encouraging an active lifestyle fromchildhood onwards should be continued since beyond the initial impact that has already been felt to some extent inrecent years these initiatives will come to their full fruition in the coming decadeskeywords physical inactivity economic burden parmethod direct costs indirect burden population attributable risk the fundamental change towards a more sedentary lifestyle has a serious impact on peoples health physical inactivity is one of the most important global issues of thetwentyfirst centuryleading to an increased risk ofchronic diseases such as type diabetes cardiovascular correspondence davidpaaretkptehu1university of pecs faculty of health sciences pecs hungaryfull list of author information is available at the end of the disease certain types of cancer rectal colon breastobesity and osteoporosis these diseases may even become the cause of death the world health anizationwho has also identified these medical conditions as themost burdensome noncommunicable diseases of todaysdeveloped world regular moderate physical activity reduces the risk of the most common of these diseases andcontributes to an increased sense of wellbeing [ ] acinactivity ranks as thecording to the who physical 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 0c¡cs bmc public health 20suppl page of fourth most significant mortality factor in the world with million deaths a year worldwide [ ]another study suggests that there is a lower likelihoodof health problems among people engaging in regularphysical activity than among those leading a sedentarylifestyle furthermore there is convincing evidence thatregular physical activity increases life expectancy and reduces the likelihood of developing coronary and cardiovascular problems of suffering a stroke or developingcolon cancer inactive and sedentary lifestyles directly affect metabolism bone mineral composition and magnify the healtheffects of cardiovascular disease furthermore thereis epidemiological evidence to suggest that a sedentarylifestyle increases the risk of cancer obesity metabolicand psychosocial problems according to oecd data the average life expectancyof hungarians at birth in was years which is years below the oecd average actually one of the lowest on the list for men this value is years forwomen years both showing an increasing trend in recent years the hungarian government has made anumber of efforts to bring about significant changes in theinactive lifestyle of the hungarian population these include measures to increase the number of physical education lessons and to improve the conditions in pe lessonsat school also the development and construction of sportsfacilitiesincreased funding for sports associations andeven the use of corporate tax incentives for sporting purposes while improving the conditions alone does notresult in a change in the attitudes of the population towards sport it is certainly a prerequisite procedures that quantify the burden on the hungarianeconomy resulting from physicalinactivity are one ofthe ways of measuring the effectiveness of state intervention [ ] this study aims to contribute to this bodyof research and proposes to analyse a longer timespectrummethodsto analyze the economic burden of physical inactivity weneed to start with the burden of diseases on the nationaleconomy as physical inactivity plays a vital role in the onset of several diseases and leads to various causes of deathat a national level diseases have direct and indirect costsdirect costs of diseases include treatments medications sickpay allowances and associated ancilliary coststhat are directly related to the illness the direct costs inhungary are mainly financed by the national health insurance fund nhif since it is called the national health insurance fund administration nhifa but we must not disregard the cost of sick leave and private costs outside of nhifnhifa financing the latterof which are directly borne by members of societyamong the indirect burdens we include items that constitute a loss to the economy or to society as a result ofthe loss of work caused by a disease there was a significant change in this area during the research periodwhile in and there was a longterm loss ofproduction only in jobs on the skillsshortage list or invery special cases by the number of job vacanciesin hungary reached thousand while by july thisnumber rose to thousand people which is of theworkforce our calculations were based on the following assumptions in and in a labor marketcharacterised by an oversupply of labor a frictional unemployment of months groupbased performance expectations and the market of goods and services beingoversupplied people on average worked days a yearand loss was calculated based on gdp per capita thestudy that inspired our calculations had a similarcalculation but we replaced its assumptions with theabovementioned assumptions and we broadened andtightened formulas and corrected data that had becomefact since then however when calculating the results we had to change the assumptions about the labormarket from the previouslyoutlined conditions as by hungarian labor market had become characterizedwith overdemand therefore we had to increase frictiontime as well monthsanother economic burden is presenteesim which isthe term used for the phenomenon when a sick individual goes to work which results in poorer performanceand thus loss of productionour main goalin this research was to quantify theeconomic burden of diseases for the years and and more specifically the costs to thenational economy directly attributable to physicalinactivity in the market years and during our research we treated as relevant secondarydata eurobarometer and and nhifnhifa data from and []in the course of our resreach we examined the typesof medical conditions related to physical inactivity andtheir possible complications the factual data for whichwas obtained from nhif and nhifawith the help of par method population attributable risk the most commonly used method in international research we were able to obtain quantitativemeasurements that were used uniformly in the analysisof data for all three yearspar ¼ pexp 02 rr¾ ¾ pexp 02 rr°°¾ 02 wherepexp prevalence refers to the section of the populationwhere a given risk is present 0c¡cs bmc public health 20suppl page of rr relative risk describes the risk associated with asedentary lifestylewhen using the index it is necessary to break downthe population into physically active and inactive sections and then by determining the relative risk rate wecan estimate the number and cost of illnesses stemmingfrom a physically inactive lifestyle the physical activity indicators ofthe hungarianpopulation showed fluctuations during the period underreview the situation was the worst in when wesaw of the population as physically inactive in ouropinion the health protection effect does not manifestitself in the case of those who never excercise or only doso times a month by this figure dropped to and at the same time the ratio of those engaging inexercise at least times per week increased threefold inthe following years a more negative trend was observed as the rate of inactive people rose to although this is still significantly better than the basefigure for fig with the help of metaanalysis we calculated the relative risk ratio rr an indicator which is prevalent ininternational literature in order to estimate the futureexpenditure stemming from physical inactivity for all affected disease groups such as cardiovascular diseasestroke hypertension colon cancertype diabetesosteoporosis depression gastrointestinal complicationsobesity high triglyceride diseases and deliberate selfharm []the rr is the proportion of the applicaple diseasesamong people with inactive lifestyles divided by the proportion of the applicable diseases among people with active lifestyles on the basis of the rr values it is possibleto quantify the par indicator by disease group for eachyear table in order to allow the data to be compared over timethe data on the burden of illnesses stemming from physicalinactivity for and was recalculated to prices while the total amount of burden imposedby illnesses was recalculated to prices using thefig the ratio of physical activity and inactivity in hungary in sourcespecial eurobarometer special eurobarometer specialeurobarometer 0c¡cs bmc public health 20suppl page of table the cumulative relative risk rate and par values for theexamined disease types in disease typesheart and coronary diseasespar par rrpar strokehypertensioncolon cancertype diabetesosteoporosisdepressiongastrointestinal complicationsobesityhigh triglyceridesdeliberate selfharmsource katzmarzyk aldoori ewing andersen schuch domestic producer and consumer price index of thehungarian central statistical office hcso the economic burden ofresultsat pricesillnessesamounted to more than billion forints huf in of which the direct burden was billion forintsdirect costs accounted for of the burden of illnessesand the billion huf sickness benefit represented justover of total direct costs indirect burden representeda significantly lower percentage amounting to over billion huf the economic burden imposed by sicknessin was of hungarys gdpby the economic burden of diseases fell to billion huf at prices direct costs accounted for of the total burden of illnesses that year less than of which amounting to billion huf was forsickness allowance expenditues indirect burdens decreased to billion huf the burden of sicknessamounted to of the gdp in by the economic burden of diseases fell to billion huf at prices direct costs accounted for of the total burden that year and of it weresick allowances amounting to billion forints indirectburdens fell to billion huf the burden of sicknessdecreased to of the gdp in by the economic burden of illnesses increasedcompared to but it was still below the initial figure huf billion and it decreased in comparison with the gdp the share of direct costs dropped significantly to within which the sickness benefitrepresented to the value of billion forints atthe same time indirect burdens increased significantlyto billion huf all in all the burden of sickness decreased to of the gdp in between and the economic burden of diseases fell by billion huf which is a total decrease of corresponding to an average annual decrease of and in the meantime the countrys gdp increasedsignificantly altogether at current prices obviously the decrease is due to a number of reasons butthe effect of the increase in physical activity is an important factor among them table in the years examined in the case of disease groupslinked to physical inactivity the burden of illnesses onthe state budget excluding sickness allowance amounted to billion huf and billion hufrespectively of which the lowest value was in however only a part of these can be directly linked tophysical inactivity as many other risk factors play a rolein the development of these diseases as regards therelative weight of each disease group cardiovascular disease is the biggest burden followed by hypertension atthe same time type diabetes was only ranked the fifthfor costs in the first year but by it became thethird largest item only slightly behind high blood pressure expenditure on stroke obesity and deliberate selfharm were almost negligible compared to other diseasegroups table based on the results it can be stated that in theexpenditures in the state budgetfor the diseasegroups examined drastically decreased by approximately billion huf compared to the initial starting positionof billion huf but by the expenditures hadsurpassed the base total from by more than billion huf compared to only type two diabetesand osteoporosis showed an increase and respectively compared to although the latter is dueto the relatively low total expenditure for all other disease groups the level of expenditure declined in absoluteterms resulting in a significant decrease of billionhuf in total expenditurehowever in the case of the picture is more varied if we examine the relative position of certain diseasegroups compared to type diabetes showed themost significant increase to the tune of more than billion huf the other diseases lag behind in terms ofexpenditure cardiovascular diseases and colon cancerare next with an increase of billion forints inaddition there is an increase in the costs associated withosteoporosis stagnation or decrease was observed forthe other disease groups but this could not compensatefor the increase in the costs of the aforementioned diseases the most significant drop in expenditure is observed in hypertension billion huf and hightriglyceride diseases billion huf table focusing on the direct burden of physical inactivitywe can conclude that of the total expenditure ofthe disease groups is directly attributable to physical 0c¡cs bmc public health 20suppl page of table economic burdens of diseases in hungary in huf million in real terms in direct costs statefinancedeconomic burdens of diseasesin hungary medicationmedical aidsgeneral practitioner servicesdental servicesoutpatient carect mrimedical centers exluding vd clinicsdialysishome careinpatient carehighcost medical procedurespatient transportspa treatmentsgovernmental health careexpendituresick leavedisability rehabilitation treatmentcharged tonhifnhifanhifnhifanhifnhifanhifnhifanhifnhifanhifnhifanhifnhifanhifnhifanhifnhifanhifnhifanhifnhifanhifnhifanhifnhifanhifnhifnhifanhifnhifain totalprivate costsprivate health care expenditureindirect costsexpenditure associated withsick leavehealth insurance managementand other costsfriction due to sickness leading toloss of productionof which reduced pay due to sickpay and sick leaveof which tax loss for the statefriction due to disability leadingto loss of productionindividualemployernhifaemployer individualstateindividualstatesocietypresenteeism costsin totalemployerinactivity the major part is the cost of cardiovasculardiseases and hypertension and these were closelyfollowed by type diabetes by due to the factthat the total expenditure for stroke obesity and deliberate selfharm was also insignificant compared to otherdisease groups their expenditure related to physical inactivity is insignificant in the case of deliberate selfharm the costs cannot be measured even in the order ofone hundred thousand forints table compared to the decrease for the year ofthe direct costs stemming from physical inactivity is larger in proportion than the decrease of total expenditurethis is true of each disease group and for those twogroups type diabetes and osteoporosis where therewas an actual increase in costs the increase was less forrespectivelyphysical inactivityrelated expenses than for overall expenses the largest drop in monetary terms can be observed in the case of hypertension and cardiovasculardiseases over billion huf but there was a decreaseof and billion hufin hightriglyceriderelated diseases and colon cancer howeverpercentagewise nhif achieved the highest cost reduction for high triglycerides and colon cancer closely followed by a decrease in stroke expenditure and deliberate selfharm although inthe last two categories the low sum total spent alsomakes this decrease appear larger percentagewise thanwould be the case with larger totalscompared to the expenditure related to physicalinactivity in shows a decrease of billion huf 0ctotal amountinactivitytotal amountinactivitytotal amountinactivitycardiovascular diseasesstrokehypertensioncolon cancertype diabetesosteoporosisdepressiondigestive disordersobesityhigh triglyceridesdeliberate selfharmtotal¡cs bmc public health 20suppl page of table total cost incured by nhifa nhif of those disease groups that are associated with physical inactivity and costs directlyattributtable to physical inactivity itself in terms of prices million hufdisease typesat the level of the individual disease groups the amountsvary the most significant decline in absolute terms is inthe high blood pressure and high triglyceriderelated illness groups however the burden of type diabetes increased significantly and there was an increase in coloncancer and osteoporosis disease groups the direction andextent of the changes are mostly comparable to the totalexpenditure amounts at the overall level of the diseasegroups although the changes in the physical inactivity ratenaturally lead to differences in the specific values this isso much so that the total expenditure amounts increasedat the level of all disease groups by but overall theexpenditure related to physical inactivity shows a decreaseof table discussionwe can clearly conclude similarly to other international researches [ ] that physical activityand forms of recreational exercise have a protectiveeffect eg a preventive effect against certain types ofchronic diseases cardiovascularlocomotor disordersdiabetes and certain types of tumors the decreasein physical inactivity has a positive effect on productivity as fewer people avail themselves of sick leave atable changes in total expenditure as reported by nhifa nhif and changes in expenditure directly stemming from physicalinactivity compared to the base level expenditure in in real terms adjusted to prices million hufdisease typescardiovascular diseasesstrokehypertensioncolon cancertype diabetesosteoporosisdepressiondigestive disordersobesityhigh triglyceridesdeliberate selfharmtotaltotal amount inactivity total amount inactivity 0c¡cs bmc public health 20suppl page of study of economic development over the past centuryhas concluded that the advancement of the populations health status is responsible for about ofeconomic growth []in our comparative study we used four samplingpoints between and to demonstrate the burden of diseases at the level of the national economy forthe various loadcarriers in the period under review theeconomic burden decreased significantly overall from of the gdp to the weight of indirect burden increased however as in the currently demanddominated labormarket it is more difficult to replacelost workforce in the period of analysis the number ofemployees in hungary increased with which increased the amount of sick leave and number of sicknessdays but their gdp contribution was significantly higheralthough associated costs and burdens increased innominal terms they decreased in relation to the gdpa large part of diseases burdens are borne by the stateand society followed by households andemployers the proportions are similar to ding in european countries included hungary although we estimate that the burdens on employers arehigher and the burdens on households are lower inhungarysince the physical activity rate of the hungarianpopulation has been fluctuating but overall there is animproving tendency which is also apparent in the savings potential of the examined expenditures categoriescompared to the gdp the amount of spending dependsheavily apart from the physical inactivity rate on thenumber of employees as well as those people who arenot employed can not have sick leavefor exampleoverall government spending depends on the budget ofthe country which is connected to the overall economicsituationcardiovascular diseases accounts for most of the costof physical inactivity in hungary which coincides withmattli in switzerland however of directinactivityto depression inswitzerland while nhifas depression costs account foronly in hungaryattributablecostsarein hungary a number of measures have recently beentaken in order to integrate physical activity and sportinto peoples daily lives such measures include theintroduction of everyday physical education in schoolsor the extensive development of sports infrastructure however the effects of these measures will have amore pronounced effect in the long run several studieshave shown that high physical activity in childhood isnot yet measurable in terms of economic returns lessfrequent use of health care and a lower cost associatedwith using them as some effects such as the high costof sports injuries high rates of childhood illness haveresearch data confirm the facta negative bearing on the rate of return on investmenthowever a longterm change of attitude and opennessto physical activity at later stages in life are where thesemeasures bear a profit so any effort to support childinterhood sports is rewarding [ ] in additionnationalthatthoseparents who are themselves engaged in sport or currently do so are more likely to prefer sporting activitiesamong their children it is important to draw attentionto the fact even minimal physical activity has a healthimproving effect at any stage of life that is whysport and health policies at all times should promoterecreational activities for all ages not just young peoplewe would like to expand the scope of our current research if we could also examine how the patient numbers varied each year by disease group unfortunatelythe data was not available this would be of particularinterest for the year as the expenditure on illnessesshowed a significant increase in real terms compared to which may be due to the fact that more patientsreceived care and treatment but may also be due an increase of the normative provision per person by the government possibly to provide better quality careif we posit based on the eurobarometer data that thephysical activity rate improved compared to wecould also assume that fewer people were treated for theanalysed medical conditions in which would basically have a downward effect on total expenditure at thesame time however the picture is somewhat shaded bythe fact that even if the attitude of the population towards regular physical activity has changed in the lastfew years it is not certain that the number of illnesseswould decrease significantly in such a short period asthe negative effects of a sedentary lifestyle led for decades would not be easily offset by a few years ofexcerciseladen lifestyle this is especially true forolder age groups that is to say a reduction in the number of patients is not realised yet in patient care at thesame time the use of rapidlydeveloping medical technologies is also increasing the financial burden on thebudget as the higher costs of new technologies makemedical care per patient more expensive on the otherhandit should not be fotten that healing can bemade more effective and can lead to higher returns onhuman capitalthe study examined the development and compositionof direct and indirect burdens of disease in hungary andthe costs of physical inactivity to the state budget theseburdens fell in the examined periode which was associated with an increase of gdphowever there was an increase in the economic burinactivity which can beden associated with physical 0c¡cs bmc public health 20suppl page of attributed to the combined effect of two factors changesin total expenditure on specific disease groups whichshowed an increase in the period under review andchanges in the physical activity levels of the hungarianpopulation which showed an improvement over theperiod under review initiatives in hungary aimed at encouraging an active lifestyle from childhood onwardsshould be continued since beyond the initial impactthat has already been felt to some extent in recent years these initiatives will come to their full fruition in thecoming decadesabbreviationsct computed tomography gdp gross domestic product hcso hungariancentral statistical office huf hungarian forint mri magnetic resonanceimaging nhif national health insurance fund nhifa national healthinsurance fund administration oecd anisation for economic cooperation and development par population attributable risk rr relativerisk vd veneral disease who world health anizationacknowledgementsthe authors acknowledge to the nhifas colleagues for their help incollecting the dataset especially to mr zsolt kiss director general to drmihaly palosi head of department to petra fadgyasfreyler head ofdepartment and to valentina beitl analistthe authors would like to express their special thanks to prof attila fabianformer vice state secretary for his cooperative help during the datacollectionabout this supplementthis has been published as part of bmc public health volume supplement level and determinants of physical activity in the v4countries part the full contents of the supplement are available online athttpsbmcpublichealthbiomedcentralcomssupplementsvolume20supplement1authors contributionspa was the leader of the complete research coordinated the different coauthors work systematized the dataset summarised the literature related tothe relative risk ratios of illnesses calculated the par indices and contributedto the s dp has made calculations of par indices the direct costs ofphysical inactivity in the nhifa budget and contributed to the sfrom its results ms has made calculations of the total burdens direct andindirect of illnesses in hungary and contributed to the s from itsresults mh and psz summarised the related literature to the section ak and tsz have revised the results and contributed to the sall authors read and approved the final manuscriptfundingthe research was carried out and the publication costs funded by thesupport of hrdop36216201700003 cooperative research network ineconomy of sport recreation and health the authors declare that thefunding body does not have any role in the design of the study andcollection analysis and interpretation of data and in writing the manuscriptavailability of data and materialsthe data of the state financed direct costs that support the findings of thisstudy are available from national health insurance fund administration butrestrictions apply to the availability of these data which were used underlicense for the current study and so are not publicly available data arehowever available from the authors upon reasonable request and withpermission of national health insurance fundthe datasets of the private ind indirect costs used and analysed during thecurrent study are available from the corresponding author on reasonablerequestethics approval and consent to participatethe ethical approval was granted for the study by ethics committee ofuniversity of p©cs nr participants were informed about theresearch aim and methods before signing the informed consent form theinvestigation conforms to the principles outlined in the declaration ofhelsinkiconsent for publicationnot applicablecompeting intereststhe authors declare that they have no competing interestsauthor details1university of pecs faculty of health sciences pecs hungary 2university ofphysical education budapest hungary 3corvinus university of budapestcorvinus business school budapest hungaryreceived march accepted march published august referencessebestyen a boncz i molnar a korosi l kovi r kriszbacher i olah apentek m sandor j relationship between surgical intervention type and days mortality of elderly femoral neck fracture in the prsence of differentcomorbidities value health 2009123a66kruk j health and economic costs of physical inactivity asian pac j cancerprev reiner m niermann c jekauc d woll a longterm health benefits ofphysical activitya systematic review of longitudinal studies bmc publichealth pratt m norris j lobelo f roux l wang g the cost of physical inactivitymoving into the 21st century br j sports med who global recommendations on physical activity for health switzerlandgeneva who blair sn cheng y holder js is physical activity or physical fitness moreimportant in defining health benefits med sci sports exerc s379tremblay ms colley rc saunders tj healy gn owen n physiological andhealth implications of a sedentary lifestyle appl physiol nutr metab rishiraj n inactivity a bad habit costing our productive lifestyle int j physmed rehabil oecd health status in edited by development ofecoa ¡cs p h©cz r pa¡r d stocker m a fitts©g m©rt©ke a fizikai inaktivit¡snemzetgazdas¡gi terhei magyarorsz¡gon k¶zgazdas¡gi szemle gabnai z m¼ller a b¡cs z b¡ba ©b the economic burden of physicalinactivity at national level [a fizikai inaktivit¡s nemzetgazdas¡gi terhei]eg©szs©gfejleszt©s health dev acs p stocker m fuge k paar d olah a kovacs a economic and publichealth benefits the result of increased regular physical activity eur j integrmed hcso in hcs o editor edn stadat time series of annual data labour market distribution of job vacancies koll¡nyi z imecs o az eg©szs©gbefektet©s budapest demosmagyarorsz¡g special eurobarometer [httpeceuropaeucommfrontofficepublicopinionindexcfmresultdocdownloaddocumentky82432]accessed jan special eurobarometer [httpeceuropaeucommfrontofficepublicopinionindexcfmresultdocdownloaddocumentky82432]accessed jan special eurobarometer [httpeceuropaeucommfrontofficepublicopinionindexcfmresultdocdownloaddocumentky82432]accessed jan powell ke population attributable risk of physical inactivity phys actcardiovasc health katzmarzyk pt gledhill n shephard rj the economic burden of physicalinactivity in canada cmaj 0c¡cs bmc public health 20suppl page of aldoori wh giovannucci el rimm eb wing al willett wc use ofacetaminophen and nonsteroidal antiinflammatory drugs a prospectivestudy and the risk of symptomatic diverticular disease in men arch fammed andersen lb schnohr p schroll m hein ho allcause mortality associatedwith physical activity during leisure time work sports and c | 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 |
" the popularization of health and medical informatics yields huge amounts of data extracting clinicalevents on a temporal course is the foundation of enabling advanced applications and research it is a structure ofpresenting information in chronological order manual extraction would be extremely challenging due to thequantity and complexity of the recordsmethods we present an recurrent neural network based architecture which is able to automatically extractclinical event expressions along with each events temporal information the system is built upon the attentionbased and recursive neural networks and introduce a piecewise representation we divide the input sentences intothree pieces to better utilize the information in the sentences incorporates semantic information by utilizing wordrepresentations obtained from bioasq and wikipediaresults the system is evaluated on the thyme corpus a set of manually annotated clinical records from mayoclinic in order to further verify the effectiveness of the system the system is also evaluated on the timebank_dense corpus the experiments demonstrate that the system outperforms the current stateoftheart models thesystem also supports domain adaptation ie the system may be used in brain cancer data while its model istrained in colon cancer data our system extracts temporal expressions event expressions and link them according to actuallyoccurring sequence which may structure the key information from complicated unstructured clinical recordsfurthermore we demonstrate that combining the piecewise representation method with attention mechanism cancapture more complete features the system is flexible and can be extended to handle other document typeskeywords clinical text mining event extraction temporal extraction relation extraction piecewise representationattention mechanism precision medicine is an emerging approach for diseasetreatment and prevention it becomes the whole worldbiomedicine domain the research hot spot which needsthe support of biomedical methods eg data mining it correspondence clixjtueducn zhijing li and chen li contributed equally to this work1school of computer science and technology xian jiaotong universityxian shaanxi china2shaanxi province key laboratory of satellite and terrestrial network techrd xian jiaotong university xian shaanxi chinaassociates with key information extracted from clinicalrecords eg symptoms over a disease course the associations are often statistically concluded from the evidencecollected from the clinical records the medical bigdata mostly exists in an unstructured form eg textwhich could store useful information very well aligningbiomedical events in clinical data along the events actually occurring time is a meaningful and efficient way ofstructuring such complex data the result may assistauxiliary diagnosistreatment scheme determinationepidemic prediction and side effect discovery etc 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 0cli bmc medical informatics and decision making page of many works have been devoted in the study of application in the medical era however large data analysis ofmedicaltreatment needs to map the correspondingmedical events in the clinical records the medical eventswith temporal information are very useful in medical erathese efforts will become the foundation of understanding disease facilitating the analysis of large medical dataas well for example the clinical record in fig may bepresented in a structured manner as the occurring eventsalong with temporal information it is easier for understanding the events and corresponding time point for example using the time point april as a referencethe entity bleeding is before the time point and the entitybolus chemotherapy and nausea is after the time pointin such case the actual events and their occurring consequence becomes clear at a glancein this paper we present a novel system which is builtupon deep neural networks to automatically extractevent expressions and their related temporal expressionsfrom clinical records the system has been evaluated onthe temporal histories of your medical event thyme corpus a corpus developed by a number of professionals according to characteristics of the corpusclinical data contains very long sentences that willundoubtedlythe difficulty of processingtherefore we do not simply use neural networks wewant to make full use of the contextualinformationour proposed method anically combines piecewiserepresentation and attention mechanism by a recurrentneural network rnn and achieves the stateoftheartperformance the results show improvements in automatic extraction of clinical event expressions along witheach events temporal informationincreaserelated workextracting clinical events along with temporal information is a complicated task and the existing systems oftenaccommodate several independent components each ofwhich retrieves different parts eg events and time andassemble them together each component may use a setof hand crafted rules or be based on a pretrained mlmodel velupillai develop the blulab systeminclude the cleartk support vector machine and conditional random fields classification approach and get thefirst place in semeval2015 task clinical tempeval macavaney present the system guir includeconditional random fields and decision tree ensemblesusing lexical syntactic semantic distributional and rulebased features guir receive the best score insemeval2017 task clinical tempeval in the way oftemporal expressions extraction tourille use aneural network based approach and achieve goodperformance for both event and relation extraction insemeval2017 task clinical tempeval lin propose a recurrent neural network with multiplesemantically heterogeneous embeddings within a selftraining framework for clinical temporal relation extraction task they achieve good results for both in andcrossdomainafter event and temporal expression extractionassigning each event with the right temporal expressioninvolves more complicated process some systemsmatch event with temporal expression by a set of syntactic rules crafted by experts wang use syntacticrulebased method for automatic pathway relation information extraction from biomedicalliterature these methods are fast but not flexible enough somefig the example of the medical information extraction the texts marked by the underscores ___ are the temporal expressions the textsmarked by the dash lines _ _ _ are the event expressions 0cli bmc medical informatics and decision making page of existing methods for medical relation information extraction are based on machine learning ml models[]conditional random field crf and supper vectormachine svm are often used in the task of relation extraction lu liu propose an svm model toextract the relations between the potential named entitypairs finkel propose a crfbased informationextraction system to determine relationships deeplearning revives the popularity of neural networkswhich can learn effective relation features from the givensentences withoutengineeringsocher is the first work that employs an rnnmodel to classify relation one early work proposed byluo is based on a recurrent neural network and ableto classify relations from clinical notes compared withthe rule based methods these methods are more flexibleneural network based methods take less time by quicklyscreening out the most unlikely candidate entitypairstherefore some approaches attempt to combine bothhence we think rnn is a good choice and we adopt thernn systemcomplicated featurein recent years attention mechanism has been widelyused in various tasks of nlp based on indepth learning li propose a model that combines abidirectional long shortterm memory network with amultiattention mechanism forrelation extractionzhou propose the attentionbased bidirectional long shortterm memory networksattblstmfor relation classification and the modelresults outperforms most of the existing methods withonly word vectors on the semeval2010 relation classification task so in this paper we also introduce theattention mechanismmethodologythe system consists of three components the first component extracts temporal expressions temporal expressions enable events to be chronologically annotatedthe second component identifies the relevant medicalevents event expressions any situation relevant to thepatients clinical timeline the third component detectsthe relations between the events and the temporalexpressionsannotating clinical records are very expensive frequently only a data of disease specific type is availablebesides the regular mlbased extraction we introducedomain adaption to allow the system to be able to extract the information from one type of disease eg braincancer while it is trained on another type eg coloncancerwe show the pipeline of our system in fig thesystem is built upon an annotating pipeline adoptingthemanagementunstructuredinformationfig the dataprocessing pipeline of the system we first extracttemporal expressions and event expressions respectively frommedical records then extract the relations between themarchitecture uima framework the preprocessingincludestokenization partofspeech tagging andlemmatization which used the stanford corenlp toolkit in both time and event extractions spans oftime and event expressions are represented by the offsets in texts the automatic annotations of event expressions temporal expressions and their relations arebased on three rnn models utilizing lexical syntacticand semantic features [] at the core of deeplearning techniques for nlp lies the vector basedword representation which maps words to an ndimensional space for the choice of the word embeddings corpus we do some research work only entities time and event expressions in the clinical records can be found in the wikipedia in comparisonthe bioasq corpus which is full of biomedical information contains more than of the entities weuse word embeddings from the european projectbioasq obtained by using word2vec on pubmed abstracts [ ] and include the vectorsof distinct words each word is representedas a 200dimensional vector if a word could not befound in the bioasq corpus the embedding is generated from wikipedia by word2vec mikolov as a complement [ ]thestateinternalof rnn can demonstratedynamic timing behavior the hidden state vector can be computed by the following formulaht ¼ f wht þ uxtðþin this formula xt is the input ht is the hidden stateu and w are the weight coefficients f is the nonlinearfunction such as tanh or relu 0cli bmc medical informatics and decision making page of extraction of temporal expressions and event expressionsindependent models are trained for the extractions oftemporal expressions and event expressions figure shows the infrastructure of the system there are twoforms of temporal expressions one is numeric temporalexpressions eg etc and the other is casualtemporal expressions eg day weeks during aperiod etc firstly we generalize all the numeric temporal expressions into for example both and become which can be easily recognizedby the regular expression the regular expression is useddue to the characteristics of the data the numeric temporal expressions are not well recognized by rnn if wedo not use it secondly the casual temporal expressionsare recognized by a rnn model the casual temporalexpressions in the training set are tagged to representthe tokens position in a particular expression there are four tags in our proposed method includingb i o and e which state that the token is at thebeginning on the inside on the outside or at the end ofthe entity respectivelyin this section we propose the system arnn whichis based on a recurrent neural network combining theattention mechanism we need to predict the tokensposition tag of each word before we can train the arnnmodel to predict the type of each temporal expressionwe treat each temporal expression as an entity and eachentity is treated as a unit input we use the average valueof all the word embeddings of an entity in the nextprocess the network in the fig shows the flow chartof arnn network to predict the type of the entitythere is an example sentence from the corpus we willget a ct enterography to rule out crohns disease inthis case the given entity is enterography and thecontext words are we will get a ct to rule outcrohn disease in order to better apply the context information we employ the attention mechanism to learnthe weighted score of each context word related to thegiven entitythe higher weight the higher semantic is bound upwith the given entity 01 00αi exp vti u vhtuv is the parameter that has to be learned from fig we can see that st is the state vector that integrates theother context words information with the given entity attime t st can be computed asst ¼xihαihiwe combine st and h3 to obtain h0sent the given entity which can repre ¼ h3 þ sth0in this process the prediction of entitys type will bepredicted from the given input entity vector the methodof using regular expression are also used to match themissing temporal expressions eg similar to temporal expressions extraction the eventexpressions extraction is built on another rnn unliketime expressions event expressions are all single wordsthere is no need to sign each tokens position we usethe softmax classifier to predict the label y² of the temporal and event expressions the state vector h0 is usedas input and y² could be computed by 10 11py ¼ softmax w h0 ¼ arg maxypyyeventtime relation erer extraction is the most important task in this paperin this section we propose the novel system aprnnwhich is based on a recurrent neural network combiningthe attention mechanism and the piecewise representation the eventtime relation are regarded as a classification problem it is divided into four categories based onsome wellknown communities such as semeval orbionlp the event time relation associates the identifiedevent expressions and temporal expressions and indeedindicates the what and when of a medical event inclinical records the four types are before after beforeoverlap and overlapthe shortestsyntactic path used includespiecewise representationthe dependency parsing of each sentence has beenobtained by utilizing stanford corenlp toolkit thepreviously trained word embeddings which representeach word by a 200dimension word vector and theshortest syntactic paths are fed into the rnn modelof er extraction the word embeddings and shortestsyntactic path are used as features the informationofthewords the poss and the length we add all thesevectors as the entity feature after adding up we stillget a 200dimensional vector for each entity if theentity include severaltemporal expressions we add the vectors of each token and get theaverage vector as the entity vector the whole is divided into sentences as input units we extractall entity pairs based on the annotations given a sentence x x1 x2 ¦ xt the words are projected intoa sequence of word vectors denoted by e1 e2 etwhere t is the number of words in this part wewould like to introduce the piecewise representationtokens eg 0cli bmc medical informatics and decision making page of fig architecture of the rnn model for the extraction of temporal expressions and event expressions we propose the attentionbased rnnmodel to do the entity extraction on the left side of the figure is the details of the attention mechanism the right part of the figure is thernn modelinformation as shown in fig in other wordsthe input sentence is divided intothree parts according to the entity pair we call thisprocess piecewise representation the purpose ofpiecewise representation is to better use of the contextthe examplesentence is divided into three parts according to theentity pair will enterography the whole sentence iswe will get a ct enterography to ruleout crohnsdisease the first part is the sequence before the entity pair we the second part is the sequence between the entity pair get a ct and the third partis the sequence after the entity pair to rule¦ thereare reasons for segmenting the sentence the firstone is that in many cases some studies may choosethe sentence between the entity pair as input insteadof using the whole sentence nevertheless thiscan miss some information and some of them maybe useful only several words cannot supply enoughcontextfeatures segmented sentences can be used to extract the effectiveinformation forextractingrepresentsinformation to the greatest extent of each sequenceand avoid the absence of contextualinformationanother reason comes from the network and our corpus in the corpus the longest sentence contains words however the average length of all sentenceshas words with the rnn structure since the information of a sentence is learned word by word thefeature vector produced at the end of the sentenceactuallysentence althoughrnn has the memory in learning process but thememory time is not long accumulation by recurrentconnectionslongterm informationquickly and the feature vector atthesentence is hard to carry the information of earlysteps in model training there are many longdistancesentences more than words in the training dataso the piecewise representation can help the systembetter use the information of the sentence the syntactic analysis and pos information of the examplesentence are also shown in the fig the end ofentirethetendsto fet 0cli bmc medical informatics and decision making page of fig the flow of our proposed model aprnn on the left side of the figure is the details of the attention mechanism the right part of thefigure is the rnn model which is divided into three partsmodelthe er model contained three parts the first component the feature layer the second component thehidden layer catch the information of word sequenceand produces wordlevel features representations andthen merges wordlevel features into a sentencelevelfeature vector by selecting the most valuable featureinformation among allfeatures weshow the whole process in the fig in this part weproposeattention mechanism to obtain thethe wordleveltherepresentation of the sentence not all the words inthe context describe the er relation each word inthe context has different effects on the given entitypair therefore we introduce the attention mechanism to learn the weighted score of each context wordrelated to the entity pair for the first part the attention mechanism is used to screen the most useful information we use the bilinear operator to computethe attention weight αi for each vector h1 and h2 toreflect how the information relevant to the first entity 0cli bmc medical informatics and decision making page of in the entity pair the current state h3 the calculation method of αi and st is the same as the description in the extraction of temporal expressions andevent expressions sectionwe combine st and h3 to obtain h0 which can represent the sequence before the entity pair for the secondpart we choose the state of the last entity in the sequence ht to represent the whole sequence for thethird part the same method is used as the first part wealso use the same attention mechanism to obtain therepresentation oft the singlesentencelevel feature vector need to be obtained to represent the entire sentence for the relation classificationwe introduce the maxpooling approach as in cnnmodels to obtain the single sentencelevel feature vector the maxpooling is formulated as followsthe sequence h0htf gm ¼ maxtnext the sentencelevel feature vector m is passed tothe output layer furthermore the output layer has classes we use the softmax classifier to predict the labely² from a set of labels y from the sentence the statevector m is used as input therefore y² could be computed bypy ¼ softmax wmð ¼ arg maxypyyþin addition there are two settings in er extraction inorder to better compare our approach the first is basedon our proposed method the second is only utilize thernn network without any piecewise representation orattention mechanism as shows in fig experiment and resultsdatathe major medical data are the data of medical institutions diagnosis and treatment collection of massiveclinical data and laboratory data produced every day atall levels of hospitala golden annotated corpus marked up with temporalexpressions events and relationship between them isneeded to allow us to evaluate by our methods thethyme corpus which has been used since isone of the suitable corpora consisting of clinical andpathological notes of patients with colon cancer andbrain cancer from mayo clinic unlike other datasetsthe events in this dataset are all single words which arevery suitable for our system the notes are manually annotated by the thyme project thymehealthnlpusing an extension of isotimeml for the annotation oftemporal expressions events and temporal relations of the corpus is used for training is forfig the flow of the comparison system this is a commonrnn modeldevelopment and is for testing the developmentset is used for optimizing learning parameters thencombine it with the training set to build the system usedfor reporting results table shows the distribution ofthe thyme corpus the colon cancer data are used astraining data and are tested on both colon cancer andbrain cancer data to demonstrate its effectiveness withtable the distribution of the thyme corpus in this table weshow the different types of data in the corpusdatacolon cancertrain dev testbrain cancertrain testdocumenttemporal expressions event expressions er 0cli bmc medical informatics and decision making page of or without domain adaptation and this can reflect thatour approach is not limited to a particular field inevaluation all methods have access to the same set oftraining and testing datatable the temporal expressions extraction results on braincancer we utilize different methods to do the task the resultsare shown in part the result of previously best system isshown in part resultsthe method has been evaluated on both colon cancerdata and brain cancer data to demonstrate the effectiveness with or without domain adaptation in order to dobetter research several methods are used to de the entityextractionsix methods of temporal expression extraction arulebased method a system based on crf a system based on general rnn without any attention mechanism or context rnn a system based on rnnwith easy attention mechanism but without any contextwords rnnatt our proposed method a rnn system with attention mechanism and context words asystem combines the crf and rnn network all the results are compared part in tables and for therulebased methods firstly we find all the prepositionsaccording to our experience and experimental statisticswe extract five tokens behind their own prepositionsthrough careful observation of data we found thatmany time expressions always show up behind a preposition we then judge whether those five words are relatedto time expressions we define a time dictionary to listthe words which we think can be a part of the time exlike month week day hour maypressionsmonday morning once and so on next we contrastthe five tokens with time dictionary and findwhether it can represent a date or a precise time finallywe extract all the continuous tokens that we thoughtmay relate to the time expressions if there is a definite before those tokens extract it as well there existsome expressions do not after a preposition and onlycontain one word and most of them have the same prefix like pre post peri so we use this prefix rule tofind the remain expressions the major feature we usedfor training the crf and svm classifier is simple lexicaltable the temporal expressions extraction results on coloncancer the part shows the results of six different methodsthat we used to do the temporal expressions extraction thepart shows the result of the previously best systempart methodrulebasedcrfrnnrnnattarnnpart crfarnnblulab run prf1part methodrulebasedcrfrnnrnnattarnncrfarnnpart guirprf1features word embeddings partofspeech tag numericlower case blulab run andtype capital typeguir system are the previously best system mentionedin the section two these results are shown in tables and part in both tables and rule based methods achievethe lowest result the recalls are relatively better thanthe precisions due to the welldefined dictionary theerror analysis shows that some pre post and periare considered as time expressions while they should notbe meanwhile the rulebased method often mistakestwo independent expressions as one if they are adjacentin table the rnn systems performances are lowerthan blulab run 3a cleartk svm pipeline usingmainly simple lexical features along with informationfrom rulebased systems the rulebased information iseffective but it has limitations it can extract rules according to the characteristics of data we do not addany rules to the rnn system the observation on theerror analysis shows that without any attention mechanism and context words rnn is not very effective forsimilar combinations of numbers and letters eg h days etc because the form of the corresponding wordvectors are generated randomly and the time series contains a large number of the above type so the modelcannot learn characteristics of time series so it cannotbe correctly extracted after adding the attention mechanism and context words the arnn system achieve therelatively good results because of the good results ofcrf we combine the crf with the arnn and achievethe best result from table we can see that the rnnoutperforms the guir system which is the current bestsystem it is an ensemble of crf and decision tree withlexical syntactic semantic distributional and rulebasedfeatures the guir system can not extract the previously unseen or atypical date formats very well it is obvious that their rules are not comprehensive enoughthis problem also exists in rnn system however whenadding the attention mechanismit can extract more 0cli bmc medical informatics and decision making page of new and otherwise unknown formats the arnn andcrfarnn system achieve the best results in this partwe have two test data sets one is colon cancer anotherone is brain caner we trained all the models on thesame training data and test them on two different testdata sets except for the different test data the parameters are exactly the same the experimental results provethat our model is effective on other test data setsmeanwhilefive methods of event extraction amethod based on svm a system based on crf asystem based on general rnn without any attentionmechanism or context rnn a system based onrnn with easy attention mechanism but without anycontext words rnnatt our proposed method arnn system with attention mechanism and contextwords all the results are evaluated part in tables and for event extraction the svm and crf modelobtain the relatively good results in colon data and perform poorly in brain colon data compared to the bestsystem limsi however rnn achieves preferably results in the two sets of test data even higher than thebest system limsi as shown in both tables and when adding the attention mechanism and contextwords the results are improvedas for the er extraction which is the key point of thepaper first we compare our proposed model with thefollowing methods a general rnn system withoutany attention mechanism or piecewise representationwe use the sentence between the entity pair as the inputrnn a general rnn system without any attentionmechanism or piecewise representation we use thewhole sentence as the input rnnwhole we can seethe results of rnnwhole is better than the results ofrnn it means that the sentence length can affect theperformance of the system therefore we use the sentence between the entity pair as the input for other system a general rnn system with attention mechanismbut without piecewise representation rnnatt ageneral rnn system without attention mechanism butwith piecewise representation rnnpie our proposed system aprnn but only use the word embeddings trained from wikipedia aprnnwiki ourtable the event extraction results on colon cancer different methods are utilized by us all the results are shown inpar1 the part shows the result of the previously best systemf1methodsvmpart prcrfrnnrnnattpart arnnblulab run table the event extraction results on brain cancer we adopt methods to do the task the results can be compared in part the result of the best system is shown in part part part methodsvmcrfrnnrnnattarnnlimsiprf1proposed system aprnn but only use the word embeddings trained from bioasq aprnnbioasq our proposed system which is based on a recurrentneural network combining the attention mechanism andthe piecewise representation all these results are evaluated part in tables and except for model and the word embeddings for other models are from bothwikipedia and bioasq from the results we can seethat both attention mechanism and piecewise representation are useful they can improve the results to someextent we can directly compare the value of attentionin two groups of experiments result and result result and result the result and result result and result can directly demonstrate the performance with and without segmentation the difference between model and is that model is missing thepiecewise representation and the difference betweenmodel and is without or with the attention mechanism the result has been improved with the piecewiserepresentation the experiment and are aboutlooking at the impact of word embeddings the result and result show that different word embeddings canlead to different results after combining the two corpuswikipedia and bioasq the results increase slightlytable the er classification results on colon cancer part shows the results of the relevant methods we used the otherrelated works which achieved the very good results are shownin part part part methodrnnrnnwholernnattrnnpieaprnnwikiaprnnbioasqaprnnblulab run svmattblstmprf1 0cli bmc medical informatics and decision making page of table the er classification results on brain cancer the resultsof our proposed methods are shown in part part shows theresults of other relatedpart part methodrnnrnn wholernnattrnnpieaprnnwikiaprnnbioasqaprnnlimsisvmattblstmprf1aprnn different factors that may affect the resultsare verified from experimental results eg piecewise representation attention mechanism word embeddings allthese factors are utilized to make better use of contextual informationwe compare our work with other related work thelimsi system which achieves the best score on the ertask in semeval2017 task li rao and zhang proposed the litway which is a system that has adopteda hybrid approach that uses the libsvm classifier with arulebased method for relation extraction theyachieve the best score in the seedev task of bionlpst thus we use their approach as a benchmark forour system the bilstmattention networks proposedby zhou were chosen as another benchmarking model attblstm which outperforms most of theexisting methods they designed a bidirectional attention mechanism to extract wordlevel features from thesentence the features for the attentionbased model include word vector | Colon_Cancer |
advances in technology hardware and computing have created new opportunities to improve the quality of cancer care and research by leveraging informatics innovations digital health machine learning and precision oncology are key areas where there are noteworthy advances in the field of cancer informatics as the adoption of smartphones and wearable technologies increases patientgenerated health data such as physical activity and electronic patientreported outcomes pros are being leveraged for prevention and in interventions for remote monitoring during treatment and survivorship and to predict health outcomes moving beyond cancer clinical trials health systems are increasingly seeking to integrate pros into routine cancer care processes as part of the national cancer institutes nci cancer moonshot initiative six healthcare systems will systematically integrate pros into clinical workflows and electronic health records ehrs additionally much attention surrounds the hype and challenges of the application of machine learning algorithms in clinical care including for a number of oncology use cases however there are concerns around biases and homogeneous training data sets thus machine learning has had limited implementation into clinical care considerable progress in genomics and computational medicine has ushered in a new era in precision oncology generating vast amounts of data much of which is publicly accessible for example the nci precision medicine initiativeoncology has had recent success defining genetically targeted therapies understanding tumor treatment resistance and developing a us cancer knowledge system while the opportunities within cancer informatics have much potential for positive impact if not applied thoughtfully these innovations risk propagating disparities and inequities operationalizing these informatics discoveries into routine cancer care also continues to present a diverse set of challenges objectivesgiven the rapid pace of cancer informatics research our objective was to conduct a survey of the literature for advancements over the past several years in three key areas of growth digital health machine learning and precision oncology we also highlight the ethical implications challenges and opportunities for each topic methodsa literature search was conducted in two electronic databases pubmed and google scholar to identify peerreviewed s and conference proceedings search terms included variations of the following neoplasms[mesh] informatics[mesh] cancer oncology clinical cancer informatics medical cancer informatics the search covered the period from october to october imia yearbook of medical informatics imia and ge thieme verlag kg 0c and returned too many s for practical review from pubmed and from google scholar thus we narrowed the search to key pubmedindexed informatics s and proceedings such as the of the american medical informatics association jamia jamia open applied clinical informatics of medical internet research bioinformatics of biomedical informatics jco clinical cancer informatics and nature digital medicine we also performed a manual review of cancer informatics s that were not yet indexed in pubmed additionally we searched the proceedings of the amia annual symposia and amia informatics summits given the sheer number of results we focused on manuscripts that demonstrated a clear focus on clinical or translational cancer informatics manuscripts were selected based on methodological rigor scientific impact innovation and contributions towards cancer informatics as a field or on impact on cancer care the reviewed manuscripts were grouped into three topics including digital health machine learning and precision oncology for each topic we describe the current state implications challenges and opportunities results digital healthdigital health is the intersection of technologies with health and wellness to enhance communication and delivery of health services it includes mobile health health information technologies wearable devices telehealth or virtual health and personalized medicine as health systems transition from traditional officebased care to more continuous interactions with patients and caregivers emerging technologies are key drivers of the capture of patientgenerated health data currently it is still challenging to bring together disparate data streams generated by patients from technologies such as smartphones social media platforms wearable activity trackers and internet of things iot devices such as smart home sensors however multiple studies have begun to demonstrate the value of employing digital health technologies to improve cancer prevention and care for example apps and webbased interventions have been used to support exercise and in digital outreach efforts focused on cancer screening in a webbased exercise intervention to mitigate the effects of chemotherapy breast cancer patients demonstrated improvements on the sixminute walk test and abdominal back lower body strength compared to the control group over the eightweek study one study demonstrated that google search and facebook advertisements could be used to promote cancer screening these advertising campaigns were associated with increased visits to institutional websites and scheduled screening exams compared to the week before and after the campaign these studies reveal the potential for digital health apps to engage patients reach broad populations and improve physical activity outcomes in addition to being a vehicle to deliver interventions social media can be used for behavioral surveillance several studies explored communications within patient communities with the most common posts or tweets on the topics of clinical trials treatment and support taylor found evidence of differing levels of emotional informational and social support for patients with lung cancer across platforms ie facebook twitter macmillanuk which may be due to differences in user characteristics or the nature of selfmoderating communities in terms of engagement cho examined the relationship between melanomarelated instagram posts and engagement outcomes ie likes comments social support they found that posts about physical consequences decreased the number of likes but increased comments and perceived emotional support and that the inclusion of images increased the number of comments made about the posts overall these highlighted studies contribute to an understanding of participative engagement and experiences on social media platforms for written and visual cancerrelated content that is usergenerated and suggest that patients may have varied experiences related to social support across platforms and communitiesthere is mounting evidence that using pros for cancer care symptom assessments can improve survival quality of life and help patients remain longer on treatment when compared to usual care while there is a wide range of pros that could be assessed during cancer treatment and in survivorship several efforts have aimed to identify key domains relevant to improving outcomes in cancer care for example a clinical advisory group identified priority domains and measures to enhance cancer care which included distress and symptoms and unmet needs girgis then developed clinical recommendations from these domains mapped them into wellbeing categories and demonstrated that these recommendations could algorithmically provide automated clinical feedback there is also concern that the use of structured questionnaires to assess pros may limit the ability to capture the broad range of symptoms experienced by patients during treatment chung et al examined whether patiententered freetext narratives of symptomatic adverse events could be mapped to established terminologies from the national cancer institute nci patientreported outcomes version of the common terminology criteria for adverse events proctcae among patients enrolled in three multicenter cancer trials over half provided unstructured freetext entries and most of these entries could be mapped post hoc to a proctcae or medical dictionary for regulatory activities meddra term this suggests that mapping to existing terminologies is not only feasible but also provides the opportunity to supplement trialspecific questionnaires to better capture diverse patient experiences during cancer treatmentsuse of wearable activity trackers and other iot sensors to capture additional patientgenerated health data is expanding several feasibility studies have had favorable results although sample size was generally small studies noted that higher daily steps recorded from activity trackers were correlated with pros such as increased performance status lower distress [ ] fatigue depressive symptoms [ ] adverse events hospitalizations and death despite high dropoff rates for imia yearbook of medical informatics 2020griffin 0cnetwork to classify topics within an online health community focused on breast cancer and found that patients with different cancer stages have different topics of conversations lee et al assessed conversations about living with cancer among sexual minority women using latent dirichl location which revealed that the most common topic was about coping and connecting to others with shared gender identities utilizing nlp techniques to analyze and understand topics in online communities could help to optimize interventions and deliver valuable supportrecognizing the ongoing challenges with concept extraction and the scarcity of annotated datasets researchers have employed open science efforts to foster interdisciplinary collaborations in several areas for example temporal history of your medical events thyme corpora [ ] informatics for integrating biology the bedside i2b2 challenge sets and medical information mart of intensive care mimiciii contain collections of deidentified clinical text for research dream challenges have also hosted several cancerfocused competitions that yielded a number of r packages and established benchmarks for prognostic models [ ]in medical imaging ml has been deployed for early detection improved diagnosis and better prognostic accuracy ardila used lowdose chest computed tomography images to perform endtoend lung cancer screening with high accuracy similarly predictive models to calculate breast cancer risk from mammograms demonstrated improved risk discrimination over current clinical standards in dermatology several deep learning methods were found to be superior to dermatologists in diagnosing and detecting skin cancers [ ] wei also found employing deep learning approaches to classify lung pathology images yielded more accurate results when compared to pathologists additionally advancements have been made in mlbased cds tools for classification of skin lesions estimates of colon cancer outcomes based on various therapies and management of uterine cervical abnormalities given the lack of large imaging datasets for training models investigators have also focused on producing realistic synthetic data [ ] research by senaras revealed that generative adversarial networks are a promising technique to generate labeled synthetic immunohistochemical stained breast tissue for precision cancer care genomics tools have also demonstrated the potential to advance the discovery of novel targeted cancer therapies deepminds alphafold tool has shown promise in predicting protein structure from its genetic sequence crisprcas9 screens have also facilitated tremendous progress in identifying essential genes across cancer cell lines several studies have used ml to identify cancer subtypes using gene expression data and pathological images for example coudray established that ml algorithms could predict six of the most commonly mutated genes within adenocarcinoma lung cancer ml could also be used to assist pathologists in detecting gene mutations or cancer subtypes given the variety of data sources necessary to compare personalized cancer care pathways incorporating genetic clinical imaging cost and quality of life data into multifaceted cds tools could aid in reducing the cognitive load of clinicians precision oncologyprecision medicine is an evolving set of diagnostic and treatment paradigms which seeks to optimize and tailor safe and effective care for patients based on their individual characteristics precision medicine has been advanced considerably by cancer genomics increasingly discoveries using genomic technologies that have fueled translational research are impacting clinical settings single cell level assays are proving transformational in the understanding of tumor heterogeneity disease biology and treatment resistance and will likely play an increasingly important role in clinical research and precision medicine applications several studies have demonstrated the feasibility of large cohorts prospectively assayed using genomic technologies with turnaround times short enough to impact wear time for activity trackers [ ] this research highlights that wearable devices could provide accurate assessments of performance status and physical function to inform treatment selection and could serve as a viable proxy for pros machine learningrecent advancements in machine learning ml reveal opportunities for ml to transform cancer care the field has seen progress in early detection of cancers and improved diagnostic accuracy personalized therapeutics and clinical workflows in the last two years the field has also experienced substantial growth in fda approvals for algorithms including oncology applications for the detection of suspicious lesions and clinical decision support cds recent reviews have highlighted a number of promising mlbased knowledge extractions from unstructured and semistructured oncology data from ehrs social media platforms and online health communities [ ] using ehr clinical documents savova created a natural language processing nlp system deepphe to generate cancer phenotypes which could help reduce time spent reviewing clinical documents guan used nlp techniques to extract genomic sequencing information from clinical progress notes and then classified the notes on whether sequencing led to a treatment change qiu also leveraged nlp to extract the primary site of tumor origin from pathology reports given that cancer stage and origin are rarely captured in structured format within ehrs many of the recent approaches have focused on nlp to extract these data however seneviratne used the observational medical outcomes partnership omop common data model to create a feature vector framework to classify patients with prostate cancer by stage using structured ehr data this approach may be more generalizable than nlp methods since clinical notes vary widely in content depending on the type and author there have also been a number of studies that apply nlp to online health community and social media datasets [ ] zhang used a convolutional neural imia yearbook of medical informatics 2020from patient engagement to precision oncology leveraging informatics to advance cancer care 0cclinical decisions [ ] for example in an institutional precision oncology initiative at memorial sloan kettering cancer center mskccimpact approximately patients sequenced at a rate of more than patients per month underwent paired tumor and germline dna sequencing of a panel of approximately known cancer genes turnaround time to final report was less than days on average most of the somatic variants were not present in the large public database of somatic variants called the catalogue of somatic mutations in cancer cosmic at the time of the study suggesting a gap between finding a variant and interpreting its relevance to a patients clinical context as with other similar studies researchers relied on publicly available data resources such as my cancer genome oncokb clinical interpretation of variants in cancer civic hemonc and communitybased resources to facilitate interpretation of results the clinical impact of largescale precision oncology sequencing efforts suggest mixed results an overview suggests that only a minority of sequenced patients have at least one actionable molecular finding specifically only and of patients respectively from the ncimatch approximately patients and the mskccimpact trials received targeted therapies [ ] increasing the number and effectiveness of available therapeutics remains a central challenge to broadening the impact of genomic precision medicine however it will also be critical to identify treatments that may yield low therapeutic benefitbiomarker definition is another growth area in precision oncology precision immunooncologics a class of relatively new therapeutics have shown remarkable efficacy for some cancers several defined prognostic and predictive indices suggest which patients are most likely to benefit from for example immune checkpoint inhibitors [ ] for chimeric antigen receptor tcell cart therapy there are over trials targeting at least different biomarkers in multiple cancer types public health projects are also beginning to adopt strategies to capitalize on biomarkers and genomic information to enhance precision medicine applications for instance the us centers for disease control and prevention maintains the public health genomics knowledge base phgkb which includes cancerspecific resources to provide systematically curated and updated information that bridges populationbased research on genomics with clinical and public health applications the nci has also created an online resource that announces and coordinates precision cancer surveillance efforts including the addition of patientlevel genomic linkages into cancer registries discussioninnovations in cancer informatics are abundant in the areas of digital health machine learning and precision oncology and have great potential in improving cancer care while there are considerable challenges there are also many opportunities for future research to advance cancer informatics as a field opportunities and implications for digital health in cancer caregiven the ubiquitous adoption of smartphones and growing ownership of wearable and iot devices digital health technologies have the unique capability to gather longitudinal data outside of clinical settings that could yield deeper insights into a patients symptom experience or help identify modifiable risk factors for cancer interoperability standards and numerous us initiatives such as the 21st century cures act and the promoting interoperability program formerly named the meaningful use program are transforming the way individuals access medical services and manage their health thereby accelerating the meaningful application of digital health technologiesthe evidence base for digital health in cancer care is currently limited by small sample size short study duration and limited focus on digital accessibility underserved or underrepresented populations or the needs and psychosocial outcomes of caregivers of patients with cancer patients with cognitive or physical disabilities may not be able to use some technologies and different modalities are needed to reach patients based on their abilities preferences cultural norms level of training and lived environment caregivers may be an important bridge to reach patients who have technical physical or cognitive limitations but this is an understudied area many studies specifically exclude participants based on nonownership of a smartphone lack of internet access severity of medical conditions being on chemotherapy treatment or a recurrence of cancer yet these populations may be those who could benefit the most from digital health technologies and interventions as companies are increasingly engaged in acquiring and sharing individuals healthrelated information there are noteworthy concerns about data privacy security and the ethical use of digital health data given the growing number of partnerships between health systems pharmaceutical companies insurers and technology companies and the increased access by third parties to cloudbased patient data and associated analytical tools there is growing unease about data linkages and the potential for misuse and data breaches for example google recently sparked a federal investigation following their partnership with ascension due to concerns for sharing protected health information and a department of justice inquiry over their acquisition agreement for fitbit apple has several partnerships with insurers and pharmaceutical companies such as aetna and eli lilly and fitbit has partnerships with bristolmyers squibbpfizer alliance and humana recently it was revealed that the uks national health service has given amazon free use of all health data under an alexa advice deal consumers may be unaware or inadequately informed about how their data are being used and by whom suggesting the need for greater transparency of privacy and data sharing practices and policies to be consumable at the patient level although the fda regulates some digital health apps or technologies the majority of apps available in the marketplace are not validated through rigorous research additionally it remains challenging for patients and caregivers to determine which apps are trustworthy effective and usefulimia yearbook of medical informatics 2020griffin 0c opportunities and implications for ml in cancer caremachine learning research has had meaningful advances in cancer prevention diagnostics and prediction this progress has been driven by increased availability of data and scalable computing infrastructures cloudbased platforms such as microsoft azure databricks google cloud automl and amazon web services sagemaker have simplified building training and deploying models though developers should be cognizant of the computational and environmental costs associated with cloudbased platforms realtime nlp and ml innovations within ehr systems are also being explored by companies like nuance and epic which could lead to better capture of tumor staging information as structured data elements through ambient computing despite the promise of ml to improve cancer care there are substantial challenges that must be addressed before ml is implemented into routine cancer care for instance women missed screening mammograms in the uk due to algorithm failure the ibm watson for oncology project highlighted a number of issues in the ml and oncology communities related to lack of validation and benchmarking data quality and subjectivity in interpretation of results data quality and availability challenges in healthcare are an acknowledged barrier to research particularly the paucity of data for underrepresented populations this may lead to biases since training data does not reflect the attributes of these populations yet may be applied in clinical care there are also growing concerns about the deskilling of physicians that could occur when some or all of the tasks become automated such as a drop in a clinicians diagnostic accuracy this has future implications for graduate medical education and how curricula may require transformation to address these emerging issuesgiven the large volume and diversity of training data needed for ml research federated learning approaches which do not require direct data sharing have strong potential but remain difficult to implement due to privacy preservation challenges furthermore the black box nature of many algorithms renders interpretation and benchmarking performance difficult to improve algorithm transparency price proposed a threestep framework for validating black box algorithms which involves having high quality training data and development procedures testing algorithm performance against independent test data and evaluating performance continuously even with benchmarking appropriate selection of realistic and biascontrolled test cases may still be an issue the intrinsic uncertainty in medicine introduces variations in results interpretation which also suggests that model performance criteria should be use case specific rather than based on standard scoring metrics in the era of deepfake ethical issues are inevitable if there are no appropriate regulatory frameworks for the deployment of ml algorithms health disparities could be widened due to lack of representative training data and the possible consequences from algorithm failures limit the current utility of implementing ml algorithms in realworld cancer care settings governance and policies for deployment audits of performance and implementation of best practices will be critical for safe implementation but these are not yet widely used within health systems to mitigate some of the challenges facing the field of ml several anizations have proposed ethical and regulatory frameworks the fda has proposed policies for ensuring safe and effective use of mlbased software for medical purposes including regulatory frameworks for software as a medical device for cds as well as a precertification program in realworld deployment into clinical care it is critical to have rigorous change protocols for algorithm modifications to ensure safety and to provide transparency to users during updates of algorithms however these may not be routinely implemented while mlbased cds tools can assist in automated detection classification or reporting safeguards are essential to proactively mitigate errors that may arise from these complex systems professional societies such as the american college of radiology along with several other us and international radiology anizations have released an exemplar consensus and guidance document on the importance of developing ethical standards for ml toolkits such as the american college of radiologys ailab framework that promote a vendorneutral approach to develop algorithms based on patient populations may also allow for extensibility of algorithms opportunities and implications for precision oncology precision medicine utilizes characteristics of individual patients but relies on populations of patients to actually guide treatment decisions even when highquality data are available experts may disagree about interpretation and actionability applied within a clinical setting perhaps more problematic is the bias that can arise when extrapolating findings to ethnic racial or age groups not wellrepresented in research cohorts [ ] despite the growing application of genomic testing in research and precision oncology use cases are still limited by lack of availability of codified data elements from molecular testing on tumor samples germline dna and serum biomarkers even for healthcare systems with advanced ehrs and cancer research efforts currently there are numerous opensource cancer genomics tools and ml training platforms that have the potential to accelerate cancer informatics research such as oncosim oncowiki and google collaboratory [ ] the nci informatics technology for cancer research program has a number of openaccess tools that support the analysis of genomic imaging and clinical data yet despite the momentum in precision oncology discoveries and actionability of results their application to the context of the treatment selection for individual patients remains an open challenge in accelerating the implementation of precision oncology conclusionsbringing informatics innovations within digital health machine learning and precision oncology into cancer care will require thoughtful approaches to operationalize the collection and meaningful summarization imia yearbook of medical informatics 2020from patient engagement to precision oncology leveraging informatics to advance cancer care 0cof disparate data sources to actualize the promise of cancer informatics moving from patient engagement in collecting and sharing health data with care teams and researchers to the delivery of precision cancer care necessitates leveraging informatics innovations the research highlighted in this survey paper reflects the fastpaced everevolving field and its challenges as we move discoveries into cancer careacknowledgementsashley griffin is supported by nlm training grant 5t15lm01250003 references simpro study team electronic symptom management implementation of patient reported outcomes in oncology research available from httpswwwesymcancermoonshot [accessed nov ] us national cancer institute nci and the precision medicine initiative available from httpswwwcancergovresearchareastreatmentpmioncology [accessed nov ] us food and drug administration digital health available from httpswwwfdagovmedicaldevicesdigitalhealth [accessed nov ] chung ae jensen re basch em leveraging emerging technologies and the internet of things to improve the quality of cancer care j oncol pract arizagarcia a lozanolozano m galianocastillo n postigomartin p arroyomorales m cantarerovillanueva i a webbased exercise system ecuidatechemo to counter the side effects of chemotherapy in patients with breast cancer randomized controlled trial j med internet res 2019217e14418 jessup dl glover iv m daye d banzi l jones p choy g implementation of digital awareness strategies to engage patients and providers in a lung cancer screening program retrospective study j med internet res 2018202e52 sedrak ms salgia mm decat bergerot c ashinggiwa k cotta bn adashek jj examining public communication about kidney cancer on twitter jco clin cancer inform chen l wang x peng tq nature and diffusion of gynecologic cancerrelated misinformation on social media analysis of tweets j med internet res 20182010e11515 taylor j pagliari c the social dynamics of lung cancer talk on twitter facebook and macmillanuk npj digit med cho h silver n na k adams d luong kt song c visual cancer communication on social media an examination of content and effects of melanomasucks j med internet res 2018209e10501 basch e deal am dueck ac scher hi kris mg hudis c overall survival results of a trial assessing patientreported outcomes for symptom monitoring during routine cancer treatment jama roth aj kornblith ab batelcopel l peabody e scher hi holland jc rapid screening for psychologic distress in men with prostate carcinoma a pilot study cancer national comprehensive cancer network nccn clinical practice guidelines in oncology distress management available from httpswwwnccnprofessionalsphysician_glsdefaultaspx [accessed nov ] richardson l jones g a review of the reliability and validity of the edmonton symptom assessment system curr oncol girgis a durcinoska i koh es ng w arnold a delaney gp development of health pathways to standardize cancer care pathways informed by patientreported outcomes and clinical practice guidelines jco clin cancer inform chung ae shoenbill k mitchell sa dueck ac schrag d bruner dw et al patient free text reporting of symptomatic adverse events in cancer clinical research using the national cancer institutes patientreported outcomes version of the common terminology criteria for adverse events proctcae j am med inform assoc gresham g hendifar ae spiegel b neeman e tuli r rimel bj wearable activity monitors to assess performance status and predict clinical outcomes in advanced cancer patients npj digit med gupta a stewart t bhulani n dong y rahimi z crane k feasibility of wearable physical activity monitors in patients with cancer jco clin cancer inform chung iy jung m | 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 |
to machine learning with python a guide for data scientists oreilly media inc california demÅ¡ar j statistical comparisons of classifiers over multiple data sets j mach learn res yates a the ensembl rest api ensembl data for any language bioinformatics kim e r chang d k colorectal cancer in inflammatory bowel disease the risk pathogenesis prevention and diagnosis world j gastroenterol diovasc dis schulte d small dense ldl cholesterol in human subjects with different chronic inflammatory diseases nutr metab car smedley d biomartbiological queries made easy bmc genom quinlan a r hall i m bedtools a flexible suite of utilities for comparing genomic features bioinformatics 101093bioin forma ticsbtq03 rom¡n j evaluation of responsive gene expression as a sensitive and specific biomarker in patients with ulcerative colitis inflamm bowel dis 101002ibd23020 song r identification and analysis of key genes associated with ulcerative colitis based on dna microarray data medicine baltimore e10658 101097md00000 schwegmann k detection of early murine colorectal cancer by mmp29guided fluorescence endoscopy inflamm bowel dis 101097mib00000 oliveira l g d positive correlation between disease activity index and matrix metalloproteinases activity in a rat model of colitis arq gastroenterol 101590s0004 shin js antiinflammatory effect of a standardized triterpenoidrich fraction isolated from rubus coreanus on dextran sodium sulfateinduced acute colitis in mice and lpsinduced macrophages j ethnopharmacol 158pt a 101016jjep201410044 owens d w lane e b keratin mutations and intestinal pathology j pathol 101002path1646 macfie t s duox2 and duoxa2 form the predominant enzyme system capable of producing the reactive oxygen species h2o2 in active ulcerative colitis and are modulated by 5aminosalicylic acid inflamm bowel dis 10109701mib00004 0e palmer n p concordance between gene expression in peripheral whole blood and colonic tissue in children with inflammatory bowel disease one e0222952 101371journ alpone02229 wei z large sample size wide variant spectrum and advanced machinelearning technique boost risk prediction for inflammatory bowel disease am j hum genet amrhein v greenland s mcshane b scientists rise up against statistical significance nature wasserstein r a0l schirm a a0l lazar n a0a moving to a world beyond p maeda y fully automated diagnostic system with artificial intelligence using endocytoscopy to identify the presence of histologic inflammation associated with ulcerative colitis with video gastrointest endosc 101016jgie201809024 acknowledgementsthis research was partially supported by grants from the natural sciences and engineering research council of canada nserc to hu grant number rgpin and to lpc grant number rgpin hmk was partially supported by funding from memorial universitys school of graduate studiesauthor a0contributionsconceptualization hu and lpc methodology hmk hu and lpc analysis hmk and lpc writing hmk hu and lpc experiments hmk supervision hu and lpccompeting interests the authors declare no competing interestsadditional informationcorrespondence and requests for materials should be addressed to hu a0or a0lpcreprints and permissions information is available at wwwnaturecomreprintspublishers note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliationsscientific reports 101038s41598020705830vol1234567890wwwnaturecomscientificreports 0copen access 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 license and indicate if changes 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 if material 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 obtain permission directly from the copyright holder to view a copy of this license visit httpcreat iveco mmons licen sesby40 the authors scientific reports 101038s41598020705830vol0123456789wwwnaturecomscientificreports 0c' | Colon_Cancer |
mgat5 knockout ko in hek293 cells induces metabolic changes resulting in increased intracellular udpglcnac increasedglycolysis enhanced spare respiratory capacity and higher citrate ï¬ux from the mitochondria mgat5 ko cells express constitutively high mica mainly regulated onthe transcriptional level through opening of the chromatin at the mica promoter mica expression in mgat5 ko cells is dependent on citrate turnover and histoneacetylation blocking citrate ï¬ux inhibits mica expression in numerous cancer cell lines and we propose that this is a central metabolic regulation of mica andimmune surveillanceintroductionnatural killer nk and cd8 t cells monitor autologouscells for markers of tumorigenesis and stress these immunecells express the nkg2d receptor that recognizes nkg2dligands nkg2dls upregulated on the surface of transformedcells nkg2dl expression is in many ways a doubleedged sword upregulation of nkg2dls on cancer cellsenhance nk cell ltration and promote cancer cytotoxicity conversely numerous cancer cells maintain chronicnkg2dl expression and evade immune elimination bydownmodulating and impairing nkg2d receptor signalingcancer cells that block nkg2dl surface expression toevade immune recognition and clearance can be treated withstressinducers such as histone deacetylase inhibitors hdacisheatshock or shortchain fatty acids scfas that upregulatenkg2dls to date studies have primarily focused ondelineating transient nkg2dl induction whereas not much isknown about regulation of their constitutive expressionmetabolic reprogramming is a central hallmark of cancercancer cells use aerobic glycolysis that was initially believedto be a result of dysfunctional mitochondria howeverlater advances have shown that cancer cells often use aerobicglycolysis alongside mitochondrial oxidative phosphorylationoxphos mitochondria are not merely the powerhouseof the cell but also provide metabolites for anabolic pathwaysnecessary for cell growth citrate can be exported from thetricarboxylic acid tca cycle for biosynthetic purposes inthe cytosol citrate is cleaved by atp citrate lyase acly togenerate acetylcoa and oxaloacetate oaa citrateis an inhibitor of glycolysis thus to maintain high aerobicglycolysis cancer cells require low cytoplasmic citrate moreover conversion of citrate by acly is a critical regulatorof gene transcription by producing acetylcoa for histoneacetylation several of these cancerassociated metabolicfrontiers in immunology wwwfrontiersinaugust volume 0cm¸ller citrate facilitates mica expressionproperties are shared with other highly proliferating cells suchas activated t cellsexpression of nkg2dls is associated with hyperproliferation and thus with highly active metabolism two studies havelinked nkg2dl expression to active glycolysis whereasone study reports that inhibition of glycolysis increased basalnkg2dl expression in breast cancer cell lines thesestudies emphasize a link to proliferative cell metabolism andsuggest that the role of glycolysis in nkg2dl regulation iscontextspeciï¬cnkg2dls fall into two groups the ul16 binding protein ulbp16 and the mhc class i chainrelated proteins aand b mica and micb surface expression of each nkg2dlis regulated individually and at all levels of protein biogenesis we have previously shown that surface expression ofspeciï¬c mica alleles depends on nglycosylation nacetylglucosaminyltransferase v mgat5 is an oncoproteincatalyzing the formation of 16branched nglycans thatpromote surface retention of glycoproteins but it is notknown if mgat5 regulates surface expression of mica growthfactor receptors are examples of mgat5 substrates and mgat5overexpression is associated with growth adhesion invasionand metastasis of cancer inhibition of mgat5 reducestumor growth enhances the antitumor responses by cd4 tcells and macrophages and promotes th1 diï¬erentiation in this study we examine the metabolic regulation of thenkg2dl mica we discover that mica was increased aftermgat5 knockout ko in a metabolically dependent way anduse this as a model to investigate the regulatory mechanismsof constitutive mica expression we ï¬nd that glycolysis andmitochondrial export of citrate promotes constitutive micatranscription in mgat5 ko cells a regulation that was alsoshown in several micaexpressing cancer cells in particularincreased mica transcription was associated with alteredchromatin accessibility of the mica promoter our ï¬ndingssuggest that citrate drives a metabolic stress that modulateschromatin accessibility to facilitate basal mica transcription andthereby regulate immune surveillancematerials and methodsanimalsfemale nmri mice to 10weeks old taconic lille skensveddenmark were used and all studies were performed inaccordance with the danish act on animal experimentationwhich implements directive 201063eu on the protection ofanimals in scientiï¬c research the studies were approved by theanimal experimentation inspectorate ministry of environmentand food denmark license no healthmonitoring was carried out in accordance with federation forlaboratory animal science associations guidelinesreagents pharmacological inhibitorsand dna constructspharmacologicalfrom sigmaaldrich werecompoundsnacetyldglucosamine glcnac a3286 pugnac a7229carbonylcyanide2dg d61345aminoimidazole4carboxamide2deoxydglucosetriï¬uoromethoxyphenylhydrazone fccp c2920 uk5099pz0160 bis25phenylacetamido134thiadiazol2ylethylsulï¬de bptes sml0601 potassium hydroxycitrate tribasicmonohydrate hc sodium dihydrogencitrate sodium acetate s5636 oxaloacetic acid oaa o41266mercaptopurine monohydrate 6mp azaserinea4142ribonucleotideaicar a9978 nacetylcysteine nac a9165 sodiumpropionate p1880 sodium butyrate b5887 dmso d2438pbs d8537 etomoxir sodium salt was purchased fromcayman chemicals ann arbor mi united states bristol bms303141 wasunited kingdom the gfpmycmica and mica vectors containingthe coding sequences of mica or mica alleledownstream of a generic leader a gfp cassette and a myctag were provided by dr m wills university of cambridgecambridge united kingdom pgl3basic pgl3bluciferase vector was purchased from promega promegamadison wi united states e1751 micaï¬reï¬y luciferasepromoter vectors and sv40renilla luciferase promoter vectorwere provided by prof c ocallaghan university of oxfordoxford united kingdom from tocris biosciencepuriï¬cation of peripheral bloodlymphocyteshuman peripheral blood mononuclear cells pbmcs wereisolated by histopaque1077 sigmaaldrich st louis mounited states separation from buï¬y coats obtainedfrom healthy blood donors the capital region blood bankcopenhagen university hospital copenhagen denmark toobtain peripheral blood lymphocytes pbls pbmcs weredepleted from monocytes by incubation with dynabeadsinvitrogen carlsbad ca united states as previouslydescribed pbls were activated in rpmi1640 withoutglucose gibco gaithersburg md united states supplemented with dialyzed fetal bovine serumfbs f9665 mm penicillinstreptomycin p4333 mmlglutamine g7513 mm sodium pyruvate s8636 and either mm dglucose g8769 or mm dgalactose g6404all purchased from sigmaaldrich pbls were activated withcd3cd28 beads invitrogen 11132d and 20uml hil2peprotech rocky hill nj united states for dayson day pbls were treated with ngml fr901228 nationalcancer institute bethesda md united states for hline pc3 and the keratinocytederived cellcell line cultivation and proliferationhuman embryonic kidneyderived hek293 cells the prostatecancer celllinehacat were purchased from american type culture collectionatcc manassas va united states nkg2d reporter cellct312 and the 2b4 parental cellline were kindly providedby chiwen chang trowsdale lab cambridge universitylines mdamb231 and mcf7 werethe breast cancer cellprovided by dr jos moreira departmentfor veterinaryfrontiers in immunology wwwfrontiersinaugust volume 0cm¸ller citrate facilitates mica expressiondisease university of copenhagen denmark and henrikleï¬ers the state hospital copenhagen denmark respectivelythe cervical cancer cellline hela was provided by jesperjurlander the state hospital copenhagen denmark themelanoma cells skmel28 fm55m1 fm78 and fm86 andthe human colon adenocarcinoma cell lines ht29 and sw480were provided by dr per thor straten herlev universityhospital denmark hek293 mdamb231 and mcf7 cellswere cultured in dmem with glutamax gibco hela hacat pc3 fm55m1 fm78 fm86 skmel28 andsw480 were cultured in rpmi1640 sigmaaldrich r5886and ht29 were cultured in mccoys 5a medium sigmaaldrich m8403 media were supplemented with fbs and mm penicillinstreptomycin mm lglutamine was addedto rpmi1640 and mccoys 5a for longterm cell culture inglucosegalactose cells were cultured in dmem medium withoutglucose gibco supplemented with dialyzedfbs mm penicillinstreptomycin mm sodium pyruvate and mm glucosegalactose all cells were kept at culture conditions¦c and co2 and were passaged every daysfor proliferation assay wt and mgat5 ko cells wereseeded in or cellswell for each experimentcells were counted in triplicate wells after and h usingthe biorad tc20 automated cell counter biorad herculesca united statesgene editingmgat5 ko cells were generated by zinc ï¬nger nucleasetargeting in hek293 cells and subsequent cloning and selectionwas performed as described previously hek293cells were transfected with mrna sigmaaldrich or µgof endotoxin free plasmid dna using nucleofection on anamaxa nucleofector lonza copenhagen denmark mgat5ko clones were selected by loss of reactivity with lphaand clones were conï¬rmed to have mgat5 mutations usingpcr and sequencinglentiviralmediated gene transfer was performed with anmgat5 encoding vector constructed by inserting the mgat5sequence generated as a bluntend pcr product from a vectorfrom hw university of copenhagen copenhagen denmarkinto an entry vector system using the pentr directionaltopo cloning kit invitrogen k243520k350020 followingmanufacturers protocol topo clonal reaction entry vectorswere transformed into macht1 chemically competent e coliusing heatshock and soc medium followed by selectionpcr inserts were conï¬rmed by sequencing at euroï¬ns mwgoperons luxembourg colonies were ampliï¬ed and plasmidswere puriï¬ed with nucleobond xtra midi kit machereynagelduren germany mgat5 sequences were insertedinto plx302 lentiviral destination vector with lr clonase iienzyme mix invitrogen after proteinase k treatmentconstructs were transformed into dh5α using heatshock andsoc medium selected clones were ampliï¬ed and dna waspuriï¬ed using nucleobond xtra midi kit destination vectorswere checked for insertion using bsrgi digestion at ¦cmgat5coding lentiviral ps were packaged in hek293tcells transfected with a mix of µg pspax2 vector packagingvector µg pcmvvsvg envelope vector µg plx302vector carrying mgat5 and µl cacl2 to a ï¬nal volume of µl the dna mixture was complexed with µl 2x hbsunder constant air ï¬ow and the transfection mix was addeddropwise to hek293t cells in antibioticfree mediumcell culture medium was harvested days after transfection andviral p preparations were prepared by centrifugation at g for min lentiviral ps were added to cells andincubated for h cells were cultivated in puromycin µgmlselection medium for weeks functional mgat5 expressionwas validated by lpha bindingtransient transfectiontransient transfections were performed as described previouslyusing amaxa nucleofector device lonza dna wasintroduced to cells in µl nucleofector solution vlonza vca1003 and pulsed using the nucleofector programq001 for gfpmyctagged mica and micaconstructs cells were transfected with µg dna and analyzedthe next day transfection with shrnas or luciferase promoterconstructs was carried out by calciumphosphate transfectionbrieï¬y dnarna were prepared in µl cacl2 25mand adjusted to a ï¬nal volume of µl dna mixture wascomplexed with µl 2x hbs hepes nacl na2hpo4and added dropwise to cells scrambled sirnacontrol siidh1 and siidh2 ontarget plus smart poolswere purchased from ge healthcare dharmacon lafayetteco united statesfunctional assaysfor nkg2d downmodulation pbls were isolated as describedabove followed by depletion of cd4 cells using cd4 antibodyebioscience san diego ca united states anddynabeads mouse panigg invitrogen cd4depletedpbls were cultured in rpmi1640 sigmaaldrich r5886supplemented with human serum sigmaaldrich h3667 mm penicillinstreptomycin mm lglutamine and ngmlhil15 peprotech for days to enrich for nkcd8t cells nkg2d downmodulation assay was performed aspreviously described nkg2d ligands on eï¬ector cellshek293 wt or mgat5 ko cells were incubated with blockingnkg2dfc rd systems minneapolis mn united states1299nk or control igg1fc rd systems 110hg µgmlfor min at ¦c eï¬ector cells and target cells nkcd8t cells were mixed at indicated eï¬ectortarget ratios and spundown min g to allow conjugate formation after h cocultivation nkcd8 t cells were analyzed for nkg2d surfaceexpression by ï¬ow cytometry using accuri c6 ï¬ow cytometerbd bioscience franklin lakes nj united statesfor the reporter cell assay the nkg2dreporter cell line2b4ct312 and the parental control 2b4 cell line target cells were mixed with eï¬ector cells wt or mgat5 ko cellsthat were either blocked with nkg2dfc or control igg1fc asdescribed above eï¬ector and target cells were cocultivated atdiï¬erent et ratios for h gfp expression of target cellswas assessed with accuri c6 ï¬ow cytometer for in vivo assaytarget cells were labeled with vybrant did celllabeling solutionfrontiers in immunology wwwfrontiersinaugust volume 0cm¸ller citrate facilitates mica expressioninvitrogen v22887 according to manufacturers protocol andinjected intraperitoneally together with wt or mgat5 kocells in a ratio of each mice were usedper group target cells were harvested after approximately hwith peritoneal lavage and nkg2d activation of didpositivereporter cells were assessed as gfp expression with accuric6 ï¬ow cytometerwas assessed by accurate mass and retention time amrt plusfragment identiï¬cation at two collision energies and evdetailed acquisition methodology has been described previously udpglcnacudpgalnac detected peak screened byexpected calculated mass could be of either compound as thesetwo sugars could not be separated chromatographically hencehas been reported as a putative metabolite pending conï¬rmationlactate and dntp measurementsconcentrations of llactate was measured enzymatically withrandox colorimetric assay according to manufacturers protocolrandox crumlin united kingdom lc2389 reaction andanalysis was performed on an advia chemistry systemsiemens munich germanydntp levels were determined in cells harvested withtrypsinization and pelleted by centrifugation for g for min followed by resuspension of cell pellets in methanolfrozen in liquid nitrogen and boiled at ¦c for min sampleswere evaporated until dryness in a speedvac and whole cell levelsof dttp datp dctp and dgtp were determined using the dnapolymerase assay previously described lchrms metabolite proï¬lingto determine intracellular metabolite levels cell pellets from cells were resuspended in µl of cold methanolafter min sonication samples were prepared by svortex followed by min equilibration at room temperatureafter centrifugation at g for min at ¦c µl supernatants were collected transferred to ultrafreemccentrifugal ï¬lter devices merck millipore ltd cork irelandand centrifuged at g for min at ¦c from this µlwas transferred to lc vials and µl of each sample was pooledto a mixed qc samplelchrms was performed on a nity ii ultrahigh performance liquid chromatography uhplc systemcoupled to a ifunnel quadrupoletime of ï¬ight qtofmass spectrometer equipped with a dual ajs electrosprayionization source agilent technologies santa clara caunited states polar metabolites were separated on a sequantzichilic merck darmstadt germany column mm mm µm p size coupled to a guardcolumn mm mm µm p size and an inlineï¬lter mobile phases consisted of formic acid in water withsolvent a and formic acid in acetonitrile with solvent bthe elution gradient used was as follows isocratic step at bfor min b to b in min and maintained at bfor min then decreasing to b at min and maintainedfor min then returned to initial conditions over min and thecolumn was equilibrated at initial conditions for min the ï¬owrate was mlmin injection volume was µl and the columnoven was maintained at ¦c the acquisition was obtainedwith a mass range of mz for where full scan highresolution data is acquired at three alternating collision energies ev ev and ev positive and negative raw lchrmsï¬les were independently processed with an inhouse developedpcdl library for polar metabolites using proï¬nder version b06agilent technologies identiï¬cation of reported compoundsextracellular flux analysisthe seahorse xfe96 extracellular ï¬ux analyzeragilenttechnologies was used to measure ocr and ecar on hek293cells cells were seeded at the density cellswell ¼ hbefore the experiment one hour prior to assay run cells wererinsed and switched to xf media agilent technologies with mm sodium pyruvate and mm glucose or galactose andincubated at ¦c co2free incubator for the mitochondrialstress tests ocr was measured under basal conditions andduring sequential injection of µm oligomycin sigmaaldrich µm fccp sigmaaldrich c2920 and µmrotenone rot sigmaaldrich r8875 µm antimycina aa sigmaaldrich a8674 reported basal respiration iscalculated from the third measuring point with ocr after rotand aa subtracted atpcoupled respiration display ocr afteroligomycin subtracted from the third measuring point andmaximal respiration is ocr after fccp with ocr after rotand aa subtractedfor measuring the eï¬ect of hc ocr was assessed h after aninjection of mm hc13c6glucose tracing experiment cells were incubated for h in dmem medium withoutglucose supplemented with fbs mm sodium pyruvateand mm uniformly labeled [u13c]glucose cambridgeisotope laboratories tewksbury ma united states clm incubation medium samples were collected and cleared bycentrifugation g for min cells were washed and detachedsterically intracellular metabolites were extracted in ethanoland centrifuged at g for min ¦c to separatethe soluble extract supernatant from the insoluble componentspellet cell extracts and medium samples were lyophilizedand reconstituted in water for subsequent biochemical analysesextract samples were adjusted to ph with hcl and evaporatedto dryness under nitrogen ï¬ow analytes were extracted into ananic phase ethanolbenzene followed by derivatizationwith dmf86 mtbstfa with a modiï¬ed procedure from standards containing unlabeled metabolites of interest andcell extracts were separated and analyzed in a gas chromatographagilent technologies 7820a chromatograph jw gc columnhp5ms parts no 19091s433 coupled to a mass spectrometeragilent technologies 5977e the isotopic enrichment of themetabolites of interest was corrected for natural abundance of 13cusing the unlabeled standards and calculated according to data are presented as labeling of m x where m is the massof the unlabeled molecule and x is the number of labeled catomsin a given metabolite frontiers in immunology wwwfrontiersinaugust volume 0cm¸ller citrate facilitates mica expressionwestern blottingproteins were extracted using ripa buï¬er thermo scientiï¬cwaltham ma united states and proteinasephosphataseinhibitor cocktail thermo scientiï¬c for minon ice lysates were sonicated times for s and clearedby centrifugation at rpm for min at ¦c proteinextracts were denatured at ¦c for min in nupage samplebuï¬er and dtt sigmaaldrich proteins were resolvedusing sdspage gels invitrogen and transferred tonitrocellulose membranes invitrogen ib301001 using the iblotdevice invitrogen for total protein stain membranes werewashed in ddh2o and stained with revert protein stainsolution licor biosciences lincoln ne united states according to manufacturers protocol membranes wereblocked in tbst blocking buï¬er licor biosciences probed with primary antibodies in tbs w tween and bsa overnight on a shaker at ¦c and washed intbs tween secondary antibody was from licorlicor biosciences and signals were visualizedby the odyssey fc imaging system licor biosciencesoglcnacylation was detected with rl2 oglcnacylationantibody abcam cambridge united kingdom ab2739 atpcitrate lyase acly was detected with rabbit acly antibodycell signaling and acly phosphorylation with rabbitphosphoacly ser455 antibody cell signaling flow cytometryadherent cells were detached in pbs w mm edta invitrogen or by pipetting cell surface staining was done aspreviously described and cells were analyzed on accuric6 ï¬ow cytometer bd bioscience antibodies used for thisstudy were mica rd systems fab1300a ulbp256 rdsystems fab1298p nkg2d rd systems fab139a ulbp1rd systems fab1380p ulpb3 rd systems fab1517aulbp4 rd systems fab6285a micab bd bioscience icam1 leinco technologies c170 mouse igg1antimyctag merck millipore micb rd systemsmab1599 or igg2b isotype control rd systems mab004detected with secondary antimouse igg biolegend san diegoca united states binding of ï¬uorescently labeledaf647lpha invitrogen l32457 and fitcepha vectorlaboratories burlingame ca united states fl1121 was usedto measure surface levels of complex nglycans all isotypecontrols were purchased from bd biosciencefor staining with mitochondrial probes neutral lipid stainsor 2nbdg uptake cells seeded the day prior toexperiment were washed once in pbs and incubated for minat ¦c and co2 in warm growth medium containing nmtetramethylrhodamine methyl ester perchlorate tmrm sigmaaldrich t5428 nm mitotracker green fm invitrogenm7514 or for h in growth medium with µm 2nbdginvitrogen n13195 bodipy invitrogen d3922 wasdiluted in warm serumfree medium in a dilution andshaken vigorously to solubilize the lipids immediately beforeloading into the cells for min cells were washed twice inpbs fbs and detached sterically prior to analysisthe soluble nkg2dfc receptor 1299nk rd systemsand igg1fc 110hg rd systems were labeled with zenonalexa fluor against human igg1 z25408 invitrogen priorto staining of melanoma cellsin forwardsidescatter plotsdata were acquired with an accuri c6 instrument usingaccuri c6 software and analyzed in flowlogic v721 inivaitechnologies mentone vic australia by gating on viablecellsfollowed bysingle cell gating by areaheightscatter plots fscafsch geometric mean ï¬uorescent intensity mfi values aredisplayed in ï¬gures as mfi or with corresponding isotype controlsubtracted as 01mfifscsscreal time pcr analysistotal rna was extracted by phase separation in trizolchlorophorm and puriï¬ed on directzol spincolumns zymoresearch irvine ca united states according to manufacturersprotocol cdna was generated using superscript cdnasynthesis kit invitrogen under standard pcr conditionsfollowing primersequences were used for quantitativertpcr with brilliant sybr green qpcr master mixkit mica mica_f tggcagacattccatgtttctgmica_r ctcgtcccaactgggtgttg ulbp2 ulbp2_f cagagcaactgcgtgacatt ulbp2_r ggccacaaccttgtcattctidh1 idh1_f ctatgatggtgacgtgcagtcg idh1_r cctctgcttctactgtcttgccidh2 idh2_f agatggcagtggtgtcaaggagidh2_r ctggatggcatactggaagcag glut1 glut1_fctgctcatcaaccgcaac glut1_r cttcttctcccgcatcatct glut2 glut2_f tacattgcggacttctgtgg glut2_r agactttcctttggtttctgg glut3glut3_f cagcgagacccagagatg glut3_r ttggaaagagccgattgtag glut4 glut4_f tgggcttcttcatcttcacc glut4_r gtgctgggtttcacctcctrplp0_fcctcgtggaagtgacatcgt rplp0_r cattcccccggatatgaggc realtime qpcr was performed on biorad cfx96 realtime thermal cycler c1000 touch and alltranscripts were normalized to housekeeping rplp0 transcripthousekeepingand rplp0asgeneluciferase reporter assaycells were transiently transfected using calciumphosphatetransfection as described above with ï¬reï¬y luciferase promotervectors µg and an sv40promoter driven renilla luciferasevector µg cells were harvested and snap frozen hpost transfection pellets were lysed in dualglo luciferasereagent promega e2920 and ï¬reï¬y luciferase activity wasanalyzed by luminometer microbeta ii perkinelmer walthamma united states renilla luciferase activity was recorded bythe instrument after subsequent addition of volume dualglo stop glo promega e2920 to correct for transfectioneï¬ciency ï¬reï¬y luciferase signals were normalized to sv40renilla luciferase signals of corresponding sampleatacseqatacseq was performed as described previously foreach cellline cells were harvested from separatefrontiers in immunology wwwfrontiersinaugust volume 0cm¸ller citrate facilitates mica expressioncultures and used to prepare tagmented chromatin replicatesof wt and replicates of mgat5 ko cell lines samplestotal quality of pcrampliï¬ed sequencing libraries was assessedusing a tapestation instrument with high sensitivity dnascreentapes agilent libraries were sequenced as paired endreads on a single lane of an illumina hiseq4000 ï¬ow cellresulting reads were aligned to the grch37hg19 referencegenome using rsubread and alignments were ï¬ltered toremove low quality duplicate and mitochondrial reads peakswere called using macs2 on merged reads from allsamples and diï¬erential peak accessibility between cell lines wasdetermined using edger with a threshold false discoveryrate of transcription factor binding motifs enriched indiï¬erentially accessible peaks were identiï¬ed using homer h3k4me3 chipseq data were downloaded from encode1 andare available under accession encff756ehfquantiï¬cation and statistical analysisresults are presented as mean ± sem diï¬erences were analyzedfor statistical signiï¬cance using prism or graphpad softwarela jolla ca united states statistical analysis was performed asstated in ï¬gure legends using unpaired ttest in 1a 1c 1e 3ef3h 5c 7a 7ef paired ttest in 4fg 7d multiple ttest in 1b1d 3d 4ab one sample ttest in 2ac 3c 4c 4e 7g twowayanova in 3a 5df 5hi 6a 6e 7hi or oneway anova in5g level of statistical signiï¬cance was determined by p p and p p resultsmgat5 knockout increases nkg2dlexpression and activates nkg2d in vitroand in vivoregulation of constitutive mica expression remains largelyunknown surface expression of certain mica alleles dependson nlinked glycosylation we questioned whetherthe cancerassociated glycosyltransferase mgat5 is required formica expression to assess the role of mgat5 in regulationof nkg2dl surface expression mgat5 ko clones weregenerated in hek293 cells remarkably mgat5 ko resultedin a permanently increased surface expression of the nkg2dlsmica micb and ulbp256 compared with parental wildtypewt cells figure 1a to conï¬rm mgat5 ko we measuredbinding of leukoagglutinin from p vulgaris lpha that bindsspeciï¬cally to mgat5modiï¬ed nglycans as expected lphabinding was reduced whereas binding of erythroagglutininfrom p vulgaris epha that interacts with mgat3modiï¬ednglycans was unaï¬ected thus verifying functional knockoutof mgat5 figure 1a modiï¬cation of mgat5 expressiontherefore associated with substantial changes in constitutiveexpression of several nkg2dlsto verify the functionality of mgat5 koinduced nkg2dlswe tested nkg2d activation in a reporter cell line expressing1httpswwwencodeprojecthuman nkg2d coupled to dap10cd3ζ signaling and nuclearfactor of activated t cells nfatcontrolled gfp ultimatelyexpressing gfp in response to nkg2d activation nkg2dgfp activation was higher after cocultivation with mgat5ko cells than with wt cells figure 1b corresponding to theincreased nkg2dl expression in mgat5 ko cells figure 1athe reporter cells without nkg2d supplementary figure s1aremained inactivated indicating that the activation was nkg2dmediated figure 1b moreover blocking nkg2dls withsoluble nkg2dfc receptor impaired the activation furthervalidating nkg2d speciï¬city supplementary figure s1bto test if mgat5 ko cells could activate nkg2d in vivowe adoptively injected nkg2d reporter cells together with wtor mgat5 ko cells into the peritoneum of nmri mice andmeasured gfp expression in reporter cells in line with ourin vitro data we observed a signiï¬cant increase in nkg2dgfpactivation by mgat5 ko cells compared with wt cells theresponse was nkg2dspeciï¬c since the control reporter cellswere unaï¬ected figure 1c these data verify that mgat5 koinduced nkg2dls maintain their functional integrity in vivonkg2d is downmodulated upon activation to furtherexamine the functionality of nkg2dl expression causedby mgat5 ko we assessed nkg2d downregulation afterreceptor activation nkg2d was further downregulated oncd4depleted peripheral blood lymphocytes pbls after cocultivation with mgat5 ko cells than with wt cells and thisdownregulation was abolished by blocking nkg2dls with asoluble nkg2dfc receptor figure 1d combined these dataindicate that ko of mgat5 upregulates mica and ulbp256resulting in nkg2d activation in vitro and in vivoto ensure that the mica upregulation was a result of mgat5ko we stably transfected mgat5 into wt and mgat5 kocells lpha binding was restored within days after transfectionconï¬rming expression of functional mgat5 interestingly ittook multiple passages for mica expression to decrease to wtlevels figure 1e and supplementary figure s1c suggestingthat mica is regulated in response to a longterm adaptation toaltered mgat5 expressionudpglcnac upregulates micaexpressionlongterm mgat5 deï¬ciency willlikely result in aberrantnglycosylation and an accumulation of the mgat5 donorsubstrate udpnacetylglucosamine udpglcnac to addressif mica was regulated by a change in nglycosylation inmgat5 ko cells we assessed the posttranslational regulationof mica by measuring surface expression of transgenicallyexpressed gfpmyctagged mica under a cytomegaloviruscmv promoter the mica alleles mica and micaare distinctly regulated posttranslationally and althoughmica was upregulated in mgat5 ko cells the regulationwas minor and unlikely to account for the profound changein endogenously expressed mica figures 1a 2a micatranscripts on the other hand were highly increased in mgat5ko cells figure 2b as well as ulbp2 mrna supplementaryfigure s2a suggesting that nkg2dls are transcriptionallyfrontiers in immunology wwwfrontiersinaugust volume 0cm¸ller citrate facilitates mica expressionfigure mgat5 knockout increases nkg2dl expression and activates nkg2d in vitro and in vivo a surface expression of nkg2d ligands and binding ofï¬uorescently labeled lpha mgat5 modiï¬cations or epha mgat3 modiï¬cations on hek293 wildtype wt and hek293 mgat5 knockout ko cells or isotypecontrol staining iso analyzed by ï¬ow cytometry data are presented as histograms representative of at least three independent experiments and in bar graphsshowing mean ï¬uorescence intensity mfi b in vitro nkg2d activation measured as gfp expression in nkg2d negative reporter cells control and nkg2dexpressing nkg2d reporter cells target cells cocultivated with wt or ko cells effector cells for h at indicated effectortarget et ratios c nkg2dactivation in vivo measured on reporter cells as in b after activation by wt or ko at a ratio in peritoneum of nmri mice for approximately h gfp expressionin didlabeled reporter cells signiï¬es nkg2d activation and is shown as gfp mfi values of cells from foursix mice per group d nkg2d downmodulation wasassessed on nkcd8 t cells target cells after cocultivation for h with wt or ko cells effector cells at indicated effectortarget ratios et nkg2dls on targetcells were blocked with nkg2dfc bl or unblocked with igg1fc un the graph depicts surface expression of nkg2d presented relative to surface nkg2dexpression on target cells alone e mica surface expression left and lphaepha surface binding right after lentiviral introduction of mgat5 into wt or kocells mfi values from antibody staining were corrected for isotype background staining 01mfi statistical analysis was performed by unpaired ttests in ace andmultiple ttest with fdr comparing wt and ko in bd p p p and p regulated in mgat5 ko cells notably we found that themgat5 substrate udpglcnac although indistinguishablefrom udpnacetylgalactosamine udpgalnac tended tobe higher in mgat5 | Colon_Cancer |
" micrornas mirnas have been reported to have important regulatory roles in the progression of several types of cancer including cervical cancer cc however the biological roles and regulatory mechanisms of mirnas in cc remain to be fully elucidated the aim of the present study was to examine the functions of mirnas in cc and the possible mechanisms using a microarray it was identified that mirna15a5p mir15a5p was one of the most downregulated mirnas in cc tissues compared with adjacent noncancerous tissues the low expression of mir15a5p was observed in cc tumor tissues with distant metastasis and in cc cell lines in addition the effects of mir15a5p upregulation on cell viability apoptosis invasion and migration of cc cells were investigated using cck flow cytometry transwell and wound healing assays respectively it was demonstrated that upregulation of mir15a5p significantly suppressed the viability migration and invasion and promoted the apoptosis of siha and c33a cells furthermore yesassociated protein yap1 a wellknown oncogene was confirmed to be directly targeted by mir15a5p and was found to be negatively regulated by mir15a5p further correlation analysis indicated that mir15a5p expression was negatively correlated with yap1 expression in cc tissues notably overexpression of yap1 abrogated the tumor suppressive effects of mir15a5p in cc cells taken together these present findings indicated that the mir15a5pyap1 axis may provide a novel strategy for the clinical treatment of cccorrespondence to professor xu chen department of obstetrics and gynaecology huashan hospital north fudan university jingpohu road baoshan shanghai pr chinaemail xuchenccx163comcontributed equallykey words cervical cancer microrna15a5p cell viability migration invasion yesassociated protein introductioncervical cancer cc is a type of malignant tumor commonly presenting in women in cc cases are diagnosed each year and it accounts for of all female cancerassociated mortalities each year worldwide despite advances in the therapeutic strategies for cc including targeted therapies and immunotherapy the prognosis of cc remains poor due to the abnormal growth of epithelial cells thus it is imperative to clarify the molecular interactions occurring during the initiation and progression of ccmicrornas mirnas are a family of short noncoding rnas with an average length of nucleotides which negatively regulate target gene expression through either translation repression or rna degradation accumulating evidence has indicated that mirnas may function as oncogenes or tumor suppressors depending on their target mrna in various types of cancer including cc for example yang reported that mir214 inhibits the growth of cc cells by the regulation of its target enhancer of zeste homolog dong demonstrated a suppressive role of mir217 in the development of cc cells via targeting rhoassociated protein kinase chen reported that mir499a promotes the proliferation cell cycle progression colony formation migration and invasion of cc cells by targeting srybox transcription factor in addition several mirnas serve as diagnostic biomarkers in patients with cc such as mir152 and mir365 despite the aforementioned findings the roles of mirnas in the development of cc require further investigationin the present study a mirna microarray was performed to investigate the expression profiles of mirnas in cc tissues and the most downregulated mirna identified mir15a5p was selected for further analysis the potential role and underlying mechanism of mir15a5p in cc cells were also investigated the present results suggest that mir15a5p may serve as a therapeutic target for ccmaterials and methodspatients and samples in total paired cervical samples tumor tissues and adjacent noncancerous tissues were 0cchen mir15a inhibits cervical cancer cell growthobtained from female patients with cc who underwent cervical surgical resection without preoperative systemic therapy at the department of obstetrics and gynecology huashan hospital north of fudan university shanghai china between may and december the median age of the patients was years range years among all patients there were patients with metastatic cc and with nonmetastatic cc the matched nontumor adjacent tissue was obtained cm beyond the boundary of cc tissue all tissue samples were immediately snapfrozen in liquid nitrogen and stored at Ëc until use the experimental protocols were approved by the ethics committee of huashan hospital north of fudan university written informed consent for participation in the study was obtained from all patientsmirna expression profiling total rna from cc tissues three randomly selected paired tumor tissues and adjacent noncancerous tissues was extracted using mirneasy mini kit qiagen gmbh the samples were assessed using the mircury lna¢ array v180 agilent technologies inc the procedure and imaging processes were performed as described previously cell culture human cc cell lines hela c33a caski and siha 293t cells and normal cervical epithelial cells ect1e6e7 were obtained from the american type culture collection all cells were cultured in dmem sigmaaldrich merck kgaa supplemented with vv fbs sigmaaldrich merck kgaa plus uml penicillinstreptomycin at Ëc with co2reverse transcriptionquantitative pcr rtqpcr total rna was extracted from tissues or cell lines using trizol reagent invitrogen thermo fisher scientific inc for mirna rt cdna was generated from ng total rna samples using taqman¢ microrna reverse transcription kit applied biosystems thermo fisher scientific inc at Ëc for min for mrna rt cdna was synthesized using primescript rt reagent kit takara bio inc at Ëc for min qpcr for mirna and mrna was performed using the sybrgreen i realtime pcr kit applied biosystems thermo fisher scientific inc on an abi system applied biosystems thermo fisher scientific inc the reaction was performed under the following conditions Ëc for min followed by cycles at Ëc for sec and Ëc for sec and a final extension at Ëc for sec the primers for qpcr analysis were as follows mir15a5p forward 'aat gtt gcc cgt aat gcc3' and reverse 'ccc aag cgg aga aag gaa3' u6 forward 'gct tcg gca gca cat ata cta aaa t3' and reverse 'cgc ttc acg aat ttg cgt gtc at3' yesassociated protein yap1 forward 'cgg tcc act tca gtc tcc3' and reverse 'gag tgt ggt gga cag gta ctg3' and gapdh forward 'gtg gtg aag acg cca gtg ga3' and reverse 'cga gcc aca tcg ctc aga ca3' the expression levels of mir15a5p and yap1 were normalized to the expression of u6 and gapdh respectively the relative expression of each gene was calculated using the cq method cell transfection the mir15a5p mimic mimic negative control nc mir15a5p inhibitor inhibitor nc yap1 overexpression plasmid pcdnayap1 and pcdnavector were all provided by guangzhou ribobio co ltd when c33a and siha cells 5x105 cellswell in 6well plates grew to confluence mir15a5p mimic nm mimic nc nm mir15a5p inhibitor nm inhibitor nc nm pcdnayap1 µg or pcdnavector µg were transfected into cells at Ëc for h using lipofectamine® invitrogen thermo fisher scientific inc the sequences were as follows mir15a5p mimic 'uag cag cac aua aug guu ugu g3' mimic nc 'uuc ucc gaa cgu guc acg utt3' mir15a5p inhibitor 'cac aaa cca uua ugu gcu gcu a3' and inhibitor nc 'cag uac uuu ugu gua gua caa3'in addition small interfering rna targeting yap1 siyap1 and the negative control targeting a nonspecific sequence siscramble were provided by thermo fisher scientific inc siha and c33a cells were transfected with the sirnas nmoll using lipofectamine invitrogen thermo fisher scientific inc the sequences of siyap1 and siscramble were as follows siyap1 'ctc agg atg gag aaa ttt a3' and siscramble 'ttc tcc gaa cgt gtc acg t3' at h posttransfection the cells were harvested for further analysis and the inhibition efficiency was determined by western blottingcell viability the c33a and siha cells were seeded in 96well plates at a density of 5x103well overnight following transfection the cell viability was measured using a cck8 assay briefly µl cck solution was added to each well and cultured for h at Ëc the absorbance of the samples at nm was detected using a microplate reader biorad laboratories inccaspase activity following transfection c33a and siha cells were harvested and the caspase3 activity was measured using a caspase3 activity assay kit beyotime institute of biotechnology according to the manufacturer's protocolcell apoptosis the apoptosis of c33a and siha cells was examined using flow cytometry following transfection c33a and siha cells were collected and the apoptotic cells were identified using an annexin vfitc apoptosis detection kit abcam according to the manufacturer's protocol after washing with cold pbs the cells were resuspended in binding buffer followed by staining with annexin v and propidium iodide for min in the dark at room temperature the fluorescence was measured using a facscan flow cytometer beckman coulter inc and then analyzed by flowjo v871 software flowjo llcimmunofluorescence assay following transfection c33a and siha cells were fixed in absolute ethyl alcohol for min at room temperature after washing twice with pbs the fixed cells were stained with primary antibody targeting cleavedcaspase3 cat no c ell signaling technology inc for h at room temperature subsequently an antirabbit conjugated antibody with fitc cat no f0382 sigmaaldrich merck kgaa was added for h in the 0cinternational journal of molecular medicine dark fluorescence images were obtained using an inverted fluorescence microscope magnification x200cell invasion assays transwell chambers 8µm pore bd biosciences coated with matrigel bd biosciences were used for the invasion assay briefly c33a and siha cells 8x104 were seeded in the top chamber with serumfree medium while the lower chamber contained culture medium with fbs following incubation for h the cells were fixed in paraformaldehyde solution beyotime institute of biotechnology for min and stained with crystal violet beyotime institute of biotechnology for min at room temperature images were captured with an inverted microscope olympus corporation magnification x100wound healing assay for the wound healing assay c33a and siha cells were seeded onto 12well plates 2x105 cellswell and h after transfection a scratch was made using a 10µl pipette tip in the confluent cell monolayer then cells were washed twice with pbs and incubated in dmem without fbs the wound healing images were captured at and h after scratching using an inverted light microscope olympus corporation magnification x100 the wound healing rate was calculated using imagej software v146 national institutes of healthdualluciferase reporter assay mirna target prediction tools including miranda httpmirandaorguk and targetscan httptargetscanorg were used to search for the putative targets of mir15a5p pgl3yap1 widetype or pgl3yap1 mutant type pgl3yap1mut promega corporation were cotransfected with mir15a5p mimics into 293t cells in 24well plates 2x105well using lipofectamine invitrogen thermo fisher scientific inc at h posttransfection the luciferase activities were analyzed using the dualluciferase reporter assay system promega corporation with renilla luciferase activity as an internal control western blot analysis western blotting was performed as previously described briefly cells were lysed using radio immunoprecipitation assay buffer beyotime institute of biotechnology and the protein concentration was determined using the bicinchoninic acid assay total protein µglane was separated by sdspage and electrophoretically transferred onto a polyvinylidene difluoride membrane emd millipore subsequently membranes were blocked with skim milk for h at Ëc overnight each membrane was probed with primary antibodies against yap1 cat no and βactin cat no at Ëc overnight all primary antibodies were obtained from cell signaling technology inc subsequently the membrane was incubated with horseradish peroxidaseconjugated goat antirabbit igg cat no abcam at room temperature for h βactin served as the loading control and for normalization of protein expression the protein bands were developed using ecl kit ge healthcare and expression levels were quantified using imagej v146 national institutes of healthstatistical analysis all data are presented as mean ± standard deviation the correlation between mir15a5p and yap1 levels was evaluated using spearman's correlation analysis pairwise comparisons were performed by student's ttest and comparisons among groups were analyzed by oneway anova followed by tukey's posthoc test p005 was considered to indicate a statistically significant differenceresultsmir15a5p is downregulated in cc to examine the potential involvement of mirnas in the development of cc microarray analysis was performed to evaluate the mirna expression profiles between cc tissues and adjacent noncancerous tissues of differently expressed mirnas identified in the tumor group mirnas exhibited decreased expression and mirnas demonstrated increased expression compared with that in adjacent noncancerous tissues fig 1a among the aberrant mirnas the present study focused on mir15a5p for subsequent experiments due to its suppressive role in a variety of other cancer types such as endometrial cancer and chronic myeloid leukemia subsequently rtqpcr was performed to detect the expression of mir15a5p in pairs of tumor tissues and adjacent noncancerous tissues the results revealed that the level of mir15a5p was significantly lower in tumor tissues compared with that in adjacent noncancerous tissues fig 1b it was also observed that mir15a5p was expressed at a significantly lower level in tumor tissues with distant metastasis compared with in tumors tissues without distant metastasis fig 1c indicating that mir15a5p downregulation is associated with cc metastasis in addition rtqpcr was used to examine the mir15a5p level in four cc cell lines hela c33a caski and siha and the normal cervical epithelial cell line ect1e6e7 which was used as a control as expected mir15a5p was significantly lower in the four cc cell lines compared with ect1e6e7 cells fig 1d siha and c33a cells were selected for further experiments as they demonstrated the lowest expression of mir15a5p among all cell lines examinedupregulation of mir15a5p inhibits cell viability and promotes cell apoptosis in an attempt to understand the biological function of mir15a5p mir15a5p expression was upregulated or downregulated in the cultured siha and c33a cells by transfection with mir15a5p mimic or inhibitor respectively mir15a5p expression was significantly increased after mir15a5p mimic transfection whereas it was significantly decreased following mir15a5p inhibitor transfection in both siha and c33a cells fig 2a the present study then investigated the effect of mir15a5p expression on cell viability and the results demonstrated that the viability of siha and c33a cells was significantly inhibited by overexpression of mir15a5p whereas it was significantly enhanced by knockdown of mir15a5p compared with the negative control group fig 2b and c to assess the effects of mir15a5p upregulation on the apoptosis of siha and c33a cells caspase3 expression level and activity were analyzed by immunofluorescence and caspase activity assays respectively as presented in fig 2d and e the expression of cleaved caspase3 and caspase3 activity was increased in siha and c33a cells transfected with 0cchen mir15a inhibits cervical cancer cell growthfigure mir15a5p is downregulated in cc tissues and cell lines a heat map presents significant differentially expressed mirnas in cc tissues and matched adjacent noncancerous tissues n3 green indicates downregulation and red indicates upregulation b mir15a5p expression was measured by rtqpcr in pairs of cc tissues and matched adjacent noncancerous tissues c mir15a5p expression was measured in tumor tissues with distant metastasis and tumors tissues without distant metastasis by rtqpcr d mir15a5p expression was detected in four cervical cancer cell lines hela c33a caski and siha and the normal cervical epithelial cells ect1e6e7 data are expressed at the mean ± standard deviation n3 of one representative experiment p001 vs ect1e6e7 cells mir microrna cc cervical cancer rtqpcr reverse transcriptionquantitative pcr mir15a5p mimic compared with the mimic nc groups furthermore the results of flow cytometry demonstrated that the extent of apoptosis was significantly increased after mir15a5p mimic transfection compared with the mimic nc groups fig 2f taken together these results indicate that overexpression of mir15a5p inhibits cell viability by inducing cell apoptosisupregulation of mir15a5p inhibits the invasion and migration of cc cells the present study further investigated whether overexpression of mir15a5p could reduce the invasiveness and migratory potential of cc cells using a transwell assay it was identified that the invasive capacities of siha and c33a cells were significantly inhibited by mir15a5p mimic whereas they were increased by mir15a5p inhibitor compared with the nc groups furthermore the wound healing assay results also demonstrated a significant reduction of cell migration in siha and c33a cells following mir15a5p overexpression however the migration of siha and c33a cells was significantly enhanced by mir15a5p inhibition fig 3c and d collectively the present data suggest that overexpression of mir15a5p suppresses the invasive and migratory abilities of cc cellsyap1 is a direct target of mir15a5p using the targetscan and miranda algorithms yap1 was found to have a putative target site of mir15a5p in its 'utr fig 4a to validate the possibility that yap1 is a direct target gene of mir15a5p a luciferase reporter assay was then performed the data revealed that mir15a5p mimic significantly inhibited the luciferase activity in the constructs containing the wildtype 0cinternational journal of molecular medicine figure overexpression of mir15a5p suppresses cell viability and promotes cell apoptosis siha and c33a cells were transfected with the mir15a5p mimic or inhibitor for h and then cells were used for analysis a transfection efficiency was assessed by reverse transcriptionquantitative pcr cell viability was measured by cck8 assay at indicated times for b siha and c c33a cells d the expression of cleaved caspase3 was determined by immunofluorescence assay magnification x200 e the caspase activity was detected by a commercial caspase activity kit f cell apoptosis was measured by flow cytometry data are expressed at the mean ± standard deviation n3 of one representative experiment p005 p001 vs mimic nc p005 p001 vs inhibitor nc mir microrna nc negative control od optical density pi propidium iodidebinding site of yap13'utr while it had no evident effects on the activity of yap13'utrmut by contrast mir15a5p inhibitor significantly increased luciferase activity without any evident effects on yap13'utrmut activity fig 4b subsequently to further detect the potential regulation of yap1 by mir15a5p the expression of yap1 protein was measured in cc cells by western blotting as presented in fig 4c the expression of yap1 was significantly decreased upon ectopic expression of mir15a5p suggesting that high expression of yap1 was partly due to the downregulation of mir15a5p in cc cells in addition it was identified that the mrna level of yap1 was significantly increased in cervical cancer compared with the control and inversely correlated with mir15a5p expression levels in cancer tissues fig 4d and e these results indicated that yap1 is a downstream gene of mir15a5p in cc 0cchen mir15a inhibits cervical cancer cell growthfigure overexpression of mir15a5p suppresses cell invasion and migration siha and c33a cells were transfected with the mir15a5p mimic or inhibitor for h and then cells were used for analysis invasion of a siha and b c33a cells was measured by a transwell assay magnification x200 the migration of c siha and d c33a cells was assessed by a wound healing assay the images were taken at and h after gaps were generated wound healing was quantified by the distance of the wounded region with an absence of cells data are expressed at the mean ± standard deviation n3 of one representative experiment p001 vs mimics nc p001 vs inhibitor nc mir microrna nc negative controlyap1 inhibition suppresses cell viability promotes cell apoptosis and inhibits invasion and migration previous evidence has shown that yap1 exerts an oncogenic function in several types of human cancer such as breast and lung cancer as the findings of the present study revealed that yap1 is upregulated in cc it was hypothesized that yap1 may act as an oncogenic gene in cc to confirm this hypothesis siha and c33a cells were transfected with siyap1 or siscramble western blot assay revealed that yap1 was notably downregulated following transfection with siyap1 fig 5a functionally yap1knockdown significantly suppressed the cell viability and induced cell apoptosis compared with the siscramble group fig 5b and c furthermore knockdown of yap1 significantly suppressed the invasive and migratory abilities of siha and c33a cells fig 5d and e suggesting that yap1 may play an oncogene role in the development of ccoverexpression of yap1 moderates the negative functions of mir15a5p on cell viability migration and invasion to ascertain whether yap1 is involved in the inhibitory effects of mir15a5p on cc cells the present study cotransfected pcdnayap1 andor mir15a5p mimic as well as their controls into siha and c33a cells the overexpression efficiency was verified by western blotting as shown in fig 6a yap1 was notably increased in siha and c33a cells after pcdnayap1 transfection subsequently the cell viability apoptosis invasion and migration were evaluated overexpression of yap1 significantly abolished the inhibitory effects of mir15a5p upregulation on the viability of siha and c33a cells fig 6b the increased apoptosis induced by mir15a5p overexpression was also reversed by overexpression of yap1 fig 6c furthermore overexpression of yap1 significantly reversed the inhibitory effects of mir15a5p on cell invasion and migration fig 6d and e in addition it was identified that overexpression of yap1 alone significantly promoted cc cell viability inhibited cell apoptosis and enhanced the invasion and migration compared with blank control group suggesting the oncogenic role of yap1 in cc cells these results indicate that mir15a5p exerts its tumor suppressive role in cc at least partially through yap1 0cinternational journal of molecular medicine figure yap1 is a direct target of mir15a5p a schematic of the yap1 'utr containing the mir15a5p binding sites b luciferase assay of 293t cells cotransfected with firefly luciferase constructs containing the yap1 wt or mut 'utrs and mir15a5p mimics mimics nc mir15a5p inhibitor or inhibitor nc as indicated n3 p001 c siha and c33a cells were transfected with the mir15a5p mimic and mimic nc for h and the expression levels of yap1 protein were determined by western blotting p001 vs mimic nc d yap1 expression was measured by reverse transcriptionquantitative pcr in cc tissues and matched adjacent noncancerous tissues n40 p001 e spearman's analysis was used to analyze the correlation between the expression of yap1 and the expression of mir15a5p expression in cervical cancer tissues r p001 data are expressed at the mean ± standard deviation n3 of one representative experiment yap1 yesassociated protein mir microrna 'utr 'untranslated region wt wildtype mut mutant nc negative controldiscussionin the present study mir15a5p was shown to be decreased in cc tissues and cell lines and associated with cc metastasis furthermore overexpression of mir15a5p inhibited the cc cell viability invasion and migration and promoted cell apoptosis while inhibition of mir15a5p demonstrated the opposite effects additionally yap1 was confirmed as a functional target of mir15a5p ectopic expression of which significantly reversed suppression of mir15a5p the present data indicated that mir15a5p may function as a tumor suppressor in cc progression by inhibiting yap1 expressiona number of studies have shown that mirnas participate in the development of cc for example xia reported that mir374b overexpression suppresses cell proliferative and invasive abilities via affecting forkhead box m1 expression yao also demonstrated that mir641 upregulation restricts cc cell growth in vitro and in vivo xu reported that mir2185p suppresses the progression of cc via the lynnfκb signaling pathway yuan demonstrated that overexpression of mir138 suppresses cc cell growth in vivo these findings suggest that targeting mirnas may be an effective therapeutic strategy for cc in the present study based on microarray expression data it was identified that mir15a5p is one of the most markedly downregulated mirnas in cc tissues notably previous studies have reported that mir15a5p functions as a tumor suppressor in several human cancer types although mir15a5p has been found to be downregulated in cc to the best of our knowledge the tumorigenic role and mechanism remain unknown therefore the present study focused on mir15a5p in cc for molecular analyses in the 0cchen mir15a inhibits cervical cancer cell growthfigure yap1 inhibition suppresses cell viability promotes cell apoptosis and inhibits invasion and migration siha and c33a cells were transfected with siyap1 or siscramble and then cells were harvested for further study a the expression of yap1 was measured by western blotting b cell viability was measured by cck assay c the cell apoptosis was assessed by flow cytometry d cell invasion was measured by transwell assay e cell migration assessed by a wound healing assay data are expressed at the mean ± standard deviation n3 of one representative experiment p001 vs siscramble yap1 yesassociated protein mir microrna si small interfering rnafigure mir15a5p inhibits cell viability and induces cell apoptosis by targeting yap1 a siha and c33a cells were transfected with the pcdnayap1 plasmid for h and then the protein expression of yap1 was measured by western blotting subsequently siha and c33a cells were cotransfected with the pcdnayap1 plasmid and mir15a5p mimic for h and then cells were used for analysis b viability of siha and c33a cells was measured by cck8 assay at indicated times c the cell apoptosis was assessed by flow cytometry d cell invasion was measured by transwell assay e cell migration was measured by a wound healing assay data are expressed at the mean ± standard deviation n3 of one representative experiment p005 p001 vs blank group p001 mir microrna yap1 yesassociated protein microarray expression data the expression levels of numerous mirnas exhibited significant changes such as mir137 which demonstrated the most significant upregulation in cc tissues miao reported that mir137 upregulation inhibits cc cell invasion migration and epithelialmesenchymal transition by suppressing the tgfβsmad pathway 0cinternational journal of molecular medicine notably mir15a3p has also reported to exhibit differential expression and induce apoptosis in human cc cells although the present study did not detect the expression change of mir15a3p in the microarray expression data the expression of mir15a3p in four cc cell lines was examined and the results demonstrated that mir15a3p was also downregulated in cc cells compared with ect1e6e7 cells data not shown however the role and regulatory mechanisms of mir15a3p on invasion and migration remain unclear the function of more mirnas in cc will be investigated in the futureprevious studies have reported that mir15a5p has the potential to suppress cell growth and inhibit the progression of human cancers by regulating its downstream target genes for example luo demonstrated that overexpression of mir15a5p causes cellular growth inhibition and suppression of migration by targeting cyclin e1 in breast cancer wu and guo found that mir15a overexpression suppressed the cell proliferation and invasion by suppression of bmi1 translation in gastric cancer gc as well as pancreatic cancer pc of note several studies have reported aberrant expression of mir15a5p in cc tissues or cells however the role and mechanism of mir15a5p in cc remain largely unknown the present results demonstrated that overexpression of mir15a5p inhibited cell viability cell migration and invasion and induced cell apoptosis in siha and c33a cells while inhibition of mir15a5p demonstrated the opposite effects indicating that mir15a5p may serve as tumor suppressive role in cc yap1 a transcriptional coactivator and oncogene has been found to play an important role in different types of carcinoma for example liu reported that yap1 overexpression promotes the invasion migration and growth of colon cancer cells yu demonstrated that knockdown of yap1 causes a significant inhibition of the growth and migration of renal cell carcinoma cells in vitro and in vivo notably yap1 has been verified to target mir15a5p to suppress cell growth and metastasis in gastric adenocarcinoma and colon cancer however whether yap1 is a target of mir15a5p in cc remains unclear in the present study yap1 was confirmed to be a target of mir15a5p and its protein expression levels were negatively regulated by mir15a5p further investigation indicated that yap1 was significantly increased in cc tissues and inversely correlated with mir15a5p in cc tissues furthermore yap1 was confirmed to act as an oncogene gene in cc cells and its overexpression partly abrogated the inhibitory effect induced by enhanced expression of mir15a5p in cc cells taken together the present study demonstrates that mir15a5p exerts its tumor suppressive role in cc cells by targeting yap1due to the limitation in experimental conditions and funds further research in the future is required to investigate whether mir15a5p serves its role via other downstream targets in addition the present study investigated the cellular function of mir15a5p and its underlying mechanism in cc however in vivo studies and clinical trial data are required to validate the preliminary in vitro results obtained therefore the function of mir15a5p in cc needs to be further investigated in vivoin conclusion the present results demonstrated that mir15a5p suppresses the viability migration and invasion of cc cells by directly targeting yap1 based on these findings it is proposed that the mir15a5pyap1 axis may serve as a novel biomarker for new targets in cc therapyacknowledgementsnot applicablefundingfunding was received from the scientific research project of shanghai science and technology commission grant nos and availability of data and materialsthe datasets used andor analysed during the current study are available from the corresponding author on reasonable request authors' contributionsrc hl tz xy and sx performed the experiments contributed to data analysis and wrote the paper rc hl tz xy and sx analysed the data xc conceptualized the study design and contributed to data analysis and experimental materials all authors read and approved the final manuscriptethics approval and consent to participateall individuals provided written informed consent for the use of human specimens for clinical research the experimental protocols were approved by the ethics committee of huashan hospital north of fudan university patient consent for publicationnot applicablecompeting intereststhe authors declare that they have no competing interests references alldredge jk and tewari ks clinical trials of antiangiogenesis therapy in recurrentpersistent and metastatic cervical cancer oncologist tsikouras p zervoudis s manav b tomara e iatrakis g romanidis c bothou a and galazios g cervical cancer screening diagnosis and staging j buon fang j zhang h and jin s epigenetics and cervical cancer from pathogenesis to therapy tumour biol wang j liu y wang x li j we j wang y song w and zhang z mir1266 promotes cell proliferation migration and invasion in cervical cancer by targeting dab2ip biochim biophys acta mol basis dis zhu l zhu l s | 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 |
" exosomes are extracellular vesicles containing a variety of biological molecules including micrornasmirnas we have recently demonstrated that certain mirna species are selectively and highly enriched inpancreatic cancer exosomes with mir1246 being the most abundant exosome mirnas have been shown tomediate intercellular communication in the tumor microenvironment and promote cancer progression thereforeunderstanding how exosomes selectively enrich specific mirnas to initiate exosome mirna signaling in cancercells is critical to advancing cancer exosome biologyresults the aim of this study was to identify rna binding proteins responsible for selective enrichment ofexosome mirnas in cancer cells a biotinlabeled mir1246 probe was used to capture rna binding proteins rbpsfrom panc1 cells among the rbps identified through proteomic analysis srsf1 eif3b and tia1 were highlyassociated with the mir1246 probe rna immunoprecipitation rip and electrophoretic mobility shift assay emsaconfirmed the binding of srsf1 to mir1246 lentivirus shrna knockdown of srsf1 in pancreatic cancer cellsselectively reduced exosome mirna enrichment whereas gfpsrsf1 overexpression enhanced the enrichment asanalyzed by next generation small rna sequencing and qrtpcr mirna sequence motif analysis identified acommon motif shared by of srsf1associated exosome mirnas emsa confirmed that shared motif decoysinhibit the binding of srsf1 to the mir1246 sequences we conclude that srsf1 mediates selective exosome mirna enrichment in pancreatic cancer cells bybinding to a commonly shared mirna sequence motifkeywords srsf1 exosome mirna mir1246 pancreatic cancer exosomes are endosomederived extracellular vesiclesevs that can be transferred from cancer cells tostromal cells in the tumor microenvironment [ ]these membrane vesicles are nm in size andcontain proteinsincludinglipids and nucleic acids correspondence weiqundingouhscedu1department of pathology university of oklahoma health sciences centeroklahoma city stanton l young blvd bmsb 401a oklahoma city ok usa6stephenson cancer center university of oklahoma health sciences centeroklahoma city ok usafull list of author information is available at the end of the small rnas such as micrornas mirnas[ ]exosomemediated intercellular communication betweencancer cells endothelial cells [ ] fibroblasts [ ] orimmune cells [ ] can facilitate tumor progressionfurthermore cancer exosomes are released into the circulation and contribute to premetastatic niche formation in distant ans [ ]how cancer exosomes interact with stromal cells topromote tumor progression has been extensively investisignaling eventgated one criticalin the tumormicroenvironmentisthe exosome mirnamediatedintercellular communication [ ] studies haveshown that exosome mirna signaling promotes tumor 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 0cxu cell communication and signaling page of progression in various model systems [ ] notablyit has been reported that mirnas contained in exosomes are delivered to recipient cells in the tumormicroenvironment or distant ans where they canregulate target gene expression and promote tumorangiogenesis and metastasis [ ]in the context of exosome mirna signaling we andothers have reported that certain mirna species areselectively enriched in cancer exosomes as compared toexosomes derived from normal epithelial cells [ ] results from several studies have also indicated thatselective enrichment of exosome mirnas is relevant totumor progression for example exosome sortingof mir193a was found to promote colon cancer progression likewise mir122 a cancer exosomeenriched mirna [ ] was shown to reprogram glucose metabolism in a premetastatic niche to facilitatemetastasis in a breast cancer model system moreover the exosome enriched mir1246 was reportedto promote tumor invasion in both breast cancer and oral squamous cell carcinoma it seems clearthat selective enrichment of exosome mirnas drivescancer exosome mirna signaling in the tumor microenvironment which in turn reinforces tumor invasiveness and progression however how exosome mirnasare enriched or how exosome mirna signaling is initiated in cancer cells remains largely unknown elucidating the mechanisms ofselective exosome mirnaenrichment in cancer cells may help identify new cancertherapeutic opportunities that are urgently neededrecentreports have indicated that certain rnabinding proteinsrbps are involved in exosomemirna sorting in eukaryotic cells and the type ofrbps involved seems to differ among various modelsystems [ ] suggesting that exosome mirnasorting is a tissue or celltype specific processfurthermore there have been no reports on the identification of rbps that regulate exosome mirna sorting in pancreatic cancer cells we have recentlycharacterized the biogenesis of exosome mir1246 which is the most highly enriched mirna inpancreatic cancer cellderived exosomes the aimof this study was to utilize our established cell modelsystems to identify rbps that are involved in exosomemirna loading in pancreatic cancer cells using alabeled mir1246 probe as bait we fished out several rbpsincludingserine and arginine rich splicing factor srsf1eukaryotic translation initiation factor subunit beif3b and t cellrestricted intracellular antigen tia1 we found that srsf1 a recently claimedoncoproteininregulating exosome mirna enrichment in pancreaticcancer model systemsfrom pancreatic cancer cellspredominantlyinvolved ismethodscell culturelines panc1the human pancreatic cancer celllinemiapaca2 and bxpc3 and breast cancer cellmdamb231 were obtained from the american typeculture collection atcc manassas va usa cellswere cultured following atccs instructions except thatexosomedepleted fetal bovine serum fbs and horseserum wereapplied whenever needed exosomedepleted fbs and horse serum were prepared by pelleting the serum exosomes at g for h at °ccells were routinely incubated in a humidified environment at °c and co2exosome isolationexosomes were isolated from the culture medium utilizing a combination of centrifugation ultracentrifugationand filtration as we recently described [ ] withminor modifications in brief the culture medium ofpanc1 cells was precleared by g centrifugationfor min at °c and the resulting supernatant was filtered through a μm pvdf centrifuge filter thelarge size evs were trapped in the filter and recovered inpbs the filtered supernatant was then applied to a μm pvdf centrifuge filter the medium size evswere trapped in the second filter and resuspended inpbs the small size evs exosomes in the final supernatant were recovered by ultracentrifugation g min at °c the isolated exosomes were verified bywestern blot detecting positive and negative exosomemarker proteins and nanop analysis nanosightns300 system malvern instruments uk measuringboth sizes and concentrations of the isolated exosomesfig mirna binding protein pulldownpulldown experiment was performed using the pierce¢magnetic rnaprotein pulldown kit thermo fisherscientific briefly pmol of biotinlabeled mir1246or polya rna oligonucleotides integrated dna technologies were hybridized to μl streptavidin magnetic beads prod1862766 thermo fisher scientificthe mir1246biotinstreptavidin beads were incubatedwith panc1 lysate for min at °c the lysatebeadmixture was washed three times with washing bufferfrom the abovementioned kit to elute bound proteins μl of elution buffer was applied and a magnetic separator was applied to separate the beads from the elutedprotein following the manufacturers protocol pierce¢magnetic rnaprotein pulldown kit thermo fisherscientific proteins were separated by sdspage beforemass spectrometry ms analysis 0cxu cell communication and signaling page of fig verification of the exosomes derived from panc1 cells a representative western blot analysis of cd63 nonreducing condition cd81flotillin and calnexin in the evs isolated from panc1 cells positive exosome markers are only detected in small evs exosomes b representativenanop tracking analysis of exosomes small evs derived from control and srsf1 knockdown panc1 cells three individual experimentswere performed for both a and bliquid chromatographymass spectrometry lcmsmassspectrometry ms measurementthe experiment was performed by the laboratory formolecular biology and cytometry research core facilityat ouhsc proteins were digested with trypsin according to the fasp protocol briefly the eluate was buffer exchanged in m urea the proteins were reducedwith mm dithiothreitol and then alkylated with mm iodoacetamide the peptides were eluted dried andresuspended liquid chromatography tandem mass spectrometry was performed by coupling a nanaoacquityuplc waters corp manchester uk to a qtofsynapt g2s instrument waters corp manchesteruk each protein digest about ng of peptide wasdelivered to a trap column μm mm nanoacquity uplc nanoease column μm beh c18 waterscorp manchester uk at a flow rate of μlmin in solvent a mm ammonium formate ph inhplc grade water tandem mass spectra were generated in the trapping region of the ion mobility cell byusing a collisional energy ramp from v low massstartend to v high mass startend the pusherionmobility synchronization for the hdmse method wasperformed using masslynx v41 and driftscope v24lockspray of glufibrinopeptideb mz wasacquired every s and lock mass correction was appliedpost acquisitionprotein identificationraw ms data were processed by plgs proteinlynxglobal server waters corp manchester uk for peptide and protein identification msms spectra weresearched against the uniprot human database containing reviewed sequences with the followingsearch parametersfull tryptic specificity up to twomissed cleavage sites carbamidomethylation of cysteineresidues was set as a fixed modification and nterminalprotein acetylation and methionine oxidation were set asvariable modificationssmall rna library preparation and next generationsequencingtotal rna was extracted from cell and exosome pelletsusing the trizol reagent invitrogenlife technologiescarlsbad california the small rna libraries were constructed and run on the illumina miseq platform as werecently described [ ]rna immunoprecipitation assaypanc1 cells or mdamb231 celllysates were prepared using ip buffer mm trishcl ph mmnacl mm edta mm pmsf and triton x the lysate was sonicated for min on ice andinsoluble material was removed by centrifugation supernatants were collected and protein concentrations weremeasured the supernatant was precleared by proteing dynabeads¢thermo fisher scientific and thenmixed with antibody srsf1 santa cruz sc33652eif3b santa cruz sc137214 tia1 santa cruz sc gapdh promab and igg santacruz sc2025 in a ratio of at °c overnight withgentle rotation to capture the antibodyproteinrnacomplexes μl of protein g magnetic beads wereadded and the complexes were rotated for h at °cthe sample was separated by magnetic separation trizol reagent invitrogenlife technologies was appliedto isolated rna from the complex the mirna expression was analyzed by qrtpcr 0cxu cell communication and signaling page of coimmunoprecipitation coipcoimmunoprecipitation coip using panc1 cell lysate and antibody of srsf1 santa cruz sc33652eif3b santa cruz sc137214 tia1 santa cruz sc gapdh promab and igg santacruz sc2025 was performed as described previously and the protein complex was detected by westernblotwestern blot analysiswestern blot was performed as we recently described[ ] primary antibodies raised against srsf1 santacruz sc33652 eif3b santa cruz sc137214 tia1santa cruz sc166247 betaactin a5441 and glyceraldehyde 3phosphate dehydrogenase gapdh santacruz sc47724 were used for detection nuclear andcytoplasmic protein extraction was extracted followingrockland nuclear cytoplasmic extract protocol and verified by histoneh3 cst 4499s andgapdh santa cruz sc47724 detection antibodiesused for exosome marker detection include cd63cd81 santa cruz bio technology inc ca usaflotillin1 and calnexin cell signaling technology incma usaquantitative realtime reverse transcription polymerasechain reaction qrtpcrqrtpcr was performed as we described [ ] withspecific primers cel54 ²gcgcgcccgtaatcttcataatcc3² mir1246 ²gcgcgatggatttttggagcag3² mir320c ²gcaaaagcuggguugagagggu3² and mir320d ²gcgaaaagcuggguugagagga3²srsf1 shrna expression plasmid constructiontarget specific oligonucleotides were designed using online tool rnai codex cold spring harbor laboratoryand were synthesized integrated dna technologieswith the addition of overhangs according to the cuttingsite of bamh1 and ecori the shrna expression plasmid was constructed by annealing the oligonucleotidesto psihh1 vector following the user manual of psihh1 shrna system sbi system bioscience the oligonucleotide sequences for shrna of srsf1 eif3b ortia1 are provided in supplemental table 3rd generation packaging plasmidslentivirus transductionlentiviral ps were produced as previously described using the shrna expression plasmid andthepmd2gaddgene plasmid pmdlrregp addgeneplasmid and prsvrev addgene plasmid the packaging plasmids were cotransfectedwith the lentiviral expression vector into t cellsusing the polyethyleneimine polysciences inc to produce replication deficient lentivirus after transfectionthe supernatant was pooled and filtered with a μmmembrane and concentrated by ultracentrifugation toacquire lentivirus infection was performed by usinglentivirus in the presence of μgml polybrene sigmaaldrich approximately h postinfection cells wereselected by treating with μgml puromycin invivogen san diego cagfpsrsf1 expressionthe gfpsrsf1 expression plasmid was a gift from drmassimo caputi dna transfection was performedusing lipofectamine thermo fisher scientific topanc1 cells and the expression of gfpsrsf1 wasverified by western blotgstsrsf1 protein purificationbl21 thermofisher scientific c600003 competentcells transformed with pgex6psrsf1 dna addgeneplasmid were cultured at °c for hand after od600 reached to bacteria weretreated with mm isopropyl βd1thiogalactopyranoside for h at °c gsttaggedsrsf1 was purifiedwith glutathione sepharose beads ge health carethe purity of the recombinant proteins was determinedby sdspage with coomassie blue stainingelectrophoretic mobility shift assay emsaird800 labeled mir1246 μm integrated dnatechnologies was mixed with μl of gst slurry stsrsf1 in binding buffer tris ph mm kcl mm mgcl2 mm np40 dtt mm glycerol and incubated at room temperature for minavoiding light 5x loading buffer kcl mm tris ph mm glycerol xylene cyanol bromophenol blue was then added and the complexwas separated on a native gel polyacrylamide m tris ph m glycine m edta apstemed at voltage for min the signal was detected using the licor odyssey imaging systemlicor inc usadesign of decoy motif mimicsthe decoy motif mimics were designed by permutationand combination of the identified motif sequences in thelength of nucleotides the secondary structure of thedesigned sequences was analyzed in rnafold webserveruniversityselfcomplementary were selected decoy mimics ²uuggacuaggacuaggau3² decoy mimics ²aggaaggaaggaagga3²sequences withoutof vienna 0cxu cell communication and signaling page of bioinformatics analysisthe mirna motif analysis was performed using memesuite the protein profile analysis for the result ofmass spectrometry was performed using david bioinformatics abcc at saicfrederick inc the rnabinding protein and mirna sequence binding analysiswas performed using the database of rnabindingspecificities rbpdb srsf1 expression in cancertissues was examined using oncomine thecorrelation of gene expression with cancer patient survival was extracted from the human protein atlasscilifelab sweden statisticsstatistical analyses were performed using graphpadprism software graphpad software inc la jolla causa the heatmap was made in rstudio rstudio incwith the ggplot2 package students ttest was applied to determine significant differences among controland experimental groupsresultsidentification of mir1246 associated proteinsbecause rbps are involved in exosome mirna sortingwe first sought to identify proteins that bind to mirnashighly enriched in cancer exosomes mir1246 the mosthighly enriched mirna in pancreatic cancer exosomeswas biotinlabeled and incubated with a cellular lysatefrom panc1 cells the biotinmir1246 probe wascaptured with streptavidincoated magnetic beads biotinlabeled polya mimics were used as control themirnaprotein complexes were eluted and the proteinswere analyzed by liquid chromatographymass spectrometry in triplicate table there were total of proteins specifically pulled down by the mir1246 probeinterestingly about half of the proteins that associatewith mir1246 are vesicleassociated proteins supplement fig 1a based on the intensity of detection rnabinding property and cancer relevance we ranked therbps using the database for annotation visualizationand integrated discovery david this resulted in tencandidate rbps that complex with the mir1246 sequenceexosomestable among them srsf1 also called sfrs1 waspredictedsequencethe mir1246and arerelevantto eukaryotictobindtotable over view of the result of mass spectrometryexperiments conditionpoly a panc1number of proteins detectedpoly a mdamb231mir1246 panc1mir1246 mdamb231table mir1246 rna binding protein candidates obtainedfrom the mass spectrometric analysisprotein full nameprotein symbolsrsf1serineargininerich splicing factor park7eif3bthoc4acocddx5tia1if5a1eif2aimdh2parkinson disease protein eukaryotic translation initiation factor subunit btho complex subunit alyref export factorcytoplasmic aconitate hydrataseprobable atpdependent rna helicase ddx5tcellrestricted intracellular antigen1eukaryotic translation initiation factor 5a1eukaryotic translation initiation factor 2ainosine5²monophosphate dehydrogenase supplement fig by in silico analysis using the database of rnabinding specificities rbpdb levelsverification of srsf1 binding to mir1246rna immunoprecipitation rip was performed to verifythe association of several identified rbps with mir1246including srsf1 eif3b and tia1 igg and gapdhantibody was used as controls for immunoprecipitationas shown in fig 2a mir1246 expression is more than12fold higher in the srsf1precipitants as compared tothat of igg precipitants indicating a specific associationof srsf1 with mir1246 mir1246 expression wasmoderately increased in the tia1precipitants and nearigg controlin the eif3b precipitants coimmunoprecipitation coip experiments were performed to verify the immunoprecipitation proceduresdata not shown to directly determine the binding ofsrsf1 to the mir1246 sequence glutathionestransferase gst conjugated human srsf1 protein wasexpressed in bl21 competent e coli captured by glutathione sepharose beads and eluted by glutathione thepurity of eluted gstsrsf1 protein was shown by sdspage and coomassie blue staining supplement fig the binding of gstsrsf1 to a fluorescenttaggedmir1246 probe was determined by rna emsa asshown in fig 2b binding of the labeled probe was specific to gstsrsf1 but not gst and increased withgreater protein input the specific binding of gstsrsf1 to the mir1246 probe was evident as the unlabeled mir1246 probe effectively competed with thelabeled mir1246 probe in a concentrationdependentmanner fig 2c the detected bands were semi quantified and the kd was calculated from the detected signalsfig 2d these data confirmed the direct binding ofsrsf1 to the mir1246 sequence 0cxu cell communication and signaling page of fig srsf1 binds to mir1246 a qrtpcr detection of mir1246 in igg gapdh srsf1 eif3b and tia1 immunoprecipitants of panc1 lysaten p student ttest bc emsa detection of the srsf1mir1246 complex hot probe ird800 labeled mir1246 mimics cold probemir1246 mimics n direct binding of gstsrsf1 and mir1246 b and concentrationdependent competition between the cold and hotmir1246 probe for binding to gstsrsf1 c d semiquantification of srsf1 and mir1246 binding in c and calculated dissociationconstant n exosome mirna enrichment by srsf1 in cancer cellsbecause srsf1 is a key splicing factor that is essential toeukaryotic cells a knockout model could not beestablished thereforeto determine whether srsf1mirna binding activity is relevant to exosome mirnaenrichment we established a lentivirus srsf1 shrnaconstruct to knockdown srsf1 expression in panc1cells fig 3a interestingly though srsf1 protein wasdetected both in the nucleus and cytoplasm the knockdown was more pronounced in the cytoplasm fig 3bknockdown of srsf1 did not significantly alter the concentration and size distribution of the exosomes releasedby panc1 cells fig 1b cellular and exosome rnafrom control and srsf1shrna cells were isolated andsmall rna sequencing was performed among the highly enriched panc1 exosome mirnas expressionof mirnas was significantly downregulatedin exosomes derived from srsf1shrna panc1 cellsas compared to exosomes derived from control panc1cells fig 3c strongly indicating the involvement ofsrsf1 in exosome mirna enrichment a heatmapshowing the expression of the top mirnas enrichedin panc1 exosomes demonstrates the dramatic dropin expression levels of mirnasin srsf1shrnapanc1 exosomes compared to panc1 exosomesfig 3d notably mir1246 was the highest enrichedexosome mirna data not shown and its expressionin exosomes wassignificantly reduced by srsf1knockdown fig 3d on the other hand among of the mirnas less enriched in exosomes only were expressed at lower levels in exosomesderived from srsf1 knockdown cells as compared toexosomes derived from wild type panc1 cells fig3c suggesting that srsf1 knockdown mainly affectsexosome enriched mirnasto further confirm the effect of srsf1 knockdown onexosome mirna enrichment the expression levels ofseveral representative mirnas were quantified by qrtpcr srsf1 knockdown in panc1 cells significantlyreduced exosome levels of mir1246 mir320c andmir320d confirming the small rna sequencing resultsfig 3e in contrast knockdown of eif3b or tia1 didnot reduce exosome mir1246 expression suggestingthat these rbps may not promote exosome mirna 0cxu cell communication and signaling page of fig see legend on next page 0cxu cell communication and signaling page of see figure on previous pagefig cellular and exosome mirna profiles after srsf1 knockdown in panc1 cells a detection of srsf1 knockdown by shrnas in panc1 cellsb panc1 srsf1 protein levels in nuclear and cytoplasmic fractions nc normal control c venn diagram of overlap of mirnas detected by nextgeneration small rna sequencing in srsf1 knockdown and control panc1 cells and exosomes d heatmap showing the expression of top exosome mirnas in cells and exosomes after srsf1 knockdown e qrtpcr analysis of mir1246 mir320c and mir320d in exosomes derivedfrom control and srsf1 knockdown panc1 cells p students ttest shown are representatives of three independent experiments aeenrichment supplement fig and our observationswere extended to two additional pancreatic cancer celllines miapaca2 and bxpc3 fig 4af in additionexpression of let7c which is less enriched in exosomeswas unchanged in exosomes after srsf1 knockdowndata not shownto verify the involvement of srsf1 in exosomemirna enrichment in cancer cells we also exogenouslyoverexpressed srsf1 in panc1 cells a gfpsrsf1 expression plasmid was introduced into panc1 cells andsrsf1 overexpression was confirmed by western blotfig 5a expression of mir1246 mir320c and mir320d in the exosomes derived from gfpsrsf1 panc1cells was analyzed by qrtpcr fig 5bc as shown infig 5b overexpression of gfpsrsf1 increased exosome expression of mir1246 and rescued mir1246levels in exosomes derived from srsf1shrna cellslevels of mir320c and mir320d were also increased inexosomes derived from the gfpsrsf1 cellsfurthersupporting the involvement of srsf1 in exosomemirna enrichment fig 5cdidentification of rna sequence motifs involved inexosome mirna enrichmentaccording to the rbpdb srsf1 binds specifically to amotif present in the mir1246 sequence supplementfig qrtpcr analysis of mir1246 mir320c mir320d expression in exosomes derived from srsf1 knockdown bxpc3 and miapaca2 cells ac qrtpcr detection of mir1246 mir320c and mir320d in exosomes derived from srsf1 knockdown bxpc3 cells n p student ttest df qrtpcr detection of mir1246 mir320c and mir320d in exosomes derived from srsf1 knockdown miapaca2 cells n p students ttest 0cxu cell communication and signaling page of fig qrtpcr analysis of exosome enriched mirnas derived from srsf1 overexpression panc1 cells a confirmation of gfpsrsf1overexpression in panc1 cells b qrtpcr detection of mir1246 in exosomes derived from wild type and srsf1 knockdown panc1 cells withgfpsrsf1 overexpression c qrtpcr detection of mir320c in exosomes derived from gfpsrsf1 overexpression panc1 cells d qrtpcrdetection of mir320d in exosomes derived from gfpsrsf1 overexpression panc1 cells p students ttest n for bdfig to understand the contribution of specific rnamotifs involved in exosome mirna enrichment we applied an unbiased approach to identify the rna motifsthat contribute to exosome mirna enrichment for thispurpose we analyzed the rna sequences of the mirnas highly enriched in cancer exosomes and regulatedby srsf1 using the bioinformatics tool meme suite a 6bp length motif was found to be shared in of the exosome enriched mirnasincluding mir fig ac to test whether the binding of srsf1to mir1246 depends on this motif two decoy mimicswere designed according to the shared motif sequencesand their secondary structure determined with thernafold webserver httprnatbiunivieacatcgibinrnawebsuiternafoldcgi the binding of the decoymimics to srsf1 protein was determined by rnaemsa analysis addition of decoy motif did not alterthe binding of srsf1 to the mir1246 probe fig 6dwhereas decoy motif competed with mir1246 binding to srsf1 in a concentrationdependent manner fig6df indicating that srsf1 directly interacts with thissequence motifdiscussionthe role of exosome mirna signaling in promotingcancer progression has been intensely investigated andwell recognized in recent years [ ] the higher enrichment of certain mirnas in cancer exosomes [] indicates that exosome mirna encapsulation isan active cellular process that initiates exosome mirnasignaling in the tumor microenvironment however thespecificselectivecellular processresponsiblefor 0cxu cell communication and signaling page of fig see legend on next page 0cxu cell communication and signaling page of see figure on previous pagefig srsf1associated exosome mirna sequence motif analysis a the motif commonly shared among srsf1associated exosome mirnas bvenn diagram showing the number of srsf1associated exosome mirnas that share the motif c list of mirnas sharing the common motif demsa analysis demonstrating the inhibition of gstsrsf1 binding to mir1246 by rna decoys d1 decoy ²uuggacuaggacuaggau3² d2decoy ²aggaaggaaggaagga3² e concentrationdependent inhibition of gstsrsf1 binding to mir1246 by d2 f semiquantification ofthe detected bands in fig 5e and the calculated dissociation constantexosome mirna enrichment has not been well established in eukaryotic cells the most significant findingfrom the present study is that we have identified srsf1as a mediator of exosome mirna enrichment in pancreatic cancer cells a specific mirna sequence motif wasalso identified that may be involved in the exosomemirna enrichment process these findings provide newinsight into how mirnas are enriched in cancer cellexosomesexosomemediated mirnasignalinginitiatetowe recently reported that exosome mir1246themost highly enriched mirna in pancreatic cancer cellderived exosomes is derived from rnu2 a smallnuclear rna important for mrna splicing alongthis line of our research we sought to determine howthis mirna is enriched in cancer exosomes using ourestablished model systems in the present study we haveprovided severallines of evidence demonstrating thatsrsf1 a vital splicing factor and established oncoprotein is significantly involved in exosome mirnaenrichment in pancreatic cancer cells the first line ofevidence indicating srsf1 involvementin exosomemirna enrichment was obtained from the biotinlabeled mir1246 pulldown experimentfollowed byproteomic analysis among the rbps identified severalwere selected based on their detection intensity relevance to extracellular vesicles and reported connectionsto human cancer including srsf1 eif3b andtia1 of note srsf1 was the only rbp among themthat was also predicted by the rbpdb to bind to a motifin the mir1246 sequence furthermore the direct binding of srsf1 to the mir1246 sequence was verified byrip and rna emsa analysis strongly indicating thephysical interaction of srsf1 and not eif3b or tia1with the mir1246 sequence the most convincing evidence demonstrating the involvement of srsf1 in cancer exosome mirna enrichment was the observationthat knockdown of srsf1 significantly reduces exosomemirna enrichmentfor a majority of the selectivelyenriched exosome mirnas without altering the expression levels of less enriched exosome mirnas these results were based on small rna sequencing andconfirmed by rtpcr analysis the observations werealso extended to additional human pancreatic cancer celllines including miapaca2 and bxpc3srsf1 was initially identified as a splicing factor ineukaryotic cells but srsf1 was later revealed toindependent ofshuttle between the nucleus and cytoplasm to regulate rna metabolism mirna procession and othercellular eventsthe mrna splicingprocess importantly srsf1 is overexpressed indifferent cancer types and is considered a potent oncogene [ ] moreover srsf1 over expression in different types of cancer is associated with worse prognosissupplement fig while the full spectrum of srsf1function remains to be determined our results revealthat srsf1 binds to specific mirnas and is significantlyinvolved in exosome mirna enrichment in cancer cellsthis function is likely independent ofthe splicingprocess as the reduced expression of the detected exosome mirnas after srsf1 knockdown is greater thantheir expression change in the cells because exosomemirna signaling contributes to tumor developmentthrough intercellular communication in the tumormicroenvironmentthe involvement ofsrsf1 in exosome mirna signaling initiation likelyrepresents a part of its oncogenic action which maylead to new therapeutic strategies to intervene withexosome mirna signaling in cancer several rbpshave been previously identified as mediators of exosome mirna sorting in various model systemsincluding major vault protein in colon cancer cells hnrnpa2b1 in t cells and ybx1 in hek293tcells the identification of srsf1 involvement inexosome mirna enrichmentin pancreatic cancercells further supports the notion that the cellular exosome mirna sorting process in eukaryotic cells maydiffer among different cell types[ ]we have also identified a mirna motif commonlyshared by the srsf1associated exosome mirnasusing the meme suite program memesuitethis motif was specifically bound by srsf1 as evidenced by our rna emsa analysis a similar motifalbeit slightly shorter was identified in our recentreport that describes exosome mir1246 enrichmentin pancreatic cancer cells our results reinforcethe concept that specific mirn | Colon_Cancer |
cancer is the second leading cause of death in the united states cancer screenings candetect precancerous cells and allow for earlier diagnosis and treatment our purpose was tobetter understand risk factors for cancer screenings and assess the effect of cancer screenings on changes of cardiovascular health cvh measures before and after cancer screenings among patientsmethodswe used the guideline advantage tgaamerican heart association ambulatory qualityclinical data registry of electronic health record data n patients to investigateassociations between timeseries cvh measures and receipt of breast cervical and coloncancer screenings long shortterm memory lstm neural networks was employed to predict receipt of cancer screenings we also compared the distributions of cvh factorsbetween patients who received cancer screenings and those who did not finally we examined and quantified changes in cvh measures among the screened and nonscreenedgroupsresultsmodel performance was evaluated by the area under the receiver operator curve aurocthe average auroc of curves was for breast for cervical and for coloncancer screening distribution comparison found that screened patients had a higher prevalence of poor cvh categories cvh submetrics were improved for patients after cancerscreeningsa1111111111a1111111111a1111111111a1111111111a1111111111open accesscitation guo a drake bf khan ym langabeer iijr foraker re timeseries cardiovascularrisk factors and receipt of screening for breastcervical and colon cancer the guidelineadvantage one e0236836 101371 pone0236836editor antonio palazo´nbru universidad miguelhernandez de elche spainreceived 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 pone0236836copyright guo 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 are ownedby a third party and the authors do not havepermission to share the data requesting access tothe guideline advantage tga data must be done one 101371 pone0236836 august one 0ccardiovascular risk factors and receipt of cancer screeningsby contacting the american heart association viaemail qualityresearchheart the python coderelated to the analyses can be found in githubrepository githubcomaixiaguopythoncodefunding the authors received no specificfunding for this workcompeting interests the authors have declaredthat no competing interests existdeep learning algorithm could be used to investigate the associations between timeseriescvh measures and cancer screenings in an ambulatory population patients with moreadverse cvh profiles tend to be screened for cancers and cancer screening may alsoprompt favorable changes in cvh cancer screenings may increase patient cvh healththus potentially decreasing burden of disease and costs for the health system eg cardiovascular diseases and cancersintroductioncancer is the second leading cause of death for both men and women in the united statesus breast cancer is the second leading cause of cancer death among women colorectal cancer ranks second among men and third among women while cervical cancer ranksas a major cause of cancer death among women regular cancer screenings for breast cervical and colorectal cancers can help to diagnose cancers early and reduce cancer deaths for example in the past years the number of deaths caused by cervical cancer has significantly decreased thanks to pap tests which can find abnormal cervical cells before they turn tocancer similarly colonoscopy removes noncancerous colon polyps before becomingmalignant and regular mammography screening can identify breast cancer in an earliermore treatable stage thus breast cancer screening bcs cervical cancer screening cecsand colorectal cancer screening cocs are very important for early detection and treatmentfactors associated with cancer screenings include demographic factors health insurancecoverage education level smoking status obesity and cholesterol testing for example receiptof mammography is associated with modifiable factors such as weight smoking and other lifestyle factors [] receipt of cecs is associated with healthier weight lower cardiovascular disease occurrence and lower cholesterol some studies suggest that smokingsedentary lifestyle high body mass index and high comorbidity are associated with a higherpercentage of cocs participation [] traditionally data for such studies originate fromquestionnaires claims data and telephone surveys and statistical analysis methods such aslogistic regression models are applied to examine the associations between the risk factors andcancer screenings electronic health records ehr contain longitudinal healthcare information and data including diagnoses medications procedures lab tests and images andtherefore can be used to discover new patterns and relationships from the rich data deeplearning algorithms have been widely and successfully used in bioinformatics and healthcarefields as they can effectively capture features and patterns in longitudinal data in this study we investigated associations between longitudinal cvh risk factors and thereceipt of cancer screenings using ehr data by the long shortterm memory lstm model we then studied the distribution of cvh factors between patients who did and did notreceive cancer screenings to further investigate the associations finally we compared measures of cvh longitudinally within those who did and did not receive screening to betterunderstand the effect of cancer screenings on cvh measuresmaterials and methodsethics statementall the data were fully anonymized before we accessed them our study was approved by theinstitutional review board at the washington university school of medicine in st louis we one 101371 pone0236836 august one 0ccardiovascular risk factors and receipt of cancer screeningsobtained a written acknowledgement of proprietary rights and nondisclosure and data useagreement from the american heart association the washington university_nda_dua_contractid 158065_20190426_kdata source and study populationthe guideline advantage tga is a clinical data registry established in by the americancancer society the american diabetes association and the american heart associationaha ehr data has been collected from over clinics across the us by the tga totrack and monitor disease management and outpatient preventative care we used longitudinal tga data to predict three types of cancer screenings among unique patientswe used a 6year range to identify female patients in the year oldage group who received bcs female patients in the year old age group who receivedcecs and patients in the year old age group who received cocs if patientsreceived multiple types of cancer screening we only considered the first using the same criteria for gender and age we randomly selected a comparison group of patients who did notreceive cancer screenings for bcs for cecs and for cocswe utilized the following cvh measures defined by the aha smoking status body massindex bmi blood pressure bp hemoglobin a1c a1c and cholesterol lowdensitylipoprotein ldl in our dataset we then classified them into three categories ideal intermediate or poor according to table we utilized the multum drug database as a template to convert the drug names in our dataset to their corresponding drug classes thelevenshtein distance algorithm was employed for the conversion by comparing the drugnames in our dataset to the multum drug database template the conversion was consideredsuccessful and medications were considered as treatments for bp a1c or ldl table if thedistance between the two compared strings was less than five all cvh measurements prior tothe date of cancer screening were considered in the analysis for those who received screeningand all cvh measurements in the data set were considered in the analysis for those who didnot receive screeningfor the primary analysis we selected patients who had at least one measure of cvh for bcs for cecs and for cocs in the comparison groups there were availabledata for bcs cecs and cocsstatistical analysiswe first studied the lstm prediction of cancer screening from timeseries cvh factors wedivided each cvh factor into its submetric of ideal intermediate or poor according totable for example if a patient had a measure of ideal blood pressure then that featuretable measures of cvh which are available in the tga adapted from lloydjones poor healthintermediate healthideal healthhealth behaviorssmoking statusyesformer � monthsbody mass index� kgm2 kgm2health factorsnever or quit months kgm2ldl� mgdl mgdl or treated to goal mgdlblood pressurefasting plasmaglucosesystolic � mm hg or diastolic � mmhgsystolic mm hg or diastolic mm hg or treated togoalsystolic mm hgdiastolic mm hg� mgdl mgdl or treated to goal mgdl101371 pone0236836t001 one 101371 pone0236836 august one 0ccardiovascular risk factors and receipt of cancer screeningswas called blood pressure ideal all features were then embedded to a 32dimensional vectorspace by word2vec for each type of cancer screenings the python genism word2vecmodel used the following hyperparameters size embedding dimension was window themaximum distance between a target word and all words around it was min_count theminimum number of words counted when training the model was sg the training algorithm was cbow the continuous bag of words time information for each measure wasadded and was calculated by the difference in days between each visit date and the most recentvisit date thus each feature was associated with its own time point in the unit of daysthe resulting embedded vectors and associated time points were fed to the lstm modeldue to the comparison group being much larger than the number of patients with cancerscreening we randomly selected patients for bcs patients for cecs and patientsfor cocs and repeated this process for times to account for the imbalance betweenscreened and unscreened groups each time the data set for each type of cancer screening wassplit into a training data set and a test data set we trained the lstm model onthe training data and tested the trained model on the test data we utilized the average of thearea under the receiver operator curve auroc to evaluate the performance of our lstmmodel for each type of cancer evaluatedour lstm model comprised an input layer one hidden layer with dimensions andan output layer the hyperparameter used in the model was as follows a sigmoid function wasused as the activation function in the output layer a binary crossentropy was used as the lossfunction adam optimizer was used to optimize the model with a minibatch size of sampleswe then investigated whether distributions of cvhcounts and percentages for each submetricdiffered between patients who did and who did not receive cancer screenings by chisquared test finally we studied changes in cvh factors within screening group for the samepatients who received screening and for those who did not within screening group we compared cvh measures from before and on the day of the screening to the cvh measures collected after the screening for the patients who did not receive screening we compared cvhmeasures before and after the midpoint of the visit dates if patients only had a single visitthen they were not included in the before and after analysis analyses were conducted by usingthe libraries of scikitlearn scipy matplotlib with python version in resultsthe majority of our study population was white with a mean of age of approximately yearsfor bcs years for cecs and years for cocs table the nonwhite study populationwas predominantly africanamerican the average number of measures avg amongpatients who received screening was higher than that of patients who were not screened forexample the average number of bp measurements for patients with bcs was for cecsand for cocs compared to for bcs for cecs and for cocs for patients who werenot screenedfig displays the performance of lstm cancer screening predictions in terms of repeated aurocs for each type of screening the average auroc of curves was forbcs for cecs and for cocstable lists the numbers and proportions of patients in ideal intermediate and poor categories for each submetric for the comparison between patients who received cancer screeningand those who did not we applied a chisquared test to check if the frequencies herepercentages between screening groups were significantly different from one other within eachcvh submetric as shown in table patients who received cancer screening had a higher one 101371 pone0236836 august one 0ctable characteristics [mean sd or n ] of the study population by receipt of cancer screeningcardiovascular risk factors and receipt of cancer screeningscancer screeningsbcsdemographicsage mean std yearwhite race n nonwhite race n unknown race n cvh factors mean std avg measuresa1cldl mgdlbmi kgm2systolic blood pressure sbp mmhgdiastolic blood pressure dbp mmhgcurrent smoking n cecsdemographicsage mean std yearwhite race n nonwhite race n unknown race n cvh factors mean std avg measuresa1cldl mgdlbmi kgm2systolic blood pressure sbp mmhgdiastolic blood pressure dbp mmhgcurrent smoking n cocsdemographicsage mean std yearwhite race n nonwhite race n unknown race n cvh factors mean std avg measuresa1cldl mgdlbmi kgm2systolic blood pressure sbp mmhgdiastolic blood pressure dbp mmhgcurrent smoking n n n n yesnon � n n avg avg avg avg avg avg � the percentages may not add up to due to rounding101371 pone0236836t002prevalence of poor a1c for bcs for cecs and for cocs compared topatients who did not receive screening for bcs for cecs and for cocsfig shows changes in cvh submetrics within the same patient screening groups fig a2c show the changes in cvh submetrics for the patients who were screened while fig2e and 2f show the changes in cvh for patients who were not screened one 101371 pone0236836 august one 0ccardiovascular risk factors and receipt of cancer screenings one 101371 pone0236836 august one 0ccardiovascular risk factors and receipt of cancer screeningsfig the area under the curve auc are shown for lstm cancer screening predictions from timeseries cvh factors which were repeated times withdifferent comparison patients for bcs a cecs b and cocs c101371 pone0236836g001from the first column of fig we can see that the prevalence of poor submetricsdecreased after cancer screenings for example all five submetrics improved after bcs fig a while bp and a1c improved after cecs fig 2b and bp a1c and smoking improvedafter cocs fig 2c notably for the prevalence of poor a1c decreased for all patients whoreceived cancer screenings in bcs in cecs and in cocs on the other handfrom the second column of fig we can see that the prevalence of poor a1c increased forall comparison patientsdiscussionin this study we demonstrated associations between timeseries cvh risk factor measuresand receipt of three types of cancer screenings ie breast cervical and colon cancer screenings by using a nationally representative datasettga data the tga data enabled us toexamine multiple sites cvh submetrics and types of cancer screenings using advanced deeplearning models an advantage of our study was that all cvh submetrics were investigatedsimultaneously for an association with different cancer screenings on a unique nationallyrepresentative dataset of patients ie the large tga data set which contains longitudinaltable comparison cvh factors between patients with cancer screening or without [n ]patients with bcs n chisquared pvalueidealintermediatepoorpatients without bcs n idealintermediatepoorpatients with cecs n chisquared pvalueidealintermediatepoorpatients without cecs n idealintermediatepoorpatients with cocs n chisquared pvalueidealintermediatepoorpatients without cocs n idealintermediatepoorbmi bmi bmi 101371 pone0236836t003bp bp bp a1c a1c a1c ldl ldl ldl smoking smoking smoking one 101371 pone0236836 august one 0ccardiovascular risk factors and receipt of cancer screenings one 101371 pone0236836 august one 0ccardiovascular risk factors and receipt of cancer screeningsfig the plots of percentages for poor cvh factors for the same patients before and after time points of cancer screening for patients with screeningsac and before and after middle time points for patients without cancer screenings df the first row is for bcs second row is for cecs andthe third is for cocs101371 pone0236836g002cvh measurements and cancer screening patterns from more than different clinics in theusthe comparison of different cvh measure distributions between patients who receivedcancer screenings and those who did not showed that patients with poorer cvh especiallypoor a1c were more likely to receive cancer screenings specifically patients with poorera1c were more likely to receive cancer screenings some recent studies have showed that individuals with diabetes had higher incidence of certain cancers and also were more likely tobe diagnosed with advancedstage tumors [] thus providers might be more likely torecommend patients with diabetes to uptake cancer screenings for early prevention of developing cancers which may lead to more individuals with diabetes to participate in cancerscreeningsmoreover we investigated the effects of cancer screenings on the changes of cvh measuresof the patients to better understand if the screenings had potential associations with theimprovement of cvh measures our results indicated that patients who received cancerscreenings appeared to have better control of cvh factors especially a1c than patients whodid not receive cancer screenings specifically a1c levels were improved after patientsreceived any type of screening while a1c levels worsened among patients who did not receivecancer screening a similar trend could be observed for bmi it became better after patientsreceived any type of screening while bmi became worse among patients without bcs orcocs levels of bp were improved after patients received bcs or cocs screenings and worsened among patients without bcs or cocs poor levels of ldl decreased among patientsafter receipt of bcs and among those without bcs however ldl improvements were muchgreater among patients after receipt of bcs decrease in ldl than those without bcs decrease in ldl after receipt of bcs and cocs current smoking declined comparedto the increase observed among those without the screeningsin summary our analyses showed that patients with poor cvh measures were more likelyto receive cancer screenings patients with receipt of cancer screenings appeared to haveimproved cvh measures after the screening as compared to before one possible reason forthis was that patients might receive more attention and through care from providers to detectand manage cvh by virtue of reviewing cancer screening and other risk factor data at thepopulation level better cvh is associated with a lower risk of cardiovascular disease cvdand cancers thus cancer screenings may indirectly decrease burden and cost on thehealth system eg cvd and cancers by improving patient cvh healthlimitationsthere were some limitations in our analyses we used values of auroc to evaluate associations between timeseries cvh measurements and receipt of cancer screenings higherauroc values indicated stronger associations between predictors and the binary outcomes however our observed auroc values were relatively low and thus have limited clinicalutility at this time cancer screenings are potentially affected by cvh and other factors weacknowledge that we had relatively few patients with receipt of cancer screening specificallythere were relatively few patients who received cancer screenings compared to patients whodid not within the same age and gender groups this limitation likely affected the accuracy of one 101371 pone0236836 august one 0ccardiovascular risk factors and receipt of cancer screeningsour prediction models the prediction accuracy of our models could be improved if morepatients in our data set had received cancer screeningswe demonstrated that deep learning lstm models can effectively predict the associationsbetween timeseries cvh measures and receipt of cancer screening poor cvh especiallypoor a1c may prompt providers to recommend cancer screening for their patients andpatients who received cancer screening may also receive better care for andor have improvedselfmanagement of cvh especially a1c overall these findings suggest that unhealthierpatients are screened for cancers and that cancer screening may also prompt favorablechanges in cvhauthor contributionsconceptualization randi e forakerformal analysis aixia guosupervision randi e forakerwriting original draft aixia guowriting review editing bettina f drake yosef m khan james r langabeer ii randi eforakerwwwmedicalnewstodaycoms282929phpreferences humphrey ll helfand m chan bks woolf sh breast cancer screening a summary of the evidencefor the us preventive services task force annals of internal medicine 107326000348191375_part_120020903000012 american cancer society cancer facts figures am cancer soc 10 pmid wwwcdcgovcancercervicalstatisticsindexhtmwwwcdcgovcancerdcpcpreventionscreeninghtmwwwcdcgovcancercervicalstatisticsindexhtm1 edwards qt li ax pike mc kolonel ln ursin g henderson be ethnic differences in the use ofregular mammography the multiethnic cohort breast cancer res treat 101007s1054900800497 pmid bynum jpw braunstein jb sharkey p haddad k wu aw the influence of health status age andrace on screening mammography in elderly women arch intern med 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demographic and psychosocial factors associated with perceived risk forcolorectal cancer cancer epidemiology biomarkers and prevention robb ka miles a wardle j perceived risk of colorectal cancer sources of risk judgments cancer epidemiol biomarkers prev 10115810559965epi060151 pmid gimeno garca az factors influencing colorectal cancer screening participation gastroenterologyresearch and practice 1011552012483417 pmid jensen pb jensen lj brunak s mining electronic health records towards better research applications and clinical care nature reviews genetics 101038nrg3208 pmid goodfellow i bengio y courville a deep learning mit press 101533 rajkomar a oren e chen k dai am hajaj n hardt m scalable and accurate deep learning withelectronic health records npj digit med 101038s4174601800291 pmid hochreiter s s long shortterm memory neural comput bufalino v bauman ma shubrook jh evolution of the guideline advantage lessons learnedfrom the front lines of outpatient performance measurement ca cancer j clin 103322caac21233 pmid wwwscrippssparkleassetsdocumentsheart_rhythm_factspdf shickel b tighe pj bihorac a rashidi p deep ehr a survey of recent advances in deep learningtechniques for electronic health record ehr analysis ieee j biomed heal informatics 101109jbhi20172767063 pmid levenshtein vi binary codes capable of correcting deletions insertions and reversals sov phys dokl citeulikeid311174lloydjones dm hong y labarthe d mozaffarian d appel lj van horn l defining and settingnational goals for cardiovascular health promotion and disease reduction circulation 101161circulationaha109192703 pmid mikolov t corrado g chen k dean j word2vec proc int conf learn represent iclr kingma dp ba j adam a method for stochastic optimization corr 2015abs14126 pearson k on the criterion that a given system of deviations from the probable in the case of a correlated system of variables is such that it can be reasonably supposed to have arisen from random sampling philos mag tsilidis kk 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cardiovascularhealth and incident cardiovascular disease and cancer the womens health initiative am j prev med 101016jamepre201507039 pmid yin j using the roc curve to measure association and evaluate prediction accuracy for a binary outcome biometrics biostat int j 1015406bbij20170500134 one 101371 pone0236836 august one 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 |
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 |
overexpression of epithelial cell adhesion molecule epcam has been associated with chemotherapeutic resistanceleads to aggressive tumor behavior and results in an adverse clinical outcome the molecular mechanism by whichepcam enrichment is linked to therapeutic resistance via nrf2 a key regulator of antioxidant genes is unknown wehave investigated the link between epcam and the nrf2 pathway in light of therapeutic resistance using head andneck squamous cell carcinoma hnscc patient tumor samples and cell lines we report that epcam was highlyexpressed in nrf2positive and hpvnegative hnscc cells in addition cisplatinresistant tumor cells consisted of ahigher proportion of epcamhigh cells compared to the cisplatin sensitive counterpart epcamhigh populationsexhibited resistance to cisplatin a higher efï¬ciency in colony formation sphere growth and invasion capacity anddemonstrated reduced reactive oxygen species ros activity furthermore nrf2 expression was significantly higher inepcamhigh populations mechanistically expression of nrf2 and its target genes were most prominently observed inepcamhigh populations silencing of epcam expression resulted in the attenuation of expressions of nrf2 and sod1concomitant with a reduction of sox2 expression on the other hand silencing of nrf2 expression rendered epcamhighpopulations sensitive to cisplatin treatment accompanied by the inhibition of colony formation sphere formation andinvasion efï¬ciency and increased ros activity the molecular mechanistic link between epcam expression andactivation of nrf2 was found to be a concerted interaction of interleukin6 il6 and p62 silencing of p62 expressionin epcamhigh populations resulted in the attenuation of nrf2 pathway activation suggesting that nrf2 pathwayactivation promoted resistance to cisplatin in epcamhigh populations we propose that therapeutic targeting the nrf2epcam axis might be an excellent approach to modulate stress resistance and thereby survival of hnscc patientsenriched in epcamhigh populationscorrespondence syed s islam drsyedsislamgmailcom1department of biochemistry and molecular biology university of chittagongchittagong bangladesh2department of pathology mcgill university montreal qc canadafull list of author information is available at the end of the edited by b zhivotovskyintroductionhead and neck squamous cell carcinoma hnsccaffects more than patients per year12 resistanceto chemotherapeutic drugs limits the overall treatment 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 0coutcome in hnscc patients3 response to chemotherapeutic drugs is partly mediated by the keap1nrf2 signaling system4 nrf2nfe2l2 nuclear factor erythroid2like is a key transcription factor which in the normalbasal state functions as cytoprotective response to oxidative and electrophilic stress under oxidative stressstate nrf2 dissociates from cytoplasmic inhibitor keap1kelch like echassociated protein translocate into thenucleus and activates nrf2 transcriptional genes andprotects cells against oxidative stress mediates detoxiï¬cation and participates in atpdependent drug efï¬ux5abnormalities of keap1nrf2 pathwaylead to amechanism of oncogenesis and chemo and radioresistance in a variety of cancers including hnscc4inhibition of nrf2 expression by sirna augmentedcarboplatininduced tumor growth inhibition in a xenograft mouse model6 recent studies have indicated thatkeap1nrf2 pathway is engaged in sustaining csc cancerstem celllike properties in cancers and causes resistanceto therapeutic agentscscs exhibit enhanced selfrenewal properties lead todisease recurrence and most importantly exhibit thestrongest therapeutic resistance within the tumor cellspopulation7 elevated expression of nrf2 target genescontribute to therapeutic resistance and facilitate survivalof cscs10 several cell surface markers such as cd44cd133 cd24 cd49f and aldh have been proposed forthe detection and isolation of cscs from tumors1112many studies also emphasized the potential use of epithelial cell adhesion molecule epcam as a marker ofcscs due to its ubiquitous overexpression in tumors13epcam was originally identiï¬ed as a novel tumorspeciï¬ccell surface antigen and overexpressed in a large numberof cancers14 and involved in cell migration proliferation and differentiation18 due to its wide expressionepcam may be a potential target for molecular intervention for therapeutically resistant tumors and requiresfurther investigationa recentstudy reported that nrf2 knockdowninhibits the selfrenewal capacity of glioma stemcells19 furthermore nrf2 signaling is activated inspheroids in breast and colon cancer cells where highnrf2 activity in spheroids has correlated with therapeutic resistance20 however it is unknown how thenrf2 pathway and epcam interact and play roles inthe development of chemotherapeutic resistance inview of the importance of epcam and nrf2 signalingin the development of chemoresistance and the limited understanding of the link between epcam andthe nrf2 pathway we investigated the potential role ofnrf2 signaling in cscs with special emphasis onepcamenriched cells that leads to chemotherapeuticresistanceofï¬cial of the cell death differentiation associationresultscancer stem cell markers are upregulated in hpvnegativeand nrf2 overexpressing hnscc tumorsgiven the role of nrf2 signaling in chemotherapeuticresistance and csc survival2122 we ï¬rst explored theexpression of several prominent csc markers in hnsccusing cases from tcga dataset we used normalizedmrna zscores and compared several csc markers withinnrf2high and nrf2low tumors statistically significantdifferences were obtained for all csc markers comparingthe nrf2high and nrf2low groups with the most significant relationship found in epcam p fig 1asince hpv human papillomavirus has emerged as anovel risk factor for hnsccs23 we therefore comparednrf2 expression in hpvpositive and hpvnegative patientgroups from our own archived tumor samples no significant differences were noted between the hpv groupsand nrf2 expression p fig 1b a significantexpression difference was noted in epcam cd49f andstemness factor sox2 p p p fig 1cin hpvnegative versus hpvpositive groups in additioncd44 p cd49fp epcam p and sox2 p showed significantly higherexpressions in the nrf2high group fig 1d csc markersincluding sox2 were significantly increased in the tumortissue compared with matched normal tissues fig 1eepcam is expressed in cisplatin resistant cells and epcaminhibition sensitizes cells to cisplatin and inhibits hnscccell proliferationtumors cisplatin resistantnext we evaluated epcam transcript levels from agroup of cisplatin resistant n and sensitive n hnscc patienttumors showed relatively high expression of epcamcompared with cisplatin sensitive tumors fig2a this ï¬nding led us to test the cisplatin resistancein vitro for which we established a line of cisplatinresistant fadu cells termed as fadures we established acisplatinresistant fadures cells by maintaining parentalfadu cells in a series of cisplatin concentrations for weeks before these cells were stably grown in μmcisplatin as shown in supplementary fig s1a fadurescells exhibited higher resistance to cisplatin treatmentcompared to parental fadu cells we then treated faducells μm of cisplatin for days and analyzed the epcamexpression by western blot fadu cells maintained in μm of cisplatin showed higher epcam expression incontrast to untreated parental cells supplementary figs1b to assess the role of epcam in cisplatin resistanceuntreated patient tumor cells scc15 and fadu cellswere transfected with siepcam and siscramble for hwashed and followed by cisplatin treatment for an additional h and assessed for cell viability supplementary 0cnoman et al cell death and disease page of fig cancer stem cell markers cscs are upregulated in hpvnegativenrf2 overexpressing hnscc tumors a cd44 cd133 epcam andcd49f were compared between nrf2high and low groups using the tcga dataset ratios were calculated by dividing the mrna expression of thenrf2high group by that of the nrf2low group b nrf2 expression was compared between hpv and hpv group using our own dataset n ratio was calculated by dividing the expression intensity of the hpv group by that of the hpv group c cscs expression comparedbetween hpv and hpv group ratio was calculated by dividing the expression intensity of the hpv group by that of the hpv group dexpression of cscs was compared between nrf2high and nrf2low group ratios were calculated by dividing the expression intensity of the nrf2high group by that of the nrf2low group e cancer stem markers were compared between hnscc and matched normal tissues ratio was calculatedby dividing the mrna expression of the tumor sample by that of the matched normal samples in all cases whiskers indicate the maximum and theminimum values pvalues were calculated using students t testfig s2 whereas parental cells were found to be somewhat resistantto cisplatin treatment knockdown ofepcam with siepcam enhanced the sensitivity to cisplatin treatment fig 2b to examine the resistancefurther cells were treated with different concentrations ofcisplatin and determined the ec50 fig 2c furthermoresilencing epcam significantly reduced epcam transcriptlevel and inhibited cell proliferation fig 2d echemotherapeutic resistance is associated with increasednrf2 transcriptional activity and epcam overexpressionit was reported that inhibition of nrf2 reverses theresistance to cisplatin of hnscc cells22 to further assessthe role of nrf2 in chemotherapeutic resistance wecompared nrf2 target genes in cisplatin sensitive n and resistant n hnscc patients tumor cells by realtime quantitative polymerase chain reaction qrtpcrthe unsupervised heat map analysis showed that nrf2target genes sod2 slc3a1 akrc1 gclc ho1nqo1 and sod1 were highly upregulated in thecisplatinresistant tumor cells compared to the cisplatinsensitive tumor counterparts fig 3a suggesting thatcisplatin treatment potentially plays a significant role innrf2 pathway activation to establish the link betweennrf2 and epcam in resistance freshly isolated cisplatinresistant n and sensitive n patient tumor cellswere subjected to ï¬ow cytometry analysis and quantiï¬edthe epcam expression approximately epcampositive cell population was found in cisplatin resistanttumors while onlycella epcampositiveofï¬cial of the cell death differentiation association 0cnoman et al cell death and disease page of fig epcam is expressed in cisplatin resistant cells and epcam inhibition sensitizes hnscc cells to cisplatin and inhibits hnscc cellproliferation a expression of epcam mrna in hnscc patients cisplatin resistant and sensitive tumor cells b doseresponse and cell viability ofhnscc patient tumor cells top panel scc15 middle panel and fadu bottom panel cells cell viability of siepcam and scrambled sirnatransfected cells were monitored following exposures of cells to different concentrations of cisplatin c ec50 of cisplatin in parental siscrambled andsiepcam transfected patient tumor cells top panel scc15 middle panel and fadu bottom panel cells the ec50 differences between siscrambleand siepcam cells were compared d relative epcam mrna expression in hnscc patient tumor cells top panel scc15 middle panel and fadubottom panel cells following transfection of cells by siscrambled and siepcam pvalues were calculated using students t test e cell proliferationwas determined following transfection of cells by siscrambled and siepcam siscrambled and siepcam cell growth was compared on day nsdenote not significant p p p population was detectable in cisplatin sensitive tumorsfig 3b based on these epcam cell fractions in resistantand sensitive groups we hereafter termed these twopopulations epcamhigh and epcamlow immunostainingfor epcam in cisplatin resistance n tissues showedenhanced expression of epcam fig 3c fluorescenceactivated cell sorting facs sorted epcamhigh cellofï¬cial of the cell death differentiation associationfraction was highly resistant to cisplatin compared to theepcamlow cell fraction fig 3d etumorsresistantfunctionallyto cisplatin showedenhanced expression of epcam coupled with theincreased level of sox2 and abcg5 fig 3f indicatingenrichment of epcam coincident with stemlike anddrug resistant features in cells we then analyzed nrf2 0cnoman et al cell death and disease page of fig chemotherapeutic resistance is associated with increased nrf2 transcriptional activity and epcam overexpression a heat map ofhierarchically clustering based on the expression of nrf2 pathway target genes reveals distinct expressions in cisplatin resistant and sensitive patienttumor cells significantly upregulated gene expression intensity marked as red and downregulated genes are marked as blue b hnscc patient tumorcells were isolated from treatment resistant and sensitive patients and epcam positive cells identiï¬ed by ï¬ow cytometry c immunoï¬uorescenceimages of epcam expression were captured from the cultured cisplatin resistant and sensitive hnscc patient tumor cells scale bar µm d cellviability determination in parental epcamhigh and epcamlow cells after treating the cells with different cisplatin concentrations e apoptotic celldetermination in epcamhigh and epcamlow cells after cisplatin µm treatment f epcam sox2 and abcg5 protein levels were determined fromcisplatin resistant and sensitive patient tumor cells by western blotting g transcript levels of epcam and nrf2 in hnscc patient tumor cells assessedby qrtpcr values represents ±sd for three independent experiments hi transcript levels of epcam and nrf2 in scc15 and fadu cells assessed byqrtpcr values represents ±sd for three independent experiments j nrf2 and sod1 protein expression in cisplatintreated scc15 and fadu cellsand epcam transcript levels by qrtpcr and found thatboth transcripts were increased in cisplatinresistanttumor cells fig 3g suggesting that resistance to cisplatin is due in part to an increased level of nrf2 transcriptional activity and epcam overexpression toconï¬rm this ï¬nding in cell lines scc15 and fadu cellswere treated with cisplatin μm for days and wereassessed for the level of nrf2 and epcam transcriptscisplatin treatment significantly increased the nrf2 andepcam expression levels fig 3hj immunoblot analysis conï¬rmed that both nrf2 and sod1 expression werehigher in cisplatin treated cells fig 3jnrf2 pathway is predominantly activated in epcamhighcells and epcam knockdown inactivates the nrf2arepathwaytumorto explore if the nrf2 pathway is exclusively activatedin epcamhigh cells freshly isolated cisplatinresistant andsensitive patientcells were facs sortedepcamhigh cells were predominantly detected in thecisplatinresistant cell fraction compared to sensitive cellsfig 4a cells were treated either with cisplatin or vehiclefor days and analyzed by ï¬ow cytometry the resultscorroborated the results obtained in patient tumor cellsfig 4a next we cultured cisplatin treated fadu cells inofï¬cial of the cell death differentiation association 0cnoman et al cell death and disease page of fig nrf2 pathway is predominantly activated in epcamhigh cells and epcam knockdown inactivates the nrf2are pathway a epcamhighand epcamlow cells were determined in hnscc patient tumor cells scc15 and fadu cells using ï¬ow cytometry b total cellular protein levels ofepcam nrf2 and sod1 were determined in epcamhigh and epcamlow cells by western blot analysis c nrf2 transcript levels in epcamhigh andepcamlow cells p compared to epcamlow group d sod1 nqo1 and akrc1 transcript levels in epcamhigh and epcamlowcells p compared to epcamlow group e fadu cells were transfected with siepcam and scrambled sirna and epcam nrf2 sod1 and sox2 transcript levelswere determined by qrtpcr analysis in epcamhigh cells f g protein levels of epcam nrf2 sod1 and sox2 were determined by western blotanalysis in fadu and scc15 hnscc cells after silencing epcam in epcamhigh cells h a luciferase assay was used to detect reporter gene activity fromares i immunostaining of hnscc cells stained with epcam green nrf2 red and dapi blue after epcam knockdown scale bar μmgrowth supplemented csc medium for days facssorted for epcamhigh and epcamlow cells were recultured in csc medium for an additional daysepcamhigh cells overexpressed epcam nrf2 and sod1proteins and nrf2 transcripts fig 4b c in additionepcamhigh cells overexpressed sod1 nqo1 andakrc1 transcripts fig 4d these results indicated thatthe nrf2 signaling pathway is exclusively activated in theepcamhigh cells to determine whether the elevated nrf2in epcamhigh cells is epcam dependent welevelsilenced epcam expression by siepcam and observedthat epcam nrf2 sod1 and sox2 proteins and transcripts were attenuated in epcamhigh cells fig 4egthese observations prompted us to hypothesize thatepcam might regulate the expression of antioxidantfactors via the nrf2are antioxidant response elementsofï¬cial of the cell death differentiation association 0cnoman et al cell death and disease page of fig nrf2 inhibition in epcamhigh cells sensitizes cells to cisplatin treatment coupled with the abrogation of production of reactiveoxygen species ros a epcamhigh and epcamlow cells were determined in hnscc patient tumor cisplatinresistant and untreated tumor cellsusing ï¬ow cytometry legend 1untreated and 2cisplatin resistant patient tumor cells b protein levels of nrf2 and sod1 were measured insinrf2 silenced and siscrambled epcamhigh cells by western blot c epcam protein was measured in sinrf2 silenced and siscramble cells by westernblot d sox2 and abcg5 protein levels were determined in sinrf2 silenced and siscrambled epcamhigh cells by western blot e cell viability wasanalyzed after incubation of cisplatin for h in sinrf2 and siscrambled epcamhigh cells e f ros activity was measured from e patient tumor cellsand f fadu cells facs sorted epcamhigh and epcamlow cells after cisplatin or sinrf2rna treatment values represent ±sd from triplicate sampledwells p compared with untreated groupspathway this hypothesis was tested by transfection offadu and scc15 cells with an are luciferase reporterhnscc cells with or without epcam knockdown weretransiently transfected with an are luciferase reporterplasmid at h post transfection the cells were assayedfor luciferase activity epcam knockdown decreased theluciferase reporter activity with a comparable decreasedstaining intensity in epcam and nrf2 fig 4h i theseresults suggest that the inhibitory effects of epcamknockdown on cell growth and cisplatin resistance correlates with the degree of nrf2 activation in csclikeepcamhigh cellsnrf2 inhibition in epcamhigh cells sensitizes cells tocisplatin treatment coupled with the abrogation ofproduction of reactive oxygen speciesan increasing number of reports suggest that cisplatinmediated csc enrichment and resulting resistance substantially limits the positive outcome of the disease24furthermore in a group of hnscc patient tumors highexpression of epcam has been reported to correlate withtherapeutic resistance2526 to explore the possible functionallink between chemotherapeutic resistance andepcam we ï¬rst sorted epcamhigh cells by ï¬ow cytometry from cisplatin and vehicletreated fadu cells andfound that higher percentage of epcamhigh cells vs fig 5a in cisplatin treated cells knockdown ofnrf2 in epcamhigh cells attenuated the expression ofnrf2 and nrf2 target gene sod1 proteins fig 5b concomitant with the attenuation in expression of epcamsox2 and abcg5 fig 5c d additionally nrf2 silencingin epcamhigh cells showed a significant increased sensitivity to cisplatin treatment fig 5evarious antioxidant enzymes are induced by nrf2pathway activation that reduce the intracellular ros levelresulting in cells becoming drug resistant27 hence wespeculated that chemotherapeutic resistance might likelybe due to the reduction of ros in epcamhigh cells toaddress this possibility we used two approaches to analyze mitochondrial ros generation first we sortedepcamhigh and epcamlow cells from treatment naivepatient tumor cells and fadu cells treated cells withofï¬cial of the cell death differentiation association 0cnoman et al cell death and disease page of cisplatin and measured the mitochondrial ros using aï¬uorescent indicator ros activity was decreased at and μm cisplatin concentrations in epcamhigh cellswhile increased in epcamlowcells fig 5f suggesting thattherapeutic resistance was partly caused by reducing rossecondly we knocked down nrf2 by sinrf2rna incisplatintreated fadu cells and measured the ros levelros levels steadily increased in both epcamhigh andepcamlow cells fig 5gnrf2 inhibition eliminates colonyforming capacity spheregrowth and invasion capacity in epcamhigh cellswe hypothesize that cells overexpressing epcam mayacquire higher colony forming capacity increased spheregrowth and invasion capacity to test this fadu andscc15 cells were grown in growth factor supplementedcsc medium for days to allow epcam enrichmentfacs sorted and quantiï¬ed for the percent of epcamhighand epcamlow cells sorted cells were evaluated for thedegree of colonyforming capacity sphere formation andinvasiveness epcamhigh populations are highly efï¬cientin forming colonies sphere growth and invasive capacitycompared to epcamlow cells fig 6ac knockdown ofnrf2 in epcamhigh cells demonstrated reduced colonyformation sphere growth and invasive capacity as compared to scramble sirna treated cells fig 6dfinterleukin6 and p62 are involved in the activation of thenrf2 pathway and resistance to cell death in epcamhighcellsaccumulating evidence indicates that both interleukin il6 and the nrf2mediated antioxidant pathwaycontribute to chemotherapeutic resistance in oral squamous cell carcinoma2829 to conï¬rm the role of il6 inthe activation of nrf2 in epcamhigh cells we assessed il mrna transcripts from a group of hnscc patienttumors treated either with chemotherapy cisplatin n doxorubicin n or chemoradiotherapy crt n or tumors obtained after debulking surgery n without treatment il6 mrna was increased in thechemotherapy and chemoradiotherapy tumors comparedwith matched adjacent normal and surgery alone tumorfig 7a to determine the effects ofil6 on theexpression of nrf2 fadu cells were treated with eithercisplatin μm or il6 pgml alone or in a combination of cisplatin and il6 and assessed for nrf2expression by immunoï¬uorescence labeling a detectableincrease in nrf2 expression in the cytoplasm and nucleuswas observed in the cisplatintreated cells fig 7baddition of il6 significantly increased the cytoplasmicand nuclear nrf2 expression fig 7b western blot analysis showed that il6 treatment activated expression ofnrf2 in cisplatin treated cells fig 7c no changes inkeap1 mrna and protein expression levels wereofï¬cial of the cell death differentiation associationobserved fig 7d next we determined whether il6plays role in preventing or reducing ros activity undercisplatin and il6 treatment conditions we found thattreatment with il6 alone reduces ros generation whilecells treated with cisplatin and il6 in combination further reduces the level of ros fig 7e tocilizumab is ahumanized antihuman il6 receptor monoclonal antibody which has been shown to controls resistance toradiation by suppressing oxidative stress via nrf2 pathway28 cisplatintreated cells undergoing il6 and tocilizumab ngml treatment were analyzed by westernblot for the expressions of sod1 and nrf2 il6 alonetreatment enhanced sod1 expression via the nrf2 pathway while tocilizumab inhibited the expression fig 7fin addition il6 treatment significantly reduced the rosproduction while tocilizumab inhibited fig 7g suggesting that il6 is likely involved in the activation of nrf2and plays a role in therapeutic resistance by reducing rosactivityto analyze the possible involvement of p62 in nrf2activation in the chemotherapeutic resistant epcamhighcells p62 protein was analyzed by western blotting p62protein in the epcamhigh cells was increased concomitant with an increase in microtubuleassociatedprotein 1a1b light chainii lc3b fig 7h it appearslikely that the increase in p62 is directly related toepcam expression fig 7h knockdown of epcamdiminished p62 expression suggesting a correlationbetween epcam and p62 fig 7i accordingly epcamsilencing in fadu cells depleted the growth of spheresfig 7i silencing of p62 by p62sirna revealed theinhibition of nrf2 p62 and sod1 fig 7j furthermorep62 knockdown also diminished the efï¬ciency of spheregrowth fig 7j interestingly keap1 expression levelincreased following p62mediated silencing fig 7j theexpression of epcam remained unchanged after p62mediated silencing suggesting epcammediated p62upregulation in these cells fig 7j k the expression levelof lc3b was also reduced during p62mediated silencingfig 7j k silencing of p62 further caused the reductionin expression of nrf2 target genes sod2 ho1 andakrc1 fig 7l all together these results suggested thatnrf2 activation in epcamhigh csclike cells were associated with the increased levels of il6 and p62 inhnscc cellsdiscussionin this study we have shown the role of the nrf2pathway activation because the cellular response to electrophilic agents is partially mediated by this pathway andlikely plays a significant role in therapeutic resistancethrough activation of nrf2 enrichment of cscs andlowering of ros activity we report that increased nrf2activity is associated with the enrichment of cscs and 0cnoman et al cell death and disease page of fig nrf2 inhibition eliminates colony forming capacity sphere growth and invasion capacity in epcamhigh cells a colonyforming assaywas carried out and quantiï¬ed from sorted epcamhigh and epcamlow cell populations b sphereforming efï¬ciency was determined and quantiï¬edusing sphere formation assay from epcamhigh and epcamlow cell populations c invasive potential of epcamhigh and epcamlow cells wasdetermined and quantiï¬ed by transwell invasion assay pvalues were calculated using students t test p p p dfnumbers of soft agar colonies formed d sphere formation e and invasion capacity f were quantiï¬ed in sinrf2rna and siscramble epcamhighcells scale bar μm values represent mean ± sd from three independent experiments p p p pvalues werecalculated using students t testdemonstrated a previously unknown link betweenepcam and the nrf2 pathway a leading cause of chemotherapeutic resistancerecent studies have highlighted the association betweenthe nrf2 pathway and cscs for examplein neuralstemprogenitor cells nrf2 overexpression modulatedofï¬cial of the cell death differentiation association 0cnoman et al cell death and disease page of fig il6 interleukin6 and p62 are involved in the activation of the nrf2 pathway and cell death resistance in epcamhigh cells a realtime pcr analysis of il6 expression in hnscc tumor tissues from matched adjacent normal n untreated surgery only n cisplatintreated n doxorubicin n and chemoradiotherapy n treated tumor tissues transcripts levels were normalized to betaactin b theimmunoï¬uorescence images of cytoplasmic and nuclear nrf2 in fadu cells after 5day cisplatin treatment with or without pgml il6 legend cytoplasmic and 2nuclear nrf2 scale bar μm c nrf2 protein levels after 5day posttreatment with cisplatin or combination of cisplatin and pgml of il6 analyzed by western blotting d keap1 mrna and protein expression in fadu cells 5day posttreatment with cisplatin alone orwith pgml of il6 e ros level was determined in fadu and scc15 cells after treating cells with cisplatin il6 and combination of il6 andcisplatin f after h cisplatin treatment with vehicle or pgml il6 or combination of il6 and ngml tocilizumab cell lysates were subjectedto western blotting and the levels of sod1 and nrf2 proteins were determined g after h cisplatin treatment with vehicle or pgml il6 or il6with ngml tocilizumab the ros production was analyzed h p62 and lc3b were measured in epcamlow and epcamhigh fadu cells by westernblotting i p62 protein was determined in epcamlow and epcamhigh fadu cells following scrambled sirna and epcamsirna transfectionquantiï¬cation and representative images of spheres formed by siscrambled and epcamsirna transfected epcamhigh cells are presented scale bar μm values represent three separate experiments p compared with siscramble group j epcamhigh cells were transfected withscrambled or p62sirna and protein levels of nrf2 p62 keap1 sod1 epcam and lc3b were assessed quantiï¬cation analysis and representativeimages of spheres formed by siscrambled and p62sirna transfected epcamhigh cells are presented scale bar μm values represent threeseparate experiments p compared with sicontrol group k l transcript levels of epcam lc3b sod1 ho1 and akrc1 were determined insiscrambled and p62sirna transfected cells by qrtpcr values represent mean ± sd from three separate experiments p p valuescompared with sicontrol groupneurosphere formation efï¬ciency as well as neural differentiation30 in addition nrf2 knockdown in primaryhuman glioblastoma cells decreased the selfrenewalcapacity of glioma stem cells31 as additional evidencecscschemotherapies and are considered alternative causes ofconventionalare highlyresistanttotumor relapse and aggressiveness cd44 cd133 cd24and aldh activity are frequently used for the detectionand isolation of cscs from tumor tissues and manycancer cell lines epcam has evolved as a potential cscmarker due to its involvement in cell signaling migrationmetastasis and therapeutic resistance the link betweenofï¬cial of the cell death differentiation association 0cnoman et al cell death and disease page of in the clinicalepcam and acquisition of csclike properties is supported by epcam inhibition activation of wntbetacatenin signaling enriched the epcam cell populationwhereas rna interferencebased blockage of epcamattenuated csc activities in cancer cells32 these reportshighlight a critical role for epcam in the development ofcsclike featuressetting epcamexpression is associated with an unfavorable prognosis inbreast cancer33 furthermore low ros levels are correlated with the maintenance of a subpopulation of drugresistant cscs within tumors34 since the mechanisticinsights into the functions of epcam have only beenrecently explored the relationship between epcam andan association with regulation of the nrf2 pathway hasnever been described moreover thus far no studies haveexplored the association between the nrf2 pathway andepcam expression in the context of csclike featuresand drug resistance as a molecular mechanism of differential antioxidant capacity and stress resistance ofcscs we identiï¬ed a direct association between epcamand nrf2 signaling with respect to drug resistance andenrichment of csclike features in hnscc cellsseveral noteworthy ï¬ndings have emerged from ourstudy first epcam was highly expressed in hpvnegative tumors nrf2positive tumors were highly enriched in a epcam cell population and epcam was highlyexpressed in hnscc tumors compared to normalcounterparts these observations suggest a direct association between epcam and the nrf2 pathway in concordance we found that nrf2 and its target genes weresignificantly upregulated in the cisplatinresistant hnscctumors compared to cisplatin sensitive tumors thefunctional implication is that nrf2 activation led to theinduction of stemness and drug resistance features byoverexpressing sox2 and abcg5 proteins in epcamhighcell population while knockdown of epcam by sirnaattenuated the expression of nrf2 sod1 and sox2secon | Colon_Cancer |
"tea is the second most popular beverage consumed in theworld next to water green tea is a kind of nonfermentedtea produced from the plant camellia sinensis it is favoredby people for its fresh ï¬avor and health beneï¬ts and consumed worldwide especially in east asian countriesgreen tea contains caï¬eine and polyphenolic compoundsknown as catechins catechins are the primary bioactivesubstances and present significant biological propertiestea leaves drycatechins constitute up to ofweight among that egcg is the main and the most abundant catechin [ ] egcg has been traditionally regardedas beneï¬cial mainly ascribed to its antioxidant action the antioxidant eï¬ects of egcg are manifested in scavenging free radicals in the body and inhibiting the formation ofros the results of earlier studies suggested that egcgcould decrease the risk of several human disorders associatedwith oxidative stress on the other hand egcg also displays significantprooxidant eï¬ects usually under highdose conditions theprooxidant actions of egcg play a dual role being both beneï¬cial and harmful while achieving desired outcomes inchronic disease prevention and treatment reports about thetoxicity of egcg are also emerging a growing body ofevidence continues to demonstrate a variety of harmfuleï¬ects from excessive consumption of green tea or oraladministration of highdose egcg supplement highdoses of egcg not only cause cytotoxicity in vitro but alsoresult in living body hepatotoxicity nephrotoxicity andgastrointestinal disorders vomiting and diarrhea the oral bioavailability of egcg is not so profound inhealthy humans as it was only of the total ingestion most of the ingested egcg is absorbed in the smallintestine and substantially degraded in the large intestine bymicrobiota action the eï¬ective dosage of egcg mightbe close to or higher than the toxic dosage in practical applications considering its low bioavailability therefore it is 0coxidative medicine and cellular longevitynecessary to understand the potential toxicity doses andusage of egcg in this review the prooxidant eï¬ects ofegcg in health beneï¬ts and adverse eï¬ects were discussedespecially concerning their underlying mechanisms involvedand doses used this review is aimed at harnessing the prooxidant eï¬ects of egcg for human health maintenance whileavoiding toxicity thereby better guiding the safety consumption of green tea and egcg chemical structure andautooxidation of egcgbasic catechins contain two or more aromatic ringshydroxyl group on carbon three position andor the higherdegree of hydroxylation of the b ring would be primarilyresponsible for the potent antioxidant activities of catechinsfigure 1a previous structureactivity relationshipstudies of catechins have demonstrated the importance ofthe number and location of the phenolic hydroxyl groupson antioxidative capacity egcg has the remarkablepotential to scavenge radicals and chelate metal ion theseabilities could be ascribed to the presence of dihydroxyand trihydroxy groups in a ring b ring and d ringfigure 1b the catechol structure of egcg makes it susceptible todegradation via autooxidation figure under normal°physiological conditions ph c egcg is autooxidized and converted to oquinone through nonenzymaticaldehydrogenation of phenolic hydroxyl groups at b ring when the cell culture medium is exposed in the airegcg could be oxidized by oxygen and yields superoxide andanion radicals o2
egcg are essential intermediate products in egcg autoox and oxygen could function as oxidants for furidation o2ther oxidation of egcg ï¬nally resulting in the formationof oquinone accompanying the generation of hydrogen could also form substantial amountsperoxide h2o2 o2of h2o2 via disproportionation reaction one egcgmolecule could produce more than two h2o2 molecules inphosphate buï¬er at neutral ph and egcg radicals
egcg o2autooxidation of egcg generates substantial ros theros comprises singlet oxygen hydroxyl radicals superoxideperoxides and h2o2 h2o2 is in a dominant position andusually is regarded as a toxic agent when the ros levelexceeds cellular antioxidant capacity oxidative stress willoccur in other words this is the result of an imbalancebetween prooxidant and antioxidant eï¬ects inclusion ofantioxidant defense enzymes such as catalase cat andsuperoxide dismutase sod could minimize h2o2 levelwhich is essential to maintain the redox balancethe concentration of egcg in the cell environmentseems to be a primary factor in explaining its prooxidanteï¬ects for example egcg treatment alone diminisheddna strand breakage of human blood lymphocytes at lowconcentrations μm while it induced dna strandbreakage at higher concentration μm thusegcg acts as an eï¬ective antioxidant at low doses withinthe range of high nanomolar and low micromolar levelswhile egcg represents a prooxidant at high doses howeverthis blurred boundary might vary depending on the type ofradical stimulants cellular environment and duration ofexposure to egcg health benefitsuntil now egcg has been a major research subject withinthe ï¬eld of healthpromoting eï¬ects the potential role ofthe prooxidant eï¬ects of egcg in cancer and obesity prevention and treatment as well as the antibacterial actionsachieved demonstrable results in previous studies prooxidant eï¬ects and anticancer activity of egcgcancer is one of the most common and lifethreateningdiseases occurring among humankind egcg as a naturalproduct has drawn a great deal of attention from boththe scientiï¬c community and the general public indeedegcg has shown both prophylactic and therapeutic eï¬cacy in multiple human cancers several mechanisms havebeen proposed to accountfor the inhibitory action ofegcg against cancer formation and growth the prooxidant eï¬ects of egcg were thought to be potential mechanisms for anticancer action the anticancer mechanismsvaried depending on the cell type dose andor time oftreatment table []apoptosis is the bestdescribed form of programmed celldeath the induction of apoptosis represents a universal andideal therapeutic strategy for cancer control cell apoptosiscould be triggered by either the intrinsic mitochondrial pathway or the external death receptor pathway the mitochondrial pathway could be induced by intracellularstresses such as oxidative stressthe apoptosistriggering eï¬ects of ros have beennoted in vitro table egcg inhibited cell growth ina dosedependent manner and the decrease in the numberof viable cells was mainly due to apoptosis caused by theegcginduced intracellular ros as early as the last century scientists found that egcg induced h2o2 formationin human lung cancer celllines h661 and 21bes andexogenously added cat completely prevented egcginduced cell apoptosis which suggested that h2o2 isinvolved in the apoptosis process provoked by egcg similar actions were also found in various cancersand tumor cells table thioredoxin trx and thioredoxin reductase trxr are pivotal regulators of cellularredox homeostasis decreased trxtrxr activity mightcontribute to the increased ros level high concentrationof egcg inactivated trxtrxr via the formation of egcgtrx1 and egcgtrxr conjugates which was linked to theelevation of ros level in hela cells and further promotedcancer cell death moreover one of the biochemical hallmarks of apoptosis is genomic dna fragmentation chen performed the dna fragmentation assay in theskov3 cells indicating that egcg induced apoptosis bycausing dna damage this result was consistent withother studies in ovarian and cervical cancer cells [ ]in terms of molecular mechanisms intrinsic apoptosisleads to the release of mitochondrial cytochrome c afterbeing released into the cytoplasm cytochrome c stimulates 0coxidative medicine and cellular longevityohbohohohhoohbohohohohdohocoaohobhoocaohafigure a basic structure of catechins b chemical structure of egcgohbegcgohohautooxidationph75 °cohboautooxidation·egcgoho2h2o2ohboooquinonefigure superoxidemediated chain reaction the formation of oquinoneapoptosome formation followed by activation of caspasecascades egcgmediated mitochondrial ros couldpromote cytochrome c release to the cytosol the antiproliferative action of egcg on prostate cancer and breast cancer is mediated through apoptosis as evident from caspase9[ ] the cells susceptible to egcginduced apoptosisalso showed activation of caspase3 moreover theincreased ros level was observed to result in the stimulationof mitogenactivated protein kinase mapk themapk signaling pathwayincluding extracellular signalregulated kinase erk jun nterminal kinase jnks andp38 plays a vital contribution in cell proliferation diï¬erentiation apoptosis and stress response egcg induced oxidative stress via generation of ros and thereafter activatedthe jnk pathway leading to changes in mitochondrial membrane potential and release of cytochrome c in ht29 humancolon adenocarcinoma cells and mia paca2 pancreaticcancer cells [ ] together these results suggest thategcginduced apoptosis is mediated through ros generation and might subsequently activate the cell intrinsic pathway in the presence of transition metals such as copper andiron h2o2 could convert to a potent oxidant hydroxyl radical via the fenton reaction nakagawa found that egcg μm produced h2o2 and triggered fenton reactionto form highly toxic hydroxyl radicals which resulted in lymphoblastic leukemia jurkat cell death in the presence offeiii and cuii egcg μm induced dna damagein hl60 cells as 8oxo78dihydro2²deoxyguanosine oxodg content increased which was a characteristic ofoxidative dna damage nevertheless no significantincrease in 8oxodg was observed in h2o2resistant colonhp100 cells suggesting that h2o2 was involved in cellulardna damage egcg could inhibit cell proliferation andinduce apoptosis through cellular dna breakage in diï¬erentcancer cell lines such dna breakage involved the mobilization of endogenous copper ions and the generation ofros moreover the observation of site speciï¬city of dnadamage by egcg is valuable cuiimediated dna damageby egcg occurred most frequently at t and g residues egcg was able to mobilize endogenous copper ions andgenerate hydroxyl radicals in situ hydroxyl radicalsserved as the proximal dna cleaving agentleading todna breakage in the nuclei this result was possibly due tothe interaction of egcg with chromatinbound copper ionsand then the nondiï¬usible hydroxyl radicals were formed atthe binding site hence hydroxyl radical generated nearbydna was well established as the cause of strand scissionbecause the concentration of copper is significantly very highin various malignancies egcg could induce cancer celldeath through the metal iondependent pathway thispathway was independent of mitochondriamediated programmed cell deaths such action involved in metal ionmediated dna cleavage would be an important mechanismof anticancer properties of egcgin addition to being transported into the cell egcgcould also function on the cell membrane fraction to regulatethe surface growth factor receptor earlier studies foundthat autooxidation of egcg led to epidermal growth factorreceptor egfr inactivation in human esophageal cancer 0ccell linesbladder cancernbtiibreast cancermcf7mcf7cervical cancerhelahelacolon canceroxidative medicine and cellular longevitytable role of prooxidant eï¬ects in the anticancer activity of egcg based on cell culture studiesegcgconcentrationtimebiological eï¬ectsreferences μm hinduced early apoptosis through dna damage μgml hinduced cell growth inhibition and apoptosis by downregulating survivinexpression via suppressing the akt pathway and activating caspase9 μm hinduced apoptosis at low doses via activation of jnk caspase9 and caspase3while inducing necrosis at high doses which is related to diï¬erences in rosgeneration and atp levels μm μm and h hincreased cell death through dna damageinduced cell death through generation of ros and inactivation of trxtrxrhct116 μm hinduced apoptosis through induction of ros and epigenetic modulation ofapoptosisrelated gene expressionht29 μm hendometrial carcinomaishikawa μm hinduced apoptotic cell death via activating the jnk pathway accompanyingmitochondrial transmembrane potential transition and cytochrome c releaseic50 was μminduced apoptosis via ros generation and p38 map kinase activationic50 was μmesophageal cancerkyse lung cancer μm hinactivated egfr by superoxide generated from autooxidation of egcg μm μm h h h μm hdisplayed strong growth inhibitory eï¬ects against lung tumor cell linesinhibited cell growth through induction of ros ic50 was μmic50 was μminduced apoptosis via h2o2 production and hydroxyl radical formationinduced apoptosis by modifying the redox systemh661 and h1299 μmh1299lymphoblastic leukemiajurkatmyelomaim9 rpmi8226and u266oral cancerscc25 andscc9ovarian cancer μm hreduced cell viability by inducing mitochondrialocalized ros and decreasingsirt3 expressionskov3 μgml dpancreatic cancerpanc1 μm hmia paca2 μm hinhibited cell proliferation and induced apoptosis by inhibiting cell cycle arrest andinducing dna damageinduced apoptosis through generation of ros as well as caspase3 andcaspase9 activationinduced stress signals by damaging mitochondria and rosmediatedjnk activationprimary eï¬usionlymphomabcbl1 and bcprostate cancer μgml hinduced apoptosis and autophagy through ros generationpc3 and μm hreduced cell survival and increased apoptosis caused a significant alteration incaspase9 alternative splicing 0coxidative medicine and cellular longevitycell line kyse one possible explanation is thath2o2 produced from egcg autooxidation in the cell culturemedium could attack and inactivate egfr leading to theinhibition of egfr phosphorylationand preferentialit is worth considering whether high amounts of egcgcould cause damage to normal cells egcgmediated rosproduction was particularly observed in cancer cells compared with normal cells the selectivity of egcginducedapoptosis in cancer cells might be due to the diï¬erentialinducibility of rosexpression ofapoptosisrelated genes moreover tao found thategcg induced diï¬erential mitochondrial dysfunction andoxidative stress in normal and oral cancer cells these eï¬ectswere related to the diï¬erential modulation of sirtuin sirt3 and its downstream targets including glutathionegsh and sod considering the cytotoxicity of egcgin normal cells the ic50 value in normal cells was checkedand showed to be more than μm while that for thecorresponding cancer cells was μm these resultssuggested that cancer cells are more sensitive to egcg thannormal cells and ros might be selectively toxic to cancercellsin addition to being used as preventive agents individually egcg could also be used as adjuvant therapies generally cooperative interaction of two or more agents couldtarget more signaling pathways thus eï¬ectively improvingagent chemosensitivity reducing untoward eï¬ects of treatment expanding the scope of action and showing highertherapeutic outcomes drug resistance is a dauntingchallenge in cancers prooxidant activities of egcg wereproposed to contribute to overcoming drug resistancehighlighted by the fact that h2o2 production induced byegcg increased the potency of cisplatin in ovarian cancercells by three to sixfold in contrast cisplatin alone washighly resistant to the treatment in some cancer cell linescopper transporter ctr1 is a critical determinant toincrease cisplatin uptake egcg could upregulate ctr1expression through the stimulation of ros simultaneous treatment of arsenic trioxide ato with egcgshowed oxidativemediated induction of apoptosis in leukemia cancer cells egcg acted as a prooxidant andincreased intracellular h2o2 and atoinduced hemeoxygenase1 ho1 provided ferrous iron to increase thefenton reaction in both cases cellular oxidative damageeventually occurredin general under typical cell culture conditions egcghas been known to generate i extracellular ros via autooxidative reaction in the cell culture medium ii ros in cellular mitochondria and iii intracellular ros through thefenton reaction upon cell entry figure these three pathways contribute diï¬erently to cancer cells but eventuallycause cell damage and death cancer initiation and progression are generally divided into several stages when egcgacts as an antioxidant it might more eï¬ectively enhance antioxidant capacity at the cancer prevention stage whereaswhen egcg acts as a prooxidant it might be more criticalat suppressing tumor growth stage one possible suppositionis that tumor cells may be more susceptible to oxidativestress because their increased growth rate and metabolismcause a heightened basal ros level the degree of cell proliferation and diï¬erentiation seems to be one factor aï¬ectingthe ros production ability of egcg future research willbe required to determine if egcg is a much more potentros inducer in diï¬erentiated than in undiï¬erentiated cancercells although a limited amount of data has shown that theseprooxidant eï¬ects can occur in vivo it is essential to understand when and to what extent the antioxidant or prooxidanteï¬ects of egcg are working in diï¬erent stages of cancers inanimal models prooxidant and antiobesity eï¬ects of egcg obesity is ametabolic disease characterized by abnormal or excessive fataccumulation it is generally associated with an increased riskof chronic diseases including diabetes hypertension anddyslipidemia a large and growing body of studies hasinvestigated the antiobesity eï¬ects of egcg in cellular andanimal experiments and the underlying mechanismsthe clinical manifestations of obesity are adipocytehyperplasia and hypertrophy in vitro studies have well demonstrated that egcg could inhibit adipocyte growth andinduce adipocyte death through its prooxidant eï¬ects hung reported that high concentrations of egcg μm reduced the cell viability of preadipocytes by induced the appearance of dna fragmentation andincreased the activity of the apoptotic enzyme caspase3 egcg was demonstrated to raise ros level anddescend gsh level in preadipocytes and adipocytes whichinduced oxidative stress thus resulting in decreased cell number ²ampregulated protein kinase ampk represents ametabolitesensing protein kinase hwang foundthat the release of ros by egcg stimulation could furtheractivate ampk rapidly in 3t3l1 adipocytes a recent studyalso proved that ampk was activated by exogenous h2o2and this activation was not through direct redox signalingto ampk but was a secondary consequence of redox eï¬ectson other processes egcg activates ampk via the generation of ros subsequently blocks anabolic pathways and promotes the catabolicpathway and suppresses gluconeogenesis and adipogenesisconsequently leading to body weight reduction and metabolic syndrome alleviation figure the activation ofampk modulates the expression of genes and proteinsinvolved in lipid metabolism downregulates the expressionof fat synthesis proteins and upregulates lipid catabolismproteins it was shown that egcg inhibited the expressions of glucose 6phosphatase g6pase for gluconeogenesis phosphoenolpyruvate carboxykinaseforgluconeogenesis fatty acid synthase fas for fatty acid synthesis acetylcoa carboxylase acc for fatty acid synthesis hydroxymethylglutarylcoa reductase hmgrforregulatory elementbinding proteinscholesterolsrebpsfor sterol synthesis peroxisome proliferatoractivated receptor gamma pparγ for lipid synthesis andstorage and ccaatenhancerbinding protein alphacebpα for adipogenesis as well as enhanced the expression of acylcoa oxidase aco for fatty acid oxidationperoxisome proliferatoractivated receptor alpha pparαpepcksterol 0coxidative medicine and cellular longevityautooxidationrosegcgcellrosfe2cu2fentonreaction·ohegfrcytochrome ccell damagecell deathcaspase9caspase3cell culture mediumfigure prooxidant eï¬ects of egcg in cell cultureegcggeneraterosactivateampkmodulateg6pase pepck fasacc hmgr srebpspparð¾ cebpð¼aco pparð¼ cpt1acad pgc1ð¼ucps atglfat synthesislipid catabolismantiobesityfigure eï¬ects of egcg on lipid metabolism via ros and ampkfor fatty acid oxidation carnitine palmitoyltransferase1cpt1 for fatty acid oxidation acylcoa dehydrogenaseacad for fatty acid oxidation peroxisome proliferatoractivated receptor gamma coactivator1α pgc1α for fattyacid oxidation uncoupling proteins ucps for thermogenesis and adipose triglyceride lipase atgl for triglyceridehydrolysis []accordingly the prooxidant eï¬ects of egcg play avital role in preventing the initiation and progression ofobesity egcg could cause oxidative stress thus damagingadipocyte directly and activating ampk and then aï¬ectingrelative genes and protein expression and signal transduction in various tissues indirectly however the increase ofoxidative stress in fat accumulation might be an importantpathogenic mechanism of obesityrelated metabolic syndrome such as diabetes firm conclusions as to whetherprooxidant eï¬ects of egcg could perform on body weightbody fat and adipose weight in humans will require morethorough clinical studies prooxidant and antibacterial eï¬ects of egcg egcgexhibits a broad spectrum of bactericidal activity against various bacteria its bactericidal eï¬ects include damage to thebacterial cell membrane and inhibition of fatty acid synthesisand enzymatic activity h2o2 which is generated byegcg appears to play an indispensable role in the bactericidal actions of egcg the bactericidal action of egcgwas related to h2o2 level as bactericidal action was inhibitedby the increase of cat concentration egcg was foundto have bactericidal activity at higher concentrations in thesalmonella assay highly correlated with h2o2 production egcg showed a dosedependent μm inhibition on escherichia coli e coli op50 strain growth this inhibitory action was associated with a profoundincrease in intracellular oxidative stress caused by egcghence the use of egcg as a prooxidant is well supportedby these studiesegcg was shown to have broad antibacterial spectrumeï¬ects on both grampositive and gramnegative bacterianevertheless egcg might function through diï¬erent mechanisms against grampositive and gramnegative bacteriaintracellular ros level was determined by ï¬ow cytometrythe results indicated that damage on gramnegative e colicell walls was induced by egcg depending on h2o2 release 0coxidative medicine and cellular longevity in contrast the damages on grampositive staphylococcus aureus resulted from a combination between egcg andpeptidoglycan layer because the outer membrane ofgramnegative bacteria was mainly composed of negativelycharged lipopolysaccharides which could resist the destruction of egcg they are less susceptible to egcg thangrampositive bacteria bacterial cell membrane damage not only prevents thebinding of bacteria to host cells but also inhibits the abilityof the bacteria to combine with each other and form bioï¬lms egcg was known to attack the lipid bilayer of bacterialcell membranes leading to physical disruption of the membrane as for the cell walls results from atomic forcemicroscopy suggested that the subminimum inhibitory concentrations of egcg treatment mgl to e colio157h7 strains could lead to temporary changes in the cellwalls cui such changes were due to the damagecaused by h2o2 generated from egcg moreover egcgcaused cell membrane damage via increased intracellularros level and led to potassium leakage these are potentiallyconducive to the antibioï¬lm eï¬cacy of egcg against vibriomimicus which is a foodborne pathogen in seafood andwater in addition egcg also regulates the expression of oxidative stressrelated genes oxyr and soxrs systems are activated upon exposure to oxidative stress oxyr induces katgand soxrs induce soda strongly when cells are stressed byexogenous h2o2 egcg treatment upregulated katgand soda expression in e coli these results veriï¬ed the roleof ros in egcgmediated bacterial inhibition the cpxsystem is thought to control protein homeostasis in the cellenvelope when e coli was exposed to egcg apoptosis happened because of ros formation by the cpx system rpos is a general stress regulator in response to oxidativestress egcg could cause a reduction in the expression forrpos indicating that egcg induced oxidative stress in bacterium models the potential prooxidant properties of egcg could beattributedin part to its suppressive eï¬ects on bacteriamore broadly research is also needed to determine relativesignaling pathways and proteomic factors egcg is superexcellent natural products it could increase the eï¬cacy ofbactericidal eï¬ect when it aids other fungicides morerecent attention has been focused on the impact of greentea and green tea polyphenols on the intestinal microï¬orawhether egcg intervention would change the diversity ofmicrobiota and lead to microbiota death is also in need offurther investigation adverse effectsin recent years egcg has become one of the most aggressively promoted food supplement products in daily lifeegcg entered the market and its safety has raised queriesthe prooxidant eï¬ect of egcg is not necessarily advantageous they might have implications regarding potential toxicity hence it is necessary to systematically explore theharmful eï¬ects of egcg and the mechanisms prooxidant and hepatotoxicity eï¬ects of egcg a considerable amount of literature has been published on hepatotoxicity of green teaderived products it is noteworthythat the hepatotoxicity of green tea and its derived productswas initially found in some diet products in after beingthe cause of liver injury in subjects france and spain governments have suspended the marketing of exolise whichwas a weightloss phytotherapeutical drug in the pasttwo decades reports on liver disorders caused by green teaingestion with overdose of egcg content have graduallyemerged the liver is a major drug metabolic organ in the bodythe bioavailability of egcg in rats was determined after min of oral administration mgkg by detecting theconcentration of egcg in plasma and diï¬erent tissuesincluding the liver the results showed that the concentrationof egcg in the liver μmolkg was four times higherthan in that in the blood plasma μmolkg moreover utilizing anatomy egcg could trigger liver damagewhereas no visible abnormalities were found in other tissuesand organs [ ] hence it could be preliminarily concluded that the liver is the toxic target organ of egcgat the cellular level egcg demonstrated cytotoxic eï¬ectin cultured rat hepatocytes it was shown that μm egcgtreatment on freshly isolated rat hepatocytes caused timedependent cytotoxicity the hepatocyte was incubatedwith egcg for h resulting in liver cell function reduceddose dependently the decrease of lactate dehydrogenase ldh a marker of cell membrane damage wasobserved in rat hepatocytes egcg also caused damageto the outer mitochondrial membrane by the fact that mitochondrial membrane potential collapsed in animal experiments table the severity of egcginduced toxicity is relevant with dose route of administration and period of treatment [ ] biochemicaland histopathological analysis showed that liver samples ofmice displayed diï¬erent degrees of liver injury liver functionindexes of plasma alanine aminotransferase alt andaspartate aminotransferase ast activity increased in adosedependent mannermalondialdehyde mda and 4hydroxynonenal hne are ï¬nal products of lipid peroxidation present biochemical markers of oxidative stress metallothionein mtand γhistone 2ax γh2ax are molecular markers of oxidative stress oral high dose of egcg mgkgd to cf mice for two days significantly enhanced the formation ofmda in the liver and elevated the expression of hepaticmt and γh2ax protein and increased positive staining for4hne in liver samples intraperitoneal administrationof egcg or mgkgd for ï¬ve days raised serum hne level and western blot analysis showed that hepaticγh2ax was markedly increased all these biomarkersillustrated that egcgtriggered hepatotoxicity in vivo wasinduced by oxidative stressprevious pharmacological studies have shown that undernormal physiological conditions egcg is metabolizedthrough methylation sulfation and glucuronidation andthen excreted in urine subsequently whereas at toxicdoses these pathways might be saturated and the excessive 0canimal typefemale swissalbino micemalekunmingmiceegcgdosagemgkgd andmalekunmingmice and and male nd4micemale cf1micewistar rats ofboth sexesmale cd1micemicefemaleswisswebster miceoxidative medicine and cellular longevitytable hepatotoxicity of egcg based on animal modelsroute ofadministrationdurationresultsreferenceip and po dip treatment increased serum bilirubin markers po treatment didnot show any dosedependent changes except alt marker dtolerable dose of egcg was mgkg for ip and mgkg foripipigigpo d dserum alt ast 4hne il2 il6 and il10 and hepatic γh2axwere raised hepatic nrf2target gene expression was increasedthe fatality rate was single doseserum alt ast 4hne il6 and il10 and hepatic γh2ax wereraised hepatic nuclear and cytosolic nrf2 proteins were suppressed d dmouse growth was not aï¬ected the dosage was considered asmaximum tolerable dosehepatotoxicity occurred major hepatic antioxidant enzymes weresuppressed nrf2mediated rescue response was inducedsingle dosemice died in a dosedependent manner andthe nrf2 pathway was not activated nrf2 and its target genes were h dsuppressedalt was slightly increased histopathology of the liver showedcongestion of sinusoids and central and portal veinssingle dosealt was markedly increased histopathology of the liver showeddegenerative hepatocytes and a small number of vacuoles d dmouse survival was reduced by mouse survival was reduced by hepatic mda mt and γh2axwere increasedsingle dosealt was increased by 108fold mouse survival was reduced by egcg2²cysteine and egcg2³cysteine were detected in theurineposingle dosemice were lethargic and their respiration was labored and andipipipsingle doseplasma alt was increased mice died within h h degcg thiol conjugates egcg2²cysteinyl and egcg2³cysteinyl were detected in the urine of mice died plasma alt activity was elevated severe hepaticnecrosis occurredamount of egcg would be oxidized to form oquinonewhich could react with gsh to form egcg thiol conjugates therefore it could be inferred that high dose of egcgresults in the accumulation of oquinones and the metabolites of oquinones are biomarkers of oxidative stress twoegcg thiol conjugates egcg2²cysteinyl and egcg2²²cysteinyl were detected in the pooled h urine of micetreated with a dose of or mgkg intraperitonealip injection of egcg however egcg thiol conjugateswere not found when the dose was or mgkg bwip when cf1 mice were treated with a single doseof mgkg intragastric ig administration of egcgboth egcg2²cysteinecysteine weredetected in the pooled h urine gsh conjugate ofand egcg2²²egcg was also detected in hepatocytes incubated withegcg these ï¬ndings indicated that the formation ofdetectable amounts of egcg thiol conjugates appears toresult from the administration of toxic doses of egcgnuclear factor erythroidrelated factor nrf2 an essential antioxidant transcription factor regulates the expressionof many antioxidant and phase ii detoxifying enzyme genessuch as ho1 and nadphquinone oxidoreductase1nqo1 through antioxidant response element are undernormal metabolic and physiologic states nrf2 is repressed inthe cytoplasm by kelchlike echassociated protein1keap1 while under oxidative stress conditions nrf2 dissociates from keap1 and translocates to the nucleus to bind toare the activation of the nrf2are signaling pathway 0coxidative medicine and cellular longevityrepresenting a major cellular defense against oxidative stresscould stimulate the expression of downstream antioxidantenzymes a previous study revealed that toxic doses ofegcg and mgkg ip inhibited hepatic antioxidantenzymes sod cat and glutathione peroxidase and exacerbated oxidative damage in hepatocytes after treatmentwith egcg the expression of nrf2 decreased in the cytosoland increased in the nucleus indicative of nrf2 activationas a result mrna expression of ho1 nqo1 and otherhepatic nrf2target genes was induced in a dosedependentmanner accordingly a conclusion could be made that themolecular mechanisms underlying highdose egcg potentialtoxicity involve activation of the nrf2are signaling pathwayand suppression o | Colon_Cancer |
"tea is the second most popular beverage consumed in theworld next to water green tea is a kind of nonfermentedtea produced from the plant camellia sinensis it is favoredby people for its fresh ï¬avor and health beneï¬ts and consumed worldwide especially in east asian countriesgreen tea contains caï¬eine and polyphenolic compoundsknown as catechins catechins are the primary bioactivesubstances and present significant biological propertiestea leaves drycatechins constitute up to ofweight among that egcg is the main and the most abundant catechin [ ] egcg has been traditionally regardedas beneï¬cial mainly ascribed to its antioxidant action the antioxidant eï¬ects of egcg are manifested in scavenging free radicals in the body and inhibiting the formation ofros the results of earlier studies suggested that egcgcould decrease the risk of several human disorders associatedwith oxidative stress on the other hand egcg also displays significantprooxidant eï¬ects usually under highdose conditions theprooxidant actions of egcg play a dual role being both beneï¬cial and harmful while achieving desired outcomes inchronic disease prevention and treatment reports about thetoxicity of egcg are also emerging a growing body ofevidence continues to demonstrate a variety of harmfuleï¬ects from excessive consumption of green tea or oraladministration of highdose egcg supplement highdoses of egcg not only cause cytotoxicity in vitro but alsoresult in living body hepatotoxicity nephrotoxicity andgastrointestinal disorders vomiting and diarrhea the oral bioavailability of egcg is not so profound inhealthy humans as it was only of the total ingestion most of the ingested egcg is absorbed in the smallintestine and substantially degraded in the large intestine bymicrobiota action the eï¬ective dosage of egcg mightbe close to or higher than the toxic dosage in practical applications considering its low bioavailability therefore it is 0coxidative medicine and cellular longevitynecessary to understand the potential toxicity doses andusage of egcg in this review the prooxidant eï¬ects ofegcg in health beneï¬ts and adverse eï¬ects were discussedespecially concerning their underlying mechanisms involvedand doses used this review is aimed at harnessing the prooxidant eï¬ects of egcg for human health maintenance whileavoiding toxicity thereby better guiding the safety consumption of green tea and egcg chemical structure andautooxidation of egcgbasic catechins contain two or more aromatic ringshydroxyl group on carbon three position andor the higherdegree of hydroxylation of the b ring would be primarilyresponsible for the potent antioxidant activities of catechinsfigure 1a previous structureactivity relationshipstudies of catechins have demonstrated the importance ofthe number and location of the phenolic hydroxyl groupson antioxidative capacity egcg has the remarkablepotential to scavenge radicals and chelate metal ion theseabilities could be ascribed to the presence of dihydroxyand trihydroxy groups in a ring b ring and d ringfigure 1b the catechol structure of egcg makes it susceptible todegradation via autooxidation figure under normal°physiological conditions ph c egcg is autooxidized and converted to oquinone through nonenzymaticaldehydrogenation of phenolic hydroxyl groups at b ring when the cell culture medium is exposed in the airegcg could be oxidized by oxygen and yields superoxide andanion radicals o2
egcg are essential intermediate products in egcg autoox and oxygen could function as oxidants for furidation o2ther oxidation of egcg ï¬nally resulting in the formationof oquinone accompanying the generation of hydrogen could also form substantial amountsperoxide h2o2 o2of h2o2 via disproportionation reaction one egcgmolecule could produce more than two h2o2 molecules inphosphate buï¬er at neutral ph and egcg radicals
egcg o2autooxidation of egcg generates substantial ros theros comprises singlet oxygen hydroxyl radicals superoxideperoxides and h2o2 h2o2 is in a dominant position andusually is regarded as a toxic agent when the ros levelexceeds cellular antioxidant capacity oxidative stress willoccur in other words this is the result of an imbalancebetween prooxidant and antioxidant eï¬ects inclusion ofantioxidant defense enzymes such as catalase cat andsuperoxide dismutase sod could minimize h2o2 levelwhich is essential to maintain the redox balancethe concentration of egcg in the cell environmentseems to be a primary factor in explaining its prooxidanteï¬ects for example egcg treatment alone diminisheddna strand breakage of human blood lymphocytes at lowconcentrations μm while it induced dna strandbreakage at higher concentration μm thusegcg acts as an eï¬ective antioxidant at low doses withinthe range of high nanomolar and low micromolar levelswhile egcg represents a prooxidant at high doses howeverthis blurred boundary might vary depending on the type ofradical stimulants cellular environment and duration ofexposure to egcg health benefitsuntil now egcg has been a major research subject withinthe ï¬eld of healthpromoting eï¬ects the potential role ofthe prooxidant eï¬ects of egcg in cancer and obesity prevention and treatment as well as the antibacterial actionsachieved demonstrable results in previous studies prooxidant eï¬ects and anticancer activity of egcgcancer is one of the most common and lifethreateningdiseases occurring among humankind egcg as a naturalproduct has drawn a great deal of attention from boththe scientiï¬c community and the general public indeedegcg has shown both prophylactic and therapeutic eï¬cacy in multiple human cancers several mechanisms havebeen proposed to accountfor the inhibitory action ofegcg against cancer formation and growth the prooxidant eï¬ects of egcg were thought to be potential mechanisms for anticancer action the anticancer mechanismsvaried depending on the cell type dose andor time oftreatment table []apoptosis is the bestdescribed form of programmed celldeath the induction of apoptosis represents a universal andideal therapeutic strategy for cancer control cell apoptosiscould be triggered by either the intrinsic mitochondrial pathway or the external death receptor pathway the mitochondrial pathway could be induced by intracellularstresses such as oxidative stressthe apoptosistriggering eï¬ects of ros have beennoted in vitro table egcg inhibited cell growth ina dosedependent manner and the decrease in the numberof viable cells was mainly due to apoptosis caused by theegcginduced intracellular ros as early as the last century scientists found that egcg induced h2o2 formationin human lung cancer celllines h661 and 21bes andexogenously added cat completely prevented egcginduced cell apoptosis which suggested that h2o2 isinvolved in the apoptosis process provoked by egcg similar actions were also found in various cancersand tumor cells table thioredoxin trx and thioredoxin reductase trxr are pivotal regulators of cellularredox homeostasis decreased trxtrxr activity mightcontribute to the increased ros level high concentrationof egcg inactivated trxtrxr via the formation of egcgtrx1 and egcgtrxr conjugates which was linked to theelevation of ros level in hela cells and further promotedcancer cell death moreover one of the biochemical hallmarks of apoptosis is genomic dna fragmentation chen performed the dna fragmentation assay in theskov3 cells indicating that egcg induced apoptosis bycausing dna damage this result was consistent withother studies in ovarian and cervical cancer cells [ ]in terms of molecular mechanisms intrinsic apoptosisleads to the release of mitochondrial cytochrome c afterbeing released into the cytoplasm cytochrome c stimulates 0coxidative medicine and cellular longevityohbohohohhoohbohohohohdohocoaohobhoocaohafigure a basic structure of catechins b chemical structure of egcgohbegcgohohautooxidationph75 °cohboautooxidation·egcgoho2h2o2ohboooquinonefigure superoxidemediated chain reaction the formation of oquinoneapoptosome formation followed by activation of caspasecascades egcgmediated mitochondrial ros couldpromote cytochrome c release to the cytosol the antiproliferative action of egcg on prostate cancer and breast cancer is mediated through apoptosis as evident from caspase9[ ] the cells susceptible to egcginduced apoptosisalso showed activation of caspase3 moreover theincreased ros level was observed to result in the stimulationof mitogenactivated protein kinase mapk themapk signaling pathwayincluding extracellular signalregulated kinase erk jun nterminal kinase jnks andp38 plays a vital contribution in cell proliferation diï¬erentiation apoptosis and stress response egcg induced oxidative stress via generation of ros and thereafter activatedthe jnk pathway leading to changes in mitochondrial membrane potential and release of cytochrome c in ht29 humancolon adenocarcinoma cells and mia paca2 pancreaticcancer cells [ ] together these results suggest thategcginduced apoptosis is mediated through ros generation and might subsequently activate the cell intrinsic pathway in the presence of transition metals such as copper andiron h2o2 could convert to a potent oxidant hydroxyl radical via the fenton reaction nakagawa found that egcg μm produced h2o2 and triggered fenton reactionto form highly toxic hydroxyl radicals which resulted in lymphoblastic leukemia jurkat cell death in the presence offeiii and cuii egcg μm induced dna damagein hl60 cells as 8oxo78dihydro2²deoxyguanosine oxodg content increased which was a characteristic ofoxidative dna damage nevertheless no significantincrease in 8oxodg was observed in h2o2resistant colonhp100 cells suggesting that h2o2 was involved in cellulardna damage egcg could inhibit cell proliferation andinduce apoptosis through cellular dna breakage in diï¬erentcancer cell lines such dna breakage involved the mobilization of endogenous copper ions and the generation ofros moreover the observation of site speciï¬city of dnadamage by egcg is valuable cuiimediated dna damageby egcg occurred most frequently at t and g residues egcg was able to mobilize endogenous copper ions andgenerate hydroxyl radicals in situ hydroxyl radicalsserved as the proximal dna cleaving agentleading todna breakage in the nuclei this result was possibly due tothe interaction of egcg with chromatinbound copper ionsand then the nondiï¬usible hydroxyl radicals were formed atthe binding site hence hydroxyl radical generated nearbydna was well established as the cause of strand scissionbecause the concentration of copper is significantly very highin various malignancies egcg could induce cancer celldeath through the metal iondependent pathway thispathway was independent of mitochondriamediated programmed cell deaths such action involved in metal ionmediated dna cleavage would be an important mechanismof anticancer properties of egcgin addition to being transported into the cell egcgcould also function on the cell membrane fraction to regulatethe surface growth factor receptor earlier studies foundthat autooxidation of egcg led to epidermal growth factorreceptor egfr inactivation in human esophageal cancer 0ccell linesbladder cancernbtiibreast cancermcf7mcf7cervical cancerhelahelacolon canceroxidative medicine and cellular longevitytable role of prooxidant eï¬ects in the anticancer activity of egcg based on cell culture studiesegcgconcentrationtimebiological eï¬ectsreferences μm hinduced early apoptosis through dna damage μgml hinduced cell growth inhibition and apoptosis by downregulating survivinexpression via suppressing the akt pathway and activating caspase9 μm hinduced apoptosis at low doses via activation of jnk caspase9 and caspase3while inducing necrosis at high doses which is related to diï¬erences in rosgeneration and atp levels μm μm and h hincreased cell death through dna damageinduced cell death through generation of ros and inactivation of trxtrxrhct116 μm hinduced apoptosis through induction of ros and epigenetic modulation ofapoptosisrelated gene expressionht29 μm hendometrial carcinomaishikawa μm hinduced apoptotic cell death via activating the jnk pathway accompanyingmitochondrial transmembrane potential transition and cytochrome c releaseic50 was μminduced apoptosis via ros generation and p38 map kinase activationic50 was μmesophageal cancerkyse lung cancer μm hinactivated egfr by superoxide generated from autooxidation of egcg μm μm h h h μm hdisplayed strong growth inhibitory eï¬ects against lung tumor cell linesinhibited cell growth through induction of ros ic50 was μmic50 was μminduced apoptosis via h2o2 production and hydroxyl radical formationinduced apoptosis by modifying the redox systemh661 and h1299 μmh1299lymphoblastic leukemiajurkatmyelomaim9 rpmi8226and u266oral cancerscc25 andscc9ovarian cancer μm hreduced cell viability by inducing mitochondrialocalized ros and decreasingsirt3 expressionskov3 μgml dpancreatic cancerpanc1 μm hmia paca2 μm hinhibited cell proliferation and induced apoptosis by inhibiting cell cycle arrest andinducing dna damageinduced apoptosis through generation of ros as well as caspase3 andcaspase9 activationinduced stress signals by damaging mitochondria and rosmediatedjnk activationprimary eï¬usionlymphomabcbl1 and bcprostate cancer μgml hinduced apoptosis and autophagy through ros generationpc3 and μm hreduced cell survival and increased apoptosis caused a significant alteration incaspase9 alternative splicing 0coxidative medicine and cellular longevitycell line kyse one possible explanation is thath2o2 produced from egcg autooxidation in the cell culturemedium could attack and inactivate egfr leading to theinhibition of egfr phosphorylationand preferentialit is worth considering whether high amounts of egcgcould cause damage to normal cells egcgmediated rosproduction was particularly observed in cancer cells compared with normal cells the selectivity of egcginducedapoptosis in cancer cells might be due to the diï¬erentialinducibility of rosexpression ofapoptosisrelated genes moreover tao found thategcg induced diï¬erential mitochondrial dysfunction andoxidative stress in normal and oral cancer cells these eï¬ectswere related to the diï¬erential modulation of sirtuin sirt3 and its downstream targets including glutathionegsh and sod considering the cytotoxicity of egcgin normal cells the ic50 value in normal cells was checkedand showed to be more than μm while that for thecorresponding cancer cells was μm these resultssuggested that cancer cells are more sensitive to egcg thannormal cells and ros might be selectively toxic to cancercellsin addition to being used as preventive agents individually egcg could also be used as adjuvant therapies generally cooperative interaction of two or more agents couldtarget more signaling pathways thus eï¬ectively improvingagent chemosensitivity reducing untoward eï¬ects of treatment expanding the scope of action and showing highertherapeutic outcomes drug resistance is a dauntingchallenge in cancers prooxidant activities of egcg wereproposed to contribute to overcoming drug resistancehighlighted by the fact that h2o2 production induced byegcg increased the potency of cisplatin in ovarian cancercells by three to sixfold in contrast cisplatin alone washighly resistant to the treatment in some cancer cell linescopper transporter ctr1 is a critical determinant toincrease cisplatin uptake egcg could upregulate ctr1expression through the stimulation of ros simultaneous treatment of arsenic trioxide ato with egcgshowed oxidativemediated induction of apoptosis in leukemia cancer cells egcg acted as a prooxidant andincreased intracellular h2o2 and atoinduced hemeoxygenase1 ho1 provided ferrous iron to increase thefenton reaction in both cases cellular oxidative damageeventually occurredin general under typical cell culture conditions egcghas been known to generate i extracellular ros via autooxidative reaction in the cell culture medium ii ros in cellular mitochondria and iii intracellular ros through thefenton reaction upon cell entry figure these three pathways contribute diï¬erently to cancer cells but eventuallycause cell damage and death cancer initiation and progression are generally divided into several stages when egcgacts as an antioxidant it might more eï¬ectively enhance antioxidant capacity at the cancer prevention stage whereaswhen egcg acts as a prooxidant it might be more criticalat suppressing tumor growth stage one possible suppositionis that tumor cells may be more susceptible to oxidativestress because their increased growth rate and metabolismcause a heightened basal ros level the degree of cell proliferation and diï¬erentiation seems to be one factor aï¬ectingthe ros production ability of egcg future research willbe required to determine if egcg is a much more potentros inducer in diï¬erentiated than in undiï¬erentiated cancercells although a limited amount of data has shown that theseprooxidant eï¬ects can occur in vivo it is essential to understand when and to what extent the antioxidant or prooxidanteï¬ects of egcg are working in diï¬erent stages of cancers inanimal models prooxidant and antiobesity eï¬ects of egcg obesity is ametabolic disease characterized by abnormal or excessive fataccumulation it is generally associated with an increased riskof chronic diseases including diabetes hypertension anddyslipidemia a large and growing body of studies hasinvestigated the antiobesity eï¬ects of egcg in cellular andanimal experiments and the underlying mechanismsthe clinical manifestations of obesity are adipocytehyperplasia and hypertrophy in vitro studies have well demonstrated that egcg could inhibit adipocyte growth andinduce adipocyte death through its prooxidant eï¬ects hung reported that high concentrations of egcg μm reduced the cell viability of preadipocytes by induced the appearance of dna fragmentation andincreased the activity of the apoptotic enzyme caspase3 egcg was demonstrated to raise ros level anddescend gsh level in preadipocytes and adipocytes whichinduced oxidative stress thus resulting in decreased cell number ²ampregulated protein kinase ampk represents ametabolitesensing protein kinase hwang foundthat the release of ros by egcg stimulation could furtheractivate ampk rapidly in 3t3l1 adipocytes a recent studyalso proved that ampk was activated by exogenous h2o2and this activation was not through direct redox signalingto ampk but was a secondary consequence of redox eï¬ectson other processes egcg activates ampk via the generation of ros subsequently blocks anabolic pathways and promotes the catabolicpathway and suppresses gluconeogenesis and adipogenesisconsequently leading to body weight reduction and metabolic syndrome alleviation figure the activation ofampk modulates the expression of genes and proteinsinvolved in lipid metabolism downregulates the expressionof fat synthesis proteins and upregulates lipid catabolismproteins it was shown that egcg inhibited the expressions of glucose 6phosphatase g6pase for gluconeogenesis phosphoenolpyruvate carboxykinaseforgluconeogenesis fatty acid synthase fas for fatty acid synthesis acetylcoa carboxylase acc for fatty acid synthesis hydroxymethylglutarylcoa reductase hmgrforregulatory elementbinding proteinscholesterolsrebpsfor sterol synthesis peroxisome proliferatoractivated receptor gamma pparγ for lipid synthesis andstorage and ccaatenhancerbinding protein alphacebpα for adipogenesis as well as enhanced the expression of acylcoa oxidase aco for fatty acid oxidationperoxisome proliferatoractivated receptor alpha pparαpepcksterol 0coxidative medicine and cellular longevityautooxidationrosegcgcellrosfe2cu2fentonreaction·ohegfrcytochrome ccell damagecell deathcaspase9caspase3cell culture mediumfigure prooxidant eï¬ects of egcg in cell cultureegcggeneraterosactivateampkmodulateg6pase pepck fasacc hmgr srebpspparð¾ cebpð¼aco pparð¼ cpt1acad pgc1ð¼ucps atglfat synthesislipid catabolismantiobesityfigure eï¬ects of egcg on lipid metabolism via ros and ampkfor fatty acid oxidation carnitine palmitoyltransferase1cpt1 for fatty acid oxidation acylcoa dehydrogenaseacad for fatty acid oxidation peroxisome proliferatoractivated receptor gamma coactivator1α pgc1α for fattyacid oxidation uncoupling proteins ucps for thermogenesis and adipose triglyceride lipase atgl for triglyceridehydrolysis []accordingly the prooxidant eï¬ects of egcg play avital role in preventing the initiation and progression ofobesity egcg could cause oxidative stress thus damagingadipocyte directly and activating ampk and then aï¬ectingrelative genes and protein expression and signal transduction in various tissues indirectly however the increase ofoxidative stress in fat accumulation might be an importantpathogenic mechanism of obesityrelated metabolic syndrome such as diabetes firm conclusions as to whetherprooxidant eï¬ects of egcg could perform on body weightbody fat and adipose weight in humans will require morethorough clinical studies prooxidant and antibacterial eï¬ects of egcg egcgexhibits a broad spectrum of bactericidal activity against various bacteria its bactericidal eï¬ects include damage to thebacterial cell membrane and inhibition of fatty acid synthesisand enzymatic activity h2o2 which is generated byegcg appears to play an indispensable role in the bactericidal actions of egcg the bactericidal action of egcgwas related to h2o2 level as bactericidal action was inhibitedby the increase of cat concentration egcg was foundto have bactericidal activity at higher concentrations in thesalmonella assay highly correlated with h2o2 production egcg showed a dosedependent μm inhibition on escherichia coli e coli op50 strain growth this inhibitory action was associated with a profoundincrease in intracellular oxidative stress caused by egcghence the use of egcg as a prooxidant is well supportedby these studiesegcg was shown to have broad antibacterial spectrumeï¬ects on both grampositive and gramnegative bacterianevertheless egcg might function through diï¬erent mechanisms against grampositive and gramnegative bacteriaintracellular ros level was determined by ï¬ow cytometrythe results indicated that damage on gramnegative e colicell walls was induced by egcg depending on h2o2 release 0coxidative medicine and cellular longevity in contrast the damages on grampositive staphylococcus aureus resulted from a combination between egcg andpeptidoglycan layer because the outer membrane ofgramnegative bacteria was mainly composed of negativelycharged lipopolysaccharides which could resist the destruction of egcg they are less susceptible to egcg thangrampositive bacteria bacterial cell membrane damage not only prevents thebinding of bacteria to host cells but also inhibits the abilityof the bacteria to combine with each other and form bioï¬lms egcg was known to attack the lipid bilayer of bacterialcell membranes leading to physical disruption of the membrane as for the cell walls results from atomic forcemicroscopy suggested that the subminimum inhibitory concentrations of egcg treatment mgl to e colio157h7 strains could lead to temporary changes in the cellwalls cui such changes were due to the damagecaused by h2o2 generated from egcg moreover egcgcaused cell membrane damage via increased intracellularros level and led to potassium leakage these are potentiallyconducive to the antibioï¬lm eï¬cacy of egcg against vibriomimicus which is a foodborne pathogen in seafood andwater in addition egcg also regulates the expression of oxidative stressrelated genes oxyr and soxrs systems are activated upon exposure to oxidative stress oxyr induces katgand soxrs induce soda strongly when cells are stressed byexogenous h2o2 egcg treatment upregulated katgand soda expression in e coli these results veriï¬ed the roleof ros in egcgmediated bacterial inhibition the cpxsystem is thought to control protein homeostasis in the cellenvelope when e coli was exposed to egcg apoptosis happened because of ros formation by the cpx system rpos is a general stress regulator in response to oxidativestress egcg could cause a reduction in the expression forrpos indicating that egcg induced oxidative stress in bacterium models the potential prooxidant properties of egcg could beattributedin part to its suppressive eï¬ects on bacteriamore broadly research is also needed to determine relativesignaling pathways and proteomic factors egcg is superexcellent natural products it could increase the eï¬cacy ofbactericidal eï¬ect when it aids other fungicides morerecent attention has been focused on the impact of greentea and green tea polyphenols on the intestinal microï¬orawhether egcg intervention would change the diversity ofmicrobiota and lead to microbiota death is also in need offurther investigation adverse effectsin recent years egcg has become one of the most aggressively promoted food supplement products in daily lifeegcg entered the market and its safety has raised queriesthe prooxidant eï¬ect of egcg is not necessarily advantageous they might have implications regarding potential toxicity hence it is necessary to systematically explore theharmful eï¬ects of egcg and the mechanisms prooxidant and hepatotoxicity eï¬ects of egcg a considerable amount of literature has been published on hepatotoxicity of green teaderived products it is noteworthythat the hepatotoxicity of green tea and its derived productswas initially found in some diet products in after beingthe cause of liver injury in subjects france and spain governments have suspended the marketing of exolise whichwas a weightloss phytotherapeutical drug in the pasttwo decades reports on liver disorders caused by green teaingestion with overdose of egcg content have graduallyemerged the liver is a major drug metabolic organ in the bodythe bioavailability of egcg in rats was determined after min of oral administration mgkg by detecting theconcentration of egcg in plasma and diï¬erent tissuesincluding the liver the results showed that the concentrationof egcg in the liver μmolkg was four times higherthan in that in the blood plasma μmolkg moreover utilizing anatomy egcg could trigger liver damagewhereas no visible abnormalities were found in other tissuesand organs [ ] hence it could be preliminarily concluded that the liver is the toxic target organ of egcgat the cellular level egcg demonstrated cytotoxic eï¬ectin cultured rat hepatocytes it was shown that μm egcgtreatment on freshly isolated rat hepatocytes caused timedependent cytotoxicity the hepatocyte was incubatedwith egcg for h resulting in liver cell function reduceddose dependently the decrease of lactate dehydrogenase ldh a marker of cell membrane damage wasobserved in rat hepatocytes egcg also caused damageto the outer mitochondrial membrane by the fact that mitochondrial membrane potential collapsed in animal experiments table the severity of egcginduced toxicity is relevant with dose route of administration and period of treatment [ ] biochemicaland histopathological analysis showed that liver samples ofmice displayed diï¬erent degrees of liver injury liver functionindexes of plasma alanine aminotransferase alt andaspartate aminotransferase ast activity increased in adosedependent mannermalondialdehyde mda and 4hydroxynonenal hne are ï¬nal products of lipid peroxidation present biochemical markers of oxidative stress metallothionein mtand γhistone 2ax γh2ax are molecular markers of oxidative stress oral high dose of egcg mgkgd to cf mice for two days significantly enhanced the formation ofmda in the liver and elevated the expression of hepaticmt and γh2ax protein and increased positive staining for4hne in liver samples intraperitoneal administrationof egcg or mgkgd for ï¬ve days raised serum hne level and western blot analysis showed that hepaticγh2ax was markedly increased all these biomarkersillustrated that egcgtriggered hepatotoxicity in vivo wasinduced by oxidative stressprevious pharmacological studies have shown that undernormal physiological conditions egcg is metabolizedthrough methylation sulfation and glucuronidation andthen excreted in urine subsequently whereas at toxicdoses these pathways might be saturated and the excessive 0canimal typefemale swissalbino micemalekunmingmiceegcgdosagemgkgd andmalekunmingmice and and male nd4micemale cf1micewistar rats ofboth sexesmale cd1micemicefemaleswisswebster miceoxidative medicine and cellular longevitytable hepatotoxicity of egcg based on animal modelsroute ofadministrationdurationresultsreferenceip and po dip treatment increased serum bilirubin markers po treatment didnot show any dosedependent changes except alt marker dtolerable dose of egcg was mgkg for ip and mgkg foripipigigpo d dserum alt ast 4hne il2 il6 and il10 and hepatic γh2axwere raised hepatic nrf2target gene expression was increasedthe fatality rate was single doseserum alt ast 4hne il6 and il10 and hepatic γh2ax wereraised hepatic nuclear and cytosolic nrf2 proteins were suppressed d dmouse growth was not aï¬ected the dosage was considered asmaximum tolerable dosehepatotoxicity occurred major hepatic antioxidant enzymes weresuppressed nrf2mediated rescue response was inducedsingle dosemice died in a dosedependent manner andthe nrf2 pathway was not activated nrf2 and its target genes were h dsuppressedalt was slightly increased histopathology of the liver showedcongestion of sinusoids and central and portal veinssingle dosealt was markedly increased histopathology of the liver showeddegenerative hepatocytes and a small number of vacuoles d dmouse survival was reduced by mouse survival was reduced by hepatic mda mt and γh2axwere increasedsingle dosealt was increased by 108fold mouse survival was reduced by egcg2²cysteine and egcg2³cysteine were detected in theurineposingle dosemice were lethargic and their respiration was labored and andipipipsingle doseplasma alt was increased mice died within h h degcg thiol conjugates egcg2²cysteinyl and egcg2³cysteinyl were detected in the urine of mice died plasma alt activity was elevated severe hepaticnecrosis occurredamount of egcg would be oxidized to form oquinonewhich could react with gsh to form egcg thiol conjugates therefore it could be inferred that high dose of egcgresults in the accumulation of oquinones and the metabolites of oquinones are biomarkers of oxidative stress twoegcg thiol conjugates egcg2²cysteinyl and egcg2²²cysteinyl were detected in the pooled h urine of micetreated with a dose of or mgkg intraperitonealip injection of egcg however egcg thiol conjugateswere not found when the dose was or mgkg bwip when cf1 mice were treated with a single doseof mgkg intragastric ig administration of egcgboth egcg2²cysteinecysteine weredetected in the pooled h urine gsh conjugate ofand egcg2²²egcg was also detected in hepatocytes incubated withegcg these ï¬ndings indicated that the formation ofdetectable amounts of egcg thiol conjugates appears toresult from the administration of toxic doses of egcgnuclear factor erythroidrelated factor nrf2 an essential antioxidant transcription factor regulates the expressionof many antioxidant and phase ii detoxifying enzyme genessuch as ho1 and nadphquinone oxidoreductase1nqo1 through antioxidant response element are undernormal metabolic and physiologic states nrf2 is repressed inthe cytoplasm by kelchlike echassociated protein1keap1 while under oxidative stress conditions nrf2 dissociates from keap1 and translocates to the nucleus to bind toare the activation of the nrf2are signaling pathway 0coxidative medicine and cellular longevityrepresenting a major cellular defense against oxidative stresscould stimulate the expression of downstream antioxidantenzymes a previous study revealed that toxic doses ofegcg and mgkg ip inhibited hepatic antioxidantenzymes sod cat and glutathione peroxidase and exacerbated oxidative damage in hepatocytes after treatmentwith egcg the expression of nrf2 decreased in the cytosoland increased in the nucleus indicative of nrf2 activationas a result mrna expression of ho1 nqo1 and otherhepatic nrf2target genes was induced in a dosedependentmanner accordingly a conclusion could be made that themolecular mechanisms underlying highdose egcg potentialtoxicity involve activation of the nrf2are signaling pathwayand suppression o | Colon_Cancer |
" inflammatory pseudotumour has been used to describe an inflammatory or fibrosing tumoral processof an undetermined cause that may involve a variety of an systems including the lungs spleen liver lymphnodes pancreas and extrahepatic bile duct with potential for recurrence and persistent local growth in this we report a patient with a big mass of uncertain nature and behaviorcase presentation a 60yearold woman presented with a 1week history of abdominal pain fever and jaundicesix months before she had had right upper quadrant pain that was interpreted as biliary colic a contrastenhancedct scan showed a big mass of soft tissue with diffuse infiltration of the gallbladder displacement of the transversecolon hepatic flexure and duodenum for diagnostic distinction between a chronic inflammatory disease or aneoplasm exploratory laparotomy was required intraoperative exploration disclosed a big mass of hard textureinvolving the gallbladder with multiple concrements hepatoduodenal ligament right and transverse mesocolonstomach and duodenumcholecystectomy was performed preserving adjacent ans with macroscopic desmoplastic reactionhistopathologic examination of the gallbladder showed a spindle cell proliferation with diffuse chronicinflammatory infiltrate of lymphocytes plasma cells and hyalinized fibrous stroma no vascular invasion or cellularatypia were evident inflammatory pseudotumour is a rare condition and diagnostic distinction from a chronicinflammatory disease or other neoplasm is only possible by histopathologic examination there is a limited numberof case reports in the literature indicating tumor location in the gallbladderkeywords inflammatory pseudotumor gallbladder inflammatory pseudotumour is a rare lesion that hasbeen described in various ans and tissues intraabdominal variants of the disease are reported to occurmost frequently in the liver spleen mesentery and extrahepatic bile duct the location of the gallbladder iseven more uncommon correspondence acd3202yahooesdepartment of surgery and pathology puerto real university hospital c¡dizspainmalignant transformations and recurrences of inflammatory pseudotumour have been reported years aftersurgery therefore longterm followup is necessary evenfor patients successfully treated by surgical resectionelevated igg4 serum levels have been reported in association with this illness as well as abundant igg4 positivity in tumor infiltrating plasma cells signs suggestiveof an igg4related disease a high serum igg4 concentrations thus provides a useful means of distinguishingthis disorder from other differential diagnoses 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 0ccalvo bmc gastroenterology page of pharmacologic treatments have also been reported forigg4associated inflammatory pseudotumor and thereare even cases of complete resolution of the disease withsteroids treatments however in the presented case thiscondition did not occur so treatment was exclusivelysurgicalcase presentationa 60yearold woman presented to the emergencyroom with abdominal pain fever pruritus and jaundicesince week the patient had a history of smoking anda family history of pancreatic canceron physical examination a hard and painful mass wasidentified on the right hypochondrium blood laboratoryexamination showed extrahepatic cholestasis enzymestotal bilirubin mgdl direct bilirubin mgdlast ul alt ul ggt ul ldh ul alkaline phosphatase ul in anamnesis thepatient referred to abdominal pain occurring during thelast months located in the right upper quadrant whichhad been interpreted as biliary colic by her generalpractitionertumour markers and blood count showed no alterations viral serology autoimmunity antibodies metanephrines and urine normetanephrine were within thenormal rangea large mass associated with the gallbladder was identified by abdominal ultrasound contrastenhanced ctscan disclosed a large soft tissue mass originating fromthe gallblader with homogenous contrast enhancementand without clear infiltration of the hepatic parenchymathe mass displaced the transverse colon hepatic flexureand duodenum no lymphadenopathies were identifiedin the hepatoduodenal ligament pancreas retroduodenum or celiac axis fig 1a b the gallbladder was distended contained stones and had a regular lumenwhile there was slight dilatation of the intrahepatic bileduct on magnetic resonance imaging fig 1csmallerwith these findings a diagnostic distinction between a chronic inflammatory disease or a neoplasticprocess was necessary the biopsy of the mass wasperformed under ultrasonographic control histopathologic examination showed spindle cells and some inflammatory cells ofsize and absence ofxanthic cells the tumor showed a mesenchymal aspect that was confirmed by absence of epithelial cellson pancytokeratin staining while some histiocyteswere recognized in summary the pathology diagnosiswas a mesenchymal process that could be reactive ormalignant the microbiological study of the bile obtained from gallbladder punctured showed a nonpurulent grams stain and negative cultures for bothaerobic and anaerobic germs the oral endoscopy andbiopsies of the second part of the duodenum didntshow any pathological conditionexploratory laparotomy was decided and cholecystectomy could be performed preserving the adjacent ans with macroscopic desmoplastic reaction the masswas peeled off the transverse colon first and the secondpart of the duodenum and common bile duct fig the histopathological examination offibrousspecimen disclosed sclerosingthe resectiontissue withfig axial and coronal computed tomography images showing a large mass of diffuse soft tissues originating from the gallbladder anddisplacing the duodenum transverse colon and hepatic flexure a b in magnetic resonance imaging the gallbladder was distended andcontained stones associated with a slight dilatation of the intrahepatic bile duct c 0ccalvo bmc gastroenterology page of stains were performedto achieve a definitive classification complementaryimmunohistochemicalandshowed positive staining for smooth muscle actin in themuscular layer ofthe gallbladder and vessel wallscd34 in the vascular lumen cd68 in histiocytes andremained negative for anaplastic lymphoma kinasealk and pancytokeratin panck massons trichrome stain showed intense positivity on collagen fibers less than of the tumor cells sample were ki67positive and plasma cells were igg4 positive per highpower field taken together these findings confirmed thediagnosis of inflammatory pseudotumor of the gallbladder with sclerosing cholangitis associated with a normallevel of serum of immunoglobulin g4 of mgdl mgdlno local recurrence was detected at the threeyearsfollowup on ct scandiscussion and sthe term inflammatory pseudotumour has been used todescribe an inflammatory or fibrosing tumoural processof undetermined cause that may involve a variety ofan systems including the lungs spleen liver lymphnodes pancreas and extrahepatic bile duct with potentialfor recurrence and persistent local growth [ ] thereis a limited number of case reports in the literature indicating gallbladder location [ ]fig on laparotomy a large and hard mass was identified in thegallbladder with stones displacing but not infiltrating right andtransverse mesocolon stomach duodenum andhepatoduodenal ligamenthistiocytes chronic lymphocytic inflammatory infiltrateand plasma cells with isolated eosinophils and no epithelial malignancy the mass presented as an expansivegrowth from the outer portion of the muscular layer ofthe gallbladder to the surrounding fatty tissuethe definitive pathological diagnosis was inflammatorypseudotumour of the gallbladder with chronic sclerosingcholangitis fig a bfig histopathologic examination disclosed a thickened gallbladder wall a with spindle cells and proliferation of connective fibrous tissuewithout signs of celular atypia b and inflammatory cells including lymphocites plasma cells and hyalinized fibrous tissue without vascularinvasion c few plasma cells were igg4 positive in relation to the whole inflammatory cell infiltrate d 0ccalvo bmc gastroenterology page of consent for publicationwe confirm in this statement that a written consent to publish thisinformation was obtained from study participant and the proof of consentto publish from study participants can be provided at any timethe authors have in their possession the informed consent of the patientbiomed central consent formcompeting intereststhe authors declare that they have no competing interestsreceived january accepted august referencesbehranwala ka straker p wan a fisher c thompson jn inflammatorymyofibroblastic tumour of the gallbladder world j surg oncol sinha l hasan a sngh ak bhadani pp jha an singh pk kumar minflammatory myofibroblastic tumor involving liver gallbladder pylorus andduodenum a rare case presentation int j surg case rep badea r veres aa andreica v inflammatory myofibroblastic tumor ofthe gallbladder imaging aspects j med ultrason abrantes cf silva mr oliveira rc eloy c cipriano ma castro lpinflammatory myofibroblastic tumour arising incidentally as a polypoidlesion in the gallbladder j bras patol med lab v51 n p koea jb broadhurst gw rodgers ms inflammatory pseudotumor ofthe liver demographics diagnosis and the case for nonoperativemanegement j am coll surg sato y kojima m takata k immunoglobulin g4relatedlymphadenopathy with inflammatory pseudotumorlike features med molmorphol hamano h kawa s horiuchi a high serum igg4 concentrations inpatients with sclerosing pancreatitis n engl j med aldhahab h mcnabbbaltar j albusafi s barkun an immunoglobulin g4related pancreatic and biliary disease can j gastroenterol lee ys lee sh lee mg immunoglobulin g4related diseasemimicking unresectable gallbladder c¡ncer gut liver muduly d deo sv shukla nk inflammatory myofibroblastic tumor ofgall bladder trop gastroenterol publishers notespringer nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliationsinflammatory pseudotumour appear to be more common in noneuropean populations they usually occur ininfancy and young adults but can occur in the elderly []the igg4 serum level should be determined due to acommon association of elevated serum igg4 with this illness and some authors describe abundant igg4 positivityin plasma cells as suggestive of igg4related disease []high serum igg4 concentrations might provide afromuseful means of distinguishing this disorderother lesions []the histopathological examinations showed sclerosingfibrous tissue with histiocytes chronic lymphocytic inflammatory infiltrate and plasma cells with isolated eosinophils compatible with igg4related disease howeveras igg4 serum levels were within normal range and theigg4 tissue expression was very weak there is no strongevidence of a clear association with igg4related diseaseinflammatory pseudotumour can generally be considered to be a relatively rare disease of undefined originwith a great variety of symptoms causing diagnosticchallenges in the distinction of chronic inflammatorydisease and neoplasminflammatory pseudotumoris defined as nonneoplastic but is currently considered as a tumour withlowgrade malignant transformations it has been reported in the liver urinary bladder kidney breast stomach pancreas spleen and retroperitoneum there is alsoa limited number of case reports in the literature indicating the gallbladder location these patients must beobserved with close and regular longterm followup asrecurrences have been reported to occur four to yearsafter surgery []acknowledgementsangela hens head of the pathology department of puerto real universityhospital for providing her pathological knowledgemario bruno for providing the new corrections in the translation of thismanuscriptpd dr med ulrich f wellner consultant surgeon pancreatic surgery andresearch clinic of surgery uksh campus l¼beck germany for providingthe latest english language correctionsauthors contributionsac the first author directed the operation and wrote the paper js hasparticiped in the design of the report and copy edited the manuscript adhas participated in the operation mc has participated in the operation gmhas made the pathological diagnosis and part of the literature review allauthors read and approved the final version of the manuscript all authors ofthis manuscript are in agreement with its content and are not beingpublished or under consideration in another scientific journalfundingnot applicableavailability of data and materialsdata sharing is not applicable to this as no data sets were generatedor analysed during the current studyethics approval and consent to participatenot applicable 0c" | Colon_Cancer |
"as metastasis is a major cause of death in cancer patients new antimetastatic strategies are needed to improvecancer therapy outcomes numerous pathways have been shown to contribute to migration and invasion ofmalignant tumors aspartate hydroxylase asph is a key player in the malignant transformation of solid tumorsby enhancing cell proliferation migration and invasion asph also promotes tumor growth by stimulation ofangiogenesis and immunosuppression these effects are mainly achieved via the activation of notch and srcsignaling pathways asph expression is upregulated by growth factors and hypoxia in different human tumors andits inactivation may have broad clinical impact therefore small molecule inhibitors of asph enzymatic activity havebeen developed and their antimetastatic effect confirmed in preclinical mouse models asph can also be targetedby monoclonal antibodies and has also been used as a tumorassociated antigen to induce both cluster ofdifferentiation cd and cd4 t cells in mice the pan3011 vaccine against asph has already been tested in aphase clinical trial in patients with prostate cancer in summary asph is a promising target for antitumor andantimetastatic therapy based on inactivation of catalytic activity andor immunotherapykeywords asph small molecule inhibitor metastasis immunotherapy cancer is a multifactorial disease with an approximate million fatalities in worldwide it is the secondleading cause of death the complex modifications inthe genome affected by the interactions between hostand environment lead to cancer development and progression despite advancements in characterizing themolecular mechanisms of oncogenesis tumor progression and metastasis delayed cancer detection limitedsurgical options therapeutic resistance and tumor recurrence are serious obstacles in decreasing the prevalence and fatality rate of cancer since metastasis is theprimary cause of deaths from cancer the design oftherapeutictarget mechanisms oftumorcell migration and invasiveness is essential in thisapproachesthat correspondence smahelmnaturcunicz1department of genetics and microbiology faculty of science charlesuniversity biocev vestec czech republicfull list of author information is available at the end of the regard a growing number of investigations of signalingpathways involving products of oncogenes and tumorsuppressor genes in human carcinomas has helped toelucidate the mechanisms underlying malignant transformation of cells and facilitated the development ofnew and more efficient therapeutic methodsaspartate hydroxylase asph has been identified asone of the cell surface proteins associated with malignant transformation of tumor cells [ ] asph belongsamong the most important biological targets to controlmigration and invasion of tumor cells as its overexpression has been observed in of human solid tumors [] the overexpressed asph is transportedfrom the endoplasmic reticulum to the plasma membrane which results in exposure of the cterminal regionto the extracellular environment where it is accessible toantibody binding recently molecular targeted therapyhas been developed against this target using small molecule inhibitors smi that can inhibit the catalytic site 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 0ckanwal of experimental clinical cancer research page of in the cterminal region moreover as antigenic epitopes that reside on the asph protein can efficientlystimulate cluster of differentiation cd and cd8 tcell responses unique to tumor cells harboring asphthis enzyme can be used as a tumor associated antigentaa in immunotherapy [ ]thedioxygenasesstructure of the asph gene and isoformsasph is a type ii transmembrane protein of approxito the family of αmately kda that belongsketoglutaratedependenthydroxylated products of asph hydroxylation were firstdetected in blood coagulation proteins [] asphwas initially identified in the bovine liver as an enzymeresponsible for catalyzing the hydroxylation of aspartyland asparaginyl residues in calcium binding epidermalgrowth factor cbegflike domains of various proteins fig thereafter the human asph gene wascloned and characterized this gene spanning base pairs long region of genomic dna and containing exons is located at the position q123 of thehuman chromosome the asph sequence is highlyconserved in mammalian evolution the sequence of thehuman protein is from about identical to the sequences of rat and mouse analogs and the catalytic siteis quite conserved among proteins of these three species the whole asph protein consists of five domainsan nterminal cytoplasmic a universal transmembranea positively charged luminal a calcium binding and a cterminal catalytic domain tissue specific transcription is directed from two putative promoters p1 and p2which differ in their regulation sequences [ ] whilethe transcription from the p1 promoter was observed inmost human tissues the p2 promoter is activated by thecalciumdependent transcription factor myocyte enhancer factor mef2 particularly in muscle tissues the asph gene undergoes extensive alternative splicingresulting in four protein isoforms ie asph humbugjunctate and junctin [ ] these proteins vary in thecterminal region which affects their function [ ]the two longest asph transcript variantsthat aretranscribed from the p1 and p2 promoters and differ inthe length of the ²untranslated region encode the fulllength asph protein this protein contains the catalyticcterminal domain that catalyzes the posttranslationalhydroxylation in the cbegflike domains of numerousproteins supplementary fig including receptors receptor ligands and extracellular adhesion moleculesthat influence cell motility and invasiveness [ ] thetruncated isoforms humbug junctate and junctin sharethe nterminal part with the asph protein but lackcatalytic function they are involved in calcium homeostasis humbug has a potential role in cell adhesionand calcium flux and similar to asph its overexpressionhas been correlated with aggressive tumorcell behavior reticulummembranebound protein that is known for its functionin the regulation of the intracellular ca2 concentrationjunctin is a structural membrane protein and as an integral part of the complex consisting of the ryanodine receptor calsequestrin and triadin influences calciumrelease from the sarcoplasmic reticulum [ ]sarcoendoplasmicjunctateisalocalization in cells tissue distribution andexpression regulationasph is predominantly a cellsurface protein that isalso localized in the endoplasmic and sarcoplasmicreticulum furthermore a recent study identifiedmitochondriallocalization of asph in hepatocellularcarcinoma hcc in that study asph overexpressioncorrelated with an instability of mitochondrial dna andmitochondrial dysfunction that may lead to more aggressive pathological outcomes in hcc asph is abundantly expressed in proliferating placental trophoblastic cells [ ] and in decidua and endometrial glands and has a potential role in placentalimplantation and fetal growth on the contrary theasph expression in normal adult tissues is relativelylow or negligible however asph expression is inappropriately activated during oncogenesis when asph is required for generation of malignant and metastaticphenotypes the elevated expression of asph at bothfig asph catalytic reaction aspartyl and asparaginyl residues in cbegflike domains are hydroxylated 0ckanwal of experimental clinical cancer research page of transcription and translation levels has been shown in awide range of transformed cell lines as well as humancarcinoma tissues including hepatocellular pancreaticcolon prostate lung breast ovarian and cervical carcinoma cholangiocarcinoma neuroblastoma and gastriccancer table the first study that demonstrated thesignificantly higher expression of both asph mrnaand protein in hcc and cholangiocarcinoma relative totheir normal adjacent tissue counterparts was by lavaissiere subsequently they verified the role of upregulated asph protein production and its enzymaticfunction in the malignant transformation on biliary epithelium the nih3 t3 cell line and animal models the level of asph also correlated with cell motility andinvasiveness in in vitro experiments [ ] in thestudy by maeda the overexpression of theasph protein was in accordance with worse clinical andhistopathological characteristics of the intrahepatic cholangiocarcinomas and prognosis of patients similar findings were obtained in other studies for hepatocellular[ ] nonsmall cell lung and colon carcinomas and glioblastoma multiforme recentlytheprognostic significance of 2oxoglutaratedependent oxygenase expression was demonstrated by analysis of expression profile datasets of tumor samples and nontumor samples asph has been identified asone of the genes which upregulated expression couldserve for risk stratification of patients with cancertypes in glioblastoma the prognostic significance ofasph was suggested by profiling of alternative mrnasplicing asph gene expression is upregulated via wntcatenin and insulininsulinlike growth factor igf1insulin receptor substrate irs1 signaling [ ]through extracellular signalregulated kinaseerkmitogenactivated protein kinase mapk andphosphatidylinositol3kinaseprotein kinase b pi3kakt pathways fig for review see ref insulinigf1irs1 signaling affects cell growth and survival andcan be involved in oncogenesis in various human tumorstable summary of the studies which have identified the elevated asph expression in human tumor tissuesstudypositive cases of studied samples nntumor tissuesdetectionmethodihcantibody recognized region ofasph proteinfb50 ab nterminusfb50 ab nterminusfb50 ab nterminusfb50 ab nterminusfb50 ab nterminus or 15c7 abcatalytic domainfb50 ab nterminusmab g3 hybridomapolyclonalfb50 mab nterminusihcihcihcihcrtqpcrihcrtqpcrihcihcihcrtqpcrihcfb50 ab nterminuslavaissiere hepatocellularcholangiocarcinomabreastcolonpalumbo pancreatic adenocarcinomasepe primitive neuroectodermalmedulloblastoma neuroblastomamaeda cantarini cholangiocarcinomahepatocellularmonte hepatocellularyang wang dong tang lin types of tumor tissuesahepatocellularpancreatic cancerhepatocellularbreast 75fold higher level of mrnacompared to normal tissue 7fold higher level of mrnacompared to normal tissuepancreatic ductal adenocarcinomaogawa aliver kidney breast cervical ovarian fallopian tube laryngeal lung thyroid pancreatic thymic prostate bladder esophagus gastric gall bladder colon andrectum cancer and cholangiocarcinomafb50 ab nterminusihc 0ckanwal of experimental clinical cancer research page of fig regulation of asph expression and asph involvement in signaling pathways the expression of the asph protein can be regulated atseveral levels the asph gene can be amplified in tumor cells and its transcription activated by inigf1 and wnt catenin pathways or inducedby hypoxia at the posttranscriptional level mir200a and mir135a can downregulate asph expression stability of the asph protein can bereduced by phosphorylation with gsk3 conversely asph can enhance gsk3 activity by inhibition of its phosphorylation with akt and p38kinases asph also supports cell proliferation epithelialmesenchymal transition migration invasion and angiogenesis and consequently tumrowth and metastasis by hydroxylation of the notch receptor and ligands ex jag and interaction with prb vimentin and adams finallyinactivation of nk cells by asph has been demonstrated green arrow activation signal red bar inhibitory signal the catenindependent wnt pathway regulatescell proliferation motility and differentiation and is oneof the most frequently modified pathways in humanmalignancies upon aberrant activation of wnt signalingcatenin is accumulated in the cytoplasm and subsequently translocated to the nucleus where an interaction between catenin and tcellfactorlymphoidenhancerbinding factor tcflef proteins forms atranscriptional regulatory complex which enhances theexpression of wnt target genes including irs1 asph was proposed as a common link between wntcatenin and insulinigf1irs1 pathways and downstream signaling the regulation of asph gene expression in tumorsmight also be affected by a copy number variation inthe study by kadota the asph gene locus hasbeen identified as one of the dna regions with focalamplification in primary breast cancer in colorectal cancer asph gain or amplification was found in ofsamples next a suppressant role of the micrornamir200a in posttranscription regulation of the asphexpression in hepatoma cells has been found mir200a belongs to mir200 family which plays significantrole in preventing cancer initiation and metastasis forreview see ref similarly mir135a has beenshown to suppress asph in endometrial cancer moreover consistent with the protein sequence analysis that recognized numerous prospective phosphorylation sites of glycogen synthase kinase3 gsk3casein kinase ck2 protein kinase a pka and protein kinase c pkc on asph several studies demonstrated that phosphorylation can regulate the asphprotein expression [ ] inhibition of the gsk3activity did not modify mrna expression but increased 0ckanwal of experimental clinical cancer research page of the asph protein level direct phosphorylation ofasph by gsk3 probably decreases asph stability andthus reduces cell mobility asph protein expressionwas also increased by inhibitors of pka pkc and ck2 mutational analysis of potential sites of phosphorylation demonstrated complex and nonuniform effects ofasph phosphorylation on protein expression enzymaticactivity and subcellular localization [ ] thereforeasph phosphorylation probably regulates the functionof this protein by various mechanismsasph expression can also be regulated by hypoxia andoxidative stress in human neuronal cells this effect wasmediated by hypoxia inducible factor alpha hif1αthat is stabilized under hypoxiaoxidative stress whenthe prolyl hydroxylase domain phd proteins and factorinhibiting hif fih are inactivated consequently thehif1 heterodimer made up of subunits hif1α andhif1 functions as a transcription factor likely enhancing asph expression by binding to hypoxiaresponsiveelements in hypoxic regions of glioblastoma bothhif1α and asph were highly expressed particularly inmore aggressive mesenchymal subtype of glioblastomasuggesting a possible involvement of asph in mesenchymal transition brewitz showed reducedasph hydroxylation activity at low oxygen concentrations and suggested an asph role in oxygen hypoxiasensing asph upregulation induced by hypoxia couldcompensate for reduced enzymatic activity moreover a recent study reported an oxidative stress state ofthe castrationresistant prostate cancer cells upon asphoverexpression which was reversed by silencing asphexpression or generating hypoxic conditions resulting inimpaired cell proliferation and invasion asph protein interactions and signaling pathwaysthe asph hydroxylation consensus sequence is confined within cbegflike domains that are found in proteins of diverse function including notch receptors andligands clotting factors structural proteins of the extracellular matrix and ligands of the tyro3axl family ofreceptor tyrosine kinases the notch signaling cascade is a remarkably conservedpathway notch proteins notch1 notch4 are singlepasscell surface receptors that mediate communication betweencells and their expression is crucial for proper embryonicdevelopment notch signaling mainly results in cell differentiation but also plays a significant role in proliferationapoptosis and the maintenance and selfrenewal of stemcells dysregulation of the notch pathway is directly linkedto cancer vascular disorders and congenital defects in mammals notch signaling activated by binding ofone of two families of canonical notch ligandsjaggedjag1 and jag2 and delta like dll1 dll3 and dll4leads to the generation of the cleaved notch intracellulardomain nicd fragment and its nuclear translocation inthe nucleus the nicd fragment interacts with the dnabinding complex csl cbf1rbpjκ suh lag1 thiscomplex is then converted from a repressor into an activator leading to increased transcription of target genes suchas hes family bhlh transcription factor hes1 heswith yrpw motif hey1 cd44 epithelial cell adhesionmolecule epcam cmyc protooncogene matrix metallopeptidase mmp29 cyclin d1 cyclooxygenase vascular endothelial growth factor vegf and proliferatingcell nuclear antigen pcna [ ]upregulation of asph results in enzymatic modification of the cbegflike repeats in the notch receptorextracellular domain and its ligands which promotes thereceptor interaction with the ligands and the activationof notch signaling [ ] furthermore the interactionof asph with a disintegrin and metallopeptidase domainadam stabilizes this complex and enhances thes2 cleavage ofthe notch receptors and subsequentnicd fragment release the activation of the targetgenes in malignant cells increases cell proliferation migration and invasion through the epithelialtomesenchymal transition emt that is probably upregulated by the interaction of asph with vimentin consequentlythis activation supports tumor growthand metastasis the asphnotch axis also stimulatesthe release of exosomes that transfer proteins involvedin invasion metastasis metabolism and immunosuppression [ ]the src kinase pathway is another important pathwayin malignant cell transformation that regulates a complex signaling network promoting angiogenesis invadopodia formation and maturation and metastasis asph has been identified as an src pathway activatoroverexpressed asph directly interacts with adam12 and strengthens the src activation by these proteinswhich promotes mmpmediated extracellular matrixdegradation and tumor invasiveness asph can also contribute to malignant phenotype ofcells by interaction with other proteins iwagami revealed the interaction of asph with gsk3 that prevents gsk3 inactivation by phosphorylation with upstream kinases this mechanism was confirmed ina castrationresistant prostate cancer model gsk3 is a multifunctional kinase that is involved in variousprocesses including glycogen metabolism cell divisionand cell fate determination some types of tumors aresensitive to gsk3 inhibitors recently huang elucidated a direct binding of asph with retinoblastoma protein prb leading to prb phosphorylation they also showed that this effect was mediated byincreased binding of cyclindependent kinase cdk cdk4 and cyclins d1 and e with prb and wasdependentasasph enzymaticonactivity 0ckanwal of experimental clinical cancer research page of phosphorylation of prb inactivates its tumorsuppressorfunction asph can contribute to the progression of cellcycle via interaction with prbeffect of asph on an immune systemtumor generation and progression are influenced bycancer immunoediting that involves immunosurveillanceand escape from a host immune system in theseprocesses various mechanisms of both innate and adaptive immunity are included immune cells that infiltrate developing tumors are initially antitumorigenicbut in tumor microenvironment they can be modifiedinto cells with protumorigenic properties as potential targets of asph hydroxylation are alsoexpressed on immune cells this enzyme could affect thefunction of immune system particularly in tumor microenvironment when asph is overexpressed on cancercells indeed such effect was demonstrated for humannatural killer nk cells by using recombinant asphwhich reduced viability and cytotoxicity of these cells viaenhancing caspase signaling and decreasing the surfaceexpression of activating receptorsrespectively antibodies against asph inhibited these effectsinteraction of asph with other immune cells has notbeen studied however we suppose possible influence ofasph on different tumorinfiltrating cells this assumption comes from the involvement of notch signaling indifferentiation and function of various immune cells fibroblasts mesenchymal cells and endothelial cells forinstance notch activation contributed to stimulation ofproinflammatoryantitumorigenic m1 polarization inboth bone marrowderived primary macrophages and tumorassociated macrophages whennotch signaling was abrogated protumorigenic m2polarization was induced even by stimulators of m1polarization mir125a has been identified as adownstream mediator of notch signaling in macrophages similarly the notch pathway plays an important role in differentiation of other types of myeloidcells and probably all subsets of cd4 and cd8 t cells different notch receptors and their interactionwith different ligands contribute to these processes moreover noncanonical notch signaling is implicatedin regulation of immune cells while activation ofnotch signaling in some cells eg t helper cells cytotoxic cd8 t cells and m1 macrophages supports induction ofincluding antitumorimmunity in other cells particularly regulatory t cellsit leads to immunosuppression thus immunostimulatory effect of notch signaling is often inhibited intumor microenvironment to enable the tumor cells toescape from the host immunity therapeutics affecting notch signaling in malignant diseases are being developed and tested in clinical trials but their effects onimmune reactionsimmune reactions and possible combination with immunotherapy have not been properly studiedasph as a therapeutic targetoncogenic abilities of asph have been experimentallydemonstrated using tumor cell lines and mouse and ratmodels of different types of human tumors with asphoverexpression including cholangiocarcinoma [ ] hepatocellular carcinoma [ ]neuroblastoma pancreatic cancer [ ] glioma breast carcinoma castrationresistant prostatecancer and colorectal cancer in studies analyzingasph function various approaches were utilized to revealsignaling pathways affected by asph particularly asphexpression was diminished by using small interfering rnas[ ] short hairpin rnas [ ] or thecrisprcas9 system [ ] the importance of asphenzymatic activity in these processes was shown by the sitedirected mutagenesis [ ] or treatment by smis [ ] in vitro assays showed asph involvement in cell proliferation migration and invasion cellularalterations included emtinhibition of apoptosis andstemness acquisition tumor growth and invasivenesscould further be supported by asphinduced extracellularmatrix degradation angiogenesis and transendothelial migration notch and src signaling are probably major pathways influenced by asph fig and contributing toincreased aggressiveness of tumor cells that was verified inin vivo models thus these studies also demonstrated thatasph is a suitable target for cancer treatment especially bysmis or immunotherapysmall molecule inhibitorssmis of asph fig have been developed and used totest the role of asph in a wide range of cancer modelsincluding subcutaneous orthotopic and patient derivedxenograft in vivo models [ ] a small orallybioavailable inhibitor has severalintrinsic advantagesover immunotherapy approaches not only can these inhibitors inhibit the catalytic activity of asph unlike conventional antibodies that simply bind to the protein butthey can also penetrate into the cell and inhibit asphcatalytic activity in the endoplasmic reticulum differentcancers have different asph expression patterns andwhile surface expression is quite common in pancreaticcancer and hepatocellular carcinoma intracellular overexpression patterns have also been observed the first asph smis published were the tetronimidesmoi500 and moi1100 tetronimides were originallysynthesized in by dahn and are redoxactivemimics of ascorbic acid and 2oxoglutarate moi500 isa mixed inhibitor that inhibits both asph and the fatmass and obesity protein fto and is not onlyorally bioavailable but also can penetrate the blood 0ckanwal of experimental clinical cancer research page of fig small molecule inhibitors of asphthereand kinasesbrain barrier moi1100 is a more potent inhibitor ofasph and is also more selective despite investigation against a wide range ofirondependent dioxygenasesare no other knownenzymatic targets for moi1100 enhanced activity wasobserved by replacing the chlorine with a trifluoromethylgroup as in moi1151 and even a greater improvement in in vivo activity was found by replacing the trifluoromethyl group with a carboxymethyl group as inmoi1182 although it is not yet clear if the nature ofthis enhancement is due to increased inhibitory activityorenhanced solubility parameters moi1182 isreported to have the ability to suppress invasive activityat a concentration of nm smis of asph have acharacteristic in vitro concentration dependent profilewhere the activity of the smi plateaus at value around viability emphasizing the noncytotoxic properties of this class of inhibitorsnatural products and inhibitors of other enzymes thathave been repurposed as asph inhibitors have alsorecently been reported in the patent literature includingbosutinibcepharanthinecn2019101414327 and guaianolides related to nortrilobolidecn2019104575886cn2019101414219cn2019101414187 0ckanwal of experimental clinical cancer research page of sesquiterpene with complex anticancerbosutinib is a wellknown inhibitor of bcrabl and srctyrosine kinases approved for the treatment of chronic myelogenous leukemia cepharanthine is a natural productactivityincluding ampactivated protein kinase ampk activationand nuclear factor kappa b nfκb inhibition nortrilobolide and related compounds are reported to be potentcytotoxic agents with subnanomolar sarcoendoplasmicreticulum calcium atpase serca inhibition recently a family of potent pyridine dicarboxylates have alsobeen published utilizing a mass spectrometrybasedinhibition assay these compounds are related toknown irondependent dioxygenase inhibitors 23pyridinedicarboxylate 24pyridine dicarboxylate and 26pyridinedicarboxylate the synthesized pyridine dicarboxylateswere assayed for activity against a range of other enzymesto include phd2 fih and lysinespecific demethylase 4ekdm4e in addition to asph with varying degrees of selectivity however while cellbased activities have not beenevaluated the dicarboxylate nature of the compounds maybe useful for cell surface asph inhibitors that may nothave cell penetrating activity immunity as a target of humoralimmunotherapyasph can be used not only as a target of the inhibitors inactivating its enzymatic activity but also as a target of immune reactions leading to destruction of tumor cells andtumor growth suppression since asph is cell surface displayed on tumor cells it represents a tumorassociatedantigen that can be targeted by both cellmediated andhumoralimmunityasph on the surface of cancer cells can be bound by antibodies that mediate antibodydependent cellular cytotoxicity adcc complement dependent cytotoxicity cdcor antibodydependent cellular phagocytosis adcp when the asph antigen is processed in tumor cells orantigen presenting cells antigenic peptides are presentedon these cells by human leukocyte antigen hla class ior class ii molecules and recognized by cd8 or cd4 tlymphocytes respectively that can be stimulated byimmunization breaking tolerance to selfantigens induction of asphspecific cd4 and cd8 t cells wasexamined in blood samples of hcc patients using synthetic peptides derived from asph after prediction of hlaclass i and hla class iirestricted epitopes it has beenfound that asph is a highly immunogenic protein that activates both types of analyzed t cells thus efficientantitumor reactions could be stimulated by immunizationthe first vaccine against asph was based on matureddendritic cells dc loaded with the asph protein andtested in an orthotopic rat model of intrahepatic cholangiocarcinoma this study showed that vaccinationstimulated cytotoxicity against cancer cells in an in vitroassay and decreased tumor growth and metastasis bothcd8 and cd4 cells contributed to an antitumor effectinduced in a mouse model of hcc by immunizationwith asphloaded dcs the next antiasph vaccine was based on a bacteriophage lambda display system the viral capsid proteingpd was fused with the n or cterminus of asph andimmunogenicity ofthese nanopforming constructs was verified in two mouse tumor models the vaccine pan3011 containing these constructs hasalready been examined in a phase clinical trial in patients with biochemically relapsed prostate cancer this study demonstrated safety and immunogenicity of pan3011 and indicated an antitumor effect interms of the reduction of prostate specific antigen psaor psa doubling time asphspecific immune responseswere mediated by both antibodies and t lymphocytesas asph is a type ii transmembrane protein its cterminus carrying the enzymatic domain is exposed outside cells and can be bound by antibodies that can be usedfor diagnostic and therapeutic purposes development ofasphspecific antibodies has been described in several s [] the human igg1 pan622 recognizesthe catalytic domain of asph this antibody is not directly cytotoxic for tumor cells but is internalized and candeliver cytotoxic moieties into cells in the subsequent study with a mouse model of metastatic breast cancerandradioimmunotherapy with promising results mouseigg1 monoclonal antibody binding to the cterminalasph domain mediated adcc by human nk cells recently a secondgeneration antibody approach hasbeen disclosed the prepared antibody binds to the extreme cterminus of asph us that is involved in specific substrate recognition thereforethis antibody has direct asph inhibitory activity anddoes not require any radioisotope or cytotoxic payloadfor potential therapeutic activitypan622 wasbioimagingusedforconclusionsasph is an important enzyme in malignant transformationof cells it stimulates tumor cell proliferation migration andinvasion but it can also affect other cells in tumor microenvironment two main pathways notch and srcthrough which asph promotes the tumor growth havebeen identified it has also been shown that asph expression is induced by some growth factors and hypoxia and isregulated at various levels the overexpression of asphand its downstream targets has been detected in numeroushuman malignancies since asph is not expressed in appreciable level in normal adult tissues and the catalytic domain is localized on the cell surface it has been proposedas one of the most exciting potential therapeutic targetsfig small inhibitory molecules orally bioavailable havebeen developed and successfully tested in several cancer 0ckanwal of experimental clinical cancer research page of fig asph as a therapeutic target asph expression is upregulated by growth factors and hypoxia its enzymatic activity can be inhibited bysmis or monoclonal antibodies which results in reduction of cell proliferation angiogenesis immunosuppression and cell migration and invasionconsequently tumor growth and metastasis are also reducedmodels but they have not yet advanced into clinical trialsadditionally as asph was identified as a tumorassociatedantigen immunotherapy approaches vaccines and monoclonal antibodies were tested with promising results in preclinical experiments and results of pha | 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 |
"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 |
laparoscopic surgery for rectal cancer is commonly performed in china however compared with open surgerythe effectiveness of laparoscopic surgery especially the longterm survival has not been sufï¬ciently provedmethods data of eligible patients with nonmetastatic rectal cancer at nanfang hospital of southern medical university andguangdong provincial hospital of chinese medicine between and were retrospectively reviewed longterm survival outcomes and shortterm surgical safety were analysed with propensity score matching between groupsresults of cases collated from two institutes matched pairs were analysed after propensity score matching the estimated blood loss during laparoscopic surgery was significantly less than that during open surgery p¼ and the operativetime and hospital stay were shorter in the laparoscopic group both p the postoperative complications rate was inthe laparoscopic group and in the open group p¼ no significant difference was observed between the laparoscopicgroup and the open group in the 5year overall survival rate vs p¼ 5year relapsefree survival rate vs p¼ or 5year cancerspeciï¬c survival rate vs p¼ an elevated carcinoembryonic antigen harvested lymph nodes and perineural invasion were independent prognostic factors affecting overall survival and relapsefreesurvivals our ï¬ndings suggest that open surgery should still be the priority recommendation but laparoscopic surgery isalso an acceptable treatment for nonmetastatic rectal cancerkey words laparoscopic surgery open surgery propensity score matching rectal cancersubmitted february revised april accepted june vc the authors published by oxford university press and sixth afï¬liated hospital of sun yatsen universitythis 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 citedfor commercial reuse please contact spermissionsoupcom 0c kl tan introductioncolorectal cancer is the second most commonly diagnosedcancer and the fifth leading cause of cancerrelated death forboth sexes in china in a multidisciplinary approach thatcombines chemotherapy with radiotherapy for the treatmentof colorectal cancer surgery remains the major approach thefirst successful use of laparoscopy in colorectal surgery waspublished in by jacobs laparoscopic surgery hasbeen performed widely in colon cancer all over the world andseveral randomizedcontrolled trials have demonstrated thatlaparoscopic surgery for colon cancer is safe and feasible withbetter shortterm outcomes including a decrease in postoperative pain a shorter hospital stay and earlier recoveryand equivalent longterm results compared to open surgery[] however laparoscopic surgery for rectal cancer is morearduous than that for colon cancer so the early clinical trialsexcluded rectal cancer [] although a few clinical trialshave shown the advantages of laparoscopic rectalcancer resection compared with open surgery [] both the acosogz6051 and alacart trials did not support the use of laparoscopic surgery for rectal cancer [ ] it is still controversialwhether laparoscopic surgery is suitable for rectal cancer especially for low rectal cancer therefore we conducted thisretrospective cohort study to compare longterm survival outcomes and shortterm surgical safety between laparoscopicand open surgery for nonmetastatic rectal cancer in thechinese population propensity score matching psm was performed for the study designpatients and methodsstudy designall consecutive eligible patients with rectal cancer were confirmed from the department of general surgery of nanfanghospital of southern medical university and the department ofproctology of guangdong provincial hospital of chinesemedicine between january and december these twocenters were members of the southern chinese laparoscopiccolorectal surgery study group demographic clinical pathologic and imaging features together with the management andoutcomes were carefully reviewed written informed consentwas acquired from patients preceding the surgical proceduresthis study was approved by the ethical committee of nanfanghospital and guangdong provincial hospital of chinesemedicine no ze2019052study subjectsinclusion criteria were patients with clinical stage iiii rectalcancer who underwentrectal cancerexclusion criteria were patients with i combined operationsextending to the surrounding an ii multiple cancers iiiemergency operation iv conversion to open surgery or vpatients who received neoadjuvant therapyradical surgery forall included cases were classified into two groups based onthe surgical approach which was either laparoscopic or opensurgery the surgical approach was decided by the individualcolorectal surgeon based on a combined assessment of clinicalendoscopic and imaging featuresdata collectiondata were collected in a prospectively maintained databasefrom clinical report forms the demographic and clinicopathological data included age gender body mass index bmi preoperative carcinoembryonic antigen cealocationoperative time estimated blood loss surgical procedure protective ileostomy tumor grade tumor stage and hospital staypreoperative cea was defined as cea measured closest to theoperation time tumor location was divided into the followingthree sections upper rectum above cm from the anal vergemiddle rectum cm from the anal verge and lower rectumbelow cm from the anal verge surgical procedures consistedof three categories low anterior resection abdominoperinealtumorfigure flow diagram of patient disposition 0claparoscopic vs open surgery for rectal cancer table baseline characteristics of the study populationcharacteristictotal cohortmatched cohortlaparoscopic groupn¼ open groupn¼ pvaluelaparoscopic groupn¼ open groupn ¼ pvalueage years mean sdgender n malefemalebmi kgm2 mean sdpreoperative cea n 14 ngml ngmltumor location n upper rectummiddle rectumlower rectumtumor stage n iiiiii sd standard deviation bmi body mass index cea carcinoembryonic antigentable operative and pathological results in matched cohorts variablesurgical procedure n low anterior resectionabdominoperineal resectionhartmanns procedureprotective ileostomy n operative time min median iqrintraoperative blood loss ml median iqrhospital stay day median iqrtumor grade n wellmoderatepoorothersharvested lymph nodes n 15lymphovascular invasion n perineural invasion n tumor deposits n postoperative complications n wound infectionileusurinary dysfunctionanastomosis leakageintraabdominal bleedingpneumoniacardiac eventreoperation n mortality n iqr interquartile rangelaparoscopic group n¼ open group n ¼ pvalueresection and hartmanns procedure tumor grade was dividedinto three types well differentiated moderately differentiatedand poorly differentiated including signet or mucinous adenocarcinoma tumor stage was based on the final pathologic report and preoperative imaging examinationoutcome measurementsthe primary endpoint of this study was overall survival osrelapsefree survival rfs and cancerspecific survival cssos was defined as the time from operation to death from any 0c kl tan figure survival curve after laparoscopic surgery vs open surgery in matchedcohortscause or the last followup rfs was defined as the time fromoperation to identified recurrence or any cause of death csswas defined as the time from operation to death due to rectalcancer the last followup was january ileus urinary dysfunctionthe secondary endpoints were operative time estimated bloodloss hospital stay reoperation postoperative complications andmortality postoperative complications were defined as woundinfectionleakageintraabdominal bleeding pneumonia and cardiac eventsintraabdominal bleeding was defined in this study as bleeding requiring transfusion or reoperation all complications within daysafter surgery were recorded postoperative mortality was traditionally defined as any death occurring within days after surgeryanastomoticstatistical analysisdata are presented as mean standard deviation or medianwith interquartile range iqr for quantitative variables withparanormal distribution and numbers with percentages for categorical variables quantitative variables were compared usingthe students ttest or mannwhitney u test categorical variables were analysed using the chisquare test or fishers exacttest the estimates of the differences in age gender bmi preoperative cea level tumor location and tumor stage between thetwo groups were performed using psm [ ]survival rates were calculated by using the kaplanmeiermethod and comparisons between groups were performed withthe logrank test to identify the prognostic factors univariateand multivariate analyses were performed using the cox proportional hazards regression model and the results were presented as hazard ratios hrs with confidence intervalscis only factors with p in the univariate analysis wereevaluated in subsequent multivariate analysis using forwardstepwise selection for os and rfs a p was regarded asstatistically significant all statistical analyses were carried outwith ibmvr spssvr statistics version resultsbaseline characteristicsbetween january and december eligiblepatients were collected from hospitals in china of patients cases were excluded among the remaining cases underwent laparoscopic surgery and underwent open surgery after psm of pairs ofpatients were successfully matched figure baseline characteristics are outlined in table before psm there were differences in age and preoperative cea between the two groups afterpsm all variables were well balancedshortterm surgical outcomesthe perioperative and pathological results in matched cohortsare presented in table the estimated blood loss during laparoscopic surgery was significantly less than that during opensurgery p¼ in the laparoscopic group the operative timeand hospital stay were shorter than in the open groupp the incidence of postoperative complications was in the laparoscopic group and in the open groupp¼ in the open group the most common complicationswere wound infection and pneumonia followed byanastomosis leakage whereas in the laparoscopic groupthe most common complication was anastomosis leakage followed by pneumonia longterm survival outcomesin the matched cohorts the median followup period was months in the laparoscopic group iqr monthsand months in the open group iqr monthsduring the followup patients died among whom diedfrom rectal cancer and had locoregional recurrence or distantmetastasis no significant difference was observed between thelaparoscopic group and the open group in 5year os vs p¼ 5year rfs vs p¼ or 5yearcss vs p¼ figure subgroup analyses for os were conducted for gender agebmi tumor location and tumor stage compared with open surgery male patients or those with an intermediate bmi to who underwent laparoscopic surgery tended to show worseos figure 0claparoscopic vs open surgery for rectal cancer figure subgroup analysis of overall survival in matched cohortstable multivariate analysis for os and rfs in matched cohortsvariableosrfspreoperative cea vs 14 ngmlnumber of harvested lymph nodes 15 vs perineural invasion yes vs nohr cipvaluehr cipvalueos overall survival rfs relapsefree survival cea carcinoembryonic antigen hr hazard ratio ci conï¬dence intervalprognostic factors for longterm survivalprognostic factors affecting survival are presented in table univariate analyses revealed that an elevated cea ngml harvested lymph nodes perineural invasion and tumordeposits were associated with poor os and that an elevatedcea harvested lymph nodes perineural invasion and lymphovascular invasion were associated with poor rfs data notshown the surgical approach laparoscopic vs open was notassociated with os hr ci and rfs hr ci multivariate analyses testified that an elevated cea harvested lymph nodes and perineural invasionwere independent factors affecting os and rfs table discussionlaparoscopic surgery for rectal cancer is commonly performedin many countries nevertheless the evidence for laparoscopicsurgery for rectal cancer is insufficient this study focused onthe longterm survival outcomes and surgical safety of patientswho underwentlaparoscopic or open surgery for nonmetastatic rectal cancer in the chinese population in this twocenter study psm was performed to make selection balance between patients treated with laparoscopic and open surgery thesix factors of age gender bmi preoperative cea level tumor location and tumor stage were used as described in the protocolthe baseline characteristics were ideally balanced between thelaparoscopic and open groupssome studies have reported similar postoperative complications and mortality between laparoscopic surgery and opensurgery for rectal cancer [ ] and other studies have reportedfewer postoperative complications after laparoscopic surgerythan after open surgery [ ] in our study there were no significant differences in postoperative complications includingwound infectionileus urinarytract infection anastomosisleakageintraabdominal bleeding pneumonia and cardiacevent between the two groups the longer operative time is often considered a disadvantage of laparoscopic surgery according to some previous reports [ ] in contrast our studyshowed that the operative time of laparoscopic surgery wasshorter than that of open surgery the clasicc trial and colorii trial both showed that hospital stay was significantly shorterin the laparoscopic group [ ] similarly our study alsoshowed that the hospital stay for laparoscopic surgery wasshorter than for open surgerywith regard to longterm survival no largescale clinical trials have demonstrated a statistically significant difference between laparoscopic and open surgery for rectal cancer thecolor ii trial indicated no statistically significant differences indfs and os between laparoscopic and open surgeries inthe corean study dfs in laparoscopic surgery is noninferiorcompared to that in open surgery for mid or low rectal cancer consistently with previous studies os rfs and css didnot differ in both groups in our study interestingly subgroupanalyses for os showed that male and intermediate bmi to kgm2 subgroups were associated with unfavorable outcomes in the laparoscopicsurgery group vs the opensurgerygroup chinese male populations have a narrow pelvis whichmight affect the visualization of and access to the deep pelvic 0c kl tan anatomy during laparoscopic surgery kitano foundthat laparoscopic surgery might affect longterm outcomes inthe highbmi kgm2 subgroup in the current study thebmi subgroup unfavorable for laparoscopic surgery that weidentified was intermediate bmi not high bmi it might be dueto lower bmi in the chinese population compared to that in thewestern population and the small proportion of patientswith high bmi in our cohort further evaluation will be neededto determine which subgroups of patients require additional attention when undergoing laparoscopic surgerylymphovascularwe evaluated several possible prognostic factors that mayinfluence survival in patients with rectal cancer including tumor location tumor stage tumor grade surgical approach preoperative cea levelinvasion perineuralinvasion and tumor deposits [] as expected our studyshowed that perineural invasion was the significant prognosticfactor affecting os and rfs perineural invasion refers to the invasion of cancer cells into any of the layers of the nerve sheatha higher grade of perineural invasion was related to local recurrence and metastasis in distant ans such as the liver lungand peritoneum all patients in this study underwent radical surgery with lymphnode dissection a minimum of harvested lymph nodes is recommended to ensure adequatestaging and oncologic resection for colorectal cancer themore lymph nodes harvested the better the prognosis [ ]in this study the average number of harvested lymph nodeswas we found that patients with 15 harvested lymphnodes had better os and rfs than those with harvestedlymph nodes several studies have shown that elevated preoperative cea was a poor prognostic factor in colorectal cancer[] in our study we also found that patients with an elevated preoperative cea had poorer os and rfsour study has several limitations first a selection biasexisted due to its retrospective design to reduce this the twogroups were matched carefully using psm second the statistical power is insufficient because the number of patients enrolled may not be sufficient after matching third data aboutadjuvant therapy after surgery were not collected which mightbe different between both groups and thus have influenced survival outcomes fourth the exclusion of converted cases mayintroduce a bias in favor of laparoscopic surgery finally thebowelrecovery data could not be exactly assessed due to thelack of records in this retrospective study therefore further research with a large population is still awaitedanydifferencesinin our study revealed the benefit of laparoscopicsurgery on shortterm outcomes including less blood lossshorter operative time and shorter hospital stay we did notfindcomplicationslaparoscopic surgery was similar to open surgery in terms ofos rfs and css for patients however male patients and thosewith an intermediate bmi in the laparoscopic group tended toshow worse os than those in the open group findings fromthis study suggest that open surgery should still be the priorityrecommendation but laparoscopic surgery is also an acceptabletreatment for nonmetastatic rectal cancer in the chinesepopulationpostoperativeauthors contributionsklt hjd zqc and tym collected and analysed the dataklt hl and rsx performed statistical analysis klt andhjd drafted the manuscript gxl and xhf performed theprocedure conceived of and designed the study and criticallyrevised all the intellectual content of the manuscript allauthors read and approved the final manuscriptfundingthis work was supported by clinical research of guangdongprovincial hospital of chinese medicine [no yn10101902]and a scientific research project of guangdong provincialacademy of chinese medical sciences [no yn2018ml11]acknowledgementsthe authors thank the patients and their families for making this retrospective study possible we also thank all theinvestigators and staff who contributed to the patientfollowup and data collection in nanfang hospital ofsouthern medical university and guangdong provincialhospital of chinese medicineconflicts of interestthe authors declare that there is no conï¬ict of interests inthis studyreferences feng rm zong yn cao sm current cancer situation inchina good or bad news from the global cancerstatistics cancer commun jacobs m verdeja jc goldstein hs minimally invasive colonresection laparoscopic colectomy surg laparosc endosc hewett pj allardyce ra bagshaw pf shortterm outcomes of the australasian randomized clinical study comparing laparoscopic and conventional open surgical treatmentsfor colon cancer the alccas trial ann surg buunen m veldkamp r hop wc survival after laparoscopic surgery versus open surgery for colon cancer longterm outcome of a randomised clinical trial lancet oncol clinical outcomes of surgical therapy study group a comparison of laparoscopically assisted and open colectomy forcolon cancer n engl j med bonjer hj haglind e jeekel i laparoscopic surgery versusopen surgery for colon cancer shortterm outcomes of arandomised trial lancet oncol fleshman j sargent dj green e laparoscopic colectomyfor cancer is not inferior to open surgery based on 5year datafrom the cost study group trial ann surg van der pas mh haglind e cuesta ma laparoscopic versus open surgery for rectal cancer color ii shortterm outcomes of a randomised phase trial lancet oncol kang sb park jw jeong sy open versus laparoscopicsurgery for mid or low rectal cancer after neoadjuvant chemoradiotherapy corean trial shortterm outcomes of anopenlabel randomised controlled trial lancet oncol zhou zx zhao ly lin t longterm oncologic outcomesof laparoscopic vs open surgery for stages ii and iii rectal 0ccancer a retrospective cohort study world j gastroenteroliii colon cancer jcog0404 a phase randomised controlledtrial lancet gastroenterol hepatol laparoscopic vs open surgery for rectal cancer fleshman j branda m sargent dj effect of laparoscopicassisted resection vs open resection of stage ii or iii rectalcancer on pathologic outcomes the acosog z6051 randomized clinical trial jama stevenson ar solomon mj lumley jw effect oflaparoscopicassisted resection vs open resection on pathological outcomes in rectal cancer the alacart randomizedclinical trial jama austin pc an introduction to propensity score methods forreducing the effects of confounding in observational studiesmultivariate behav res wang h chen x liu h laparoscopyassisted colectomyas an oncologically safe alternative for patients with stage t4colon cancer a propensitymatched cohort study bmc cancer bonjer hj deijen cl abis ga a randomized trial of laparoscopic versus open surgery for rectal cancer n engl j med lujan j valero g biondo s laparoscopic versus opensurgery for rectal cancer results of a prospective multicentreanalysis of patients surg endosc landi f vallribera f rivera jp morbidity after laparoscopic and open rectal cancer surgery a comparative analysisof morbidity in octogenarians and younger patientscolorectal dis toda s kuroyanagi h laparoscopic surgery for rectal cancercurrent status and future perspective asian j endosc surg guillou pj quirke p thorpe h shortterm endpoints ofconventionalinpatients with colorectal cancer mrc clasicc trial multicentre randomised controlled trial lancet laparoscopicassisted surgeryversus jeong sy park jw nam bh open versus laparoscopicsurgery for midrectal or lowrectal cancer after neoadjuvantchemoradiotherapy corean trial survival outcomes of anopenlabel noninferiorityrandomised controlled triallancet oncol kitano s inomata m mizusawa j survival outcomes following laparoscopic versus open d3 dissection for stage ii or mehrkhani f nasiri s donboli k prognostic factors insurvival of colorectal cancer patients after surgery colorectaldis wiratkapun s kraemer m seowchoen f high preoperative serum carcinoembryonic antigen predicts metastatic recurrence in potentially curative colonic cancer results of aï¬veyear study dis colon rectum huang qs qin hb xiao j association of tumor differentiation and prognosis in patients with rectal cancer undergoingneoadjuvant chemoradiation therapy gastroenterol rep liebig c ayala g wilks ja perineural invasion is an independent predictor of outcome in colorectal cancer j clin oncol ueno h shirouzu k eishi y study group for perineuralinvasion projected by the japanese society for cancer of thecolon and rectum jsccr characterization of perineural invasion as a component of colorectal cancer staging am j surgpathol amin mb edge sb greene fl eds ajcc cancer stagingmanual 8th edn new york springer rosenberg r engel j bruns c the prognostic value oflymph node ratio in a populationbased collective of colorectal cancer patients ann surg sjo oh merok ma svindland a prognostic impact oflymph node harvest and lymph node ratio in patients withcolon cancer dis colon rectum tarantino i warschkow r worni m elevated preoperative cea is associated with worse survival in stage iiii rectalcancer patients br j cancer huang sh tsai ws you jf preoperative carcinoembryonic antigen as a poor prognostic factor in stage iiii colorectal cancer after curativeintent resection a propensity scorematching analysis ann surg oncol konishi t shimada y hsu m association of preoperativeand postoperative serum carcinoembryonic antigen and colon cancer outcome jama oncol 0c' | Colon_Cancer |
of hydrophobic fragments into its structure allowed the preparation of waterinsoluble modified dtpa complexes21 the original substance of the modified dtpa dtpamod was synthesized in tomsk polytechnic university preparation of colloid solution dtpamod was produced using the following method a sample of modified dtpa with the mass of a0mg was quantitatively transferred to a volumetric flask of a0ml and dissolved in a0ml of nahco3 solution by heating to a0°c after that the volume was adjusted with the same solvent up to the mark in order to reduce the p size the container with suspension was heated in water to a0°c and treated with ultrasound for a0min 1tomsk polytechnic university lenina avenue tomsk russia 2tomsk national research medical center russian academy of sciences kooperativny street tomsk russia email sadkintpuruscientific reports 101038s41598020709912vol0123456789wwwnaturecomscientificreports 0cfigure a0 scheme for determining the sentinel lymph node using nanocolloid radiopharmaceuticals radiopharmaceutical sentinel lymph node detectorfigure a0 the general scheme for the synthesis of 99mtcdtpamodwhich reduced the average p radius up to a0nm the general scheme for the synthesis of 99mtcdtpamod is shown in fig a0the second type of colloids is iron nanops coated with a carbon shell of fec fig a03a these ps were obtained from the institute of metal physics urb ras ekaterinburg russia in order to impart lipophilic properties to ironcarbon ps and to increase their stability in solution in the form of a colloid a technique for preliminary deposition of anic radicals aryldiazonium tosylates adt onto the surface of these ps has been developed an effective method for the synthesis of adt followed by their application onto the carbon surface of ps was developed at the tomsk polytechnic university22 the general scheme for the synthesis of fec ps and their subsequent interaction with 99mtc is shown in fig a03bin the third type of colloids technetium99m was adsorbed on aluminum oxide powder a powder of lowtemperature cubic modification of gammaoxide al2o3 prepared from aluminum hydroxide powder aloh3 by its calcination in a muffle furnace was used the substance was synthesized in tomsk polytechnic universitya reducing agenttin ii chloride dihydrate was used in order to obtain complexes of 99mtc with colloidsgelatin powdered ph eur uspnf pure pharma grade cas number was purchased from applichem gmbh darmstadt germanymethods method for preparation of 99mtc labeled nanocells the introduction of the radioactive label 99mtc into a colloidal substance was carried out by mixing of the selected substance with the reducing agent sncl22h2o a0mgml in different ratios and then adding a a0ml of eluate of 99mtc a0mbqml to the mixtures the mixtures were incubated for a0min at a temperature of a0°c the preparation is ready for use after cooling at room temperature the reducing agent sncl22h2o was used as a hydrochloric acid solution the amount of a0g of tin chloride ii is added to the vial and a0ml of a0m hydrochloric acid hcl scientific reports 101038s41598020709912vol1234567890wwwnaturecomscientificreports 0cfigure a0 a carbon encapsulated iron nanop b the general scheme for the synthesis of fec psis then added for its preparation after dissolution the volume is adjusted with distilled water to a0ml dissolution was carried out in an inert gas argon mediumdetermination of the size of 99mtc labeled colloidal ps the determination of the size of the labeled nanocolloids was carried out by spectroscopy on a nanophox p size analyzer sympatec gmbh germany and also by a technique based on measuring the activity of the suspension before and after filtering it successively through filters with predetermined pore sizes and a0nm three samples were taken with a volume of a0μl from each initial solution and filtrates for the subsequent measurement of their activity calculations of the yield of products with different p sizes were determined according to the formulas given belowc220 avc a1avc c100 a1 a2a1 c50 a2 a3a2where avc is the activity of the initial suspension prior to filtration a1 is the activity measured after filtration through a a0nm filter a2 is the activity after filtration through a0nm filter a3 is the activity measured after filtration through a0nm filterin parallel determination of the radiochemical purity rcp of preparations by thin layer chromatography method was carried outthinlayer chromatography tlc procedure to determine radiochemical purity of 99mtcnanocolloid a0 µl of prepared sample was spotted on silicagel impregnated strip sorbfil russia a0 cm to determine scientific reports 101038s41598020709912vol0123456789wwwnaturecomscientificreports 0camount of sncl22h2o mga[sn99mtc]a atcviia table change in relative activities of the complex [sn99mtc] and 99mtc viipertechnetate content of the radiopharmaceutical sample first strip was developed using acetone as the mobile phase time of chromatography a0min in this system pertechnetate migrated with the front of the mobile phase rf to determine the colloid content of the preparations the second strip was developed using ethanolwaterammonium hydroxide as the mobile phase time of chromatography a0min in this system the 99mtcnanocolloid migrated with the front of the mobile phase rf stability the stability of 99mtcnanocolloid was studied in a0vitro by mixing of a0ml of normal serum and a0ml of 99mtcnanocolloid following by incubation at a0°c for a0h at different time points a0h a0h and a0h a0ml aliquots of complex were removed and evaluated for radiochemical purity using tlc24determination of the functional suitability of preparations for scintigraphic detection of sln a study to assess the functional suitability of new nanocolloid rps was performed in series of experiments involving white wistar male rats weighing a0g injection of rp in a dose of a0mbq was performed between the first and second fingers of the rats hind paw the animals were anesthetized with ether before the subcutaneous injection and during the scintigraphic study since the introduction the kinetics of radiopharmaceutical distribution throughout ans and tissues was recorded by a framebyframe recording for a0min one frame a0s in a pixel matrix static scintigraphy was performed after and a0h in the anterior and posterior projections in a matrix of with a set of pulses scintigraphy of animals was performed on an ecam signature gamma camera siemens germany the results of scintigraphic studies determined the percentage of accumulation of rp in the lymph node and the injection site the maintenance and participation of the animals in the experiment was carried out in accordance with the rules adopted by the european convention for the protection of vertebrates used for experiments or other scientific purposes strasbourg the experimental protocols were approved by cancer research institute biomedical ethics committee protocol number all invasive manipulations with animals were performed using inhalation or drug anesthesiastatistical analysis all mean values are expressed as idg ± sd data were analyzed statistically using methods of general statistics with a commercially available software package statistics for windows statsoft inc version results and discussionto carry out the labeling of colloids 99mtc extracted from a standard generator in the form of pertechnetate ions contained in a nacl solution was used it has a higher degree of oxidation vii in this chemical form and is not prone to complex formation a reducing agenttin ii chloride dihydrate widely used for the preparation of various compounds labeled with 99mtc to was used to reduce its valence state in order to obtain complexes with nanoscale ps25 as a result of the reaction of these components the appearance of an untargeted colloid is also possible due to the hydrolysis of excess sncl2·2h2o or the additional formation of a complex of reduced 99mtc with tin26 all this required preliminary experimental studies to establish the necessary and sufficient amount of sncl2·2h2o in the reaction mixtureduring the experiments it was found that the optimal concentration of sn ii in the composition of the reaction mixture when it interacts with 99mtc should be in the range of a0mgml table a0it was found that when the eluate with the preliminarily reduced 99mtc vii was added to the nanops the sn ii concentration introduced in the rp was csn a0mgml almost the entire amount of 99mtc has time to enter the composition of the largesize complex with tin even before its mixing with nanops this means that the sequence of the introduction and mixing of the reagents has a great influence on the labeling process especially it concerns the introduction of the sn ii solution into the reaction mixture in this connection the reduction of 99mtc with tin ii was carried out in the presence of the selected substance in this case we can observe a competitive redistribution of the radionuclide between the substance and the tin complex the technique of applying of the 99mtc label to the surface of nanosized ps is given in the previous sectionas a result of the studies reagent compositions and conditions for obtaining three nanocolloid rps were determined table a0 shows their components as well as the radiochemical purity and yield of the target colloid with p sizes of a0nmproceeding from the chromatograms it follows that the content of radiochemical impurity of unreduced 99mtc vii in the obtained preparations is preliminary tests of these preparations on experimental animals showed that accumulation in lymph nodes is practically not observed although colloids have p sizes in the required range from to a0nm scintigrams of rats obtained after subcutaneous administration of a technetium99m labeled nanocolloid based on aluminum oxide are shown in fig a0scientific reports 101038s41598020709912vol1234567890wwwnaturecomscientificreports 0ccomposition of the preparation per a0mldtpamod a0mg 99mtc a0mbq sncl22h2o a0mg n fec a0mg 99mtc a0mbq sncl22h2o a0mg n al2o3 a0mg 99mtc a0mbq sncl22h2o a0mg n colloid yield a0nm rcp ± ± ± table composition of reagents for production of technocium99 a0m nanocolloidsfigure a0 distribution of the preparation in the rat when the preparation is administered [al2o3 99mtc sn ii] a immediately after the administration of the drug b a0min after the administration c a0min after the administrationcomposition of preparations per a0mlal2o3 a0mg 99mtc a0mbq sncl22h2o a0mg g a0mg n dtpamod a0mg 99mtc a0mbq sncl22h2o a0mg g a0mg n fec a0mg 99mtc a0mbq sncl22h2o a0mg g a0mg n yield of colloid a0nm rcp ± ± ± table indicators of rcp and the yield of a colloid with a fraction of a0nm after the introduction of gelatin in the reagentsscintigrams showed that the drug remains at the injection point for a0h without significant accumulation of 99mtc in the blood of animals which indicates a strong fixation of the radionuclide on the surface of the nanocolloid along with this positive point it should be noted that accumulation of the preparation in the lymph nodes is not observed gelatin g was introduced into the reaction mixture in this connection to increase the mobility of the labeled ps and increase the speed of their movement through the lymph system matrix systems based on gelatin provide a fairly uniform distribution of the immobilized substance and in this case prevents the formation of a large tin colloid with 99mtc the results of the experiments showed that the addition of gelatin a0mgml to the reagent additionally provides an increase in the yield of the target colloid with p sizes of a0nm table a0in addition the size of these ps was determined on a nanophox p analyzer the obtained dependence of the change in the density of the distribution of the number of ps on their size constructed from the results of a threedimensional measurement of the preparations is shown in fig a0 a b c the average p size diameter is and a0nm respectivelystability test showed that complex 99mtcnanocolloid was stable in normal serum at least for a0h radiochemical purity of the tracer at the end of the experiment was ± a study of the functional suitability of the obtained radioactive colloids for the scintigraphic imaging of the sentinel lymph nodes showed that these preparations provide a good level of accumulation in the sentinel lymph nodes fig a0 table a0 displays the al2o3 99mtc dtpamod 99mtc fec 99mtc biodistribution data at different time points postinjectionthe level of accumulation of preparations in the lymph nodes is of the total injected activityconclusionas a result of the studies the composition of the reagents and the conditions for the synthesis of three nanocolloid rps were determined an experimental dependence of the change in the content of 99mtc vii impurities on the concentration of tin ii was established and its minimum amount a0mgml was determined to reach a rhp greater than in this case the yield of the target colloid with p sizes of ± a0nm is preliminary tests of the developed preparations on experimental animals showed that accumulation of rp in lymph nodes is practically not observed although the sizes of colloidal ps are in the required range increase in the speed of transportation of colloids through the lymphatic system was achieved by the introduction of gelatin in the composition a0mgml in addition there was an increase in the yield of the colloid scientific reports 101038s41598020709912vol0123456789wwwnaturecomscientificreports 0cfigure a0 change in the density of the distribution of the number of ps from their size in radiopharmaceuticals a 99mtcal2o3 b 99mtcfec c 99mtcdtpamodwith p sizes of a0nm to with radiochemical purity of the preparations of repeated studies in experimental animals have shown that all synthesized nanocolloid preparations provide a good level of scientific reports 101038s41598020709912vol1234567890wwwnaturecomscientificreports 0cstomachtime h99mtcal2o399mtcdtpamod99mtcfec ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± figure a0 distribution of the preparation in the rat with injection of suspension [al2o3 99mtc sn ii gelatin] a immediately after the administration of the preparation b a0min after the administration c a0min after the administration d a0min after the administration the numbers indicate lymph node site of preparation administrationg ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± liver ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± g ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± spleen ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± g ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± bloodmlheart ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± g ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± lungs ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± g ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± table biodistribution data up to a0h after injection of a0mbq of 99mtc in healthy male rats data represent the average value n accumulation in the sln thus the level of accumulation of rp 99mtcdtpamod and rp 99mtcfecadt in the sln is and respectively at the same time the accumulation level of the preparation based on aluminum oxide is of the total input activityreceived march accepted july references jakobsen j k sentinel node biopsy in urooncology a history of the development of a promising concept urol oncol weixler b et al sentinel lymph node mapping with isosulfan blue or indocyanine green in colon cancer shows comparable results and identifies patients with decreased survival a prospective singlecenter trial world j surg 101007s0026 beasley g m et al sentinel lymph node biopsy for recurrent elanoma a multicenter study ann surg oncol moser j et al sentinel node biopsy in melanoma a singlecentre experience with consecutive patients br j dermatol 101245s1043 buda a et al optimizing strategies for sentinel lymph node mapping in earlystage cervical and endometrial cancer comparison of realtime fluorescence with indocyanine green and methylene blue int j gynecol cancer scientific reports 101038s41598020709912vol0123456789wwwnaturecomscientificreports 0c sahbai s et al pericervical injection of 99mtcnanocolloid is superior to peritumoral injection for sentinel lymph node detection of endometrial cancer in spectct clin nucl med hoogendam j p et al 99mtcnanocolloid spectmri fusion for the selective assessment of nonenlarged sentinel lymph nodes in patients with earlystage cervical cancer j nucl med stoffels i leyh j p¶ppel t schadendorf d klode j evaluation of a radioactive and fluorescent hybrid tracer for sentinel lymph node biopsy in head and neck malignancies prospective randomized clinical trial to compare icg99mtcnanocolloid hybrid tracer versus 99mtcnanocolloid eur j nucl med mol imaging beisani m et al initial experience in sentinel lymph node detection in pancreatic cancer rev esp med nucl imagen mol schubert t uphoff j henke r p wawroschek f winter a reliability of radioisotopeguided sentinel lymph node biopsy in penile cancer verification in consideration of the european guidelines bmc urol jaukovic l et al lymphoscintigraphy and sentinel lymph node biopsy in cutaneous melanoma staging and treatment decisions hell j nucl med subramanian s pandey u shah s rangarajan v samuel g an indigenous singlevial kit formulation of human serum albumin nanocolloid for use in sentinel lymph node detection nucl med commun ruizdomnguez j m ibarzservio l garcade manuel g calaf peris© o intraoperative injection of 99mtcnanocolloid for localization of nonpalpable intratesticular tumours in ansparing surgery actas urol schauer a j specific developments in sentinel node labling using 99mtccolloids in the sentinel lymph node concept springer berlin wang y et al gasphase chemistry of technetium carbonyl complexes chem phys oconnor m k et al comparison of tc99m maraciclatide and tc99m sestamibi molecular breast imaging in patients with wang j zhang r evaluation of 99mtcmibi in thyroid gland imaging for the diagnosis of amiodaroneinduced thyrotoxicosis suspected breast cancer ejnmmi res br j radiol costa p et al scintigraphic imaging with technetium99mlabelled ceftizoxime is a reliable technique for the diagnosis of deep sternal wound infection in rats acta cir bras vera d r wallace a m hoh c k mattrey r f a synthetic macromolecule for sentinel node detection 99mtcdtpamannosyldextran j nucl med hoh c k wallace a m vera d r preclinical studies of [99mtc]dtpamannosyldextran nucl med biol skuridin v et al modified dtpa moleculebased nanocolloid radiopharmaceuticals j radioanal nucl chem filimonov v d et al unusually stable versatile and pure arenediazonium tosylates their preparation structures and synthetic applicability lett lukasz k thin layer chromatography in drug analysis crc press london skuridin v et al radiopharmaceutical drug based on aluminum oxide indian j sci technol 1017485 ijst2015v8i36 sazonova s i et al synthesis and experimental study of norfloxacin labeled with technecium99m as a potential agent for infection imaging iran j nucl med skuridin v s et al synthesis and biological characterization of 99mtclabeled ciprofloxacin pharm chem j acknowledgementsthis work was financially supported by ministry of education and science of the russian federation rfmefi57514x0034author contributionsvs conducting experimental research analysis and interpretation of the data final approval for manuscript publication vs development of the concept and direction of research analysis and interpretation of the data validation of critical intellectual content final approval for manuscript publication en development of the concept and direction of research analysis and interpretation of the data validation of critical intellectual content final approval for manuscript publication es development of the concept and direction of research experimental research development of analytical control methods for the developed kits and radiopharmaceuticals analysis and interpretation of the data validation of critical intellectual content final approval for manuscript publication ar conducting experimental research analysis and interpretation of the data final approval for manuscript publication nv conducting experimental research analysis and interpretation of the data final approval for manuscript publication rz conducting tests of the functional suitability of drugs preparation of the section evaluation of the functional suitability of the preparation by determining its pharmacokinetic characteristics and figures final approval of the manuscript for publication of the manuscriptcompeting interests the authors declare no competing interestsadditional informationcorrespondence and requests for materials should be addressed to vsreprints and permissions information is available at wwwnaturecomreprintspublishers note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliationsscientific reports 101038s41598020709912vol1234567890wwwnaturecomscientificreports 0copen access 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 license and indicate if changes 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 if material 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 obtain permission directly from the copyright holder to view a copy of this license visit httpcreat iveco mmons licen sesby40 the authors scientific reports 101038s41598020709912vol0123456789wwwnaturecomscientificreports 0c' | Colon_Cancer |
" introduction it is well known that immune system is highly specific to protect the body against various environmental pathogens the concept of conventional vaccination has overcome the pandemic situation of several infectious diseases outbreak area covered the recent idea of immunization through oral route edible vaccine is vital alternatives over conventional vaccines edible vaccines are composed of antigenic protein introduced into the plant cells which induce these altered plants to produce the encoded protein edible vaccine has no way of forming infection and safety is assured as it only composed of antigenic protein and is devoid of pathogenic genes edible vaccines have significant role in stimulating mucosal immunity as they come in contact with digestive tract lining they are safe costeffective easytoadminister and have reduced manufacturing cost preproofhence have a dramatic impact on health care in developing countries expert opinion the edible vaccine might be the solution for the potential hazard associated with the parenteral vaccines in this review we discuss the detailed study of pros cons mechanism of immune stimulation various outbreaks that might be controlled by edible vaccines with the possible future research and applied application of edible vaccine keywords edible vaccine outbreak transformation mucosal immunity adjuvants conventional vaccine probiotic 0c preproof introduction the immune system is a dynamic structure in our body that protects us from various pathogens immune system continuous tracking of molecules which circulate within the body to detect substances which negatively affect our health once the foreign body pathogens are identified the immune system attacks to neutralize them with the help of antibodies1 human immune cells are extremely complex and quickly adjusted to overcome every days challenges literarily the immune system can be described as a complex collection of cells tissues ans and process working together to prevent disease the immune system targets microanism like a virus bacteria pathogen parasitic worm toxins allergens and even sometimes own cell that show unusual characteristics as microanism rapidly evolves in a very short period the immune system has to be prepared to handle massive diversity of antigens which are commonly identified as an agent that activates immune system2 generally each molecule may be an antigen but researchers have found that carbohydrate and proteins offer the best response whereas lipids and nucleic acids are poor antigens the preproofsensitivity and the specificity of the immune system should be taken in to account for the developments of highly specific chemical and cellular tools as it is known that immune system is complex but well anized as a result this destroys or kills the invading pathogens ensuring longterm protection against pathogens and immunological memory for the body that immediately reacts to subsequent encounters with the same antigen3sometimes body immune system is unable to combat with these pathogens or microanisms may be due to the resistance causes in the microbe over the long period in which they modified their internal external structure modify receptors that present on their surface or due to the new strain that does not attack before to the human body to overcome this problem scientists develops vaccines usually contains whole microbe either killed or as a live form microbe small parts a protein molecule or toxins that mimic the diseasecausing pathogen these vaccine elements are used to activate the body's immune responses to identify kill or prevent further attacks of pathogens such as viruses bacteria fungi or other foreign bodies4 in both infectious and noninfectious disease vaccines play a crucial role to prevent it nowadays conventional approaches have successfully developed a vaccine against many infectious diseases particularly by attenuating the pathogen inactivation of microbes or by subunits of microbes still the effectiveness stability cost and safety of these current conventional vaccines remain important considerations in the production of vaccine delivery storage and availability of vaccine therefore it is important to create a new vaccine that is more costeffective and safer for the benefits of people relative to the current vaccine hence 0c preproofa new alternative approach comes into the picture nowadays that is edible vaccine refer to the use of edible plantvegetable parts or probiotics5 live microanisms as a vaccine which is taken orally as whole or parts of plantvegetable the concept of edible vaccine first given by charles arntzen in as per the new research the probiotics and plant may be genetically modified with the help of gene transfer or transformation and plant virus that contains the human pathogen proteins for the development of an edible vaccine against various lifethreatening disease such as cholera chickenpox aids malaria foot and mouth disease fmd hepatitis b and c etc some of these diseases require booster vaccination or multiple antigens to promote and retain protective immunity the plants can express more than one transgene which allows delivery of multiple antigens for repeated inoculation8 mostly the oral vaccine is associated with degradation of proteins in the stomach due to the action of the gastric enzyme and low ph and gut which restricts immune response but due to the rigid protective plant cell wall these proteins get protection from the stomach acids and enzymes as human enzymes are incapable of breaking down glycosidic bonds of plant cell wall carbohydrates9but subsequently release proteins at the gut lumen due to the action of microbial enzymes which break the plant cell wall bioencapsulation lactobacillus species preproofare considered intrinsically resistant to acid and the survival rate of probiotic lactobacilli increased in acidic condition when given with glucose10the resistant of probiotic and by encapsulation of antigensin plants stomach acid or at low ph will provide extra future prospects and tools to over come the challenges that will be faced by edible vaccines regarding its degradation in the stomach due to gastric enzyme now fda has approved plant cells for costeffective production of protein drugs pds in large scale current good manufacturing practice cgmp hydroponic growth facilities11 in freezedry lyophilized plant cells proteindrugs pds are more stable at ambient temperature for a longer period and maintain their folding and efficacy11 outbreak of diseases an outbreak occurs when several cases of a particular disease increase more than expected in a specific region over a specific period in the last three decades numbers of highly transmissible or pathogenic infectious disease like zika ebola monkeypox sars measles polio cholera diphtheria cases have increased in many parts of the globe12 this trend further continues with zoonotic spillover events expected to continue as a result of population expansion or overcrowding of cities unhygienic condition pollution and migration to uninhabited areas and effect of global warming on vector distribution due to the presence of these factors it decreases the ability of the individual to combat these diseases in recent 0c preproofyear's demand for pharmaceutical products vaccine increase over the world to treat these diseases however some counties are unable to manufacture these vaccines due to poor infrastructure or buy these vaccines at higher prices in these countries the edible vaccine may be a better option to combat diseases as edible vaccines are cheap and stable for a longer time table criteria for the selection of plant or plant part for the development of an edible vaccine edible vaccines are the form of medication taken as food by the humans and animals to boost their immunity thus the criteria needed in selecting plants for the production of edible vaccine must meet specific requirements the far most important criteria of selecting plant is its life cycle13 it shouldnt be longer as if it takes long time for maturation then their production and maintenance cost increase the next criteria are the choice of plant for antigen incorporation which should not contain any biomolecule that will interact with it next many plants have been identified and studied for the edible vaccine which was transformed to express antigen for rotavirus gastroenteritis cholera autoimmune diseases and rabies moreover several experiments have used vegetable potato but potatoes may not be the ideal choice for edible vaccines since frying or boiling will degrade certain antigenic proteins certain foods such as bananas tomato carrots peanuts corn and tobacco have a more promising potential as edible vaccines as it can be eaten raw not only because they are commonly available but since genetic engineering is efficiently developed these kinds of vegetable plants the following plant list contains edible vaccines previously studied in animals and which are required to be approved in both human and animal use potato preproofimportant one is the stability of the antigen under high temperature as all vegetable or plant parts are not eaten raw eg potato rice pea sometime they are boiled or fried at higher temperature plant and plant parts that can be consumed raw are preferable for human immunization however banana might be the ideal source of edible vaccines because they are consumed raw even by infants and are a major crop in many developing counties8 the one disadvantage of both banana and tomato is the low protein content of the fruit which might limit the amount of antigen that can be expressed13 in contrast seeds of the plants such as maize and soybean have higher protein content and have been reported to express very high level of foreign proteins edible vaccine from the plant source 0c preproofmason et al was the first person who conducts the vaccinebased assay produced in potato solanum tuberosum to fight against ltb stain produced by e coli in mice14 in that same year in rats and human volunteers in the same year the effectiveness of the antigens produced from potatoes solanum tuberosum towards the nontoxic subunit of vibrio cholera endotoxin and the norwalk virus capsid pathogen was identified in rats and human volunteers1215thanavalas'group proposed in that potatoes could play a role in human hepatitis b as an oral reinforcement since injectable vaccine cause redness swelling or itching at the site of administration also the edible vaccine for the animal has now been developed to replace the injectable vaccine for animal protection12 tomatotobacco model species for the edible vaccine1 cherry tomatillos tomato solanum lycopersicum an appropriate candidate for vaccine development for coronavirus that causes a highly acute respiratory syndrome sars for the development of recombinant sarscoronavirus cov vaccine sspike protein sprotein and its truncated fragment are considered as the best choice912 the genome of tomato and tobacco is incorporated with nterminal fragment of sarscov protein s1 used to develop the safe effective and inexpensive vaccine when these plantbased vaccines for sars give to mice shows significant increase level of sarscovspecific iga after oral ingestion of tomato expressing s1 protein whereas tobaccoderived s1protein indicate the presence of sarscovspecific igg detect by elisa analysis and western blot16tobacco is not an edible plant but play a major role in the development of the vaccine as it is used as a proofofconcept preprooffor hbsag gene of hepatitis b lines of transgenic cherry tomatillos have been grownthe expression of genes was seen through the whole plant but it was maximum in the fresh leaves weight of ngg and with fresh fruit weight of ngg12 lettuce lactuca sativa express the bsubunit of the thermolabile protein of e coli responsible for both human and animal enteric disease show the possibility of this vegetable as an edible vaccine in the typical swine fear hog pest virus glycoprotein e2 was expressed by lettuce in poland the transgenic lettuce that shows effect against hepatitis b virus is in the development stage17 soybean in the study e coli bacteria bsubunit of thermolabile toxin expression was performed in the endoplasmic reticulum er of soybean glycine max which yielded a total antigen level 0c preproofof up to of the total soybean seed protein without any problem during drying for further processing moreover when this protein is given orally to rats leads to a rise in systemic igg and iga1819 algae chlamydomonas reinhardtii green algae has been used as a tool to achieve a large number of proteins specific to both animal and humans for therapeutic purpose1819the use of algae for the production of vaccines is optimistic as algae have a very high growth rate the entire system can be used as a raw material for the development of edible vaccines besides to facilitate the already rapidly growing algae can be cultivated in bioreactors c reinhardtii contains one chloroplast which facilitates the stability of the desired antigens in the algal line notably the effectiveness of algal vaccines after lyophilization is unchanged which might based on the expression of the capsid protein norwalk virus the transgenic plant was developed protein deposition in the unripened fruit with a lower accumulation in red fruit was reported up to of soluble protein expression in seeds allowed the storage of antigenic peptides thus creating a plant with a high yield of proteins with an average protein content of about which would preclude intensive purification procedure by pharmaceutical industries1220in addition to the expression of hemagglutinin protein h a pa against rinder pest virus rpv pea plants were used the total soluble protein level of expression in leaves was observed to be determined by western blot even more preproofpromote global delivery of edible algae vaccine12 pea studies are also required to improve the expression of a protein in transgenic peas banana in banana plants hbsag expression was reported with four distinct cassettes pher phb pefeher and pefehbs at the different level expression of hbsag were studied with pcr reverse transcription pcr and southern hybridization method expression levels reached a height of ngg in the plant and the antigen was found in banana leaves however because of the long period required the shrub needs to grow the use of this vaccine was denied21 papaya in a papaya carica papaya vaccine was developed to counter cysticercosis caused by taenia solium by expressing synthetic peptides in transgenic papaya clones vaccine was tested in rats with an immunogenic response of per cent in vaccinated animals these 0c preproofedible vaccines may offer good relief both in humans and in pigs which are the two major carriers of the disease22 carrot in an experiment carrot along with a thaliana was utilized to develop an edible vaccine for surface hiv antigen expression and in the study it was reported that rats showed more positive effect compared to those nontreated animals25 carrot daucus carota has a positive effect in the treatment of hiv not only because carrots are nutritious and tasty but because of carrot main chemical constituent carotenoids which on consumption by rats increases monocytes lymphocytes and other immune defence thus people with a weakened immune system might benefit from the use of this potential edible antihiv vaccine26 the efficacy of this antihiv vaccine must be confirmed by a clinical trial in it has been reported that the ureb subunit of helicobacter pylori was used in transgenic carrots as a potential vaccine transgenic carrot expressing the b subunit from e coli thermolabile toxininduced iga and igg production and occurred at the intestinal and systemic level in the rat2728 rice preproofa research in found that transgenic rice oryza sativa plants expressing the b subunit of e coli induces significant number of antibodies to this subunit in the same year an immune response was found to be caused in chicken by transgenic rice that is a result of the vp2 antigenic protein from infectious bursitis in pcr and southern blot analysis confirmed the functional expression of hbsag in rice seeds2930 in addition transgenic rice was developed in in parallel to express the subunit b of the e coli thermolabile toxin used to convert plant cells using bioballistic approach pcr verified the expression india and china both are the world two biggest rice producer and have the capability to export these modified rice vaccine plant all over the globe31 selected gene encoding antigen genes from a pathogenic anism bacteria viruses or parasites that have already been identified and for which antibodies are easily accessible can be controlled in a twofold fashion in one scenario the whole structural gene is inserted inside the functional elements of the ' to ' plant transformation vector allowing the transcription and aggregation of the coding sequence in the plant32 for the second scenario the epitope is identified within the antigen dna fragment encoding may be used to create genes through fusion with coated protein gene with the plant virus eg cmv or tmv then the recombinant virus is then introduced into suitable healthy plants which produce several new plants 0c preprooftable method for the transformation of genedna into selected plants essentially there are two types of plant transformation methods but many other approaches have also been utilized to transformation vectorplasmid carrier system agrobacterium tumefacien mediated gene transfer agrobacterium a soil bacterium naturally occurring has been used to transfer a small fragment of dna into the plant genome and is called transformation5051 in this method the appropriate recombinant dna is inserted into the tregion of disarmed tiplasmid of agrobacterium tumefaciens plant pathogens which is cocultured with the plant cells andor the tissue that will be transformed the insertion of the exogenous genes and the infection of such a plant tissue into sufficiently modified agrobacterium tdna cell contributed to studies of the stable gene incorporation in the genome of the plant as well as transgenic protein production32 however this technique is sluggish and yield are lower but the application of this transformation is first limited to tobacco plant and too few other plant species which extend to most preproofvegetable species including leguminose and graminae microprojectile bombardment biolistic method this is a sophisticated method based on the microprojectile bombardment the selected dna sequence is precipitated on microparticles of metals eg tungsten gold and bombarded by a particle gun at an accelerated rate toward selected plant tissue5253 these metallic microparticles penetrate the cell walls and the exogenous dna is emitted into the cell where it is integrated into the nuclear genome through a process known for the photosynthetic role of cytoplasmic anelles called chloroplast comprising chlorophyll particle gun shoots adequately charged metallic particles with selected and processed dna which penetrate chloroplast and merge with its genome32 transformation of the chloroplast is an effective alternative to nuclear transformation5455 pds in chloroplast are more stable when plant cells lyophilized and when preserved at ambient temperature therefore the freezedrying method improves pds concentration and prevents bacterial contamination11 electroporation in this method dna is introduced into the cells to which the electrical pulses of high voltage are released which are intended to create transitory pores in the 0c preproofplasmalemma it requires the extra effort to weaken the cell wall as it serves as an efficient barrier to the entrance of dna into the cell cytoplasm32 mechanisms of action of edible vaccines for mucosal and peripheral immune figure response mucosal and peripheral immunity a critical issue for oral vaccination immune response to the vaccine is affected by the route of immunization the form of antigen and the active content of vaccine mediate specific tissue tropism there is now substantial evidence supporting the existence of at least two immune systems a peripheral immune system and a mucosal immune system20 these systems operate separately and simultaneously in most species including human protective immunity acquired during recuperation is usually referred to as systemic immunity but the fact is that it might be dominated by an incomplete form of immunity dictated by a specific pathogen as the systemic immunity might be a combination of mucosal and peripheral immunity lymphocyte traffic patterns regulated by selective expression of adhesion proteins in peripheral or mucosal lymphatic tissues affects the outcome of an immune response for example the same antigen may produce qualitatively different immune responses in lymph nodes spleen or peyer's patches the antigens in the lymph are presented over the fixed antigenpresenting cells in lymph nodes results in peripheral immunity characterized by the appearance of specific igg in the blood the antigen in the blood is presented in strategic preprooftissue interface in the spleen this also results in peripheral immunity however the microenvironment of the spleen is somewhat more complicated as it also accommodates circulating antigenpresenting cells and immunoreactive t and b cells from other tissues committed to either peripheral or mucosal immunity triggers of antigen in the lumens of enteric ans presented on payer's patches commitment to mucosal immunity characterized by the release of specific iga into the secretions2056 the mucosal surfaces are a popular site for delivering therapeutic small molecules due to the ease of administration and speed of uptake across the large surface areas efficacy of the mucosal route of immunization is largely based on the fact that mucous membranes constitute the largest immunogenic an of the body this interface is endowed with the wellanized lymphatic structure called malt mucosaassociated lymphoid tissue which constitute t and b cells innate and adaptive arms of the immune system oral vaccines stimulate the generation of immunity in gutassociated lymphoid tissue galt which includes lymph nodes payer's patches in which lymphocytes are the major component 0c preproof are b cells while are t cells and isolated lymphoid follicles in the gastrointestinal tract git a significant hurdle impacting protein delivery to the git that the antigens are rapidly degraded within the harsh environment of the digestive tract is the remarkable challenge for vaccine development it will also be important to consider the characteristics of the git in which several factors including proteolytic enzymes acidic ph bile salts and limited permeability that may hinder the induction of a protective immune response20 mucosal immunity system described above have a clear image that induction of mucosal immune response starts with the recognition of an antigen by specialized cells called mcells located in the mucosal membranes of lymphoid tissues such as peyers patches within the small intestine57 then the apc internalize and process the antigen as soon mcell channel antigens into the underlying tissues causing activation of cd4cells57 which leads to the pathogen this whole process is elicited in figure the antigen bioencapsulation by the plant cell which avoids degradation and conformational alterations and the enhancement of mcells uptake of the conjugation of the vaccine antigen with specific ligands will overcome the challenge faced by the conventional vaccine for mucosal immunity stimulation458 second generation edible vaccine figure the secondgeneration edible vaccines are multiplecomponent protective vaccines against multiple pathogens which can produce more than one antigenic protein by crossing two cell lines containing different antigens in the same plant the adjuvant is coexpressible with the same antigen a trivalent edible vaccine against cholera enterotoxigenic escherichia germinal center development bcells maturation and class switching to iga through cd40cd40 ligand interaction and cytokine secretion the antigenic epitopes present on apc then activate bcells with the help of tcells12 due to the expression of chemokine hormone receptors like cxcr5 or cxcr10 the bcells migrate to the mesenteric lymph nodes where they mature into plasma cells and finally migrate to the mucosal membrane and differentiate into plasma cells causing secretion of dimeric and polymeric iga12 on passing through the mucosal epithelial layer toward the lumen the iga molecules complex with membranebound secretary components to form secretary iga siga transported into the lumen the siga interacts with specific antigenic epitopes and neutralize the invading preprooffigure 0c preproofcolietec and rotavirus are the examples that could effectively initiate an immune response59 factor affecting the efficacy of an edible vaccine antigen loaded in the specific plant tissue is the principle of edible vaccines thus the efficacy and the potency of the vaccines are significantly affected by the nature of adjuvants adjuvants are the biomolecules lectins saponins that do not exhibit immunogenic response but potentiate the immune response when coadministered with an antigen adjuvants can improve immune and potentiate responses by acting as a depot to guide antigens to relevant sites protect them from degradation control release and activate apcs20 immunogenically inert biodegradable adjuvants like lipids proteins starch polysaccharides or polyesters act as the delivery vehicles for efficient availability at antigen presenting cells apcs there are major two methods to associate antigen and particles ie encapsulated by the particle by entrapment and linked to the surface by chemical conjugation or physical adsorption the choice of carrier particles is critical to maximizing the bioavailability of its complex with antigens encapsulated antigens are afforded protection against extracellular proteases at the time of transport to the target immune responsive sites20 preproofthe erosion of the carrier particles leads to the exposure of antigens to apcs including dendritic cells dcs macrophages and monocytes thus a fine balancing act is needed to avoid overprotection of the encapsulated antigen inhibiting the release of the therapeutic or premature release if protection is inadequate the above case leads to the reduced bioavailability that weakened the host immunity alternatively the conjugation or linked to the surface by chemical bonding or physical adsorption have efficient therapeutic action as it can facilitate delivery too specific immune responsive sites or cells the structures like liposomes virus like particles vlps virosomes and immunostimulatory complexes iscoms are well recognized by apcs because they have characteristics including size shape and surface properties that are similar to viral and bacterial pathogens that the immune system has evolved to attack20 beyond the factors regarding adjuvants and vehicles for the efficacy of an edible vaccine these are some factors that significantly affect the efficacy of edible vaccines \uf0b7 antigen selection \uf0b7 efficacy in the model system \uf0b7 choice of plant species \uf0b7 delivery and dosing frequency 0c preproof\uf0b7 release patterncontrolled and sustained \uf0b7 public perceptions and attitudes to genetic modification the advantage over the conventional vaccine figure due to the use of the oral route the administration of the edible vaccine is less complicated than the conventional methods that are given through im sc and intradermal thus it removes the needs of trained medical personnel and decreases the risk of infection as there is no need for the sterilization of premises and the manufacturing areas \uf0b7 such oral immunization will become a practical key strategy for effective disease prevention in lowincome countries in general \uf0b7 processing purification sterilization packaging or delivery does not require rigorous structure in edible vaccines minimizing longterm costs in relation to conventional vaccines32 \uf0b7 maintenance and distribution of edible vaccine are easier than the conventional vaccines as it enabling the preservation without the constant cold chain storage \uf0b7 improved storage possibilities for edible vaccines become possible as transgenic plant seeds have lesser moisture content heat stable and can quickly be dried6032 \uf0b7 a plant containing therapeutically active edible vaccine protein is free of toxins pathogens and do not have the risk of proteins to reform into the infectious anism8 \uf0b7 improved compliance is particularly related to children's who refuse to take injections preproof\uf0b7 most specifically in this case the immunity is activated on the mucosal surfaces of the gi including those which are the first line of defence on the mouth mucosal of the vaccine immunity \uf0b7 sophisticated equipment and machinery are not required in edible vaccines since they can easily be cultivated on rich soils and are costeffective relative to fermenters where the recombinant cell is cultured in a controlled manner32 limitation beyond the several advantages and the convenience over the conventional vaccines edible vaccines have certain challenges that have to be overcome for efficient and pure beneficial edible vaccination some of them are pointed below 0c preproof\uf0b7 uncertainty in calculating the appropriate oral dosage which might take multiple rounds for a patient to get the effective therapeutic action and raises the final expense of its application32 \uf0b7 the concentration of peptide or protein of edible vaccine varies from generationtogeneration planttoplant and fruitto fruit32 \uf0b7 patientrelated factor such as patient age and weight also affected the dose to be administered \uf0b7 repeated intakes of these antigenbearing plants which stimulate the immune system might over stimulate the immune system itse | Colon_Cancer |
"dysregulation of bcl2 is a pathophysiology observed in haematological malignancies forimplementation of available treatmentoptions it is preferred to know the relative quantificationof bcl2 mrna with appropriate reference genes for the choice of reference genesi reference genes were selected by assessing variation of genes from rnaseq datasets of haematological malignancies followed by filtering based on their go biological processannotations and proximity of their chromosomal locations to known disease translocationsselected genes were experimentally validated across various haematological malignancy samples followed by stability comparison using genorm normfinder bestkeeper and reffinderii commonly used reference genes were obtained from literature through extensive systematic review levels of bcl2 mrna was assessed by qpcr normalized either by novel reference genes from this study or gapdh the most cited reference gene in literature andcompared the analysis showed ptcd2 ppp1r3b and fbxw9 to be the most unregulatedgenes across lymphnodes bone marrow and pbmc samples unlike the reference genesused in literature bcl2 mrna level shows a consistent higher expression in haematologicalmalignancy patients when normalized by these novel reference genes as opposed togapdh the most cited reference gene these reference genes should also be applicable inqpcr platforms using taqman probes and other model systems including cell lines and rodentmodels absence of sample from healthynormal individual in diagnostic cases call for carefulselection of reference genes for relative quantification of a biomarker by qpcrbcl2 can beused as molecular diagnostics only if normalized with a set of reference genes with stable yetlow levels of expression across different types of haematological malignanciesintroductionoverexpression of bcl2 bcell lymphoma a mitochondrial membrane protein has beenobserved in several haematological malignancies due to genetic and epigenetic mechanismsa1111111111a1111111111a1111111111a1111111111a1111111111open accesscitation dwivedi n mondal s p k s t ssachdeva k bathula c relativequantification of bcl2 mrna for diagnostic usageneeds stable uncontrolled genes as reference one e0236338 101371 pone0236338editor pedro v baptista universidade nova delisboa portugalreceived may 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 pone0236338copyright dwivedi 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 pone0236338 august one 0cfunding the study is funded by glue grantscheme number btpr23078med2912532017by department of biotechnology httpdbtindiagovin govt of india awarded to sd and md thefunders had no role in study design data collectionand analysis decision to publish or preparation ofthe manuscriptcompeting interests the authors have declaredthat no competing interests existbcl2 molecular diagnostics with novel reference genesresulting in evasion of apoptosis giving the malignant cells a longer life span and survival benefits at times of nutrient deficiency hypoxia and growth factor deprivation [] estimationof level of bcl2 along with other antiapoptotic genes are essential to avail efficient treatmentoptions by rchop regimen of cyclophosphamide doxorubicin vincristine and prednisone and rituximab or venetoclax in different haematological malignancies [ ] byvisualization of chromosomal aberrations using karyotyping or fish fluorescence insituhybridization bcl2 levels can be inferred indirectly detection of expression of bcl2protein by immunohistochemistry a standard pathological testing procedure for dlbcl hasnot been adopted in the clinics for bone marrow tissues of liquid cancers due to sample inconsistency and challenging procedure of capturing low concentrations of biomarkers western blotting for the very nature of the method cannot be adopted for high throughputpathological testing elisa for detection of bcl2 in human plasma remains limited sinceonly one splice isoform of the mitochondrial membrane protein is available in soluble formthus bringing down the effectiveness of the assay bcl2 at the mrna level can be determined without ambiguity by next generation sequencing nanostring and microarray though increasing time and expense of pathological testing in clinical trials relative quantification by qpcr quantitative polymerase chain reactioncan be successfully used due tothe availability of appropriate controls in untreated or normal groups [ ] although beingtime and costeffective it suffers misinterpretation in pathological setting since the relativequantification depends only on the rg reference gene used due to the absence of normalsamplesnormalization with a rg which shows varying expression across samples can often lead towrong s as seen with the use of glyceraldehyde3phosphate dehydrogenasegapdh as rg in gene expression studies of pulmonary tuberculosis and cd8 tcellsunder inactivated or activated condition similarly abl protooncogene abl1 therecommended rg for gene expression studies with leukemic patients was found to haveextremely low expression in neutrophils making it unsuitable as rg for the specific casesuch discrepancies have prompted researchers to analyze gene expression across multiple tissues or pancancer database like tcga to propose normalization factors using multiple rg candidatesthis study through a systematic review of literature in haematological malignancies concluded that mostly conventionally used housekeeping genes are still being deployed s1table and s1 fig despite their varied expression based on cell type developmental stage andexperimental conditions with rare exceptions [ ] none of the genes thus identified could be used to relatively quantify bcl2 as molecular diagnostics since compared to thefpkm fragments per kilobase of transcript per million mapped reads value of the antiapoptotic genes across databases s2 fig most of the rgs from the literature are not onlyhigher but also varied significantly s3 and s4 figs with few exceptions inspired by genomewide search for rgs from publicly available rnaseq or microarray data in human and otheranisms [] we report here a set of novel candidate rgs obtained from an unbiasedsearch of genes in haematological malignancies to be used to normalize bcl2 andother antiapoptotic genes in qpcr as molecular diagnosticsmaterials and methodsethics statementthe study was performed in compliance with ethical practices and was approved by narayanahealth academics ethics committee narayana health hospitals ethics approval numbernhhaeccl2017152a one 101371 pone0236338 august one 0cbcl2 molecular diagnostics with novel reference genessystematic review of commonly used rgsliterature search was carried out in pubmed databasepubmed as detailed in s5 figaccording to prisma preferred reporting items for systematic reviews and metaanalysesguidelines selection of stable genes proteincoding genes identified from publicly available datasets table using ensembldb annotation package within r statistical software were categorised into four quartiles based on their median expression values across all samples geneswith median expression in middle two quartiles q2 and q3 in all datasets were consideredas q1 and q4 representing extreme ends of the expression spectrum are not preferred as rgcandidates for normalization of molecular diagnostic markersto determine the stability of a gene following statistical measures were employedi cv �xsx where �x and Ïx are mean and standard deviation of a variable x respectively and ii normality pvalue as measured by shapirowilks test where a pvalue less than signifies thatthe distribution is away from normal cv although used most frequently isnt a robustmeasure as it is affected by outliers to solve this a third parameter was used mad medianabsolute deviation medianjx 00 xj where x is the median of x after normalization withmedian mad is a better measure for understanding the spread of the distribution as itdepends on medians a parameter less prone to deviations by outlierslow or comparable statistical variation across samples represented by low values of cvand mad and a normal distribution high value of normality pvalue or low values of pvalue are characteristics of an ideal rg therefore genes with median expression values inmiddle quartiles q2 and q3 were shortlisted and clustered based on their cv mad and pvalue normalized to their respective zscores using pam partitioning around medsalgorithm required optimal number of clusters was calculated using silhouette graphicalmethod for each tissue sample the gene cluster with the lowest med value of parameters was selected and the genes at the intersection of the four clusters were shortlisted the list was further filtered by analysing and eliminating genes based on stop words in theirgo gene ontology annotation such as transcription factors nuclear receptor or other nuclearlocalization dna binding activity response to external stimuli translational and transcriptionalactivation since genes with such characteristics regulated by environmental conditions areunsuitable as rg candidates next genes were ranked in ascending order of their mean euclidqffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffifficv þ mad2 þ ð1 00 pþ2ean distance d ¼all parameters replaced by their zscores in thisthreeparameter hyperspace for each dataset average of d across four datasets was taken to calculate the mean euclidean distance �d genes with �d median were selected for furthertable list of rnaseq databasesdatasetdiseasetcgalamlamltargetaml paediatric amlgdcdlbcdlbclmmrfmmmultiplemyeloma� both primary and recurrent tumor only 1st visit recordstissuebloodbonemarrowlymphnodesbonemarrowsamples n sourcedownload location� tcga research networkwwwcancergovtcgaschmitz multiple myeloma researchfoundationgdccancergovaboutdatapublicationsdlbclresearchthemmrf fpkm data for gdcdlbc dataset was available as log2 transformed normalized value which was converted to fpkm101371 pone0236338t001 one 101371 pone0236338 august one 0cbcl2 molecular diagnostics with novel reference genesanalysis locus of genes associated with pathogenic translocations were identified [ ] andcandidate rgs in close proximity of such loci within bands in the same arm of chromosomewere eliminated by an automated method further only genes with nonzero fpkm value in allsamples from four datasets were retained then each gene was given a composite quartile ranking cqr the sum of quartile indices from each dataset and genes with cqr value median expression in 2nd quartile in at least two datasets were shortlisted s6 figdesign of primersbcl2 primers bcl2 has two known splice isoforms membranebound bcl2α and aless studied soluble bcl2β lacking the transmembrane domain at the cterminal most reported primers amplified only bcl2α or larger amplicon s2 table hence new primers were designed table rg primers primers for shortlisted genes were designed table s3 table using primerbank and idt sample detailsrna was isolated from peripheral blood or bone marrow samples from patient or normalindividuals s7 fig with their informed consent ethics approval number nhhaeccltable primers details of rgs and bcl2primeracy1accession nonm_000666ankrd26nm_014915jmjd4nm_001161465ptcd2nm_0247545ppp1r3bnm_024607fbxw9nm_032301nanpnm_1526673plekhm3nm_0010804753tsga10nm_025244nat1nm_001160174ric8bnm_018157gapdhnm_0012897453bcl2nm_0006572sequence fw 'cactgacaaccgctatatccgrv 'ctcatgcagccgttcatcgtfw 'tctcggcaagatccacaaagcrv 'aatgtagagccgtcctgttcafw 'gtctgtcaatgtctgtgggagrv 'caggtgtgtgtcgcagagt3'fw 'tatgggacactgcacatcac3'rv 'ggctgaccatcctcttgttta3'fw 'agaacctcgcatttgagaagac3'rv 'tctgaaccggcataagtgtcc3'fw 'tagggcggtgcgatgattc3'rv 'cggattttggcggactgaga3'fw 'ggtccgcctacttctattaacg3'rv 'tctctgctctccacctacaa3'fw 'gatgatatcagcccagccttag3'rv 'ggacttcctggatcccataaac3'fw 'tactcagcgacaccttgctaa3'rv 'ccagatcattgagggttccac3'fw 'gggagggtatgtttacagcac3'rv 'acatctggtatgagcgtccaa3'fw 'atagtgttcaacagtcagatggc3'rv 'gcaagcgcaagtcaaagca3'fw 'tcgacagtcagccgcatcttcttt3'rv 'gcccaatacgaccaaatccgttga3'fw 'ggaggattgtggccttcttt3'rv 'gcccaatacgaccaaatccgttga3'fw forward primer rv reverse primer101371 pone0236338t002amplicon length bptm Ëcamplification factor one 101371 pone0236338 august one 0cbcl2 molecular diagnostics with novel reference genes2017152a subjects with hepatitis bc or hiv and pregnant or lactating women wereexcluded from the studypbmcbmmc peripheral blood mononuclear cells bone marrow mononuclear cellswere separated by layering of blood or bone marrow diluted to with 1x pbs gibco¢germany above ficollpaque plus histopaque himedia india followed by centrifugation at rcf for mins with brakes off resultant buffy coat was washed twice with 1x pbs andonce with 1x penstrep himedia india before culturing at cell density of to millioncellsml of rpmi himedia india with fbs gibco¢ germany brazil origin and1x penstrep for subculturing the lymphocyte populationrna cdna and qpcrfrom ffpe formalinfixed paraffinembedded blocks curls were deparaffinized inxylene at Ëc followed by proteinase k himedia india treatment prior to rna isolationeither from lymphocytes or from deparaffinized retrospective samples rna was isolatedby trizol¢ ambion us method and quantified with qubit rna br assay kit thermofisher scientific us before converting to cdna using superscript iv ssiv thermo fisherscientific us as per manufacturers instructions with notemplate control ntc qpcrwas performed in triplicates for each sample using kapa sybr green universal reagentssigma aldrich us cdna dilution and primers in a 5μl reaction mix qpcr condition preincubation at Ëc for minutes followed by amplification for cyclesdenaturation at Ëc for sec amplification at Ëc for sec and extension at Ëc for sec inroche lightcycler ii machineoptimization of primersprimers were optimized for qpcr as required by the miqe guidelines all primers wereused at four different final concentrations forwardreverse 200nm200nm 200nm100nm100nm200nm and 100nm100nm with pooled cdna template obtained from six normalhealthy volunteers to yield single amplification product primer efficiency was checked using atwofold fivepoint dilution of the template primer efficiency was obtained from standardcurve using the formula amplication factor ¼ 00� table ��slope 00 stability analysis of candidate rgsmean of cq quantification cycle of ntc were subtracted from cq values of each gene inqpcr experiments to obtain δcq cq samplemean cq ntc and relative expression aseδcq for each replicate where e is the amplification factor of corresponding genestability of expression of the candidate rgs was analysed using three independent algorithmsgenorm normfinder and bestkeeper and the webbased reffindertool that integrates all three algorithms plus the delta ct method algorithm genorm wasrun using the slqpcr r package whereas authorsupplied r package and excel worksheet were used for normfinder and bestkeeper analysis respectively mean cq values foreach gene for all samples were used as input for bestkeeper and reffinder whereas fenorm and normfinder relative expression values were used since normfinder uses amodelbased approach to quantify inter and intragroup variations the malignant and nonneoplastic or healthynormal samples were used as two groups for normfinder analysiscomprehensive stability rank of each gene was calculated as the geometric mean of stabilityrank given by each method one 101371 pone0236338 august one 0cbcl2 molecular diagnostics with novel reference genesexpression analysis of bcl2rq relative quantification of bcl2 expression was calculated either as ratio of relativeexpression of bcl2 with relative expression of gapdh or the normalization factor which isgeometric mean of relative expression of three candidate rgsrq ðgapdhþ ¼ e 00 dcqðbcl2þe 00 dcqðgapdhþrq ðproposedþ ¼ e 00 dcqðbcl2þgeo mean e 00 dcqðptcd2 ppp1r3b fbxw9þresults and discussionquantification by qpcr could be the choice of pathology laboratories for a quick and costeffective platform for singlegene expression level with appropriate rg towards this effort macrae performed a genome wide search and statistical analysis using rnaseq datafrom leukemia patients in a more recent pancancer study publicly available geneexpression data from microarray studies were analysed to identify a few rg candidates thatshowed minimal variation between malignant and normal samples and were validated in droplet digital pcr on bone marrow samples of all patients we have used types of haematological malignancy samples encompassing bone marrow pbmc and ffpe blocks along with nonneoplastic bone marrow and healthy pbmc samples subsequent to using much wider publiclyavailable data from samples in aml dlbcl and multiple myeloma databases furtherwe have employed an improved statistical analysis including clustering technique described inmethods section instead of an ad hoc approach of selection of top few genes from the clusterswe used important biological considerations to further prune the list of candidate rgssystematic review of commonly used rgs from literaturesystematic review of s yielded rgs used in haematological malignancies througha selection of genes by different analysis methods s4 table and b usage of known rgs inqpcr s1 table fpkm values of all these rgs when examined in public databases showedvaried expression among different types of haematological malignancies s3 and s4 figs withmaybe the exception of pggt1b however since other genes selected in the literatureshowed higher expression and correlated extreme variation we could not depend on the assayand proceeded to select novel rgs with an unbiased approachselection of candidate rgsstatistical analysis stepwise filtration of the number of genes from each dataset is summarized in s6 fig and also in graphical abstract fig shows gene clusters plotted in cv normalized mad and 1pvalue hyperspace for four datasets cluster marked in green in eachfigure represents the cluster with least med value s5 table for the three parametersselected clusters in the four datasets had an overlap of genes indicating large number ofgenes involved in housekeeping processes and hence showing lesser intersample variationacross diverse datasets common genes were pruned further to by go biological processterm filtration disease association and cqr to lead to a final of genes s6 table that weretaken through experimental validation melt curve analysis and efficiency check with pooledcdna from six healthy volunteers narrowed it down to genes with stable median expression and single amplification product of expected size for each table primers for geneswhich did not qualify the efficiency check were eliminated as they failed to show single amplification peak after repeated trials with new experimental conditions and even new primersequences s3 table one 101371 pone0236338 august one 0cbcl2 molecular diagnostics with novel reference genesfig statistical analysis of candidate genes genes plotted in the cv normalized mad and pvalue hyperspace for the fourdatasets a tcgalaml b targetaml c gdcdlbc and d mmrfmm cluster shown in green represents the chosencluster with least value of meds101371 pone0236338g001expression of genes with efficient primers were analysed on samples by qpcr usingobserved cq values preliminary stability analysis of the genes were done with online reffinder tool to select top stable genes ptcd2 ppp1r3b fbxw9 nanp ric8b jmjd4plekhm3 nat1 ankrd26 tsga10 as rg candidatessssstability analysis of candidate rgs results of bestkeeper algorithm used independentlyor as part of reffinder were comparable whereas results of genorm or normfinder analysisdiffered as they used different inputs geometric mean of stability ranks assigned in each algorithm was used to create comprehensive stability ranking of all the candidate rgs s7 tableand fig the analysis shows ptcd2 ppp1r3b and fbxw9 to be most stable across all analysed patient samplesptcd2 pentatricopeptide repeatcontaining protein codes for a mitochondrial proteininvolved in rna binding maturation and respiratory chain function though its exact one 101371 pone0236338 august one 0cbcl2 molecular diagnostics with novel reference genesfig stability rank of candidate reference genes101371 pone0236338g002molecular function is not well understood [ ] ppp1r3b protein phosphatase1 regulatorysubunit3b encodes for a catalytic subunit phosphatase regulatory subunit 3b which isinvolved in hepatic glycogen dysregulation in type diabetes [] fbxw9 fboxwdrepeatcontaining protein is a cytosolic protein involved in ubiquitination and proteasomedegradation expression analysis of bcl2accurate determination of bcl2 expression among few antiapoptotic markers in patients withhaematological malignancies is emerging as a critical diagnostic test for clinicians to suggest efficacious therapy options fpkm values of rgs common and novel from the publicly availabledatabases when compared fig with bcl2 indicated the novel rgs to be better normalizationcandidate for bcl2 in qpcr assays in pathology labs due to less and stable expressioncomparison of relative expression of gapdh versus the proposed normalization facteometric mean of relative expression of the three rg candidates clearly show a large variation in gapdh expression across malignant samples fig 4a s8 table granted itspopularity the expression stability of gapdh has been proven to differ in different conditionsdue to its involvement in apoptotic cell death through ubiquitin ligase membrane trafficking upregulation in aml involvement in nonhodgkins bcell lymphomas and inconsistency in several other cancers on the other hand proposed rgs havelesser variation and their expressions are consorted with each other making them better candidate as rg compared to gapdh this behaviour is translated to bcl2 expressionrq in malignant samples when normalized with gapdh fig 4b evidently normalizationwith gapdh underestimates relative quantification of bcl2 compared to normalization withproposed rgs with a statistically significant difference in median values p wilcoxonrank sum test between the two schemes bcl2 quantification in haematological malignanciesby qpcr is overtly reliant on rg since availability of adjacent normal sample is ruled outabove results clearly demonstrate how the quantification may go off limit due to a wrongchoice of rg one 101371 pone0236338 august one 0cbcl2 molecular diagnostics with novel reference genesfig candidate reference genes in hematological malignancy datasets expression values of candidate genes in four datasets a tcgalamlb targetaml c gdcdlbc and d mmrfmm101371 pone0236338g003broader applicability of proposed reference genesthough primary objective of this study is to discover rg candidates for bcl2 diagnostics in aclinical setting the rgs may have broader utility in other experimental platforms or modelsystems in the systematic review we found a number of research s [] that haveused taqman probes instead of sybr green whereas our validation experiment was carriedout using sybr green probes however studies in different contexts such as a tropical oilseedplant or measurement of expression of various adenosine receptors in breast cancer tissue and in experiments using human reference rna sybr green pcr assays wereobserved having fair concordance with taqman pcr from these evidences we believe thatstability of proposed rgs is not likely to differ between sybr green and taqman qpcr assaysto assess variation of these stable rgs in cell lines we analyzed rpkm values of proteincoding genes across cell lines of haematopoietic and lymphoid tissue origin frombroad institute cancer cell encyclopedia and found the proposed rgs presenting muchlesser variations in expression compared to the common rgs gapdh abl1 b2m gusband actb in cell lines as well s8 figboth transgenic and wild type and occasional rat models are widely used in leukemia andlymphoma research [ ] usability of rgs common between clinical and animal studies one 101371 pone0236338 august one 0cbcl2 molecular diagnostics with novel reference genesfig relative expression of chosen reference genes and relative quantification of bcl2 a relative expression of chosen reference genes solidlines and gapdh dashed line across patient samples b relative quantitation of bcl2 expression with respect to the candidate reference genesand gapdh in malignant patient samples101371 pone0236338g004will thus be of immense advantage we find that the proposed rgsptcd2 ppp1r3b andfbxw9 have sequence similarity and identity with corresponding genes in mice andother commonly used rodent models s9 table suggesting the genes playing similar role incellular function thereby displaying stability similar to that in humans hence normalizationfactor derived from the expression of these rgs may be applicable in murine and other rodentmodels as well with suitable design of primers encompassing conserved regionsbeyond detection of gene expression at mrna level it may be worthwhile to explore theapplicability of protein counterpart of the stable rgs in western blot as control for proteindetection by design we have chosen rgs that are of moderate expression level in middlequartiles of expression among other genes and they may not be detectable by western blotunless a larger amount of sample is loaded which is often not feasible with clinical sampleshowever it may be an interesting proposition to predict stable reference proteins for use inwestern blot by statistical analysis of proteomics data and associated systematic review ofliterature one 101371 pone0236338 august one 0cbcl2 molecular diagnostics with novel reference genesour results indicate that genes ptcd2 ppp1r3b and fbxw9 render more reliability toqpcrbased diagnostic test of bcl2 in haematological malignancies the can beextended to other biomarkers in liquid cancer as well as for research with other model systemssuch as cell lines and rodentssupporting informations1 table list of reference genes in literaturedocxs2 table list of bcl2 primers from literaturedocxs3 table list of unqualified primersdocxs4 table literature explaining analysis and selection of reference genedocxs5 table zscore med valuesdocxs6 table list of selected genesdocxs7 table individual and combined stability rank and scores of candidate reference genesdocxs8 table relative expression of gapdh and the proposed normalization factordocxs9 table sequence similarity and identity with corresponding genes in mice rat andguinea pigdocxs1 fig rgs found in literature with more than one citationtiffs2 fig fpkm values of bcl2 family of antiapoptotic genes in the four datasetstiffs3 fig fpkm values of rgs found in relevant literature with more than one citationtiffs4 fig fpkm values of rgs found in relevant literature with a single citationtiffs5 fig workflow according to prisma guidelines for systematic review for commonlyused reference genestiffs6 fig statistical analysis workflowtiffs7 fig patient samples used in the studytiff one 101371 pone0236338 august one 0cbcl2 molecular diagnostics with novel reference geness8 fig variation in stable rgs in cell lines and animal modeltiffs1 graphical abstracttiffacknowledgmentsauthors acknowledge prof joy kuri chair department of electronic science and engineering indian institute of science bangalore for providing the computational resourcesauthor contributionsconceptualization sujan k dhar manjula dasdata curation nehanjali dwivedi sreejeta mondal smitha p k sowmya t kartik sachdeva christopher bathula vishnupriyan kformal analysis sujan k dharfunding acquisition sharat damodar manjula dasinvestigation nehanjali dwivedi sreejeta mondal smitha p k sowmya tmethodology nehanjali dwivedi sreejeta mondal smitha p k sowmya t vishnupriyank manjula dasproject administration manjula dasresources nataraj k s sharat damodarsoftware sujan k dharsupervision manjula dasvalidation nehanjali dwivedi sreejeta mondal smitha p k sowmya t kartik sachdevachristopher bathula vishnupriyan kvisualization manjula daswriting original draft sreejeta mondal sujan k dharwriting review editing nehanjali dwivedi sreejeta mondal smitha p k sujan kdhar manjula dasreferences perini gf ribeiro gn pinto neto jv campos lt hamerschlak n bcl2 as therapeutic target forhematological malignancies vol of hematology and oncology biomed central ltd gratiotdeans j merino r nuñez g turka la bcl2 expression during tcell development early lossand late return occur at specific stages of commitment to differentiation and survival proc natl acad sciu s a oct 101073pnas912210685 pmid merino r ding l veis dj korsmeyer sj nuñez g developmental regulation of the bcl2 protein andsusceptibility to cell death in b lymphocytes embo j feb pmid li l li y que x gao x gao q yu m prognostic significances of overexpression myc andorbcl2 in rchoptreated diffuse large bcell lymphoma a systematic review and metaanalysis scirep 101038s41598017177655 uchida a isobe y asano j uemura y hoshikawa m takagi m targeting bcl2 with venetoclaxis a promising therapeutic strategy for doubleproteinexpression lymphoma with myc and bcl2 one 101371 pone0236338 august one 0cbcl2 molecular diagnostics with novel reference genesrearrangements haematologica jun 103324haematol2018 pmid baro´ c espinet b salido m garcı´a m sa´nchez b florensa l cryptic ighbcl2 rearrangementswith variant fish patterns in follicular lymphoma leuk res feb 101016jleukres201009011 pmid hofman p heeke s alixpanabières c pantel k liquid biopsy in the era of immunooncology is itready for primetime use for cancer patients suppressed immune microenviron repert brain metastases from patients with resected nsclc fatani s h mukhtar m h ali a s correlation between serum antiapoptotic bcl2 level and its immunohistochemical expression in relation to apoptosis in gastric cancer j mol biomark diagn albitar m zijun xy wang y manman d tzankov a visco c myc and bcl2 mrna expressionas determined by ngs predicts survival in dlbcl in gcb but not in abc subgroup blood nov 134supplement_15092 derenzini e rossi a agostinelli c rossi m melle f motta g integration of nanostring profilingand functional characterization of oxidative and replicative stress biomarkers identifies poor prognosis mycbcl2 positive diffuse large bcell lymphoma subsets providing opportunities for precisiontherapies blood nov 132supplement zhang f yang b zhang k hou ml lu xc li yx ccnd1bcl2 gene network a direct target of amifostine in human acute megakaryocytic leukemia cells chem biol drug des may 101111cbdd12889 pmid patel vm balakrishnan k douglas m tibbitts t xu ey kutok jl duvelisib treatment is associated with altered expression of apoptotic regulators that helps in sensitization of chronic lymphocyticleukemia cells to venetoclax abt199 leukemia sep 101038leu2016382 pmid bomben r ferrero s dagaro t dal bo m re a evangelista a a bcell receptorrelated genesignature predicts survival in mantle cell lymphoma results from the fondazione italiana linfomi mcl trial haematologica apr 103324haematol2017184325pmid dheda k huggett jf chang js kim lu | Colon_Cancer |
liver cancer the predominant primary malignancy ranks as the fifth most commonly diagnosed cancer in male and the seventh in female around the world in the year it has been estimated thatliver cancer is the fourth leading cause of cancer death with approximated new cases were diagnosed and over patients succumbed to this malignancy the incidence of liver cancer differsamong different geographical regions and the global map of liver cancer revealed that it remains a highestincidence rates in east asia with china accounting for more than of the burden in the world multiple risk factors including chronic infection with hepatitis b virus hbv or hepatitis c virus hcvaflatoxincontaminated eatables intake heavy alcohol consumption smoking corpulence and type diabetes are suggested to be essential for the occurrence of liver cancer of the two infection diseaseshbv is suggested to be implicated in of virusassociated liver cancer while hcv is implicated in suggesting a predominant role of inflammationimmune response in the process of liver cancer chemokines also named as chemoattractant cytokines are a large subfamily of small heparinbindingproteins and originally characterized by their properties to direct and recruit the movement of variousleukocyte subsets on the basis of the position of the cysteine residues adjacent to the nterminalchemokines were classified into four conserved subfamilies namely cc cxc cx3c and c and theythese authors contributedequally to this workreceived april 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 bsr20201169101042bsr20201169table sample characteristics in a liver tissue microarraycharacteristiccancer samples casenormal samples casegendermalefemaleage yearrangemedianstageiiiiiivgradeexecute their contributions mainly through interactions with their gproteincoupled receptors identified as ccrcxcr cx3cr and cr it is generally acknowledged that chemokines and their receptors may play multifaceted roles in engendering and mediating inflammation and immune response and therefore be involving inthe pathogenesis and pathological process of various diseases recently expanding evidence highlighted thatchemokinesreceptors were constitutively expressed and responsible for various malignant progression includingtumor cell proliferation migration invasion metastasis and tumor angiogenesis in a variety of human tumor types in the current research we evaluated whether cxcl8 a member of cxc chemokines was constitutively expressedin the tissues with liver cancer and explored the underlying role and mechanism of cxcl8 in regulating malignantprogression of liver cancermaterials and methodscel line cell culture and cell transfectionthe human hepatoblastoma cell line hepg2 were provided by american type culture collection atcc usa andthe cells were cultured in rpmi1640 medium supplemented with fetal bovine serum fbs gibco thermofisher scientific inc containing unitsml penicillin and mgml streptomycin at ¦c with co2 in ahumidified atmosphere a expression plasmid carrying cxcl8 were applied to transfect cells using lipofectamine invitrogen according to the suppliers instructions and the transfection efficiency was verified using a humancxcl8 elisa kit boster biological technology wuhan china following the manufacturers recommendationsimmunohistochemistry staining assaya commercial human tissue microarray containing liver cancer and normal liver tissue samples alenabioxian china was used to estimate the expression of cxcl8 sample characteristics were shown in table afterroutine deparaffinization and hydration the microarray was treated with vv hydrogen peroxidemethanolfor min at room temperature for inactivating endogenous peroxidase activity following antigen retrieval with m citrate buffer in a microwave oven for min the microarray was blocked with normal goat serum for min at room temperature and subsequently probed with a primary antibody specific for cxcl8 dilution after rinsing with pbs the microarray was incubated with hrpconjugated secondary antibody boster biologicaltechnology wuhan hubei china at ¦c for min finally dab substrate was utilized for the visualization ofantigen and haematoxylin for a routine nuclear counterstain the expression of cxcl8 at protein level was evaluatedas the following two parameters a the expression intensity negative weak mild strong superstrong staining b the percentage of cells stained of stainingthe scores for the two parameters are summed to produce a total score a total score of was referred to as lowexpression whereas ¥ as high expression 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 bsr20201169101042bsr20201169cell proliferation assayhepg2 cells at density of per well were maintained in 96well plates in μl of rpmi medium containing fbs with or without exogenous cxcl8 following culture for up to or h a cell proliferation assaykit counting kit8 boster biological technology wuhan china was applied to determine the proliferation ability ofcells following the manufacturers recommendations the optical density od in individual wells was detected witha microplate reader at nmcell migration assaya total of hepg2 cells were individually maintained in each chamber of 8μm pore inserts containing μl ofserumfree rpmi medium and the low chamber which contained μl of rpmi medium supplementedwith fbs with or without exogenous cxcl8 was employed as attractant after h incubation the cells that hadpenetrated through the inserts and attached to lower surface of the inserts were fixed and stained using ethanolcontaining viola crystallina finally the migrating cells were photographed and counted under a light microscopeapoptosis assaycell apoptosis was detected using a commercial an apoptotichoechst staining kit beyotime shanghai chinabriefly the cells were fixed with fixative solution and the cells were stained with hoechst followed by washingwith pbs three times a confocal microscope was used to determined cell apoptosis the apoptotic rate was evaluatedby the following formulaapoptotic rate total number of cells number of apoptotic cells total number of cellswestern blotting assaytotal protein from individual cell pellets was extracted using a commercial ripa lysate buffer boster biologicaltechnology wuhan china following suppliers instructions about μg of total protein was subjected to electrophoresis in sdspage and subsequently electrotransferred onto a pvdf membrane millipore bedfordma after blocking with nonfat milk at room temperature for h the membrane was respectively incubatedovernight at ¦c with different specific antibodies directed against erk abways perk11000 abwayssurvivin santa cruz bax santa cruz caspase3 affinity and actin hrplinked secondaryantibodies and ecl reagent were used to display protein bands the relative protein levels were normalized againstactinstatistical analysisdata were analyzed using spss software in vivo the pearson correlation coefficient analysis was applied toreveal significant differences among several clinicopathological parameters in vitro data were presented as means standard deviations significant differences between two groups were compared using the students ttest p005was considered as statistical significanceresultscxcl8 was overexpressed in tissues with liver cancerthe results from immunohistochemistry assay showed that the expression of cxcl8 at protein level was markedlyincreased in liver cancer tissues figure 1ac whereas normal liver tissue showed a decreased expression ofcxcl8 protein figure 1d p00246 in addition statistical analysis form liver cancer tissues demonstrated thatupregulated level of cxcl8 was positively concerned with high clinical stage and tumor infiltration p00061 onthe contrary there was no positively association between cxcl8 expression and other clinicopathological parametersincluding pathological grade sex and age table exogenous cxcl8 regulates proliferation migration and apoptosis ofhepg2 cellsto assess the roles of cxcl8 in regulating malignant behavior of liver cancer a classic cell line namely hepg2 wasemployed for following cell experiments cck8 analyses demonstrated that the growth rate of hepg2 cells treatedwith or ngml cxcl8 was significantly elevated as compared with hepg2 cells treated with ngml cxcl8figure 2a coupled with this transwell analyses revealed that migration cells of hepg2 cells treated 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 bsr20201169101042bsr20201169figure upregulated expression of cxcl8 in tissues with liver cancer in a human liver tissue microarrayliver cancer tissues with stage ii a iii b and iv c represented strong staining of cxcl3 normal tissue d showed weak stainingof cxcl8table cxcl8 level in a liver tissue microarraycharacteristicnormal liver sampleshcc samplesclinical stagetumorinï¬ltrationtpathological gradeagesexcase of score case of score¥ ii t2iiiivt3t4 manwomanÏ2por ngml cxcl8 was significantly more than that of hepg2 cells treated with ngml cxcl8 figure 2bc inaddition apoptosis assay showed that treatment of cells with or ngml cxcl8 can significantly inhibit theapoptosis rate of cells figure 2doverexpression of cxcl8 contributes to proliferation migration andapoptosis of hepg2 cellsto reveal the role of cxcl8 in liver cancer gene transfection strategy was used to construct cxcl8overexpressinghepg2 cells and their control fluorescent detection demonstrated that cells exhibited high content of fluorescencefigure 3a moreover elisa analysis showed that the level of cxcl8 in supernatant from cxcl8overexpressioncell medium was remarkably increased as compared with supernatant from their parental and control cell mediumindicating hepg2 cells overexpressing cxcl8 have be established figure 3b cck8 and transwell assays demonstrated that the proliferation figure 3c and migration figure 3de abilities of hepg2 cells overexpressing cxcl8were markedly enhanced as compared with their parental and control cells on the contrary the apoptosis rate ofoverexpression cells was significantly lower than those of parental and control cells figure 3foverexpression of cxcl8 regulates tumorspeciï¬c protein expression ofhepg2 cellsfinally we investigated whether overexpression of cxcl8 regulates the expression of tumorrelated genes thedata from western blotting indicated that cxcl8overexpression cells presented significantly increased levelsof erk perk and survivin compared with their control cells inversely the levels of caspase3 and bax incxcl8overexpression cells were predominantly reduced than that in their control cells figure 4ab 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 bsr20201169101042bsr20201169figure the effects of exogenous cxcl8 in cell proliferation migration and apoptosis of hepg2 cells in vitroproliferation index a the number of migration b and c and apoptosis rate d from hepg2 cells following treatment with exogenous cxcl3 at different concentrations p005 p001 versus ngml of cxcl8discussioncxcl8 also known as il8 is a member of the cxc family of chemokines that is produced by many types of cellsincluding leukocytes fibroblasts endothelial cells and malignant cancer cells and play a various spectrum of biological effects in cell functions through interaction with its g proteincoupled receptors cxcr1 and cxcr2 cxcl8 are best known for their function in the initiation of the inflammatory reaction during the process ofinflammation cxcl8 recruits leukocytes to the site of infection ultimately resulting in increased neutrophil infiltration which is responsible for the damage of endothelial cells this implies that downregulation of cxcl8 is crucialfor the resistance to chronic inflammation in addition to its function in inflammation and immune response increasing evidences suggested that cxcl8 isalso implicated in diseaserelated processes including tissue damage fibronolysis angiogenesis and tumorigenesissuggesting that cxcl8 may be implicated in disease pathologies in which inflammation plays a vital role it is well accepted that inflammation as a predominant regulator is involved in the developmental processes ofnumerous human cancers and is the seventh leading of hallmark of cancer studies showed that many chronicinflammatory states obviously increase the risk of certain cancers and about of cancers are thought to be causedby chronic inflammation or inflammatory states as a proinflammatory chemokine cxcl8 belongs to elrcxc family of chemokines which shares structural homology with cxcl5 to date increasing number of studiesindicated that cxcl8 and its receptors were overexpressed in several types of human cancers including colorectalcancer prostate cancer cervical cancer and nonsmall cell lung cancer in the current investigationwe found that cxcl8 was significantly upregulated in tissues with liver cancer and its overexpression was closelycorrelated with high clinical stage and tumor infiltration in agreement with this it was demonstrated that the expression of cxcl8 was significantly increased in tissues with oesophageal squamous cell carcinoma of which stronger 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 bsr20201169101042bsr20201169figure the effects of overexpression of cxcl8 in cell proliferation migration and apoptosis of hepg2 cells in vitroa fluorescence images of cxcl8overexpression cells and their control cells b the expression of cxcl in supernatantfrom cxcl8overexpression cells parental cells and control cells was detected by elisa the proliferation index c number ofmigration d and e and apoptosis rate f from hepg2 cells overexpresssing cxcl8 parental cells and control cells p005p001 versus controlexpression of il8 predominantly connected with many advancedstage pathological characteristics including depthof invasion lymph node metastasis pathologic stage lymphatic invasion and venous invasion recently cxcl8was also proposed to be predominately overexpressed in tissues with bladder cancer its overexpression was tightlyassociated with advanced disease and the overall survival rate of patients with increased expression of cxcl8 wasobviously reduced these findings suggest a possible significance of cxcl8 in cancer development and progressionaccumulating studies have revealed that cxcl8 is a critical component that involved in tumor initiation promotion and progression in cervical cancer jia et al reported that the proliferation and migration of hela cervical cancer cells were significantly enhanced after cells treated with different concentrations of exogenous cxcl8 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 bsr20201169101042bsr20201169figure the effects of cxcl8 in regulating tumorspeciï¬c protein expressiona representative images of western blotting analysis for erk perk survivin caspase3 and bax genes b relative expressionof these genes at protein levels from western blotting analysis p005 p001 versus control accordingly cxcl8 is also responsible for cancer cells malignant behavior in an autocrine fashion for example pc3 prostate cancer cells overexpressing cxcl8 present rapidly tumorigenicity highly proliferation rateremarkably angiogenesis and exhibit incidence of lymph node metastasis this investigation also demonstrated that several genes associated with angiogenesis and metastasis including vggf mmp2 and mmp9 wereupregulated in the clones with high cxcl8 expression on the contrary one study suggested that neutralizing antibodies against cxcl8 exerted a partial inhibition of tumor growth and exhibited antiangiogenesis activity in a nudemouse xenografts model however it is not clear whether cxcl8 stimulates the malignant process of liver cancer in the present studywe demonstrated that exogenous administration of cxcl8 significantly promote hepg2 cells proliferation and migration and overexpression of cxcl8 can also facilitate to these behaviors through an autocrine pathway finallymechanism studies demonstrated that overexpression of cxcl8 in hepg2 cells regulates tumorspecific protein expression including erk12 bax and survivin the involvement of cxcl8 in tumor progression and angiogenesiswere mediated by multifaceted signaling pathways including nfκb jnk pi3kakt p38 mapk and erk []in which erk signal pathway is a crucial mediator of a variety of cancer cells fates including proliferation migrationand survival in lung cancer it was suggested that egf stimulated a significant increase of cxcl8 production in lungcancer cells and il8 production from lung cancer cells could be initiated by their own produced factors resulting inthe recruitment of inflammatory cells in the tumor microenvironment as well as the formation of inflammatory microenvironment through pi3kakt and erk pathways cxcl8 signaling has also been proposed to have a vitalrole in promoting tumor progression by regulating apoptosisrelated gene expression for example administrationof the anticancer reagent induced cxcl8 production and the expression of the receptors of cxcl8 cxcr1 andcxcr2 in addition cxcl8mediated chemoresistance to oxaliplatin was demonstrated to be mediated by induction of nfκbtranscription leading to the upregulation of various antiapoptotic genes including bcl2 and survivin conclusionswe showed that cxcl8 expression was upregulated in tissues with liver cancer exogenous administration and overexpression of cxcl8 significantly facilitated to the malignant phenotypes of hepg2 cells by regulating tumorspecificprotein expression including erk12 survivin caspase3 and bax our findings might provide a potential markerand target for the treatment and diagnosis of liver cancerdata availabilitythe supplementary information that accompanies this can be accessed via the corresponding authorcompeting intereststhe authors declare that there are no competing interests associated with the manuscript 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 bsr20201169101042bsr20201169fundingthe study was financially supported by grant from scientific research project of heilongjiang health and family planning commission [grant number ] and basic scientific research project of provincial colleges and universities in heilongjiangprovince [grant number 2019kyywf1346]author contributionyh designed the study and participated in the revision of the manuscript sy and hw participated in all the experiments andwrote the manuscript cq performed immunohistochemical experiments hs performed cell experiments and data analysisethics approvalthe study was conducted in accordance with the ethical principles for medical research on human beings established by the helsinki protocol and its later amendments or comparable ethical standards and was approved by the institutional ethicscommittee of jiamusi university no jmsu216abbreviationshbv hepatitis b virus hcv hepatitis c virus od optical 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" 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 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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 |
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 |
several immunotherapeutic strategies that harness the exquisite speciï¬city of the immune systemto eliminate tumors have emerged during the past decade these include cancer vaccines immunecheckpoint blockade and adoptive cell therapy act with the potential to revolutionize thestandard of care for a range of malignanciesto a large extent the speciï¬city of immunotherapy is dependent on the recognition of speciï¬ctumor antigens especially neoantigens neoantigens are a kind of tumor antigen derived fromtumorspeciï¬c somatic mutations and are highly restricted to tumor cells with minimal establishedimmune tolerance neoantigenbased cancer vaccines have shown promising therapeutic eï¬ectsin the clinic in addition a growing body of evidence indicates that neoantigenspeciï¬c tcells underlie the success of the recently emergent immune checkpoint inhibitor therapy adoptive transfer of autologous in vitro expanded tumorltrating lymphocytes tils wasreported to achieve dramatic clinical responses in some metastatic cancer patients especially inthose with melanoma and cervical cancer indepth studies have revealed the criticalroles of neoantigenspeciï¬c t cells in maintaining durable responses following act in support of these ï¬ndings the adoptive transfer of selected tils targeting neoantigens led tosignificant tumor regression increasing research attention has been shifted to identifyingand selecting neoantigenspeciï¬c t cells however such a precise targeting strategy posesa great challenge in terms of the identiï¬cation and isolation of neoantigenspeciï¬c t cells methodshave been proposed and developed for this purpose here we attempt to summarize the knownstrategies for isolating neoantigenspeciï¬c t cellsedited bycyrille j cohenbarilan university israelreviewed bymanel juanhospital clnic de barcelona spainrodabe n amariauniversity of texas md andersoncancer center united statescorrespondencezhenyu dingdingzhenyuscueducnspecialty sectionthis was submitted tocancer immunity and immunotherapya section of the frontiers in oncologyreceived december accepted june published august citationli q and ding zy the ways ofisolating neoantigenspeciï¬c t cellsfront oncol 103389fonc202001347frontiers in oncology wwwfrontiersinaugust volume 0cli and dingisolating neoantigenspeciï¬c t cellsidentification and isolation ofneoantigenspecific t cells fromtilsfor most metastatic patients this time frame is unacceptable toaddress these issues additional attempts have been made usingeither surface markers or t cell receptor tcr redundancyresearchers have long attempted to isolate neoantigenspeciï¬csubpopulations from the of transferred tils in earlystudies an autologous tumor cell cdna library was constructedand used as a pool to screen for neoantigenspeciï¬c t cells in a study of a melanoma patient who experienced acomplete response going beyond years following adoptive tiltransfer one t cell clone speciï¬c for a mutated antigen ppp1r3bwas identiï¬ed and shown to be responsible for the antitumoreï¬ects advanceshowever the timeconsuming and laborious process requiredto identify neoepitoperesponsive t cells has hindered theirextensive functional assessmentin nextgeneration sequencing have enabled the rapid assessment of themutational landscape of human cancers and made it possibleto identify immunogenic mutated tumor antigens throughin silico analysis rosenbergs group ï¬rst employed predictedneopeptides obtained by wholeexome sequencing and humanleucocyte antigen hla class ibinding algorithms for tilscreening using this approach they identiï¬ed neoantigensrecognized by therapeutic bulk til cultures that mediatedobjective tumor regressions in three individuals with melanoma using a similar method neoantigenspeciï¬c cd8 tilscould also be identiï¬ed in hematological malignancies such asacute lymphoblastic leukemia all prickett and stevanovic also demonstrated that neoantigenspeciï¬c t cells could be identiï¬ed from therapeutic tils byscreening tandem minigene tmg libraries encoding cancermutations identiï¬ed from patients tumors by wholeexomesequencing this ï¬nding might further facilitate the recognitionof neoantigenspeciï¬c t cells because it circumvents the needfor prediction of hlapeptide binding and synthesis of a largenumber of peptideswith the advent of these techniques the ï¬eld of act took agreat leap from bulk tils to neoantigenspeciï¬c t cells a conciseï¬owchart showing the steps involved in identifying and isolatingneoantigenspeciï¬c t cells for act is summarized in figure tran successfully performed neoantigenspeciï¬c t celltherapy in a 43yearold woman with extensively metastatic andintensively treated cholangiocarcinoma after administration ofa bulk lymphocyte population containing a high percentage ofneoantigen erbb2ipspeciï¬c cd4t cells the patient showed alonglasting objective clinical response without obvious toxicitysubsequently neoantigenspeciï¬c t cells were identiï¬ed in onecolon cancer patient and another breast cancer patient andreinfusion of these speciï¬c t cells led to a partial response inone patient and a durable complete response in another currently act with neoantigenspeciï¬c t cells is beingtested in clinical trials in both solid and hematological tumorssupplementary table howeverthe extensive expansion of neoantigenspeciï¬ct cells during preparation compromises their proliferationpotential the method involved requiressophisticated equipment and a time period of several monthsin additionapproaches based on surfacemarkerscd137 belongs to the tumor necrosis factor receptor superfamily it functions as a costimulatory molecule to promote theproliferation and survival of activated t cells cd137expression is highly restricted to transiently activated cd8 tcells but almost undetectable in resting cells upregulated cd137can be detected on stimulated cd8 t cells of all phenotypeseg na¯ve t cells as well as early and late memory eï¬ector tcells naturally occurring tumorreactive t cells stimulatedby tumor antigens also express cd137 as proven by ye in a clinical trial trial registration id nct02111863 among patients with melanoma who underwent adoptive transfer withcd137selected tils only patient achieved partial responseand the remaining progressed the study was terminatedthis approach has its pitfalls because cd137 is an activationmarker cd137 t cells obtained by largescale productionare generally overactivated and highly diï¬erentiated withlimited proliferative potential a potential solution is to obtaintcrs from these cd137t cells instead this strategy wasreported by parkhurst brieï¬y cd8 t cells werestimulated overnight with immunogenic mutated tmg rnassubsequently the cd8 t cell population with the highestcd137 expression was sorted by ï¬uorescentactivated cell sortingfacs and expanded in vitro then dominant tcr α and chains were sequenced in the enriched populations twentyseven tcrs from patients that recognized neoantigensexpressed by autologous tumor cells were identiï¬ed howeverthis process was timeconsuming monthsa simpliï¬ed protocol was proposed by seliktaroï¬r here tils but not cd8 t cells were coculturedwith autologous tumor cells cd137 t cells were isolatedby magnetic bead separation and expanded no further tcrsequencing was performed the entire process took only dayst cells stimulated with neoantigens or other tumorassociatedantigens exhibit upregulated cd137 expression therefore a cd137based selection protocol was advocated forits broad antigen coverage including both neoantigen and sharedtumor antigens without prior knowledge of epitope speciï¬cityhowever the prerequisite of the establishment of autologoustumor cell lines poses a challengedirect and indirect evidence shows thatthe interactionbetween pd1 and pdl1 inhibits t lymphocyte functionleading to evasion of persistent ammatory or autoimmunereactions howeverthis protective mechanism ishijacked by tumors to escape immune surveillance pd1 hasbeen characterized as an inhibitory receptor on chronicallystimulated tcells in the tumor microenvironment atthe tumor site tils are exposed to tumor antigensthebinding of tcr and antigen upregulates either costimulatory orcoinhibitory receptors to promote or inhibit t cell activation andfrontiers in oncology wwwfrontiersinaugust volume 0cli and dingisolating neoantigenspeciï¬c t cellsfigure the general approach of identifying and isolating neoantigenspeciï¬c tils for act the tumor cells from excised tumor tissue and matched normal cellsunderwent wholeexome sequencing wes and rna sequencing to identify nonsynonymous mutations based on the information either tandem minigenes tmgsor peptides were then synthesized these tmgs or peptides were pulsed into autologous antigen presenting cells apcs such as dendritic cells dcs or b cells andthey were processed and presented in the context of major histocompatibility complex mhc on the other side the excised tumors were minced into ¼ mm3fragments and placed in 24well plates stimulated with il2 then the tils were cocultured with these pulsed apcs the identiï¬cation of the individualneoantigenspeciï¬c t subpopulation was dependent on the ifnγ enzymelinked immunospot elispot assay and the activation of the markers such ascd13741bb or cd134ox40 on the t cell surfaces when recognizing their cognate target antigen t cells with these activation surface markers would be puriï¬ed byï¬ow cytometry then the sorted t cells were subject to rapid expansion in vitro and reinfusion to the tumorbearing patientfunction respectively therefore pd1 t cell populationsamong tils may contain a large proportion of tumorspeciï¬ct cells the ï¬ndings of inozume and ahmadzadeh that tumorresponsive t cells are enriched amongcd8pd1 lymphocytes from fresh melanoma specimensprovide direct support for this notionin another study gros demonstrated that pd1expression on cd8 tils in fresh melanoma tumor specimensenabled identiï¬cation of a diverse patientspeciï¬c repertoireof clonally expanded tumorreactive cells including mutatedneoantigenspeciï¬c cd8 lymphocytes although pd1 is aninhibitory receptor expressed on t cells studies have shownthat il2 restored the antitumor function of t cells in vitro however on antigenexperienced terminally diï¬erentiatedeï¬ector memory temra cells pd1 is either not expressed orexpressed at very low levels therefore a pd1basedenrichment strategy may not be suitable for these cellsscreening strategies based on cd137 or pd1 expressionare suitable for cd8 t cells mainly in melanoma epithelialcancers which accountfor more than of all humanmalignancies harbor fewer mutations than melanoma theyexhibit compromised capability to induce mutationspeciï¬c tcell responses together with a limited number of ltratingneoantigenspeciï¬c tils in addition cd4t cells havebeen shown to play an important role in mediating tumorregression in animal models and patients however cd137 or pd1 is expressed on cd8 cells as a solemarker therefore it may not be reliably used to enrich activatedcd4 cells cd134 is transiently expressed on cd4 t cells stimulated by antigens and can be used as a marker forthe classiï¬cation of mutant reactive t cells recently yossef reported an approach in which thetils that expressed cd134 or cd137andor pd1 were isolatedby facs thus both cd4 t and temra cells were rescuedwhich would otherwise be missed if a single marker were usedsorted cells underwent limitingdilution in microwell plates toavoid the overgrowth of nonspeciï¬c t cells cultures were testedfor the ability to recognize a 25mer peptide pool encompassingpossible neoantigens notably the highly oligoclonal natureof these t cells makes possible the convenient applicationof single cell sequencing of their tcrs in patients withmetastatic epithelial cancer this highthroughput approach ledto the detection of cd4 and cd8 t cells targeting and neoantigens respectively whereas only and neoantigenswere identiï¬ed by using the til fragment screening approachin patients in which no neoantigen was found by traditionalfrontiers in oncology wwwfrontiersinaugust volume 0cli and dingisolating neoantigenspeciï¬c t cellsscreening the novel approach identiï¬ed distinct neoantigenspeciï¬c tcr clones for one patient and a highly potent mhcclass iirestricted krasg12vreactive tcr for the other in ametastatic tumor sample from a patient with serous ovariancancer mhc class iirestricted tcrs targeting the tp53g245shotspot mutation were identiï¬edtcr frequencytcr sequence analysis is used as a tool to monitor t cellresponses to speciï¬c antigens by measuring the abundance oft cell clonotypes the advent of nextgenerationsequencing has enabled identiï¬cation of the full tcr repertoireof tils this valuable data for tcrs from tumorreactive tils could be used to modify t cells tcrt howeverthe lengthy expansion process and excessive stimulation wouldresult in tcr repertoire switching to avoid this problempasetto directly performed tcr sequencing of thefresh enzymatically digested melanoma tissues prior to in vitroexpansion as described earlier tumorreactive clonotypes wereenriched in cd8pd1 til subsets in melanoma the authors analyzed the tcr repertoire of tils in cd8cd8 cd8pd1 or cd8pd1 subsets respectivelyand found that many of the most frequently occurring tcrclonotypes present in the cd8pd1 til subset recognizedthe autologous tumor and tumor antigens included neoantigensthis report provided a much more convenient approach toeï¬ciently identify tumorreactive t cells based solely on thefrequency of tcr and pd1 expression without prior knowledgeof the speciï¬c neoantigen however this strategy must be appliedwith caution because the isolated tcr clones may be selfreactiveand result in deleterious ontarget oï¬tumor toxicities isolation of neoantigenspecific tcells from peripheral bloodlymphocytes pblsin some situations neoantigenspeciï¬c t cells were undetectablein the til compartment possibly owing to the following factorspresentation of neoantigens in a nonammatory context impaired t cell ltration because of the sparse distributionof adhesion molecules on these cells and presence ofimmunosuppressive cytokines and cells eg regulatory t cellsin the tumor microenvironment furthermore the tissuefrom which tils may be obtained poses a challenge in thisregard peripheral blood is an alternative and reliable source forneoantigenspeciï¬c t cellsthe ï¬rst attempt is considered to have been made by agroup led by lennerz in this study a systemof mixed lymphocytetumor cells mltc was establishedwherein peripheral blood mononuclear cells pbmcs froma patient with metastatic melanoma were cocultured withautologous tumor cells the mtlc system could be viewed asa simpliï¬ed in vitro simulation of the tumor microenvironmentfurthermore cytotoxic t lymphocyte ctls clone derived bylimiting dilution from the mltc system or mltc were subjectto autologous tumor cell cdna library screening t cell clonesreactive to mutated epitopes were obtainedthe use of mhcpeptide tetramers is a canonical methodto identify and study a certain antigenspeciï¬c t cell subset for act tetramers were used to isolate and expandtumor antigenspeciï¬c t cells moreoverin immunecheckpoint inhibitor icitreated cancer patients mhcpeptidetetramers have been successfully used to monitor neoantigenspeciï¬c t cells cohen used this method tosort neoantigenspeciï¬c t cells from the pbls of patients withmetastatic melanoma in brief a panel of mhcpeptide tetramersconsisting of predicted neoepitopes was synthesized and usedto screen pbls neoantigenspeciï¬c t cells targeting of the mutated epitopes identiï¬ed from tils could be isolated fromautologous peripheral blood with frequencies ranging between and in cancers with intermediate mutational loadssuch as multiple myeloma the use of mhcpeptide tetramerscould also isolate neoantigenspeciï¬c t cells from the pbls however this method was only applied to cd8 t cellsand required hlabinding prediction algorithms to guide thesynthesis of hlapeptide tetramersa previous study has shown that pd1 expression couldguide the identiï¬cation of neoantigenspeciï¬c cd8 t cellsfrom the tumor microenvironment the same strategycould be adopted for isolation from pbls in one study patients with metastatic melanoma were enrolled cd8pbls were expanded in vitro and cocultured with autologousdcs which were electroporated with in vitro transcribed tmgrna for mutant epitopes in out of patients neoantigenspeciï¬c lymphocytes could be isolated from the cd8pd lymphocyte subset but not the cd8pd1 lymphocytesubset the isolation of neoantigenspeciï¬c cells from the pblsof patients with epithelial cancer is even more challengingpreexisting antigenspeciï¬c memory t cells may represent apotential solution memory t cells including central memoryt cells tcm eï¬ector memory t cells tem and temrafrom pbls were cocultured with dcs loaded with candidateneoantigens in the tmg or peptide form after coculturingmemory cells were restimulated with dcs loaded with all tmgsand then sorted by the expression of cd134 and cd137 to enrichfor neoantigenreactive t cells the resulting cells were thenexpanded and screened against all tmgs to test for neoantigenrecognition with this highly sensitive in vitro stimulationivs method t cells targeting krasg12d and krasg12vwere successfully isolated from out of epithelial cancerpatients this new method enabled identiï¬cation and isolationof neoantigenreactive t cells from the blood circulation at verylow frequenciesthe identiï¬cation of neoantigenspeciï¬c t cells from na¯vet cells is also of interest a previous report showed that bothna¯ve and activated neoantigenspeciï¬c t cells could be expandedfrom the peripheral blood of follicular lymphoma patients bypriming with peptidepulsed dcs using the same methodneoantigenspeciï¬c t cells were successfully expanded fromthe peripheral blood of hlamatched healthy donors these preliminary results support the use of na¯ve t cells asfrontiers in oncology wwwfrontiersinaugust volume 0cli and dingisolating neoantigenspeciï¬c t cellsfigure a strategies of identifying neoantigenspeciï¬c t cells the limitations of current methods of identifying neoantigenspeciï¬c t cells and strategies toimprove neoantigenspeciï¬c t cells identiï¬cation tils tumor ltrating lymphocytes pbls peripheral blood lymphocytes pd1 programmed cell death1 temracells terminally differentiated effector memory cells tcr t cell receptor tmg tandem minigene mhc major histocompatibility complex b the blueprint ofisolating neoantigenspeciï¬c t cells from peripheral blood after neoantigentargeting vaccine after several rounds of immunization with neoantigen vaccines t cellsare collected from the patients peripheral blood and neoantigenspeciï¬c t cells are identiï¬ed and isolated from these t cells then the neoantigenspeciï¬c t cellsundergo rapid expansion rep or their tcrs are exploited to modify autologous lymphocytes the expanded neoantigenspeciï¬c t cells or modiï¬ed tcrt cells arereinfused to the patientan alternative source for act however their exceptionally lowfrequencies in peripheral blood and requirement for repeatedstimulation pose hurdles recentlyalargelibrarybased minilinesscreeningapproach was proposed which aimed to identify na¯ve antigenreactive t cells from small volumes of blood this systembegan with a smallscale culture in 96well plates with initial t cells in each well the smallscale culture underwent arapid to 5000fold expansion miniline thousands ofsuch wellscaled cultures were conducted simultaneously eacht cell clone was maintained at a frequency of in butampliï¬ed to an absolute number of cells which isa suï¬cient number for routine detection applying this highthroughput parallel t cell culture system neoantigenspeciï¬ct cells were identiï¬ed and expanded months prior to theï¬rst tumor recurrence in a patient with highgrade serousovarian cancer however the long duration of culture possiblyrendered this method more suitable as a preemptive therapeuticstrategy discussionafter decades of eï¬orts the adoptive transfer of neoantigenspeciï¬c t cells is ï¬nally close to readiness for clinical applicationhigh eï¬cacy of this immunotherapeutic strategy has beenachieved in a number of cancer patients and the prospects arepromising however these approaches are also quite costly andhard to apply to large numbers of patients the current methodsof identifying neoantigenspeciï¬c t cells are summarized infigure 2a and supplementary table more convenient andfrontiers in oncology wwwfrontiersinaugust volume 0cli and dingisolating neoantigenspeciï¬c t cellseï¬ective screening methods for neoantigenspeciï¬c t cellsremain necessary some strategies to improve neoantigenspeciï¬ct cells identiï¬cation are shown in figure 2ait is feasible to obtain neoantigentargeting t cells frompbls although their frequencies are generally much lower thantils however increasing the frequencies of thesevaluable neoantigenspeciï¬c t cells in peripheral blood remainsa challengevaccination with neopeptides has been shown to primecd4 and cd8 tcell responses in mouse models patients treated with vaccines generated neoantigenspeciï¬c tcells it could be reasonably inferred that the isolationof neoantigenreactive t cells from the peripheral bloodwould be more easily achieved following neoantigenspeciï¬cvaccination this neoantigenbased combo immunotherapy hasits advantages ï¬rst isolation and expansion of tils in vitro isnot necessary second cancer vaccines not only elicit neoantigenspeciï¬c t cell responses and amplify existing tumorspeciï¬c tcells responses but they also increase the breadth and diversityof the tumorspeciï¬c t cell response multiclonal t cellsmay thus be obtained third the relatively easy preparation ofcancer vaccines would buy time for the isolation of neoantigenspeciï¬c t cells in maintaining the performance of patients theblueprint is shown in figure 2bconclusionthe previous decade has witnessed theemergence ofimmunotherapy for cancer accumulating evidence suggests thatneoantigenspeciï¬c t cells underlie successful immunotherapytherefore the isolation of neoantigenspeciï¬c t lymphocytesrepresents the holy grail for cancer immunotherapy howevera fundamental challenge is to eï¬ectively identify and isolateneoantigenspeciï¬c t cells the developments summarizedin this review and future breakthroughs are anticipated totranslate the adoptive transfer of neoantigenspeciï¬c t cells intoa powerful weapon in our armamentarium against cancerauthor contributionsql prepared the manuscript draft zyd revised it critically forimportant intellectual content and approved the ï¬nal versionql and zyd contributed to the conception and design of thereview all authors contributed to the and approved thesubmitted versionfundingthis work was supported by the national clinical researchcentersichuanuniversity z2018b18for geriatrics west china hospitalsupplementary materialthe supplementary materialonline202001347fullsupplementarymaterialfor this can be foundhttpswwwfrontiersins103389foncatreferences hu z ott pa wu cj towards personalizedtherapeutic 101038nri2017131vaccinesforcancer natrevtumourspeciï¬cimmunolin an ipilimumabresponsive melanoma j clin oncol 31e439 101200jco2012477521 gubin mm zhang xl schuster h caron e ward jp noguchi t checkpoint blockade cancer immunotherapy targets tumourspeciï¬c mutantantigens nature 101038nature13988 chen fj zou zy du j su s shao j meng fy neoantigen identiï¬cationstrategies enable personalized immunotherapy in refractory solid tumors jclin invest 101172jci99538 le dt uram jn wang h bartlett br kemberling h eyring ad pd1blockade in tumors with mismatchrepair deï¬ciency new engl j med 101056nejmc1510353 keskin db anandappa aj sun j tirosh i mathewson nd li sq neoantigen vaccine generates intratumoral t cell responses in phase ibglioblastoma trial nature hilf n kuttruï¬coqui s frenzel k bukur v stevanovic s 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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 |
"purpose squamous cell carcinomas and adenocarcinomas are the most common types of cervical cancercompared to squamous cell carcinomas adenocarcinomas are more common in younger women and have apoorer prognosis yet so far no useful biomarkers have been developed for these two types of cancer in thefollowing study we examined the combination of cytokeratin p63 p40 and muc5ac for distinguishingsquamous cell carcinoma scc from adenocarcinoma of the cervix aecmaterials and methods a total of scc and aec were collected immunohistochemical analyses wereconducted to determine the expression of ck56 p63 p40 ck7 and muc5ac one pathologist who was blinded tothe patients clinical and pathological data interpreted the staining resultsresults muc5ac and ck7 were detected in and of aec cases compared to and of scccases p the specificity of muc5ac was higher than that of ck7 in aec p the sensitivity of muc5accombined with p40 or p63 was similar to that of ck7 but the specificity was slightly higher than that of ck7 inaec moreover the expression of muc5ac was correlated with the degree of tumor differentiation inadenocarcinomas p and was not related to the prognosis of cervical adenocarcinoma and subtypess muc5ac may be useful as a biomarker for differential diagnoses between squamous carcinoma andadenocarcinoma of the cervixkeywords cervical adenocarcinoma cervical squamous cell carcinoma muc5ac ck7 correspondence xiaofangzhangsdueducn hailing li and xiaotong jing contributed equally to this work2department of pathology school of basic medical science shandonguniversity jinan shandong p r china5department of pathology school of basic medical science shandonguniversity jinan shandong p r 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 0cli diagnostic pathology page of thelastintroductioncervical cancer is the fourth most common carcinomain women responsible for of cancerrelateddeaths worldwide [ ] squamous carcinoma is themost common type of cervical carcinoma followed byadenocarcinoma nevertheless overthreedecades a significant increase in adenocarcinoma caseshas been observed in many developed countries especiallyin younger women papsmear screening also knownas pap test is still considered the main screening methodfor cervical cancer especially for squamous carcinoma compared to squamous carcinoma the adenocarcinomaof the cervix is more common in younger women and hasa poorer prognosis therapeutic approaches includechemoradiotherapy ccrt which has been proven tobe effective for squamous carcinoma of the cervix but notfor adenocarcinoma of the cervix due to its highchemo and radioresistance therefore differentiatingadenocarcinoma from squamous carcinoma is importantin order to provide patients with most suitable therapyp63 p40 and cytokeratin 56ck56 are the mostcommon panel ofimmunochemical markers for thediagnosis of squamous carcinoma p63 and ck56are traditional markers that indicate squamous differentiation in primary lung neoplasms most squamouscarcinomas and large cell carcinomas are positive forck56 warth found that the probability of acorrect sqcc diagnosis using ck56 is p63 a transcriptional regulator has a crucial role in thedevelopment and differentiation of stratified squamousepithelium it is usually strongly expressed in the basalkeratinocytes [] vosmik analyzed patientswith cervical squamous cell carcinoma and found that had positive expression of p63 p40 is a new specific marker for distinguishing squamouscarcinomas from adenocarcinoma whose specificity isabout in lung carcinomas however the positiveexpression of ck56 p63 and p40 are only found in a fewadenocarcinomas [ ] kriegsmann suggested theuse of either ck56 or p40 over p63 in the routine diagnostic setting ck7 is expressed in many ductal andglandular epithelial cells mainly gallbladder hepatic ductsand pancreatic ducts in tissues of the female genital tractovary endometrium fallopian tube and cervix and in thebreast lung and urinary tract tissues in the normalcervical tissue and adenocarcinoma ck7 staining wasobserved in the columnar cells of endocervical glandshashiguchi found the different rates of ck7 in patientswith cervical intraepithelial neoplasia and those with invasivecarcinomas vs [ ] thus far no efficientmarkers have been developed for distinguishing squamouscell carcinoma and adenocarcinoma in the endocervixmucins are a family of large glycoproteins expressedon the epithelial cell surfaces including ducts of lacrimalglands in the eye salivary glands the lining of the respiratory gastrointestinal urothelial and reproductive tracts muc5ac belongs to gelforming mucins multiple histological studies have highlighted that muc5acis expressed in the conjunctiva middle ear nasopharynxlungs gallbladder and stomach under normal conditionswhere it provides protection to corresponding epithelialsurfaces from different factors some research hasshown that muc5ac may be a potential biomarker inpancreatic cancer tissues dimaio found thatanterior gradient homolog and muc5ac are usefulpositive markers of adenocarcinoma in the setting ofabsent or diminished p63 and cytokeratin staining inesophageal carcinoma it is also expressed in theendocervix yamanoi found that muc5ac waslargely expressed in typical legh atypical legh gasmda and gasnonmda thus we speculated thatmuc5ac could be expressed in other adenocarcinomasand might be used for the differential diagnosis of adenocarcinoma and squamous carcinoma the aim of thisstudy was to examine the combination of cytokeratin p63 p40 and muc5ac for distinguishing squamous cellcarcinoma scc from the adenocarcinoma in the cervixaecmaterials and methodstissue sampleswe analyzed poorly to moderately differentiated cervical squamous carcinoma scc and adenocarcinomasof endocervix aec all tissues were collected from thedepartment of human pathology of qilu hospitalshandong university china from to specimenswere retrieved from the pathology files of the departmentof pathology at the same hospital after collection all specimens were fixed in buffered formalin hematoxylin eosin he stains were available for review paraffin blockswere used for immunohistochemical staining all the slideswere reviewed by two experienced pathologistshistopathological and clinical variables including agetumor size differentiationinfiltrate depth and lymphnode metastasis were summarized in table followupinformation was available in aec with the followuptime ranging from to months mean monthsimmunohistochemistryfour to five micronthick paraffin sections of the cases were dewaxed rehydrated in graded alcohols andprocessed using the pv9000 detection kit zsbio commerce store beijing china briefly antigen retrieval wasperformed in a microwave oven for min in mm trisedta buffer mm tris base mm edta solution tween ph endogenous peroxidase activitywas blocked with a h2o2methanol solution for min slides were then incubated in normal goat 0cli diagnostic pathology page of table comparison of clinicopathological features between cervical squamous cell carcinoma and cervical adenocarcinomasquamous cell carcinomasn adenocarcinoman Ï p valueage¤ size cm ¥ unknowndifferentiationpoormoderatewellunknown infiltrate depth of mesenchyme¤ unknownlymph node metastasisnoyesunknown serum for min to prevent nonspecific binding sampleswere then incubated overnight at °c with a primary antibody phosphate buffered saline pbs was used instead ofthe first antibody as a negative control consequentlysamples were incubated with reagent atroomtemperatureroomfor min and reagent attemperature for min finally the tissues were stainedwith diaminobenzidine dab the antibodies used in thisstudy are listed in table scoring methodstaining results were interpreted by one pathologist whowas blinded to the patients clinical and pathologicaldata for ck56 ck7 and muc5ac more than oftumor cells with a membrane or cytoplasmic brownyellow granules were considered positive for p63 andp40 the positive standard was that more than oftumor cells have brownyellow granules in the nucleusstatistical analysisstatistical analysis was performed with spss softwareversion spss inc chicago ii usa chisquareor fishers exacttests were used when comparingfrequencies between two groups probability values lessthan were considered statistically significantresultsthe expression of ck56 p63 p40 ck7 and muc5ac inscc and aecihc for the five proteins was performed on humanprimary cervical cancersincluding scc and aec as shown in fig and fig muc5ac ck56and ck7 were mainly expressed in the cell membranetable immunohistochemical antibodiesantibodymuc5acnozm0395ck56ck7p40p63zm0313zm0071zm0472zm0406vendorzsbio commerce store beijing chinazsbio commerce store beijing chinazsbio commerce store beijing chinazsbio commerce store beijing chinazsbio commerce store beijing chinadiluationready to useready to useready to useready to useready to use 0cli diagnostic pathology page of fig the expression of ck56 p63 p40 ck7 and muc5ac in a case of poordifferentiated squamous cell carcinoma by ihc a he b ck56positive staining c p63 positive staining d p40 positive staining e ck7 positive staining f muc5ac negative staining Ãfig the expression of ck56 p63 p40 ck7 and muc5ac in case of poordifferentiated adenocarcinoma invasive stratified mucinproducingcarcinoma ismile by ihc a he b ck56 negative staining c p63 negative staining d p40 negative staining e ck7 negative staining f muc5acpositive staining à 0cli diagnostic pathology page of and cytoplasm while p40 and p63 were mainly locatedin the nucleussignificant effect on the prognosis of cervical adenocarcinoma patients p as shown in fig tumorin thecells ofwe found that muc5ac exhibited prominent immucervical aecnoreactivitymuc5ac and ck7 were detected in and of aec cases compared to and of scc casesbesides for aec the specificity of muc5ac was muchhigher than that of ck7 p moreover the sensitivity of ck56 p40 and p63 was and respectively and the specificity was and respectively in aec table through the combined detection of p40 or p63 wecompared muc5ac and ck7 again we found that thesensitivity and specificity of muc5ac in aec combinedwith p40 or p63 were and respectively and respectively the sensitivity and specificity of ck7 combined with p40 or p63 were and and respectively table thesensitivity of muc5ac combined with p40 or p63 wassimilar to that of ck7 while the specificity was slightlyhigher than that of ck7expression of muc5ac and ck7 in cervicaladenocarcinoma subtypeswe further detected the expression of muc5ac insubtypes of aec table among cases of usualtype cervical adenocarcinoma cases were muc5acpositive and cases were ck7 positive and there wasno statistical difference p in cases of mucinous adenocarcinoma nosthe expression rate ofmuc5ac and ck7 were both moreover out of cases of gastric mucinous adenocarcinomaexpressed muc5ac and of them were ck7 positivep the positive rate of the muc5ac in mucinouscarcinoma intestinal type villous tubular adenocarcinomaendometrioid adenocarcinoma clear cell carcinoma serouscarcinoma adenosquamous carcinoma and invasive stratified mucinproducing carcinoma ismile was and respectively the expressionrate of muc5ac had no statistical difference among thesesubtypes all p correlation between muc5ac expression andclinicopathological characteristics in cervicaladenocarcinomathis study further analyzed the relationship between theexpression of muc5ac and clinicopathological featuresin cervical adenocarcinoma table the expression oftumormuc5ac was correlated with the degree ofdifferentiation p a lower degree oftumordifferentiation was associated with a lower expressionrate of muc5ac there was no significant correlationbetween the expression of muc5ac protein and agetumor size depth of myometrialinvasion and lymphnode metastasis all p kaplan meier analysisrevealed that the expression of muc5ac protein had nodiscussion and sidentification of previously unutilized sensitive biomarkers is still a priority for improved differential diagnosis of cervical aec and scc at present ck56 p63p40 and ck7 are the main biomarkers for differentiatingcervical adenocarcinoma from squamous cell carcinomack56 is a kind of high molecular weight basal cellkeratin 58kda and 56kda which is mainly expressed inthe basal cells of squamous epithelium and ductal epithelium and some squamous epithelial germinal layercells myoepithelial cells and mesothelial cells butpoorly expressed in glandular epithelial cells someresearch results showed that ck56 has high sensitivityand specificity in the diagnosis ofsquamous celltable sensitivity and specificity of muc5acãck56ãck7ãp40ãp63 in cervical squamous cell carcinoma and adenocarcinomamarkerssensitivityspecificitysquamous cell carcinomasn adenocarcinoman muc5acck7ck56p40p63ck56and p40ck56and p63muc5acand p40muc5acand p63ck7and p40ck7and p63 0cli diagnostic pathology page of table the correlation of muc5ac and the clinical variants inthe cervical adenocarcinomathe expression of muc5acpositivenegativeÏ valuep valueage¤ v size cm ¥ differentiationpoorwellmoderateinfiltrate depthof mesenchyme¤ lymph nodemetastasisnoyescarcinoma [] in contrast other studies showedhigh sensitivity but low specificity when diagnosing thistype of tumor p63 is a member of the p53 family a classical tumorsuppressor gene family it is located on chromosome3q27 filho showed good sensitivity whendetecting squamous cell carcinoma with a positive rateof contrary kaufmann suggested thatp63 could also be expressed in a small number of adenocarcinoma basal cell carcinoma and transitional epithelial carcinoma moreover p63 can also be used as amarker of myoepithelial cells and prostate basal cellstherefore p63 lacks absolute specificity for squamousdifferentiationp40 is a subtype of p63 protein expressed in squamousepithelial cells including epidermis and hair folliclesurothelial cells myoepithelial cells ofthe mammarygland sweat gland and salivary gland and basal cells ofthe prostate which are highly specific in labeling squamous epithelium bishop showed that in cases of squamous cell carcinoma of the lung and cases of adenocarcinoma of the lung the sensitivity andspecificity of p63 were and respectivelythe sensitivity and specificity of p40 in the diagnosis ofsquamous cell carcinoma of the lung were and respectively therefore p40 is considered as ahighly specific and sensitive tumor biomarker of squamous epithelial origin in this study we used immunohistochemistry to detectck56 p63 and p40 in cervical squamous cell carcinomaand adenocarcinoma the sensitivity of ck56 p40 andp63 was and respectively and thespecificity was and respectivelymoreover the specificity of ck56 is slightly lower thanthat of p40 and p63 we also found that a combinationof ck56 with p40 or p63 slightly decreased the sensitivity and and increased the specificity and which in turn increased the accuracy of diagnosing squamous cell carcinomack7 is a kind of low molecular weight keratin mainlyexpressed in glandular epithelium and transitional epithelial cells of most normal tissues many studieshave found that ck7 is not only expressed in adenocarcinoma but also in squamous intraepithelial neoplasiacervical squamous cell carcinoma lung squamous cellcarcinoma and esophageal squamous cell carcinomalee found a positive expression of ck7 in fig survival analysis of muc5ac expression in cervical adenocarcinoma 0cli diagnostic pathology page of table expression of muc5ac and ck7 in differentadenocarcinoma subtypessubtypesmuc5acusual typeÏ valueck7p valuepositivenegativemucinous adenocarcinoma nospositivenegativegastric typepositivenegativeintestinal typepositivenegativevillous tubular adenocarcinomapositivenegativeendometrioid adenocarcinomapositivenegativeclear cell carcinomapositivenegativeserous carcinomapositivenegativeismilepositivenegativeadenosquamous carcinomapositivenegativeismile invasive stratified mucinproducing carcinoma cases with scc and cases withciniii furthermore yamada found that ck7expression in esophageal squamous cell carcinoma butalso in iiiaiib stage esophageal squamous cell carcinoma suggest poor tumor differentiation and thus canbe used as an independent prognostic factor ourstudy showed that the positive rate of ck7 was incervical poorly differentiated squamous cell carcinomawhich further suggested that ck7 is not an ideal markerfor differentiation between squamous cell carcinoma andadenocarcinomamucin is a high molecular weight glycosylated proteinsecreted by epithelial cells in the respiratory tractgastrointestinal tract and urogenital tract which has animportant role in the protection of epithelium cell adhesion signal transduction immune activation and inhibition at present at least mucins have been found inthe female reproductive system riethdorf and albarracin used immunohistochemistrymethods to detect the expression of muc5ac in different female reproductive system malignant tumors theyfound that muc5ac was highly expressed in cervicaladenocarcinoma and poorly expressed inendometrial adenocarcinoma all of themwere expressed in the primary ovarian mucinous tumor but not in colon adenocarcinoma therefore they concluded that muc5ac could beused as an effective marker to distinguish the origin ofpelvic tumors and distinguish primary ovarian tumorsand colorectal metastasis as well as endometrial adenocarcinoma from cervical metastasis [ ] in thisstudy we found positive expression of muc5ac in cases of cervical adenocarcinoma and in cases of squamous carcinoma which wasconsistent with riethdorfs study the sensitivity ofmuc5ac and ck7 to cervical adenocarcinoma was and respectively but the specificity ofmuc5ac was much higher than that of ck7 through the joint detection of p40 or p63 wecompared muc5ac and ck7 again and found that thesensitivity and specificity of muc5ac combined withp40 or p63 were and respectively and respectively the sensitivity and specificity ofck7 combined with p40 or p63 were and and respectively these results showed thatthe sensitivity of muc5ac combined with p40 or p63was similar to that of ck7 butthe specificity wasslightly higher than that of ck7 therefore muc5ac issuperior to ck7 in the diagnosis of cervical adenocarcinoma and squamous cell carcinomabesides we preliminarily detected the expression ofmuc5ac in different types of cervical adenocarcinomaand found no significant difference these data suggestedthat muc5ac has no diagnostic significance in the classification of cervical adenocarcinoma at the same time weanalyzed the relationship between the expression ofmuc5ac and the prognosis of cervical adenocarcinomaand the result revealed that muc5ac was not related tothe prognosis of cervical adenocarcinomaoverall our observations strongly suggest that muc5acmay be useful as a biomarker for differential diagnosesbetween squamous carcinoma and adenocarcinomaabbreviationsscc squamous cell carcinoma aec adenocarcinoma of the cervixck cytokeratin he hematoxylin eosin pbs phosphate buffered salinedab diaminobenzidine ismile invasive stratified mucinproducingcarcinoma 0cli diagnostic pathology page of authors contributionsxiaofang zhang designed the study and drafted the manuscript hailing liand xiaotong jing analyzed the data and carried out theimmunohistochemistry jie yu and tingguo zhang read the pathologicalsections jinan liu collected the clinical data and carried our followupshiming chen made the slides the authors read and approved the finalmanuscript downey p cummins r moran m gulmann c if it's not ck56 positive ttf negative it's not a squamous cell carcinoma of lung apmis warth a muley t herpel e meister m herth fj schirmacher p weichert whoffmann h schnabel pa largescale comparative analyses ofimmunomarkers for diagnostic subtyping of nonsmallcell lung cancerbiopsies histopathology fundingthis work was supported by the national natural science foundation ofchina no and technology development foundation of yantaino ws017availability of data and materialsnot applicableethics approval and consent to participateall tissue samples from patients were collected and protocols wereperformed according to the procedures approved by the research ethicscommittee of shandong medical university all patients provided informedconsentcompeting intereststhe authors declare that they have no competing interestsauthor details1department of pathology weifang traditional chinese hospital weifangshandong p r china 2department of pathology school of basic medicalscience shandong university jinan shandong p r china 3department ofpathology the fourth hospital of jinan the third affiliated hospital ofshandong first medical university jinan shandong p r china 4departmentof oncology yuhuangding hospital yantai shandong p r china5department of pathology school of basic medical science shandonguniversity jinan shandong p r chinareceived july accepted august referenceskurman rj carcangiu ml herrington cs who classification of tumours offemale reproductive ans4th ed lyon iarc press takeuchi s biology and treatment of cervical adenocarcinoma chin jcancer res young rh clement pb endocervical adenocarcinoma and its variants theirmorphology and differential diagnosis histopathology forouzanfar mh foreman kj delossantos am lozano r lopez ad murraycj naghavi m breast and cervical cancer in countries between and a systematic analysis lancet galic v herzog tj lewin sn neugut ai burke wm lu ys hershman dlwright jd prognostic significance of adenocarcinoma histology in womenwith cervical cancer gynecol oncol favero g pierobon j genta ml araujo mp miglino g del cpdm deandrade ch fukushima jt baracat ec carvalho jp laparoscopicextrafascial hysterectomy completion surgery after primary chemoradiationin patients with locally advanced cervical cancer technical aspects andoperative outcomes int j gynecol cancer rose pg java jj whitney cw stehman fb lanciano r thomas gm locallyadvanced adenocarcinoma and adenosquamous carcinomas of the cervixcompared to squamous cell carcinomas of the cervix in gynecologiconcology group trials of cisplatinbased chemoradiation gynecol oncol ma y fan m dai l kang x liu y sun y xiong h liang z yan w chen kexpression of p63 and ck56 in earlystage lung squamous cell carcinoma isnot only an early diagnostic indicator but also correlates with a goodprognosis thorac cancer kaufmann o fietze e mengs j dietel m value of p63 and cytokeratin as immunohistochemical markers for the differential diagnosis of poorlydifferentiated and undifferentiated carcinomas am j clin pathol barbieri ce pietenpol ja p63 and epithelial biology exp cell res senoo m pinto f crum cp mckeon f p63 is essential for the proliferativepotential of stem cells in stratified epithelia cell pozzi s zambelli f merico d pavesi g robert a maltere p gidrol xmantovani r vigano ma transcriptional network of p63 in humankeratinocytes plos one 200943e5008 vosmik m laco j sirak i beranek m hovorkova e vosmikova h drastikovam hodek m zoul z odrazka k prognostic significance of humanpapillomavirus hpv status and expression of selected markers her2neuegfr vegf cd34 p63 p53 and ki67mib1 on outcome after chemoradiotherapy in patients with squamous cell carcinoma of uterine cervixpathol oncol res nobre ar albergaria a schmitt f p40 a p63 isoform useful for lung cancerdiagnosis a review of the physiological and pathological role of p63 actacytol stolnicu s hoang l hankobauer o barsan i terinte c pesci a avielronens kiyokawa t alvaradocabrero i oliva e and others cervicaladenosquamous carcinoma detailed analysis of morphologyimmunohistochemical profile and clinical outcomes in cases modpathol toyoshima m momono y makino h kudo t oka n sakurada j suzuki hkodama h yoshinaga k cytokeratin 7positivecytokeratin 20negative cecaladenocarcinoma metastatic to the uterine cervix a case report world jsurg oncol hashiguchi m masuda m kai k nakao y kawaguchi a yokoyama maishima s decreased cytokeratin expression correlates with theprogression of cervical squamous cell carcinoma and poor patientoutcomes j obstet gynaecol res lee h lee h cho yk cytokeratin7 and cytokeratin19 expression in highgrade cervical intraepithelial neoplasm and squamous cell carcinoma andtheir possible association in cervical carcinogenesis diagn pathol krishn sr ganguly k kaur s batra sk ramifications of secreted mucinmuc5ac in malignant journey a holistic view carcinogenesis thornton dj rousseau k mcguckin ma structure and function ofthe polymeric mucins in airways mucus annu rev physiol rose mc voynow ja respiratory tract mucin genes and mucinglycoproteins in health and disease physiol rev balmaña m duran a gomes c llop e lópezmartos r ortiz mr barrabés sreis ca peracaula r analysis of sialyllewis x on muc5ac and muc1mucins in pancreatic cancer tissues int j biol macromol dimaio ma kwok s montgomery kd lowe aw pai rkimmunohistochemical panel for distinguishing esophageal adenocarcinomafrom squamous cell carcinoma a combination of p63 cytokeratin muc5ac and anterior gradient homolog allows optimal subtyping humpathol yamanoi k ishii k tsukamoto m asaka s nakayama j gastric gland mucinspecific oglycan expression decreases as tumor cells progress from lobularendocervical gland hyperplasia to cervical mucinous carcinoma gastrictype virchows arch reisfilho js simpson pt martins a preto a gartner f schmitt fcdistribution of p63 cytokeratins and cytokeratin in normal and neoplastic human tissue samples using tarp4 multitumor tissuemicroarray virchows arch yamada a sasaki h aoyagi k sano m fujii s daiko h nishimura myoshida t chiba t ochiai a expression of cytokeratin predicts survival instage iiiaiib squamous cell carcinoma of the esophagus oncol rep baker ac eltoum i curry ro stockard cr manne u grizzle we chhieng dmucinous expression in benign and neoplastic glandular lesions of theuterine cervix arch pathol lab med 0cli diagnostic pathology page of riethdorf l o'connell jt riethdorf s cviko a crum cp differentialexpression of muc2 and muc5ac in benign and malignant glandularlesions of the cervix uteri virchows arch albarracin ct jafri j montag ag hart j kuan sf differential expression ofmuc2 and muc5ac mucin genes in primary ovarian and metastatic coloniccarcinoma hum pathol publishers notespringer nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations 0c" | Colon_Cancer |
sarcomatoid carcinomas scs are extremely rare aggressivemalignant tumors characterized by distinct cellular morphology the features of this tumor were ï¬rst described in by snover scs can occur in a wide variety of sitesincluding the respiratory tract digestive tract genitourinary tractbreast and thyroid glands however these tumors are rarein the digestive tract especially in the stomach as of april there are only six cases of gastric sarcomatoid carcinomagsc reported in the english medical literature these previousreports focused on the pathological and clinical manifestationsthem have not systematically described the radiologic appearanceof the tumor due to the more invasive nature and poorerprognosis of gsc than pure gastric adenocarcinoma gacand gastric lymphoma gl it is clinically beneï¬cial to narrowdown the diï¬erential diagnoses by understanding the computedtomography ct characteristics of gsc the present studyanalyzed our experience in diagnosing ï¬ve patients with gscin terms of the imaging ï¬ndings and clinical features to thebest of our knowledge our study represents the largest seriesof gscs to datein addition due to the rarity of gsc the diï¬erential diagnosisbetween gsc and other types of malignant gastric tumors hasnot received much attention so we also initially explored thediï¬erential diagnosis of gsc from gac and glmaterials and methodsthe protocol was approved by the medical ethics committeeof zhengzhou universityinformed consent was obtainedfrom all patientspatient selectionfrom august to january we searched the pathologyrecords and the picture archiving and communication systemspacs of our hospital the search terms included stomachand sarcomatoid carcinomas a total of ï¬ve patients wereidentiï¬ed as having sc and were enrolled in the present study weretrospectively reviewed all clinical data demographic featureslaboratory ï¬ndings clinical interventions and the histologicï¬ndings of the ï¬ve biopsy or operation specimensct evaluationfive gsc patients underwent ct examinations the ctscans were acquired with a 64row multidetector devicediscoveryct750hd ge healthcare waukesha wiunited states conventional axial scanning was performedbefore and after an intravenous iv injection of nonioniciohexol iopromide mgml ge medical systems mlkgand mls through a dualhead pump injector medradwarrendale pa united states the imaging parameters wereas follows tube voltage kv tube current ma ï¬eldof view fov mm matrix mm and sectionthickness mm finally a 20ml saline ï¬ush was performedat a rate of mlsgastric sarcomatoid carcinomacontrastenhanced ct scans were acquired with scanningdelays of s arterial phase ap and s portal venous phasepp after the iv injection of the contrast agent started the ctdose index volume for the three phases was msvimage analysistwo experienced radiologists and years of abdominal ctexperience performed a retrospective analysis of the ct imagesall analyses were performed with an aw47 workstation gehealthcare and the radiologists were blinded to the clinicalinformation of the patients the evaluated parameters includedthe tumor location gastric cardia gastric fundus gastric bodygastric angle and gastric antrum longaxis diameter shapegrowth pattern serosa condition attenuation and enhancementcharacteristics such as the enhancement pattern and degreeof enhancement the enhancement pattern of the tumor wasclassiï¬ed as homogeneous or heterogeneous based on the apimage the degree of enhancement of the tumor was based ondynamic ct imaging using hu attenuation where obviousenhancement was deï¬ned as hu moderate enhancementas hu and mildly enhancement as huthe gscs were staged with the union for internationalcancer control uicc tnm staging standard all imagingï¬ndings were compared with the postoperative pathologicalï¬ndings the accuracy rate the number of cts coincidentwith the pathological diagnosisthe number of actual pathologicaldiagnoses pathological evaluationthree patients underwent gastrectomy and two underwentendoscopic biopsy the three gastrectomy specimens measured cm cm cm cm cm cmcm cm cm respectively in two of theseand tumors the mucosal surface of the excised specimen showedulcerative masses of approximately cm cm cmand cm cm the remaining specimen was a soft massmeasuring cm cm cm for biopsy multiple sampleswere acquired and the diameter of each sample was cmaccording to the relevant literature the diagnostic criteria fsc were generally as follows the tumor originated fromthe stomach and the tumor consisted of both carcinomatousand sarcomatoid components and the sarcomatoid componentaccounted for more than of the tissue in addition if biopsywas performed the sarcomatoid component can be seen in everysample furthermore sarcomatoid regions express epithelialmarkers such as ck or emathe specimens were fully stretched ï¬xed and soaked in formaldehyde solution for h all biopsy specimenswere analyzed the specimens underwent routine dehydrationparaï¬n embedding sectioning into µm thick sectionsand hematoxylin eosin he staining immunohistochemicalstaining was performed using a roche benchmark xt automaticimmunohistochemical detector the antibodies used in thisstudy included ae1ae3 ckl ck818 epithelial membraneantigen ema vimentin p40 p63 and antigen ki67 ki67all antibodies listed above were purchased from dako dakoglostrup denmarkfrontiers in oncology wwwfrontiersinaugust volume 0cliu gastric sarcomatoid carcinomatable comparison between gsc and gac glage median age rangemain symptomsepigastric discomfortpainintermittent vomitingacute hematemesisbloody stooldysphagialocationcardia and fundusbodyantrumthe longaxis diameter median size rangeshapefocal thickeningdiffuse thickeningmassserosal surfacebare areaclearunclearulcersyesnodensity characteristicsheterogeneoushomogeneousenhancement patterheterogeneoushomogeneouslymph node involvementyesnoliver involvementyesnotherapyresectionchemotherapyresection and chemotherapyneoadjuvant chemotherapyradiation therapygscgacgl years years years cm cm cm comprehensive comparative analysiseach patient with gsc was matched by age ± years year ofdiagnosis and sex to four patients with gac gl patients witheach disease were retrieved from pacs patients with gsc werecompared with those with gac gl in terms of demographicclinical and ct characteristics table resultspatient characteristicsthe patients included four men and one woman ranging inage from to years with a median age of years theclinical and ct features of these patients are summarized intables all patients had nonspeciï¬c symptoms includingabdominal discomfort epigastric discomfort nausea or vomitingthe other presenting symptoms included hematemesis or weightloss three patients underwent radical resectionin whichonly one patient was treated with adjuvant chemotherapyafter surgery and two patients chose to deny treatment inaddition we also reviewed the upper gastrointestinal radiographyresults figure the laboratory ï¬ndings revealed that patient was positivefor tumor abnormal protein tap and patient was positivefor carbohydrate antigen ca125 before treatmenthemoglobin and erythrocyte count decreased in three patientsfrontiers in oncology wwwfrontiersinaugust volume 0cliu gastric sarcomatoid carcinomatable clinical and pathological factors of the ï¬ve gsc patientscasesexage yearscomplaintlocationmaximumdiametertumor markercmanemiatherapymetastasismmfmmsuddenhematemesisepigastricdiscomfortintermittentvomitingepigastric painepigastric painepigastric painlesser curvatureremnant stomachcardia andfunduscardia and fundusfunduscardia and fundusnormaltap ca125 normalnarrnnonenonerc yespresentpositive noabsentnegative f female m male age in years r radical gastrectomy rn remnant gastrectomy c chemotherapyna not availabletable computed tomography features of the ï¬ve gsc patientscasegross features of the tumorulcersgrowth modedensity characteristicsenhancement patterfocal thickeningfocal thickeningmassfocal thickeningfocal thickeningintracavityintracavityintracavityintracavityintracavityheterogeneousheterogeneoushomogeneousheterogeneousheterogeneousheterogeneousheterogeneoushomogeneousheterogeneousheterogeneousmarginunclearunclearunclearclearunclear yespresentpositive noabsentnegativefigure characteristics of xray examinations of a 65yearold male patient with gsc ab reveals that there is a huge niche with irregular shapes at the smallcurvature of the stomach the niche is located inside the outline of the stomach the niche is surrounded by transparent bands with different widths that is ringembankments with irregular outlines the surrounding mucosa is thickened interrupted and the local gastric cavity is narrowedpatients and and platelet count was elevated in fourpatients patients and pathological featuresmicropathologically the gastric tumor cells showed inï¬ltrativegrowth the cytological characteristics ofthe tumor cellsshowed obvious malignant characteristics microscopicallythe spindle cell structure and the nucleus were obviouslyatypical pleomorphic and enlarged mitotic ï¬gures were visiblefigures 2ab on immunohistochemical examination thetumor cells showed positive staining for ae1ae3 cklck818 ema p40 vimentin the ki67 index was higher than figures 2ci all ï¬ve tumors were diagnosed as gscin addition the sarcomatoid component showed spindle cellsarcomatoid morphologyct findingsof the cases of gsc were in the gastric fundus and cardiafigure was in the gastric body and was in the gastricfundus of these tumors one was a recurrence in the remnantstomach the ct manifestations of this tumor included localthickening n mass formation n the longaxisfrontiers in oncology wwwfrontiersinaugust volume 0cliu gastric sarcomatoid carcinomafigure histological and immunohistochemical features of gsc ab hematoxylineosin he staining showing tumor cells demonstrated spindleshapedstructures signiï¬cant atypical nuclei pleomorphic nuclei and giant nuclei mitotic ï¬gures visible tumor cells showed inï¬ltrative growth cells were stained withhematoxylin and eosin stain magniï¬cation a b by immunohistochemistry the tumor cells were positive for ae1ae3 c ckl d ck818 e emaf p40 g and vimentin h moreover of them were positive for ki67 i the ï¬nal diagnosis was sc [magniï¬cation ci ]diameter of the lesions ranged from to cm mean size cm in addition ulcers with an irregular base and slightlyraised borders were observed in of cases among the threepatients who underwent surgery two lesions invaded the gastricserosa and the remaining lesion invaded the gastric bare areaamong the two patients with biopsyproven gsc one patientexhibited tumor invasion of the gastric bare area the majorchanges in the ct imaging characteristics were an irregularouter layer of the gastric wall and obscuration of the perigastricfat initially the ct ï¬ndings were interpreted as gac in fourcases and gl in the tumor showed predominantly inhomogeneous densityand the radiodensity values were hu in the noncontrastphase heterogeneous enhancement was seen in four casesdue to necrotic or cystic areas and the other tumor revealedhomogeneous enhancement the radiodensity values on the apimages ranged from to hu and to hu in thevenous phase after contrast medium injection two tumorsshowed obvious enhancement and moderate enhancementwas seen in the other three tumors the peak tumor valuewas observed in the portal phase one of the three patientswho underwentlymphsurgery demonstrated evidence ofin one patientthe boundary betweennode involvementthe lesion and the left lobe of the liver was unclear andthe area with low attenuation was conï¬rmed by pathologythe liver withas a metastatic lesion in the rightcircular enhancement the remaining patientshowed noevidence of metastasis among the two patients with biopsyspecimens one patient was identiï¬ed as having lymph nodemetastasis on ctlobe ofct staging versus pathological stagingof gscnone of the gscs were staged as t1t2 by ct or pathologythe accuracy of ct staging t3 and t4 gsc was and respectively the overall diagnostic accuracy of ctfor determining the t stage of gsc was none of the gscs were staged as n2n3 by ct or pathologythe accuracy of ct in staging n3 and n4 gsc was and respectively the overall diagnostic accuracy of ct fordetermining the n stage of gsc was the comparison of tn staging based on ct and pathology isshown in table frontiers in oncology wwwfrontiersinaugust volume 0cliu gastric sarcomatoid carcinomafigure sarcomatoid carcinoma of the stomach in 62yearold women a unenhanced ct image of stomach reveals an intraluminal mass of homogeneousattenuation with an irregular surface at the gastric fundus and cardiac region bd contrastenhanced ct image shows obvious homogeneous enhancement ofmass with the peak value of the tumor on the portal phase in perigastric lymph nodes an enlarged and signiï¬cantly enhancement lymph node can be seenb arterial phase of contrast enhancement image c portal phase of contrast enhancement image d portal phase of contrast enhancement coronal imagediscussiontable ct and pathological tn staging for comparisonsarcomatoid carcinoma is an extremely rare and complicatedmalignant tumor composed of malignant epithelial componentsand atypical spindle cells however the spindle cells of scsappear to show evidence of epithelial diï¬erentiationforexample showing epithelial markers or epithelial ultrastructuralinstead of a speciï¬c line of mesenchymalcharacteristicsdiï¬erentiation moreoverliteratureemphasizes that the sarcomatous components occupy ofthe elements involved in the present study our patientstumor cells displayed atypical spindle shapes that expressed theepithelial phenotypethe currentsome ofsarcomatoid carcinomas can occur in almost any an wherecarcinoma can occur in the digestive system the incidencesof scs in the esophagus and liver are relatively high but scsare exceedingly rare in the stomach we could ï¬nd only sixprevious reports in the english literature table between and patients with sc conï¬rmed by pathologywere retrospectively analyzed with only ï¬ve tumors occurringin the stomach the average age of the reported patientswas years range and that in our series was years range a previous study reported that the sexcaseno no no no no ctt4an0t3n0t3n1t3n0t4an1na not available t tumor n nodepathological staget4an1t3n0nanat4an0distribution of male to female gsc patients was and thecorresponding proportion in our patients was ithas been noticed that scs are more common in male patientsand sex is a probable risk factor gsc patients may present withepigastric pain or discomfort dysphagia nausea and vomitinghematemesis and emaciation due to thickening of the gastricwall pain or discomfort in the upper abdomen is common thesymptoms can last from a few days to several years withoutobvious speciï¬cityin the present study of the cases of gsc were recognizedin the proximal stomach and the remaining tumor was founddistal to the stomach four cases of gsc in the present study hadfrontiers in oncology wwwfrontiersinaugust volume 0cliu gastric sarcomatoid carcinomatable clinical and imaging features of six previously reported cases of gcscasegenderageyearslocationsize cmshapeulcersenhanceappearancerecurrencemetastasistherapyprognosis mmffmmremnant stomachgreater curvaturelesser curvaturegastroesophageal junctionremnant stomachdistal stomachpolypoidpolypoidpolypoidulceratedpolypoidmassnenenenenehypernenonesurgerysurgerysurgeryendoscopysurgeryna mo d mo d mo d mo d mo dthe patient succumbed to heart failure before the surgical treatment an autopsy was performed yespresentpositive noabsentnegative hyper hyperdensene no evaluate mo month d dieareregarded asa longaxis diameter less than cm and the remaining tumorhad the largest longaxis diameter among our patients cmthe location distribution and longaxis diameters of the gscs inour patients were similar to those in previous reports in the actual diagnosis processthe diagnosis of sc has always been diï¬cult for cliniciansand pathologists especially the diï¬erential diagnosis fromcarcinosarcoma carcinosarcomastrulybiphasic neoplasms composed of distinct malignant epithelialcarcinomatous and mesenchymal sarcomatous componentsthe sarcoma components show typical specialized diï¬erentiation howeverthe termssarcomatoid carcinoma and carcinosarcoma have been usedinterchangeably in some cases therefore the understanding ofthese tumors has been hampered nevertheless we can try tofocus on whether there is a diï¬erence between these tumors froma new perspective the ct ï¬nding sc in the stomach have notbeen previously scientiï¬c reported or even detailed descriptionthere are only four simple descriptions chunchao reported that a patient with a giant sc presented a mass witha cm diameter in the antrum and body of stomach whichinï¬ltrated the gastric serosa the hepatic ï¬exure of the colon andgallbladder were also involved on ct contrastenhanced ctimages showed obvious enhancement of the two lesions sato reported a patient with sc of the remnant stomachand the radiographic examination showed an elevated lesionwith a large ulcer at the gastric cardiac lesser curvature thatmeasured cm in diameter the other two reports only describeda soft tissue mass or a large tumor in the dilated stomach on the other hand within in the upper gastrointestinal tractalthough there are fewer reports of carcinosarcoma localized inthe stomach this type of tumor is still more common than sc gastric carcinosarcoma showed an elevated lesion or thickenedgastric walls in of all reviewed cases tomoaki reported a 79yearold man with gastric carcinosarcomaand his veins showed severe invasion enhanced abdominalct showed irregular thickening and slight enhancement of thegastric wall on the side of the lesser curvature with suspiciousbulky lymph nodes yoshiyuki reported a 70yearoldjapanese woman who presented with a soft tissue mass adjacentto the lesser curvature of the stomach that was lobulated andct revealed an ulcer on the lesion the contrastenhanced ctimages showed heterogeneous enhancement of the mass theï¬nal pathological diagnosis was gastric carcinosarcoma inthe present study we found that gsc showed local thickeningof the gastric wall and mass formation often accompaniedby ulcers the site of the disease was mostly in the proximalpart of the stomach but these tumors can also occur in theremnant stomach the signal of the tumor was homogeneousor heterogeneous on plain ct scans after contrast mediuminjection of tumors demonstrated heterogeneousenhancement on ap images due to cystic areas or necrosis inthe lesions in this study the enhancement degree of all tumorsreached a peak in the pp after contrast enhancement for thesetumors the enhancement degree in the delayed phase wasnot signiï¬cantly reduced the overall enhancement mode wasdelayed enhancement in addition ct showed that four patientshad invasion into the gastric serosal region or gastric bare areatwo patients had the characteristics of enlarged perigastric orretroperitoneal lymph nodes and uneven enhancement and onepatient had invasion into the adjacent liver tissue these ï¬ndingsreï¬ect the metastatic and highly invasive characteristics of gscoverall ct and contrastenhanced ct can clearly show theprimary lesion inï¬ltration range lymph node metastasis anddistant metastasis of gsctomographic diagnosis of gsc has not been attemptedbecause of the rarity of this entity according to the ï¬ndingsof our study gsc needs to be diï¬erentiated from gac andgl on ct adenocarcinoma is the most common pathologicaltype of gastric tumor and is mainly distributed in the antrumseldomly in the body and fundus of the stomach the incidenceof gac is high in men and the median patient age is years the most common ct signs of gac are localor extensive thickening of the gastric wall mass formationincluding fungoidestype polypoidtype masses rough orsmooth serous surfaces and continuous interruption of themucosal layer tumors involving the mucosal surface can appearenhanced s after injecting a contrast agent the peakvalue for tumors invading the muscular layer usually appearsafter s and after the mucosal surface is strengthenedthe duration is longer primary gl accounts for ofmalignant gastric tumors and is predominantly situated in thegastric antrum gastric body and gastric fundus the incidenceof gl is high among males with a median patient age of yearsthe clinical symptoms included epigastric pain bleeding earlysatiety and fatigue the most common ct manifestations ofgl are diï¬use thickening of the gastric wall or a homogeneousfrontiers in oncology wwwfrontiersinaugust volume 0cliu gastric sarcomatoid carcinomatissue mass with slight attenuation or an appearancesoftsimilar to that of the normal gastric wall for gl becauseof hemorrhage necrosis submucosal edema or infarction thegastric wall may be heterogenous on ct gl originates froma submucosal process and gastric mucosa is commonly intactin the early stage but shows interruptions or ulceration in thelater stage after contrast medium injection most gl showedhomogeneous and slight enhancement in the delayed phase lymphoma is considered when distant structures the mesenteryretroperitoneum or other parts of the abdomen have lymphnode metastasis the ct ï¬ndings may only reï¬ect features of gsc but cannotaccurately diagnose gsc l one explore the origin of thesarcomatous portion immunohistochemistry ihc also failedto conclusively establish the origin of gsc rodrigues usedï¬uorescence in situ hybridization fish to conï¬rm that sc andadenocarcinoma have a common origin that is the epithelium while primary gl originated from gastric submucosallymphoid tissuethe main treatment for localized lymphomas is eradicationof helicobacter pylori and surgical treatment whereas advanceddisease often requires radiation or chemotherapy alone surgery is the only treatment option for patients with gacadjuvant chemotherapy and chemoradiotherapy are also oftenused targeted therapy isin the exploration stage however there are currently no speciï¬c national comprehensivecancer network guidelines for the treatment of only gscbecause the tumor is relatively rare although complete surgicalresection is the most important treatment method for examplewhile chemotherapy is considered in clinical practice whetherchemotherapy can be applied for gsc and the eï¬cacy ofchemotherapy remain controversial domblides ï¬rstevaluated the eï¬cacy of immune checkpoint inhibitors icis forsc and found that lung sc patients exhibited high response ratesand prolonged overall survival os with icis this studyprovides a new idea for the treatment of gscbecause gl tends to be conï¬ned to the gastric wall forprolonged periods before tumor spread its prognosis is betterthan that of gac previous literature has found that scin the parotid gland lung hypopharynx liver and pancreashave poor prognoses due to metastasis or recurrence with asurvival period of a few months similarly gscpatients also died or developed metastasis or recurrence withina few months or it was already in the advanced stage at theï¬rst diagnosis all these clinical manifestations suggest that gschas a poorer prognosis than gac and gl in additiongsc can metastasize through the blood and lymph nodesand the most common sites of metastasis are the local lymphnodes and liver this conclusion is consistent with ourresearch resultsconclusionthe incidence rate of gsc is extremely low so clinicians andradiologists are not familiar with the features of this tumorbased on systematic research of this rare tumor and comparisonswith common gastric cancers we found that gsc is morecommon in men who are approximately years old and isoften accompanied by ulcers the disease is mostly located in theproximal part of the stomach and can also occur in the remnantstomach with delayed enhancement on contrastenhanced ctimages these characteristics can provide a reference for furtherresearch on gscs in the future however an accurate diagnosisof gsc depends on the combination of clinical imaging andhistopathological features due to the aggressive nature and poorprognosis of the tumor rapid clinical intervention and detailedfollowup with ct are essentialdata availability statementthe original contributions presented in the study are includedin the supplementary material further inquiries can bedirected to the corresponding authorethics statementthe studies involving human participants were reviewed andapproved by the medical ethical committee of the zhengzhouuniversity the patientsparticipants provided their writteninformed consent to participate in this study written informedconsent was obtained from the individuals for the publication ofany potentially identiï¬able images or data included in this author contributionsyl manuscript preparationliterature research and dataanalysis pl literature research and data analysis kf manuscriptreview and data collection kc guidance of pathologicalknowledge sy guidance of imaging knowledge jj imaging datacollection and analysis wl and xz manuscript editing jgstudy conception and design manuscript review and guarantor ofintegrity of the entire study all authors have read and approvedthe ï¬nal manuscriptfundingthis work was supported by the national natural and sciencefund of china no references zhu cc li mr lin tl zhao g sarcomatoid carcinoma of the stomach acase report and literature review oncol lett doi ol20153460 snover dc levine gd rosaij thymic carcinoma five distinctivehistological variants am j surg pathol zhou dk gao bq zhang w qian xh ying lx wang wl sarcomatoidcarcinoma of the pancreas a case report world j clin cases doi 1012998wjccv7i2236 xie y xiang y zhang d yao x sheng j yang y sarcomatoidthe doi 103892mmr2018and review ofthe pancreasreportcaseofcarcinomaliterature mol med repafrontiers in oncology wwwfrontiersinaugust volume 0cliu gastric sarcomatoid carcinoma sato a oki e kohso h endo y uchida h hiroshige s sarcomatoidcarcinoma of the remnant stomach report of a case surg today doi 101007s0059501204027 nakayama y murayama h iwasaki h iwanaga s kikuchi m ikeda s gastric carcinosarcoma sarcomatoid carcinoma with rhabdomyoblastic andosteoblastic diï¬erentiation pathol int doi 101111j144018271997tb04540x robeycaï¬erty ss grignon dj ro jy cleary kr ayala ag ordonezng sarcomatoid carcinoma of the stomach a report of three caseswith immunohistochemical and ultrastructural observations cancer doi 101002109701421990040165730co2n ruess da kayser c neubauer j fichtnerfeigl s hopt ut wittel uacarcinosarcoma of the pancreas case report with comprehensive literaturereview pancreas doi 101097mpa0000000000000904 fujiie m yamamoto m taguchi k iwanaga a ohgaki k egashira a gastric carcinosarcoma with rhabdomyosarcomatous diï¬erentiation a casereport and review surg case rep doi 101186s407920160176z tanimura h furuta m carcinosarcoma of the stomach am j surg doi 101016000296106790325x kitamura s study on carcinosarcoma of stomach gan kumagai k kawai k kusano h matsuo k irie j tsuchiyama h a caseof socalled carcinosarcoma of the stomach gan no rinsho bekki t fujikuni n tanabe k yonehara s amano h noriyuki t thegastric carcinosarcoma with severe venous invasion a case report surg caserep doi 101186s4079201804218 ikeda y kosugi s nishikura k ohashi m kanda t kobayashi t gastriccarcinosarcoma presenting as a huge epigastric mass gastric cancer doi 101007s1012000604054 cid³n eu cuenca ij gastric adenocarcinoma is computed tomography ctuseful in preoperative staging clin med oncol doi cmos2641 hallinan jt venkatesh sk gastric carcinoma imaging diagnosis stagingand assessment of treatment response cancer imaging doi gossios k katsimbri p tsianos e ct features of gastric lymphoma eurradiol doi 101007s003300050069 rodrigues dn hazell s miranda s crespo m fisher c de bono js sarcomatoid carcinoma of the prostate erg ï¬uorescence insituhybridization conï¬rms epithelial origin histopathology doi 101111his12493 levine ms rubesin se pantongragbrown l buck jl herlinger h nonhodgkins lymphoma of the gastrointestinal tract radiographic ï¬ndings ajram j roentgenol doi 102214ajr16818976941 russo ae strong ve gastric cancer etiology and management in asia and thewest annu rev med doi 101146annurevmed081117 domblides c leroy k monnet i mazi¨res j barlesi f gounant v eï¬cacy of immune checkpoint inhibitors in lung sarcomatoid carcinoma jthor oncol doi 101016jjtho202001014 niu x sarcomatoid carcinoma in the parotid gland a review of years ofexperience laryngoscope doi 101002lary27474 li s jiang l he q wei w wang y zhang x the prognostic signiï¬canceof jmjd3 in primary sarcomatoid carcinoma of the lung a rare subtypeof lung cancer onco targets ther doi 102147otts22 dai l fang q li p liu f zhang x oncologic outcomes of patients withsarcomatoid carcinoma of the hypopharynx front oncol doi103389fonc201900950 seo n kim mj rhee h hepatic sarcomatoid carcinoma magnetic resonanceimaging evaluation by using the liver imaging reporting and data system eurradiol doi 101007s00330019060528 shi y chen j chen h hong x sarcomatoid carcinoma of the gallbladdera case report j int med res doi conï¬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 liu liang feng chen yue ji li zhao and gao this is anopenaccess distributed under the terms of the creative commons attributionlicense cc by the use distribution or reproduction in other forums is permittedprovided the original authors and the copyright owners are credited and that theoriginal publication in this journal is cited in accordance with accepted academicpractice no use distribution or reproduction is permitted which does not complywith these termsfrontiers in oncology wwwfrontiersinaugust volume 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 |
chrysoperla nipponensis okamoto which has the unique diapause phenotype distinguishable from nondiapause adult is an ideal model anism for studying the mechanism of reproductive diapause however there is no reliable and effective reference genes used for the reproductive diapause study of c a0nipponensis therefore in this study we evaluated the expression stability of candidate reference genes tub1 arpc5 ef1a 128up rps5 rps26e gapdh arp3 actin αtub in adults under diapause and nondiapause induction conditions using four statistical algorithms including genorm normfinder bestkeeper and ct method results showed that arp3 and tub1 were the most stable reference genes in all samples and in the adult tissues group arp3 and rps5 were the most stable reference genes in the development degree group αtub and ef1a were unstable reference genes under the conditions of this study meanwhile to verify the reliability of the reference genes we evaluated the relative expression levels of vg and vgr in different treatments significant upregulation and downregulation in expression level of two genes in response to diapause termination and diapause fat body tissue was respectively observed when using arp3 as the reference gene but not when using an unstable reference gene the reference genes identified in this work provided not only the basis for future functional genomics research in diapause of c a0nipponensis and will also identify reliable normalization factors for realtime quantitative realtime polymerase chain reaction data for other related insectskey words chrysoperla nipponensis okamoto reference genes qrtpcr reproduction diapausedue to the advantages of high sensitivity rapidity specificity and accuracy bustin et a0al valasek et a0al vanguilder et a0al shakeel et a0al quantitative realtime polymerase chain reaction qrtpcr has been widely used in the study of animals plants and microanisms roy et a0al jia et a0al zhang et a0 al ding et a0 al sun et a0 al qrtpcr is the most commonly used method for the expression analysis of target genes however the reliability of qrtpcr results in different samples is determined by a variety of factors among which the use of stably expressed reference genes is an important link for accurate detection of gene expression changes by qrtpcr bustin et a0 al at present several commonly used reference genes for data normalization include tubulin actin ribosomal protein elongation factor 1α glyceraldehyde3phosphate dehydrogenase 18s ribosomal rna and other genes bustin vanguilder et a0al however more and more studies have found that these reference genes do not show consistent expression patterns under different experimental conditions and even affect the reliability of experimental results therefore in order to obtain stable and reliable normalization factors reference genes with stable expression are usually used for correction and standardization to reduce errors between samples selection and evaluation of reference genes have become a necessary step before quantifying the expression of target genes accuratelynowadays there have been many studies on the selection of reference genes for insects such as sesamia inferens helicoverpa armigera aphis gossypii myzus persicae etc lu et a0 al shakeel et a0al zhang et a0al ma et a0al kang et a0al in these studies four statistical algorithms including genorm normfinder bestkeeper and ct method were mainly used to analyze the ct values obtained by qrtpcr of reference genes under various experimental conditions shakeel et a0 al finally the stability of the candidate reference genes was determined according to the geometric mean value of each gene ranked using different the authors published by oxford university press on behalf of entomological society of americathis 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 spermissionsoupcom 0c of insect science vol no algorithms and the most suitable reference gene was selected for the target gene expression analysis xiao et a0al kang et a0al chrysoperla nipponensis okamoto as one of the important predatory natural enemies of agricultural and forestry pests prefers to eat aphids thrips and other pests okamoto nie et a0al because of its characteristically wide geographical distribution and broad range of host prey niijima syed et a0al it has good prospects for widespread application in biological control mcewen et a0 al memon et a0 al reproductive diapause is an important way for c a0nipponensis adults to escape from adverse environments xu et a0al at present there have been many reports on the diapause of c a0nipponensis xu et a0al found that the body color of c a0nipponensis was green during the reproductive period but turned brown and yellow during the diapause period chrysoperla nipponensis belongs to the photoperiodic sensitive insect the adult diapause was induced by short photoperiods xu et a0al chen et a0al found that different photoperiods affected the material content eg protein and glycogen of c a0nipponensis diapause induced by the short photoperiod was beneficial to the storage of c a0nipponensis chen et a0al we expect an exponential increase of diapause research on c a0nipponensis at the molecular level in the near future thus stable and reliable reference genes are important for accurately quantifying gene expression of c a0nipponensisribosomal proteins and ribosomal rna have been used as reference genes in previous diapause studies for example williams et a0al used ribosomal protein rp49 as a reference gene to study the natural variation of drosophila melanogaster diapause williams et a0 al and sim and denlinger used ribosomal protein large subunit rpl19 as a reference gene in a study of ovarian development of culex pipiens during overwintering diapause sim and denlinger this indicates that under the same experimental conditions the selected reference genes in different species research are also differentin this research candidate reference genes were selected including tub1 arpc5 ef1a 128up rps5 rps26e gapdh arp3 actin and αtub whose expression profiles were measured by the qrtpcr the stability was analyzed by four statistical algorithms genorm normfinder bestkeeper and ct method in different developmental stages reproductive and diapause of adults and among different tissues the optimal reference genes under different conditions were determined which contributed to the accurate expression of target genes for future researchmaterials and a0methodsinsecta stable population of c a0 nipponensis was maintained in an artificial climate chamber rsz intelligent artificial climate chamber changzhou guohua jiangsu province under the following conditions a0± °c temperature a0± relative humidity rh and long photoperiod of l d h in our laboratory the eggs were collected by cutting the stalk and incubated in fingertip tubes a0cm in diameter and a0cm in height the primary hatching larvae were fed megoura japonica matsumura whose host plant was vicia faba l a0the adults were paired immediately after emergence in a bottle a0cm in diameter and a0cm in height fed a dry brewers yeast feed mixed with sucrose in a ratio of and then minced in a mortar and sifted through mesh and honey water the diapause adults used in this study were kept under the conditions of short photoperiod of ld h in all processes from eggs larvae pupae to adults whereas the nondiapause adults were kept under the conditions of long photoperiod of ld a0hsample collection development degree samples from individuals from varying developmental stages included female adults in the diapause induction period d under the short photoperiod the diapause maintenance period d under the short photoperiod the diapause termination period d under the short photoperiod and the reproduction period d under the long photoperiod each sample which included three to four females was independently replicated three times as three biological replicates adult tissues reproduction seven different tissues were collected from reproductive adults including ovarian fat body head wings antennae front thorax and epidermis the tissues of reproductive adults under long photoperiod were collected on the 10th day after emergence each tissue required about a0mg of material and each tissue sample collection was independently replicated three times as three biological replicates adult tissues diapause seven different tissues were collected from diapause adults including ovarian fat body head wings antennae front thorax and epidermis the tissues of diapause adults under short photoperiod were collected on the 20th day after emergence each tissue required about a0 mg of material and each tissue sample collection was independently replicated three times as three biological replicates adult tissues samples from reproductive and diapause adult tissues all samples samples from group and all treatments were immediately frozen with liquid nitrogen and stored in an ultralow temperature refrigerator at °c prior to rna extractiontotal rna extraction and cdna synthesisin this study total rna was extracted using minibest universal rna extraction kit takara japan and dnase i a0 was used for digestion of the membrane rna integrity was estimated by agarose gel electrophoresis rna concentration and purity were measured with a nanodrop one spectrophotometer thermo scientific then 1μg rna was reversetranscribed into the firststrand complementary dna cdna according to the hiscript ii q rt supermix for qpcr gdna wipers vazyme nanjing china instructions and stored at °c all cdna was diluted 10fold with dnasernasefree sterile water before usecandidate reference gene selection and primer a0designaccording to several commonly used reference genes candidate reference gene sequences were obtained by screening from the existing transcriptome of c a0 nipponensis in the laboratory namely tub1 arpc5 ef1a 128up rps5 rps26e gapdh arp3 actin and αtub in order to ensure the predictive accuracy of the selected sequences we conducted blast alignment all primers were designed using primer premier based on the following criteria gc content annealing temperature °c and primers length a0 bp and the specificity of each pair of amplicons was determined by qrtpcr followed by agarose gel electrophoresis and melting curve analysis the amplification efficiency of the pcr was calculated by using the formula e a0 a0 slope the slope was obtained by the standard curve which was generated by qrtpcr of a series of continuously diluted cdna samples 0c of insect science vol no realtime qrtpcr analysisthe 20µl total reaction volume were configured according to the protocol of chamq sybr qpcr master mix vazyme contained 10µl a0à chamq sybr qpcr master mix a0µl a0μm of each gene specific primer a0µl of cdna and a0µl of ddh2o the amplification reaction program was set as follows predenaturation at °c for a0s followed by cycles of denaturation at °c for a0s annealing at °c for a0 s the melting curves were analyzed in the °c temperature range after amplification step the reaction was performed on a roche lightcycler96 instrument to obtain ct values amplification curves melting curves and standard curves all samples were carried out in four technical and three biological replicates and the negative control no template was performed in paralleldata analysisct values for all samples were exported into an excel spreadsheet and were used to analyze the stability of candidate reference gene expression by genorm normfinder bestkeeper and ct method the comprehensive ranking were performed following methods adapted from xiao et a0 al the optimal number of genes was determined by the pairwise variation vnn1 between the normalization factors calculated by genorm among the four algorithms genorm and normfinder need to convert the original ct value according to the corresponding requirements before analysis in genorm the stability ranking of genes was determined by the expression stability value m value in bestkeeper the stability ranking of genes was determined by the coefficient of variation cv and sd in normfinder the stability ranking of genes was determined by the gene expression stability value sv in ct method the stability of genes was ranked according to the sd of genes ct values in four statistical algorithms genes with the lowest value were the most stably expressedvalidation of reference a0genesin most insects vitellogenin vg and vitellogenin receptor vgr play important roles in the reproductive process of female insects vg is taken up by developing oocytes through receptormediated endocytosis rme thereby promoting the development of oocytes and the formation of eggs in this process vgr is the main receptor mediating endocytosis previous studies have shown that reproductive diapause arrests development of oogenesis and vitellogenesis tatar and yin and the expression levels of the vg and the vgr in nondiapause female were significantly higher than those in reproductive diapause female jiang et a0al in order to evaluate the effectiveness of the selected reference genes the expression levels of the target genes vg and vgr were respectively detected by qrtpcr in the different development degree and tissues of adults and the most unstable reference gene was used for comparison in parallel the reaction system and program were the same as for qrtpcr of reference genes and four technical and three biological replicates were performed for each treatment the relative expression levels of vg and vgr were respectively calculated in excel using the ct method the differences of target genes expression levels were analyzed by tukeys test using spss software spss inc under different experimental conditionsresultsselection and primer performance of candidate reference a0genesthe candidate reference genes including tub1 arpc5 ef1a 128up rps5 rps26e gapdh arp3 actin and αtub were selected to identify the normalization factors for qrtpcr analysis and the sequence information has been submitted to genbank and the accession numbers are shown in supp table online only to determine the amplification specificity of the primers agarose gel electrophoresis and melting curve analysis were performed all primer pairs showed a single band and a single peak fig a0 to obtain correlation coefficient r2 and amplification efficiency e of pcr a standard curve was generated with the 10fold dilution series of cdna the results showed that the amplification efficiency ranged from to and the correlation coefficient varied from to supp table [online a0only]expression profiling of candidate reference a0genesto evaluate expression levels of the candidate reference genes the cycle threshold ct values under different groups were obtained by qrtpcr and represented by boxplot fig a0 the results indicated that ct values of all candidate reference genes were different under different conditions and also varied under the same condition overall ct values ranged from to among them the genes with higher expression abundance were tub1 rps26e and actin followed by 128up arp3 arpc5 rps5 and ef1a the genes with lower expression abundance were αtub and gapdh according to the ct value range of each candidate reference gene the genes with relatively stable expression were tub1 and arp3 whereas the most unstable genes were ef1a actin and αtubexpression stability of candidate reference genes under different conditionsin this study four statistical algorithms were used to analyze the expression stability of the candidate reference genes under different conditions including genorm normfinder bestkeeper and ct method as different statistical algorithms would generate different ranking patterns the comprehensive ranking of genes was finally determined through the geometric mean of sequencing bestkeeper the original ct values were used for analysis evaluated the stabilities of the candidate reference genes according to the cv and sd of the ct values ct method the difference values of original ct values were used for analysis performed stability ordering according to the mean sd of ct value genorm and normfinder the original ct values were converted for analysis sequenced the candidate reference genes according to the stability values the lower the stability value the more stable the gene expressiondevelopment degree of a0adultsthe expression stability of the candidate reference genes at different periods of reproductive and diapause female showed that the top four ranked genes identified by the genorm bestkeeper normfinder and δct method were similar but the rank order was slightly different arp3 and rps5 were the first and second stably expressed genes in the four statistical algorithms supp table [online only] and comprehensive ranking analysis fig a0 as for the third and fourth ranked gene tub1 and actin identified by comprehensive ranking analysis were the same as those generated by genorm and normfinder while bestkeeper selected arpc5 and tub1 ct method selected actin and rps26e supp table [online only] αtub was ranked by genorm bestkeeper normfinder and δct method as the least stable gene among the candidate reference 0c of insect science vol no fig specificity a and product length b of qrtpcr amplification for ten candidate reference genesgenes during different development degrees of adults supp table [online a0only]adult a0tissuesthe expression stability ranking of candidate reference genes in different tissues of reproductive and diapause females was varied according to the four statistical algorithms in different tissues of reproductive females the top four genes were rps5 arp3 arpc5 and tub1 respectively fig a0 but the rank order of the four genes was significantly different among different statistical algorithms rps5 was ranked first by genorm and ct method and was ranked third and fourth by normfinder and bestkeeper respectively arp3 was ranked first by bestkeeper and was ranked second third and fifth by normfinder δct method and genorm respectively arpc5 was ranked first and second by genorm and δct method and was ranked fourth and fifth by normfinder and bestkeeper respectively tub1 was ranked first and second by normfinder and bestkeeper and was ranked fifth and sixth by δct method and genorm respectively supp table [online only] however the four statistical algorithms found that ef1a was ranked as the least stable gene in the tissues from reproductive females supp table [online only]in different tissues of diapause females the top four genes were different from those of different tissues of reproductive females tub1 was ranked first by the comprehensive ranking followed by rps26e arp3 and 128up fig a0 through analysis it was found that ct method and normfinder displayed the same rankings for expression stability of candidate reference genes under this condition supp table [online only] tub1 was identified as the most stably expressed gene by genorm ct method and normfinder although it was ranked fifth by bestkeeper rps26e ranked steadily among the four statistical algorithms was ranked second by ct method and normfinder whereas it was ranked first and third by genorm and bestkeeper respectively arp3 was ranked first by bestkeeper with the smallest coefficient of variation whereas it was ranked third by ct method and normfinder in addition to being ranked third by genorm 128up was ranked fourth by bestkeeper ct method and normfinder supp table [online only] however in tissue from diapause females actin was consistently identified as the gene with the most unstable expression by the four statistical algorithms supp table [online only]in the reproductive and diapause female tissues the expression stability of the candidate reference genes was different in order to accurately determine the expression of the target genes the expression stability of candidate reference genes under the two conditions was analyzed according to the comprehensive ranking 0c of insect science vol no fig expression profiles of candidate reference genes in c a0nipponensis expression data are displayed as ct values for each reference gene using a box and whisker plot in different experimental conditions the line across the box is the median the box indicates the 25th and 75th percentiles the whiskers represent the 10th and 90th percentilesfig expression stability and comprehensive ranking of reference gene measured by the geomean method a a0lower geomean value indicates more stable expressiontub1 and ef1a were the most stable and unstable genes respectively whereas arp3 128up and arpc5 were ranked second third and fourth respectively fig a0 tub1 was the best candidate reference gene identified by normfinder and ct method and was ranked third and sixth by bestkeeper and genorm respectively arp3 was the most suitable candidate reference gene selected by bestkeeper and was ranked second by normfinder and ct method and fifth by genorm 128up and arpc5 were the most stable candidate reference genes identified by genorm whereas they were ranked separately fifth and sixth by normfinder and ct method and seventh and fifth by bestkeeper respectively supp table [online only] ef1a was identified as the least stable gene by genorm normfinder and δct method although bestkeeper selected actin as the least stable gene supp table [online only]all a0samplesin order to determine the best reference gene suitable for the different conditions of adults the stability of the ten candidate reference genes was ranked for all samples arp3 was identified as the most stable gene by the comprehensive ranking followed by tub1 arpc5 and rps5 whereas ef1a was identified as the least stable gene fig a0 0c of insect science vol no but the most and least stable genes identified by different statistical algorithms were slightly different arp3 was selected as the most stable reference gene by bestkeeper and ct method although tub1 and arpc5 were selected as the most stable reference gene by normfinder and genorm respectively ef1a was selected as the least stable reference gene by genorm and ct method despite it being ranked ninth by bestkeeper and normfinder supp table [online only]the best combination of candidate reference genes under different conditionsaccording to the pairwise variation vnn1 between the normalization factors and cutoff value calculated by genorm the number of reference genes required for optimum normalization in each experimental condition was determined the cutoff value of vnn1 a0 suggested that n reference genes were enough to make gene expression normalization otherwise n reference genes were needed the analysis results showed that all v23 were indicated that the optimal number of reference genes under each condition was two fig a0 more specifically arp3 and rps5 were the most stable gene combinations under adult developmental stage and reproductive adult tissues conditions tub1 and rps26e were the most stable gene combinations under adult diapause tissues conditions and arp3 and tub1 were the most stable gene combinations under adult tissues and all samples groups table a0relative expression levels of target genes vg and vgr genbank mt308983 mt522179 in the whole adult and from tissues of reproductive and diapause adults respectively when the most stable reference genes arp3 andor rps5 were used as normalization factors at different periods of reproduction and diapause the expression patterns of the target genes vg and vgr were consistent with low expression in the diapause period and rich expression in the late diapause and reproduction period however when the most unstable reference gene αtub was used as a normalization factor neither the target gene vg nor vgr showed a consistent expression pattern fig a0 under different tissue conditions when the most stable reference genes tub1 andor arp3 were used as the normalization factors the expression level of the target gene vg in the fat body of the reproductive female was significantly higher than that in the ovary of the reproductive female the expression level of the target gene vgr in the fat body of the reproductive female was lower than the ovary of the diapause female while when the most unstable reference gene ef1a was used as the normalization factor the expression pattern differed with normalization by tub1 and tub1arp3 fig a0 in general when the most stable reference genes were used as the normalization factors the accurate expression pattern of the target gene could be obtainedvalidation of reference a0genesthe stability of reference gene is very important for the analysis of expression level of target gene vg and vgr which are important for insect reproduction were selected to verify the applicability of the selected reference gene we examined the discussionqrtpcr has become an important means to explore gene expression level due to its high sensitivity rapidity specificity and accuracy and was widely used in physiology studies that investigated insect diapause such as drosophila melanogaster williams fig pairwise variations vnn1 was calculated by genorm to determine the optimal number of reference genes for accurate normalization in different conditions the cut off values under indicate that no additional genes are required for the normalization 0c of insect science vol no et a0al culex pipiens sim and denlinger leptinotarsa decemlineata lehmann et a0 al chrysopa septempunctata liu et a0al and pieris melete wu et a0al however the selection of appropriate reference genes was the key to accurately analyze the gene expression level for example under conditions of injury heatstressed and experimentally varied diets table recommendation for the best combination of reference genes based on the genorm and comprehensive rankings under various experimental conditionsgroupreference genemostdevelopment degreeadult tissues reproductionadult tissues diapauseadult tissuesall samplesarp3rps5tub1tub1 arp3 rps5arp3rps26earp3tub1leastαtubef1aactinef1aef1athe best reference gene was different in drosophila melanogaster ponton et a0al under biotic factors and abiotic stress inappropriate selection of the reference genes in locusta migratoria resulted in significant differences in the expression level of the target gene chitin synthase chs1 yang et a0al diapause of most insects was mainly affected by photoperiod and temperature in past studies the screening of reference genes of drosophila melanogaster ponton et a0al leptinotarsa decemlineata shi et a0al helicoverpa armigera zhang et a0al bombyx mori guo et a0al and harmonia axyridis qu et a0al under main environmental factors was completed by genorm normfinder bestkeeper and ct method in this study the expression profiles of candidate reference genes of c a0nipponensis were analyzed under different conditions by the same four statistical algorithms genorm vandesompele et a0 al normfinder andersen et a0al bestkeeper pfaffl and ct method silver et a0al different algorithms produced different stability rankings in order to obtain statistically consistent and accurate results we finally ranked the gene based on their stabilities determined by fig validation of selected reference genes under different periods a and tissues b of reproductive and diapause female in c a0 nipponensis relative expression levels of the vg and vgr in different samples using different normalization factors the most and least stable genes asterisks indicate significant differences in the expression levels of the vg and vgr r reproduction period d1 the diapause induction period d2 the diapause maintenance period d3 the diapause termination period 0c of insect science vol no comprehensive analysis method xiao et a0 al and selected the most stable reference genes under each condition as far as we know actin which played an important role in cell contraction and cytoskeletal maintenance was found in virtually all eukaryotic cells and was considered as an ideal reference gene for many anisms sürencastillo et a0al shakeel et a0al for example actin was used as a reference gene for normalization in the determination of genes related to reproductive and nutritional signaling such as vitellogenin of chrysopa septempunctata liu et a0al however in this study three genes related to actin were selected for analysis among which actin was similar to actin of c a0septempunctata which was also a member of the neuroptera in our study actin was the most unstable gene in the diapause female tissues but the actinrelated protein arp3 which was structurally homologous with actin showed better stability arp3 was selected as the most stable reference gene in adults of different developmental levels and all samples while it was the second most stable gene in the reproductive adult tissues and all adult tissues although arp3 was ranked as the third most stable gene in the diapause adult tissues it showed relatively stable expression in the expression profile tubulin which played an essential role in maintaining cell shape movement and intracellular material transport was also often used as a reference gene but different types of tubulin have different stability for example in the study of helicoverpa armigera the expression of βtub was relatively stable compared with that of αtub under almost all conditions zhang et a0 al similarly in this study αtub showed unstable expression and was the least stably expressed gene in adults of different developmental stages whereas tub1 was considered to be the most stably expressed reference gene in diapausing adult tissues and all adult tissues and was the second most stable gene in all samples ribosomal protein rp widely distributed in various tissues played an important role in protein biosynthesis and was widely used as a reference gene in many insects lu et a0al koyama et a0 al sun et a0 al in this study rps5 was considered to be stable in the tissues of reproductive females and ranked second among different developmental stages of adults elongation factors ef was a protein factor which promoted polypeptide chain to extension during the translation of mrna and was recommended as the ideal reference gene under different conditions of a variety of insects chapuis et a0 al ponton et a0 al however some studies showed that ef1α was one of the most unstable genes under certain conditions fu et a0 al in our study ef1a was found to be the most unstable gene in the reproductive adult tissues all tissues and all samples and the second least stable gene in the adults of different developmental stages and diapause adult tissues therefore it was not suitable for the study of c a0nipponensisrecently an increasing number of studies have demonstrated the importance of using multiple stably expressed reference genes for the accuracy of qrtpcr analysis ling and salvaterra yuan et a0 al kang et a0 al however this does not mean that the more reference genes increase the reliability of the results the study has indicated that either too few o | 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 |
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 |
"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 |
alcoholic liver disease ald is a chronic alcoholinduced disorder of the liver for which there are few effectivetherapies for severe forms of ald and for those who do not achieve alcohol abstinence in this study we used asystematic drugrepositioning bioinformatics approach querying a large compendium of geneexpression proï¬les toidentify candidate us food and drug administration fdaapproved drugs to treat ald one of the top compoundspredicted to be therapeutic for ald by our approach was dimethyl fumarate dmf an nuclear factor erythroid related factor nrf2 inducer we experimentally validated dmf in liver cells and in vivo our work demonstrates thatdmf is able to significantly upregulate the nrf2 protein level increase nrf2 phosphorylation and promote nrf2nuclear localization in liver cells dmf also reduced the reactive oxygen species ros level lipid peroxidation andferroptosis furthermore dmf treatment could prevent ethanolinduced liver injury in ald mice our results provideevidence that dmf might serve as a therapeutic option for ald in humans and support the use of computationalrepositioning to discover therapeutic options for aldintroductionoxidative stress is implicated in the development ofdiverse liver disorders such as alcoholic liver diseaseald12 ald encompasses a variety of chronic liverdiseasesincluding liver steatosis fatty liver hepatitiscombined with ammation ï¬brosis cirrhosis andultimately hepatocellular carcinoma hcc3 althoughalcohol abstinence is effective for patients with mild aldsteatosis there are few effective therapies for severeforms of ald and for those who do not achieve alcoholabstinence corticosteroid is the only treatment option toimprove the shortterm survival of severe alcoholiccorrespondence yongheng chen yonghenc163com orting liu liuting818126com1department of oncology nhc key laboratory of cancer proteomics statelocal joint engineering laboratory for anticancer drugs national center feriatrics clinical research xiangya hospital central south university changsha hunan china2department of gastroenterology xiangya hospital central south university changsha hunan chinathese authors contributed equally ye zhang shuang zhaoedited by m agostinihepatitis ah patients4 however many of these patientsdo not respond to this treatment and experience severeadverse effects such as infection5 therefore there is anurgent need to develop novel targeted therapeutics totreat severe forms of ald or patients who fail to achievealcohol abstinence the computational repositioning offood and drug administration fdaapproved drugs is apromising and efï¬cient avenue for discovering new uses6given the high costs possible side effects high failurerate and long testing periods for developing new medicines an fdaapproved compound was known to begenerally safe in humans and available for clinical use7 itis possible to identify safe drugs with potentialforrepurposing in other conditions by using computationalstrategies which can eliminate the need for a phase isafety trial and expedite phase ii efï¬cacy trials analysis ofinteractions between genes and fdaapproved drugsallow the pursuit of new indications for treating diseaseswith no fdaapproved pharmacotherapiesrecent advancements in computing and the dramaticexpansion of available highthroughput datasets have 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 0cenabled the development of drug repurposing to identifynovel treatment options for ald thus in this study weaimed to identify a new therapeutic option with potential forrepositioning in ald we used a systematic computationalapproach based on both public geneexpression patterns inald and the interactions between genes and fdaapproved drugs interestingly we identiï¬ed nuclear factorerythroid 2related factor nrf2 as a novel therapeutictarget in ald8 nrf2 is a basic leucine zipper bziptranscription factor that regulates the expression of certainproteins which protect cells against oxidative stress underunstressed conditions nrf2 is kept in the cytoplasm bykelch likeechassociated protein keap1 and cullin3upon oxidative stress nrf2 is phosphorylated at ser40 andreleases from keap1 then translocates into the nucleus inthe nucleus nrf2 forms a heterodimer with one of thesmall maf proteins maff mafg and mafk binds tothe antioxidant response element are in the promoterregions of many antioxidative enzymes and regulates thetranscription of these enzymes such as glutamatecysteineligase catalytic gclc and heme oxygenase1 ho1more surprisingly we found that the fdaapproved nrf2inducer9 dimethyl fumarate dmf which has not previously been described to have a therapeutic associationwith ald was determined to have a strong therapeuticpotential for repositioning in ald we evaluated the efï¬cacy of dmf for ald in liver cells and in vivo using anethanolinduced mouse model concordant with our computational prediction the experimental results demonstratethat dmf is able to significantly ameliorate ethanolinducedliver injury compared to untreated groupsresultscomputational repositioning of fdaapproved drugs foraldto identify efï¬cient therapeutic strategies for patientswith liver diseases we downloaded drug datasets thatcontain both clinical application and animal test fromgene expression omnibus wwwncbinlmnihgovgeogse accession number gse28619 and then weused a bioinformatics approach to testthe drugrepositioning potential of fdaapproved drugs for aldfrom this approach we computed the activity score ofcandidate drugs and compared geneexpression proï¬les inresponse to these drugs in ald then we annotated theknown gene targets of the topscoring candidates andqueried fdaapproved drugbank using gene targets as aninput which displayed an output of a list of chemicalcompounds notably ald cells are known to abnormallyexpress molecules in the antioxidant response pathwaythus we aimed to study one of the ï¬ve topscored candidate genes nrf2 among nrf2compound interactionsthe main use of dmf is previously tested with some success in multiple sclerosis patients with relapsing formsofï¬cial of the cell death differentiation associationfocused on thesuggesting that dmf used in the clinic may affect the aldgeneexpression signature this analysis led us to focus ondrugs targeting molecules fig 1a the majority of knownphysiologic or pharmacological nrf2 inducers are electrophilic molecules that covalently modify by oxidation oralkylation cysteine residues presentin the thiolrichkeap1 protein10 dmf is one of the known nrf2 inducers which has been tested for the treatment of multiplesclerosis and approved in for its drug bioavailabilityand efï¬cacy11 currently mmf has been used to develop asecond generation of nrf2 inducers as prodrugs12therefore wefumarateregulationmechanism of nrf2 in liver disorders the generation oftoxic metabolites by ethanol such as lipidperoxidationproducts contributes to the pathogenesis of alcoholic liverinjury fumarates prevent ros accumulation via the nrf2pathway in liver cells therefore we used an ald mousemodel six mice a group and hepatic ï¬brosis rat modelnine rats a group to examine the role of fumarates in vivohepatic lipid accumulation was distinctively increased inethanolfed rats in order to address the role of dmf inhepatic lipid accumulation we administered ald micewith dmf at mgkgday or mgkgday for daysin order to address the role of dmf in hepatic ï¬brosis weadministered hepatic ï¬brosis rats with dmf at mgkgday or mgkgday for weeks dmf ameliorated thehepatic steatosis induced by ethanol as observed in liversections stained with hematoxylin and eosin he fig 1band supplementary fig s1a at the same time the highlycrosslinked collagen fraction increased significantly during ethanolinduced ï¬brosis progression while collagendeposition was partly reduced under dmf treatment fig1c and supplementary fig s1b to substantiate theï¬nding that dmf increases the activity of nrf2 pathwayto inhibit ald we collected liver sections from normaland ald mice and checked nrf2 and gclc proteinlevels in the mouse model we performed immunohistochemistry ihc and western blotting for nrf2 andgclc results revealed that dmf treatment significantlyincreased nrf2 and gclc protein levels in ald mouseliver when compared to the matched control groups fig1df and supplementary fig s1c ddmf and mmf activate the nrf2 pathway in liver cellsregulator ofnrf2 is an essentialthe antioxidantresponse pathway which promotes the expression of various genes in response to oxidative stress1314 fumaratesprotect neurons and astrocytes against ros damage15 todetermine whether dmf or mmf regulates the nrf2protein level in liver cells we cultured hepg2 and lo2cells under the treatment of μm dmf and mmf fordifferent lengths of time and found that both dmf andmmf increased the protein level of nrf2 in a timedependent manner fig 2a and supplementary fig s2a 0czhang et al cell death and disease page of fig see legend on next pageofï¬cial of the cell death differentiation association 0czhang et al cell death and disease page of see ï¬gure on previous pagefig computational repositioning of food and drug administration fdaapproved drugs for alcoholic liver disease ald a schematicrepresentation of the bioinformatics workï¬ow for the repositioning approach used to identify potential candidate drugs and genes for the treatmentof ald b dimethyl fumarate dmf prevents ethanolinduced hepatic steatosis mice were fed with the control diet or ethanol diet containing vv ethanol respectively followed by treatment with mgkg dmf or mgkg dmf by oral gavage for days tissue sections from the mouseliver were prepared for hematoxylin and eosin he staining scale bars are μm c dmf decreases ethanolinduced hepatic ï¬brosis mice were fedas in b tissue sections from the mouse liver were prepared for collagen staining scale bars are μm d e dmf increases endogenous nrf2 andgclc to activate the nrf2 signaling pathway in the mouse liver immunohistochemical staining of nrf2 and gclc proteins in mouse liver tissuesliver tissue sections from different groups were stained immunohistochemically with antinrf2 antibody d or antigclc antibody e as indicateddata shown are from one mouse from each group scale bars are μm f nrf2 and gclc in mouse liver sections were compared against actb bywestern blotting the statistical analysis of all samples is shownfurther results revealed that the nrf2 protein level wasupregulated with increased dmf and mmf concentrationsfig 2b and supplementary fig s2b phosphorylationserine40 is required for nrf2 activation1617 to conï¬rmthe activation of nrf2 we treated hepg2 or lo2 cellswith dmf and mmf respectively as indicated thendetermined the level of phosphorylated nrf2 protein bywestern blotting results showed that dmf and mmftreatment significantly increased the phosphorylation levelof nrf2 when we adjusted the sample loading to keep thenrf2 level constant fig 2c and supplementary fig s2cindicating that nrf2 was activatedin addition wechecked the protein levels of nrf2regulated genes15 ourdata showed that dmf and mmf treatment promoted theexpression of gclc and ho1 protein levels fig 2a bmoreover nrf2 knockdown dramatically decreasedgclc and ho1 protein upon either normal condition orfumarates treatment fig 2d and supplementary fig s2dcollectively our results demonstrate that fumarates activate the nrf2 pathway in liver cellsonce phosphorylated nrf2 can translocate into thenucleus and activate transcription of various detoxiï¬cation and antioxidant enzymes upon exposure to stresses18to examine whether fumarates regulated nrf2 nuclearlocalization in liver cells we treated hepg2 or lo2 cellswith dmf and mmf at different concentrations for hfig 2e then cells were lysed and subjected to cytosolicand nuclear fraction extraction we found that dmf fig2e left pannel and mmf fig 2e right pannel promotednrf2 nuclear accumulation in a dosedependent mannermoreover we performed immunoï¬uorescence in livercells confocal microscopy data showed that nrf2expression and nuclear localization were enhanced inhepg2 cells upon dmf and mmf treatment fig 2ftaken together our data provide evidence that fumaratesactivate nrf2 and promote its translocation from cytoplasm to the nucleusdmf and mmf reduce the ros level by activating nrf2 inliver cellsthe relative levels of gsh and gssg are associatedwith various disease aging and cell signaling events19ofï¬cial of the cell death differentiation associationto illustrate the potency offumarates as antioxidantagents we performed the reaction to convert total glutathione and the oxidized form gssg to the reducedform gsh then we measured both total glutathioneand gssg in the luminescent reaction scheme with thegsh probe the results showed that dmf and mmfinduced a dosedependent increase of intracellular gshfig 3a b doxorubicin dox an effective anticanceragent can induce the generation of ros which then leadsto oxidative damage of cellular and mitochondrial membranes2223 27dichloroï¬uorescin diacetate dcfhdais a speciï¬c indicator of ros formation24 and has beenused widely as a ï¬uorescence probe in cells2526 confocalmicroscopy data revealed that ros were accumulated inhepg2 cells with the presence of dox while dmf andmmf blocked the doxinduced accumulation of rosfig 3c and supplementary fig s3a then we performedsirna transfection in hepg2 cells to knock down nrf2and observed a significant increase of ros upon doxtreatment even in the presence of dmf and mmf fig3d and supplementary fig s3b moreover we usedmitotracker® red cmxros kit an agent which can bepassively transported through the cell membrane anddirectly gathered on the active mitochondria to test theeffect of fumarates on the mitochondrial ros level wefound a significant reduction of h2o2 or ethanolinducedmitochondrial ros under fumarates treatment fig 3eand supplementary fig s3cthese results suggest aresistant effect of fumarates in response to ros by activating the nrf2 pathwaydmf and mmf reduce rosinduced lipid peroxidation andferroptosis in liver cellsrecent studies showed accumulation of ros can lead tolipid peroxidation and ferroptosis27 therefore we speculated that fumarates regulate rosinduced ferroptosis toexamine ferroptosis in dox or ethanoltreated cells weexamined the levels of hepatic malondialdehyde mdaand nadpnadph content2829 consistent with rosinduced ferroptosis we found that dox or ethanoltreatment significantly increased lipid peroxidation fig4a b and decreased nadph content fig 4c we 0czhang et al cell death and disease page of fig see legend on next pageofï¬cial of the cell death differentiation association 0czhang et al cell death and disease page of see ï¬gure on previous pagefig dimethyl fumarate dmf and mmf activate the nrf2 pathway in liver cells a dmf or mmf treatment increases endogenous nrf2gclc and ho1 protein level in a timedependent manner hepg2 or lo2 cells were either untreated or treated with μm dmf or mmf for differentlengths of time followed by being lysed and subjected to western blotting with the indicated antibodies b dmf or mmf treatment increasesendogenous nrf2 gclc and ho1 protein level in a dosedependent manner hepg2 or lo2 cells were either untreated or treated with dmf ormmf at the indicated concentrations for h actb is shown as a loading control c dmf or mmf increases the nrf2 s40 phosphorylation levelhepg2 or lo2 cells were treated as in b analyzed by western blotting with nrf2 phospho s40 antibody and normalized against nrf2 proteinthe sample loading was adjusted to keep the nrf2 level constant d nrf2 knockdown decreases gclc and ho1 protein levels under normal orfumarates condition hepg2 or lo2 cells were transfected with sinrf2 or negative control nrf2 gclc and ho1 protein levels were determined bywestern blotting e dmf or mmf promotes nrf2 nuclear accumulation after treated with μm dmf left panel or mmf right pannel for hhepg2 or lo2 cells were subjected to cytosolic and nuclear fractionation and nrf2 protein levels were determined by western blotting histone3h3 and αtubulin were used as nuclear and cytoplasmic markers respectively while actb was used as a wholecell lysate maker f hepg2 cells weretreated with dmso μm dmf or μm mmf for h as indicated then paraformaldehyde ï¬xed blocked and processed for immunoï¬uorescencewith dapi blue or antibody against nrf2 green nrf2 staining is shown on the left and the merged nrf2 and dapi on the right bar μm relativenrf2 ï¬uorescence intensity was calculated using imagej software the ratio was quantiï¬ed mean values were calculated from the individualdistributions in ten cells per conditionobserved a decrease of mda levels and a restoration ofnadph when we added fumarates into liver cells pretreated with dox or ethanol fig 4ac more evidencewas obtained when we detected the protein level of gpx4an important ferroptosis regulator which can inhibit cellmembrane phospholipid peroxidation results showedthat compared with dmso treatment gxp4 was substantially decreased under ethanolstimulated conditionindicating a promoting role of ethanol in liver lipid peroxidation and ferroptosis however we observed arestoration of the gxp4 protein level when we addedferrostatin1 an inhibitor of ferroptosis into hepg2 andlo2 cells pretreated with ethanol fig 4d and supplementary fig s4a a similar result was detected in mouseliver primary cells ethanol treatment lead to a significantdecrease offerrostatin1restored gpx4 protein pretreated with ethanol fig 4eand supplementary fig s4b in addition we treated livercells with erastin an inducer of ferroptosis which playsthe opposite role to ferrostatin1 in ferroptosis and foundfumarates led to an accumulation of gxp4 and nrf2protein even in the presence of ethanol or erastin fig 4ef we also detected lipid peroxidation with c11bodipy undecanoic acid by measuring the ï¬uorescenceintensity in red color consistent with our previousresults an increase of ros production was observedunderthe treatment of ethanol and erastin whileferrostatin1 or fumarates can inhibit lipid peroxidationinduced by ethanol supplementary fig s4c suggestinga preventive effect of fumarates in rosinduced lipidperoxidation and ferroptosisendogenous gpx4 whiledmf inhibits ethanolinduced lipid peroxidation andferroptosis in vivothese results strongly suggest that dmf prevents rosinduced liver injury and ferroptosis via activating thenrf2 pathway we therefore studied the role of dmf inrosinduced ferroptosis in mice hepatocytes treated withofï¬cial of the cell death differentiation associationethanol or not compared to the untreated group andferrostatin1 treated group groups treated by ferroptosisinducer erastin and ethanol had smaller ruptured mitochondria fig 5a these cellular morphological featuresare characteristic of ferroptosis however dmf ameliorated the ferroptosis induced by ethanol as observed bytransmission electron microscopymore evidence was obtained when we performed western blotting and ihc compared with the normal micethe protein levels of 4hne which indicated an increasedlipidperoxidationinduced ferroptosis were higher in aldmouse livers while the gpx4 protein level was lower incontrast dmf treatment could block lipidperoxidationinduced ferroptosis by decreasing the protein levels of4hne and increasing the protein levels of gpx4 in vivofig 5bf these data further validate fumarates as inhibitors of the lipidperoxidationinduced ferroptosisdiscussionusing a computational repositioning of existing drugsbased on the publicly available geneexpression data todiscover therapies for ald we inferred that the nrf2inducer dmf could serve as a therapeutic option for aldand performed experimental validations which demonstrated the efï¬cacy of dmf in ameliorating ald in livercells and in the mouse model the precise mechanism ofaction for dmf is unknown but it is known to activatethe nrf2 antioxidant pathway although dmf has notpreviously been suggested as a therapy for ald previousstudy has shown that nrf2 prevents alcoholinducedfulminant liver injury30 in this study we found thatfumarates activate the nrf2 signaling pathway promoting nrf2 phosphorylation and nuclear localization inliver cells nrf2 further activates the transcription ofgenes encoding various detoxiï¬cation and antioxidantenzymes in response to rosoxidative stress is implicated in the development ofdiverse liver disorders such as ald nonalcoholic fatty 0czhang et al cell death and disease page of fig see legend on next pageofï¬cial of the cell death differentiation association 0czhang et al cell death and disease page of see ï¬gure on previous pagefig dimethyl fumarate dmf and mmf reduce the ros level by activating nrf2 in liver cells a b dmf or mmf enhances cellular redoxpotential by increasing gsh level hepg2 cells were treated with or without different concentrations of dmf a or mmf b for h and thenassessed for cellular gsh and gssg levels denotes p ns denotes no signiï¬cance error bars represent mean ± sd for triplicate experiments cfumarates block dox or ethanolinduced ros accumulation hepg2 cells were pretreated with dox or ethanol for h followed by treatment with μm dmf or mmf for another h as indicated cells were loaded with dcfhda μm and incubated for min at °c in the darkfluorescence images were acquired by a confocal microscope bar μm d nrf2 knockdown accumulates ros damage in liver cells either with orwithout fumarates hepg2 cells were transfected with sinrf2 and treated as in c fluorescence images were obtained e fumarates block h2o2 orethanolinduced mitochondrial ros accumulation lo2 cells were pretreated with h2o2 or ethanol for h followed by the treatment with μmdmf or mmf for another h as indicated cells were incubated with mitotracker® red cmxros red at °c in the dark images were acquired byï¬uorescence microscope bar μmliver disease nafld and hcc2 elevated cellularstresses which are induced by alcohol hepatic viruses ordrugs play a vital role in the initiation and progression ofmultiple liver pathologies31 certain stressed conditions can cause the accumulation of cellular rosuncontrolled production of ros results in oxidativestress on tissues and cells and causes lipid peroxidation34the nrf2 antioxidant pathway is a highly conservedsignal transduction pathway that allows cells tissues andans to survive under oxidative stress conditions35 ourstudy showed that fumarates activate the nrf2 signalingpathway reduce the cellular ros level and protect livercells from ethanolinduced oxidative injuryferroptosis is an iron and rosdependent form of celldeath which is characterized by the accumulation of lipidlevels3637 ros accumulationhydroperoxides to lethalcould directly react with unsaturated fatty acids whichmay lead to a destruction of the mitochondrial membrane a massive release of substances promoting apoptosisand increased ferroptosis dysregulation offerroptosis has been implicated in various pathologicalprocesses including cancer neurodegenerative diseasesacute renal failure druginduced hepatotoxicity ischemiareperfusion injury and tcell immunity3839 our studyshowed that fumarates upregulate the protein level ofgpx4 a gshdependent enzyme that reduces lipidhydroperoxides while decrease lipid peroxidation andferroptosis and thus ameliorate ethanolinduced liverinjury in the ald mouse model fig in addition theseï¬ndings support that fumarates could also be effective inother ferroptosisassociated diseasesin recent years drug repurposing has gained more andmore attention for accelerating drug development40given the high costs possible side effects high failure rateand long testing periods for developing new medicines7drug repurposing provides an attractive approach to meetthe need for improved diseases treatment for exampledisulï¬ram an old alcoholaversion drug has emerged as acandidate for treating highrisk breast cancer7 hippeastrine hydrobromide hh which has been used to preventavian uenza h5n1 has become a promising drug forinhibiting zika virus zikvinfection41 topiramate aofï¬cial of the cell death differentiation associationsafe and effective drug for treating neurological diseasesis capable of ameliorating ammatory bowel disease42in this study we demonstrate that computational repositioning of fdaapproved drugs by analyzing publicgeneexpression data can be used to infer drug therapiesfor ald and offer experimental evidence that the nrf2inducer dmf is capable of ameliorating disease pathophysiology in the ald mouse model dmf was alreadyestablished as a safe and effective drug for treating multiple sclerosis43 additional clinical investigation will beneeded to test whether dmf could beneï¬t patients suffering from aldmaterials and methodscell culture and treatmentcell culture was performed as previously described44hepg2 or lo2 cells were cultured in dmemhigh glucose medium hyclone sh3002201 or rpmi mediummodiï¬ed hyclone sh3080901 supplemented with fetal bovine serum gibco penicillin andstreptomycin gibco at °c in a humidiï¬edatmosphere containing co2 for fumarate treatmentcells were ï¬rst cultured in the medium which containedfetal bovine serum then dmf sigmaaldrich and mmf sigmaaldrich of different concentrations were added into the medium the treatmentsto increase cell oxidative stress and ferroptosis wereperformed by adding ethanolsigmaaldrich e7023 mm doxorubicindox solarbio d8740 μm anderasitin selleck s7242 μm to the culture medium for hthen we treated liver cells with fumarates orferrostatin1 sigmaaldric sml0583 μm for another h all the concentrations are ï¬nal concentrations in theculture mediumwestern blottingwestern blotting was performed as previously mentioned4546 hepg2 or lo2 cells were lysed in ripa lysisbufferbeyotime p0013b containing protease andphosphatase inhibitors cell debris was removed by centrifugation while celllysates were boiled for minand centrifuged at °c before loading on or 0czhang et al cell death and disease page of fig see legend on next pageofï¬cial of the cell death differentiation association 0czhang et al cell death and disease page of see ï¬gure on previous pagefig dimethyl fumarate dmf and mmf reduce rosinduced lipid peroxidation and ferroptosis in liver cells a b fumarates obviouslyreverse dox or ethanolinduced lipid peroxidation hepg2 a and lo2 b cells were pretreated with μm dox mm ethanol or μm erastinfor h followed by μm ferrostatin1 or μm fumarates for h thereafter cells were lysed and subjected to lipid peroxidation malondialdehydemda assay c fumarates reverse dox or ethanolinduced ferroptosis lo2 cells were pretreated with μm dox mm ethanol or μm erastinfor h followed by μm ferrostatin1 or μm fumarates for h thereafter cells were lysed and subjected to nadpnadph assay denotesp denotes p and ns denotes no signiï¬cance error bars represent mean ± sd for triplicate experiments df fumarates block lipidperoxidation and ferroptosis in liver cells hepg2 cells lo2 cells d f or mouse liver primary cells e were pretreated with mm ethanol or μmerastin for h as indicated followed by μm ferrostatin1 or μm fumarates for another h thereafter cells were lysed and subjected to westernblotting for nrf2 and gxp4 with actb as loading control the statistical analysis of all samples is shown fsdspage gels then proteins were transferred ontopvdf membranes merck millipore ltd ipvh00010 forwestern blotting analysis the primary antibodies tophosphors40 nrf2 abcam ab76026 workingdilution nrf2 proteintech 163961ap working dilution gclc proteintech 126011ap working dilution ho1 proteintech 107011ap working dilution αtubulin proteintech 660311lg working dilution gxp4 abcam ab125066 working dilution histone3 proteintech 1ap working dilution actbβactin proteintech 205361ap working dilution werecommercially obtainedrna interferenceknocking down of nrf2 was performed by rnainterference following the manufacturers instructions forlipofectamine rnaimax reagent invitrogen the knockdown efï¬ciency was determined by westernblotting synthetic sirna oligo nucleotides were obtainedcommercially from genepharma co ltd list of effectivesequences is as follows sinrf21 ²gguugagacuaccaugguutt3²sinrf22 ²ccagaacacucaguggaautt3²sinrf23 ²gccuguaaguccuggucautt3²negative control ²uucuccgaacgugucacgutt3²cytoplasmic and nuclear extractsfor nrf2 nuclear translocation experiments cells werecultured in the medium which contained fetal bovineserum then dmf and mmf of different concentrationswere added into the medium for h in all 10cmdiameter plates of hepg2 and lo2 cells were lysed andcytosolic and nuclear fractions were separated followingthe protocol provided by the nuclear and cytoplasmicextraction kit manufacturer active motif inc the nuclear pellets were washed three times with phosphate buffered saline containing freshly added proteaseand phosphatase inhibitors the cytosolic supernatantwas centrifuged to remove any nuclear contamination andtransferred to a new tube both the cytosolic and nuclearfractions were boiled separately in sds sample buffer andanalyzed by western blotofï¬cial of the cell death differentiation associationgsh analysishepg2 cells were plated into white and ï¬atbottom well plates and cultured for h at °c then we treatedcells with dmso or fumarates and incubated for another h for ï¬uorescent gsh assay we ï¬rst washed cells withhanks balanced salt solution solarbio h1045500 thendetermined the levels of reduced and oxidized gsh bygshgssg assay kit promega v6611 according to themanufacturers protocol totalrelative luminescenceunits rlu are graphed as means ± sd denotes p ns denotes no signiï¬cance graphed data represents oneof three experimental repeatsmeasurement of cell lipid peroxidation and nadpnadphassayin thefumarates erasitin μm orliver cells were plated into 60mm dishes and culturedfor h at °c the treatments to increase cell lipidperoxidation were performed by adding ethanol mmdoxorubicindox μm and erasitin μm to theculture medium for h then we treated liver cells with orwithoutferrostatin1 μm for another h all the concentrations are ï¬nalconcentrationslipidperoxidation assay and nadpnadph assay we ï¬rstwashed cells with °c precooled phosphate bufferedsaline then determined the levels of cell lipid peroxidation by mda assay kit beyotime s0131 and nadpnadph quantitation colorimetric kit biovision k347according to the manufacturers protocol the totalhepatic mda content and nadpnadph levels aregraphed as means ± sd graphed data represent one ofthree experimental repeatsculture medium forimmunoï¬uorescence staininghepg2 cells were plated into glass bottom cell culturedishes nest and pretreated with or withoutdox for h followed by addition of dmf and mmf intothe medium thereafter cells were ï¬rst ï¬xed with paraformaldehyde biosharp then permeabilized in triton x100 amresco blocked by bovine serum albumin amresco in pbs buffersigmaaldrich p5368 and lastly incubated with theindicated primary nrf2 antibody working dilution 0czhang et al cell death and disease page of fig see legend on next pageofï¬cial of the cell death differentiation association 0czhang et al cell death and disease page of see ï¬gure on previous pagefig dimethyl fumarate dmf inhibits ethanolinduced lipid peroxidation and ferroptosis in vivo a dmf prevents ethanolinducedferroptosis mice were fed as indicated on the ï¬nal day morning the mice were given alcohol liquid gkg or maltodextrin control by gavageand sacriï¬ced after h in addition ferrostatin1 mgkg and erastin mgkg were provided min before | Colon_Cancer |
" ovarian cancer is the leading cause of cancerrelated death among women complete cytoreductivesurgery followed by platinumtaxene chemotherapy has been the gold standard for a long time variouscompounds have been assessed in an attempt to combine them with conventional chemotherapy to improvesurvival rates or even overcome chemoresistance many studies have shown that an antidiabetic drug metforminhas cytotoxic activity in different cancer models however the synergism of metformin as a neoadjuvant formulaplus chemotherapy in clinical trials and basic studies remains unclear for ovarian cancermethods we applied two clinical databases to survey metformin use and ovarian cancer survival rate the cancergenome atlas dataset an l1000 microarray with gene set enrichment analysis gsea analysis western blotanalysis and an animal model were used to study the activity of the aktmtor pathway in response to thesynergistic effects of neoadjuvant metformin combined with chemotherapyresults we found that ovarian cancer patients treated with metformin had significantly longer overall survival thanpatients treated without metformin the protein profile induced by low concentration metformin in ovarian cancerpredominantly involved the aktmtor pathway in combination with chemotherapy the neoadjuvant metforminprotocol showed beneficial synergistic effects in vitro and in vivos this study shows that neoadjuvant metformin at clinically relevant dosages is efficacious in treatingovarian cancer and the results can be used to guide clinical trialskeywords neoadjuvant metformin ovarian cancer clinically relevant dosage aktmtor pathway synergisticeffects correspondence syeungmdanderson limh33mailsysueducn kuochang wen and pilin sung share first authorship9department of emergency medicine division of internal medicine theuniversity of texas md anderson cancer center houston tx usa10guangdong research institute of gastroenterology the sixth affiliatedhospital of sun yatsen university yuancun erheng rd guangzhou pr 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 0cwen of ovarian research page of ovarian cancer is the fifth leading cause of mortality in developed countries in the united states an estimated women were diagnosed with ovarian cancer in and deaths due to ovarian cancer occurred complete cytoreductive surgery followed by standard firstline platinumtaxene chemotherapy has been shown to improve the survival rate however the majority of patientsexperience relapse and the 5year survival rate is approximately chemoresistance to platinumbased treatment remains a major challenge in the successful treatmentof ovarian cancer and the mechanisms underlying platinum resistance are multifactorial various cellular processes are observed in resistant cells and activation of thepi3kakt pathway is believed to be a determinant of resistance in ovarian cancer [ ] thus the development ofan improved treatment to overcome acquired resistance incancer cells or decrease the side effects of platinumbasedtreatment is needed to treat ovarian cancermetformin n0n0dimethylbiguanide a biguanide is anoral hypoglycemia agent that is widely used as an antidiabeticdrug to treat type diabetes mellitus dm it is also widelyused to treat polycystic ovarian syndrome metformin hasbeen shown to reduce cancer development in type dm patients and inhibit growth in several cancer models [ ] either alone or in combination with cytotoxic agents [ ]the major target of metformin in cancer cells is the tumorsuppressor lkb1ampactivated protein kinase ampkpathway which serves as a metabolic checkpoint to arrestcell growth when intracellular atp levels are low such as innutrientpoor conditions after activating ampk metformin phosphorylates tuberous sclerosis complex tsc2and then binds with its obligate partner tsc1 tsc2 leadsto the accumulation of rhebgdp and the inhibition ofmtorc1 which influence eukaryotic translation initiationfactor 4ebinding protein 4ebp1 and ribosomal s6 kinases6k1 respectively shank showed that metformincan restrict the growth and proliferation of ovarian cancerstem cells yasmeen revealed that metformininduces apoptosis in ovarian cancer cell lines in an ampkindependent manner by activating caspases downregulating bcl2 and bclxl expression and upregulating baxand bad expression which results in cell cycle arrest in the sand g2m phases rattan identified metformin asan antiproliferative therapeutic that can act through bothampkdependent and independent pathways via thesepathways metformin inhibited cell proliferation in bothwildtype and ampk null mouse embryo fibroblasts as wellas in ampksilenced ovarian cancer cells in addition metformin has been shown to inhibit pi3kaktmtor signaling in lung cancer [ ] breast cancer pancreaticcancer and hepatic cancer however most studies showing that metformin alleviates cancer have used higher doses in vitro than thoseused in diabetic patients these high concentrationsmay directly cause the death of tumor cells in thepresent study we tested a low concentration as the effective dose which was a clinically relevant dose the effects of lowconcentration metformin on aktmtorsignaling in ovarian cancer remain unclearthe aim of the present study was to examine the effectsof a combination of metformin at clinically relevant dosages and chemotherapy on ovarian cancer via the aktmtor pathway we found that metformin reduced ovarian cancer death in two clinical datasets and predicted thatthe effect of metformin in ovarian cancer was mediated bythe aktmtor pathway using a bioinformatics modelthen we demonstrated that the low concentration ofmetformin inhibited the growth of a mouse ovarian surface epithelial cell line mosec and that it had a synergistic effect in combination with chemotherapy via theaktmtor pathway neoadjuvant application of metformin plus chemotherapy yielded beneficial synergistic effects both in vitro and in vivo the results provide insightinto the potential of neoadjuvant metformin to augmentthe efficacy of existing cancer therapeuticsresultsthe effect of metformin on survival in ovarian cancerpatientswe investigated the impact of metformin on humanovarian cancer by analyzing a clinical dataset in total patients were diagnosed with primary ovarian cancerin the department of gynecology and obstetrics taipeiveteran general hospital from to after theirclinical and drug histories were reviewed patientswho underwent complete surgery and were treated withcarboplatin were included in the analysis thirtytwo ofthese patients took metformin either during admissionor in the outpatient clinic os was measured from thedate of diagnosis to death or was censored at the date ofthe last followup the os of patients with metformintreatment n was significantly higher than that ofpatients without metformin n p fig 1atable figure 1b shows the ovarian cancerfree incidence of female dm patients n fromthe national health insurance taiwanese dataset ovarian cancer was less frequent among metformininsuthan among metforminlin users n insulin users n p the use of metformin or insulin may help prevent ovarian cancer in female dm patients we next investigated the cellulareffects of metformin on ovarian cancer the expressionprofiles of differentially expressed genes in response totreatment with metformin chemotherapy or both wereretrieved from the l1000 study gsea was performedusing the up and down gene expression datasets froml1000 gsea revealed that the gene expression induced 0cwen of ovarian research page of fig the effect of metformin on survival in ovarian cancer patients a kaplanmeier os of ovarian cancer patients with n or without n metformin use b the ovarian cancerfree incidence in female dm patients metformin ever usedinsulin ever used users n and metformin never usedinsulin never used users n from the national health insurance taiwanese dataset c tumorproliferation cell number 2dcolony formation and western blot analyses of the indicated proteins in the aktmtor pathway of mouse ovariancancer cells treated with different concentrations of metformin d comparison of the mortality rates between groups with low and high proteinexpression by the halfdivision approach kaplanmeier analysis assessed the correlations of the indicated proteins akt [total and pser473] mtor[total and pser2448] with the overall survival of patients data from the cbioportal tcga database tcga provisional ovarian cancer genomicsn a logrank pvalue less than indicated a significant difference in overall prognosis p p p by control and metformin treatment was similar to thatof the kegg pancreatic cancer pathway httpwwwgenomejpdbgetbinwww_bgetpathwayhsa05212fig s1 which predominantly involved the aktmtorpathway based on the proteomic and viability investigation metformin may involve the alternation of phosphorylation in the aktmtor pathway accompanyingcell retardation in ovarian cancer without affecting thetotal amount of protein fig 1c metformin inhibitedthe aktmtor pathway in a dosedependent manneras shown by western blot analysis the phenomenonwas found at both high mm and low mmdoses of metforminlow doses were closer to theclinically relevant concentration fig s2 at low dosesof metformin the alternation of phosphoampk wasnot as obvious as that in the aktmtor pathway wefurther investigated the clinical role of the aktmtorpathway using the tcga ovarian cancer dataset thepatients with upregulated phosphorprotein akt_pser473 or mtor_pser2448 had significantly poor osthe expression of total protein was not associated withclinicalimportance in ovarian cancer fig 1d thesefindings highlight the value of metformin in inhibitingovarian tumor cells via phosphorylation of the aktmtor pathway which indeed plays an essential role inthe prognosis of ovarian cancer 0cwen of ovarian research page of table the clinical characteristics of the study populationcharacteristicp valueno metformin usen metformin usen diabetes ratestageaearly i ii late iii iv histologybepithelialother typesc ± ± ± ± ca125ddeath rateoverall survivale§afigo stage international federation of gynecology and obstetrics surgicalstaging of ovarian cancer missing value not included in statistical testbmissing value not included in statistical testcincluding primary peritoneal serous carcinoma ppscdbefore surgery mean ± sd umleoverall survival months mean ± sdcalculated by chisquare testcalculated by students ttest§calculated by kaplanmeier logrank testmetformin at a clinically relevant dosage inhibits ovariancancer growth through the aktmtor pathwayto mimic the clinically relevant dosages in the humanbody we used lowdose concentrations of metformin inthe study we assessed the cell growth of metformin in mmtreated ovarian cancer cell lines to evaluate thegrowthinhibitory effect of metformin as shown infig 2ab mm metformin can reduce colony formation and cell growth both mouse and human ovariancancer cell lines manifested significantly reduced proliferation after treatment with clinically relevant doses ofmetformin fig s3a furthermore mm metformintreatment beginning from day to day reduced cellviability and cell viability recovered from day to day after discontinuing treatment fig 2c the inhibitory effect of lowdose metformin on the aktmtor pathwaywas shown during metformin treatment day to day the phosphoprotein expression was inhibited duringmetformin treatment but these expression levels wererestored when suspending metformin fig 2d theaforementioned data suggested that metformin at lowdose concentrations could inhibit cell growth in ovariancancer through inhibition of the aktmtor pathwayas supported by the gseathe synergistic effects of metformin and chemotherapyon ovarian canceralthough standard firstline platinumbased protocolsimprove survival in ovarian cancer strengthening thesechemotherapy regimens is warranted we evaluated theantiproliferative effects of different protocols beforeduring or after and doses of metformin in combinationwith chemotherapy fig 3aleft panel as shown infig 3a concurrent combination of metformin andchemotherapy yielded the strongest inhibition fortyeighthour exposure to concurrent metformin andchemotherapy resulted in a clear synergistic effect withnegative log ci values in ovarian cancer cells fig 3aright panel regarding human ovarian cancer cellslower concentrations of carboplatin μm but nothigher concentrations μm show synergy with metformin with negative log ci values fig s3b the activated forms of akt are key intracellular mediators ofgrowth cell survival and platinum response determining the activation state of the aktmtor pathway isimportant for understanding the synergistic mechanismof action of lowdose metformin combined with chemotherapy in ovarian cancer western blot analysis demonstrated that both chemotherapy alone and metforminalone reduced the levels of phosphorylated aktmtorwithout affecting the total amount of aktmtor protein fig 3b the combination of metformin and chemotherapy produced a stronger inhibition of pakt and theakt downstream effectors pmtor ser2448 ps6 kinaseser235236 and p4ebp1 thr3746 compared withmetformin or chemotherapy alone in contrast the totalamounts of mtor s6 kinase and 4ebp1 were unaffectedby treatment and ampk phosphorylation was not reduced by treatment with lowdose metformin or chemotherapy either alone or in combinationsome dm patients treated with metformin cannotachieve good blood sugar control and require other medications we investigated the cellular effects of metformintreatment on tumors in patients with poor blood sugarcontrol fig 3c left panel as shown in fig 3c middlepanel mtt assays indicated that cells cultured in highglucose medium mgl showed accelerated cell proliferation relative to that of cells in control medium mgl however metformin treatment diminished this effect induced by high glucose the combined effect of metformin and chemotherapy in a highglucose medium wasantagonistic with a positive log ci fig 3c right panelto determine whether high glucose induces the aktmtor pathway in mosecs we treated the cells with metformin alone chemotherapy alone or a combination inhighglucose medium or control medium for h fig s4highglucose medium resulted in marked increases inpakt pmtor ps6 kinase and p4ebp1 levels comparedwith the control medium treatment with metformin andcarboplatin in the highglucose medium reduced the protein level of pakt but not the protein levels of pmtorps6 kinase and p4ebp1 relative to the levels in the controlmedium these results revealed that high glucose or poorblood sugar control may diminish the antitumor effects ofmetformin and chemotherapy although they still have effects on the paktmtor pathway 0cwen of ovarian research page of fig see legend on next page 0cwen of ovarian research page of see figure on previous pagefig metformin at a clinically relevant dosage inhibits ovarian cancer growth through the aktmtor pathway ab growth of mouse ovariancancer cell line mosec in cells incubated for serial days with lowconcentration metformin mm met metformin p by twowayanova c and d cell viability and protein analyses of the aktmtor pathway under treatment with metformin mm from day to day andwhen treatment was suspended from day to day the beneficial synergistic effects of neoadjuvantmetformin under combination treatmentwe further investigated the synergistic effects of lowconcentrations of metformin and chemotherapy underdifferent treatment regimens commonly used in clinicalsettings as shown in fig 4a synergistic effects were obviously observed in both protocol neoadjuvant metformin and protocol concurrent metformin but notin protocol adjuvant metformin western blot analysis indicated that protocols reduced the activitiesof pakt pmtor ps6 kinase and p4ebp1 with protocols and having superior effects compared withprotocol fig 4b these results indicate that metformin should be used before or at least concurrent withchemotherapy to enhance the antitumor effect neoadjuvant metformin combined with chemotherapy was betterthan the combination protocolwe further investigated the in vivo antitumor activityof neoadjuvant metformin in b6 mice bearing mosecsthat were grown subcutaneously as tumor xenograftstreatment with metformin or chemotherapy as singleagents caused a decrease in tumor size relative to that ofcontrol untreated mice treatment with the neoadjuvantmetformin combined with chemotherapy significantlyreduced tumor growth fig 4c finally we selected thecases with chemotherapy from fig 1a to determine theclinical impact of metformin in chemotherapy metformin before or during chemotherapy indeed showed atrend toward better overall survival compared with single chemotherapy although this trend did not reach statistical significance fig 4dschematics of the intracellular effects of treatmentwith metformin chemotherapy or their combination onovarian cancer are shown in fig 4e neoadjuvant metformin combined with chemotherapy produced bettersynergistic effects inhibiting akt and its downstreampathway a highglucose environment such as that inpoorly controlled type dm patients may increase activated aktmtor signaling thusfor patients withpoor glucose control the combination of metformin andchemotherapy may be slightly superior to chemotherapyalone and not as efficacious as that in patients with goodglucose controldiscussiondiabetes is strongly associated with an increased incidence of cancer many studies have shown thatmetformin can reduce the risk of cancerincludingbreast colon liver and pancreatic cancers and improveoutcomes over those obtained with other antidiabetictreatments sulfonylurea insulin in diabetic patients whether metformin can reduce the risk of ovarian cancer has been investigated [] but few studies havefocused on the effects of metformin combined withcommonly used firstline chemotherapeutic drugs suchas carboplatin and the underlying mechanisms neoadjuvant metformin combined with other therapieshave been administered to treat erpositive breast cancer in a phase ii clinical trial clinicaltrialsgov identifier nct01589367 in this study we evaluated thesynergistic effects of neoadjuvant metformin combinedwith chemotherapy in ovarian cancer via the aktmtor pathway both in vitro and in vivo and found thatpoor glucose control diminished the synergistic antitumor effects of combination treatmenta previous casecontrol study that used the ukbasedgeneral practice research database revealed thatthe adjusted odds ratio of metformin use vs nonuse forovarian cancer incidence was not significant in nondiabetic patients but was significant in diabetic patientsin another recent metaanalysis which included one observational study and two clinical trials the pooled oddsratio ci of metformin use for ovarian cancer incidence was recently a study wasperformed using reimbursement databases of the national health insurance nhi to evaluate metforminuse in taiwanese women with type dm metformindecreased the incidence of ovarian cancer and the overall fully adjusted hazard ratio ci for everusersversus neverusers was in thepresent study the results were similar to those of a previous report in which ovarian cancer was less likelyto occur in metformininsulin users n than in metformininsulin users n thisprevious study similarly used reimbursement databasesof the nhi although the data were from a different timeperiod the hospital cohort data showed that ovariancancer patients treated with metformin had a significantly longer os compared with patients not treatedwith metformin this finding is similar to that of another casecontrol studyin which an association between metformin use and improved survival of ovariancancer patients was identified these results indicated that metformin may reduce ovarian cancer incidence in type dm patients and improve survival aftera diagnosis of ovarian cancer 0cwen of ovarian research page of fig the synergistic effects of metformin and chemotherapy on ovarian cancer a synergistic effects of metformin combined withchemotherapy carboplatin at different concentrations and different combination protocols concentration of metformin and mmconcentration of carboplatin and μm met metformin chemo chemotherapy b the effects of metformin alone mmchemotherapy carboplatin μm alone or combined treatment assessed by the indicated antibodies in western blot analysis c mtt assaysshowed that cells cultured in a highglucose medium mgl exhibited greater growth than those in control medium mgl greensquares represent cells cultured in highglucose medium and red solid circles represent those cultured in control medium concentration ofmetformin and mm concentration of carboplatin and μmmost studies examining the effects of metformin oncancer have used doses mm higher than those usedclinically for diabetic patients [ ] yielding metforminplasma concentrations between and μmoll fewstudies have focused on the impact of metformin at clinically relevant dosages although dr hu and colleagues 0cwen of ovarian research page of fig see legend on next page 0cwen of ovarian research page of see figure on previous pagefig the beneficial synergistic effects of neoadjuvant metformin under combination treatment a and b protocol a neoadjuvant protocolmosecs treated with metformin alone for day and then with a combination of metformin and carboplatin for days protocol a concurrentprotocol mosecs treated with both metformin and carboplatin from day for days protocol an adjuvant protocol mosecs treated withcarboplatin alone from day for day and then with a combination of metformin and carboplatin for day pink circles represent log ci valuesunder protocol green squares represent those under protocol and yellow triangles represent those under protocol concentration ofmetformin and mm concentration of carboplatin and μm met metformin chemo chemotherapy imagej analysis for relativeintensity of protein bands c mosecs were injected into b6 mice n which were divided into groups and subcutaneous tumor size wasmeasured after different treatments control metformin or carboplatin alone neoadjuvant metformin from monday combined with carboplatinfrom wednesday subcutaneous tumors were assessed at the end of the experiment control vs met p control vs chemo andmet chemo p d kaplanmeier os of ovarian cancer patients with n or without n metformin in before or duringchemotherapy e the model of synergistic inhibitory effects by neoadjuvant metformin combined with chemotherapy in the aktmtor pathwayadministered lowdose metformin mm or mmto suppress ovarian and breast cancer cell growth and survival in vitro the aim of their study was to investigatewhether lowdose metformin reprograms these cancercells into noncancerous cells in a foxo3dependentmanner which may allow patients to successfully overcome these cancers with minimal side effects howeverthey did not compare the inhibitory effects or therapyprotocol of metformin combined with chemotherapy inaddition they administered metformin via intravenous injection of metformin mgkg bw in mice which contrasts the more common oral route used in clinicalapplications for example diabetes mellitus in thepresent study we tested a low concentration of mm μmoll as the effective dose the cellular events observed in vitro suggest that this dose is safe and can betranslated to in vivo conditions the dosage of metformingiven to mice was mgkgday which is equivalent to mgday for a 60kg person according to a formulasuggested by the national institute of health usa this equivalent dosage is times lower than the maximum safe dosage of mgday recommended in thephysicians desk referencemetformin activates ampk via lkb1 which leads tothe inhibition of mtor signaling and its major downstream effectors the 4ebps and p70s6ks and the inhibition of global protein synthesis and proliferation invarious cancer cell lines [ ] in previous studies metformin tested on ovarian cancer was found to induce cellcycle arrest apoptosis angiogenesis and decreasedpmtor expression p38 mapk pathway activity and cancer stem cell activity in this study weshowed for the first time the effects of metformin on geneexpression patterns using gesa and the l1000 systemand found that the effects of metformin on gene expression in ovarian cancer are similar to those following activation of the kegg pancreatic cancer pathway fig s1in previousfollowing metformin treatmentphosphoakt levels decreased in two pancreatic cancercell lines a549 and panc1 we analyzed tcga dataand found that the phosphoaktmtor pathway is a determinant of clinical survival in ovarian cancer [ ] westudiesalso demonstrated for the first time the effects of metformin in combination with carboplatin a firstline chemotherapy drug and showed that the synergism of thesedrugs is due to the inhibition of the aktmtor pathwaywhich is independent of ampk at micromolar concentrations of metformin mm these results are consistentwith those of rattan who showed that metformin as an antiproliferative therapeutic can act throughboth ampkdependent and independent pathways metformin can be a veryveryvery inexpensive drug compared with aktmtor inhibitors and it may also haveantitumor effects knowledge of this mechanism may beuseful in clinical trials to adjust the dosage of chemotherapy or further overcome the chemoresistance to platinumin the futuremetformin can be added at different times beforeduring and after adjuvant chemotherapy and the effectsof these addition time points need to be investigated inthe present study a synergistic effect ci was observed in the neoadjuvant protocol and concurrentprotocol protocols but not in protocol adjuvantneoadjuvant metformin protocol was better than theconcurrent regimen these results are similar to those oferices although they did not assess the adjuvant use of metformin the optimal time frame of neoadjuvant metformin before chemotherapy should bedetermined in future studies or clinical trials althoughwe observed an inhibitory effect of neoadjuvant metformin day prior to chemotherapy in vitro and daysprior to chemotherapy in vivo furthermore the ovariancancer patients have a better prognosis with metformincombined with before or during chemotherapy compared with chemotherapy alone due to the small casenumber and retrospective analysis our results are notsignificant the optimal time frame for neoadjuvant metformin administration before chemotherapy will allow forthe acquisition of the most ideal results in basic experiments and clinical analyses these findings provide beneficial evidence that can guide the design of clinical trialsthe differences among protocols observed in this studymay be related to the cytotoxic effects of metformin oncancer stem cells which can enhance the efficacy of 0cwen of ovarian research page of neoadjuvant and concurrent chemotherapy by preventingthe establishment of chemoresistant clonesreducesome patients treated with metformin continue to showpoor blood sugar control and high blood glucose may diminish the antitumor effect of metformin karnevi reported that metformin can significantlytheproliferation of several pancreatic cancer cell lines under normal glucose conditions however they found that hyperglycemia reduced metformininduced growth inhibition byenhancing the igfi response and activating akt whichstimulated ampkser485 phosphorylation and impairedampkthr172 zhuang obtained similar results inbreast cancer and ovarian cancer cell lines reporting that cancer cells became less responsive to metformin when glucosewas increased to mm in a breast cancer cell line underlowglucose conditions metformin significantly decreased thephosphorylation of akt and various targets of mtorwhereas phosphoampk was not significantly altered in thepresent study we used an ovarian cancer cell line and demonstrated that a highglucose medium decreased the response tometformin the synergistic effects of carboplatin and metformin were abolished the phosphorylation of akt in lowglucose conditions mgl was substantially reduced bymetformin and phosphorylation levels of targets of mtors6k and 4ebp1 were decreased relative to those in highglucose conditions mm in ovarian cancer phosphoampk was not significantly altered the response to metformin was substantially altered in lowglucose conditions basedon previous studies and our observations we hypothesize thathigh glucose fuels glycolytic metabolism which maintains cellular atp levels when metformin blocks mitochondrial function when glucose is limiting cancer cells lack sufficient fuelto maintain glycolytic metabolism additionally mtor signaling is blocked in an ampkindependent manner enhancingmetabolic deficiency cellular atp is depleted leading to energy collapse and cell death sin lowconcentration metformin treatment ofpatients with ovarian cancer may have antitumor effectsand synergistic effects when used in combination withchemotherapy through the aktmtor pathway neoadjuvant metformin is a more preferable protocol than theconcurrent regimen future prospective clinical trials inpatients with ovarian cancer are required to investigatethe beneficial effects of neoadjuvant metformin in augmenting the efficacy of existing cancer therapeuticsmethodspatient samples from taipei veteran general hospitalmedical centera total of patients were diagnosed with primaryovarian cancer in the department of gynecology andobstetrics taipei veteran general hospital from to after a review of the patients clinical and drughistories patients who underwent complete surgeryand were treated with platinumbased therapy plus paclitaxel were included in the analysis of these patients were identified as having taken metformin eitherduring admission or in the outpatient clinic the overallsurvival os was measured from the date of diagnosisto death or was censored at the date of the last followup all documents were collected under protocols approved by the institutional review board of the hospitalpatient samples from the national health insurancetaiwanese datasetthe reimbursement data of taiwanese female patientswith a new diagnosis of type dm between and n were retrieved from the nationalhealth insurance database among these patients noneused only insulin or only metformin therefore we compared two groups those who received metforminand insulin n and those who received neiinsulin n then wether metformin norfollowed the two groups for newly diagnosed ovariancancer from to thirtyseven patients acrossthe two groups were diagnosed with ovarian cancermicroarray analysisthe microarray experiments were conducted following thel1000 operating procedure l1000 sop briefly thehuman ovarian cancer cell line es2 was left untreated control or treated with micromolar concentrations of metformin mm μm carboplatin or a combination in amicroplate after h of drug treatment the medium was removed and lysis buffer was added included in the l1000kit to the wells for min after cell lysis the lysate wasstored at °c for at least one night before being transferred to a 384well plate which was performed using theprotocol avai | Colon_Cancer |
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phylogenetic trees and networks nucleic acids res w573w579 101093nargks48 letunic i bork p interactive tree of life itol v4 recent updates and new developments nucleic acids res w256w259 saeed a i tm4 a free opensource system for microarray data management and analysis biotechniques 10214403342 mt01 weber t antismash 30a comprehensive resource for the genome mining of biosynthetic gene clusters nucleic acids res 101093nargkv43 battistuzzi f u feijao a hedges s b a genomic timescale of prokaryote evolution insights into the origin of methanogenesis phototrophy and the colonization of land bmc evol biol acknowledgementstiara padayachee and nomfundo nzuza thank the department of science and technologynational research foundation dstnrf south africa for masters scholarships grant numbers mnd190619448759 and mnd190626451135 respectively khajamohiddin syed expresses sincere gratitude to the nrf south africa for a research grant grant number and university of zululand grant number c686 the authors want to thank barbara bradley pretoria south africa for english language editingauthor contributionsks designed conceptualized and provided funding for the study all authors were involved in generation analysis and interpretation of data 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 drn a0or a0ksreprints and permissions information is available at wwwnaturecomreprintspublishers note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliationsopen 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 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chronic respiratory diseases are highly prevalent worldwide and will continue to rise in theforeseeable future despite intensive efforts over recent decades the development of novel and effectivetherapeutic approaches has been slow however there is new and increasing evidence that communities ofmicroanisms in our body the human microbiome are crucially involved in the development andprogression of chronic respiratory diseases understanding the detailed mechanisms underlying this crosstalk between host and microbiota is critical for development of microbiome or hosttargeted therapeuticsand prevention strategies here we review and discuss the most recent knowledge on the continuousreciprocal interaction between the host and microbes in health and respiratory disease furthermore wehighlight promising developments in microbiomebased therapies and discuss the need to employ moreholistic approaches of restoring both the pulmonary niche and the microbial communityreceived nov accepted after revision april copyright ers this version is distributed under the terms of the creative commons attribution noncommercial licence 10118313993003023202019eur respir j 0crespiratory basic science r gosens introductionin the past decade we have learned that the lungs previously considered sterile in fact harbour a dynamicecosystem of diverse bacteria fungi and viruses this lung microbiota is detectable in health [ ]altered in disease predictive of disease outcomes [ ] and correlates with variations in host immunity[ ] recent insights based on studies in both humans and mice are that the baseline immunetone of the lungs even in health is closely linked to the local microbial milieu this hypothesis thatthe immune tone in the airways and alveoli is at least in part regulated by the microbiota is a radicaldeparture from our conventional dichotomous understanding of lung immunity dormant in healthactivated in infection next to known changes in the inflammatory milieu of the lungs most lung diseasessuch as asthma copd cystic fibrosis idiopathic pulmonary fibrosis ipf andrecently lung cancer have been associated with a microbial dysbiosis in the lung however it isunknown if changes in the bacterial composition drive disease pathogenesis or if they are rather areflection of alterations of the ecological niche [ ] thus it is of utmost importance to understand theunderlying molecular mechanisms at the hostmicrobiome interface to develop novel targeted therapies orpreventative approacheshere we discuss the impact of hostmicrobiome crosstalk on respiratory health and disease whilefocusing on how the local microbiota and the host interact at the epithelial surface furthermore wereview current therapeutic approaches and suggest a more holistic approach for treating lung disease inthe future this review is a followup of presentations and discussions at and after the ers researchseminar crosstalk in the lung microenvironment implications for understanding chronic lung diseaseberlin february the mucosal niche in the lungall external interfaces of the human body such as gut skin reproductive and respiratory tracts arecolonised by a distinct microbial flora the microbial communities differ at the various body sites dueto local factors eg oxygen carbon dioxide ph nutrients host defence factors inhaled pollutants thatshape the niche as examples the lumen of the lower gastrointestinal tract represents a lowoxygennutrientrich environment and is thus commonly populated by highabundance communities of anaerobicanisms by contrast the skin is a lownutrient environment directly exposed to environmental oxygenand is thus more commonly populated by relatively sparse communities of oxygentolerant bacteria thusthe local environment is a crucial determinant of the formation of microbial communities early indevelopment but also at later stages antimicrobial peptidesand otherclearance production ofconsequently the lung microenvironment creates a special ecological niche for microbes that differs fromother body sites and will presumably influence nichespecific colonisation accordingly airway epithelialcells are a principle contributor in shaping this niche as they are strategically located to be the first contacttissues various mechanisms arebetween inhaled substances including microanisms and hostemployed by the airway epithelium in host defences against infectionincluding its barrier functionmucociliaryandantiinflammatory mediators and ability to transport eg polymeric iga and igm from the basal to theapical side of the epithelium through the polymeric immunoglobulin receptor pigr [] it is likelythat similar mechanisms contribute to the formation of nichespecific communities along the respiratorytract and such local conditions are crucial for allowing beneficial microbiota to persist at epithelialsurfaces the involvement of such hostmicrobiota interactions in disease is wellillustrated in cysticfibrosis in cystic fibrosis mutational dysfunction of the cystic fibrosis transmembrane regulator cftrprotein results in a reduction of anion mostly bicarbonate and chloride exchange across the epithelialsurface resulting in a dehydrated surface and sticky mucus this mucus cannot be readily cleared from theairways which helps to explain why cftr mutations are associated with alterations of the residentmicrobiota including frequent colonisation with staphylococcus aureus and pseudomonas aeruginosa asrecently reviewed in however we are still lacking knowledge on the underlying mechanisms of theniche alterations in more complex genetic lung diseases such as asthma and copdsubstances proadditionallyallowing it to mount appropriate responses or develop tolerance [ ]the epithelium transmits environmental and microbial signals to the immune systemvarious mechanisms of sensing microbial presence include pattern recognition receptors such as thetolllike receptors and other processes not mediated via classical receptormediated signalling egthe integrated stress response upon sensing microbial environmental or endogenous challengesepithelial cells mount active responses by increasing defence molecules such as antimicrobial peptidescytokines and chemokines these enhance the defence against respiratory pathogens while simultaneouslychanging the ecological niche of complex microbial communities indicating that the properties of theecological niche encountered by microanisms changes as a result of environmental exposures10118313993003023202019 0crespiratory basic science r gosens anatomical differences along the respiratory tract such as differences in epithelial cell composition shapethis ecological niche while the epithelium contributes to local conditions that shape the microbiotaepithelial exposure to microbes and their products has marked effects on its function consequentlyhuman epithelia and the microbiota have developed interactions that are of mutual benefit responding topathogens and tolerating innocuous substances this concept is important to understand how inhaledenvironmental triggers regulate immunity since microbial components contribute to the response toenvironmental exposures such as farm and geogenic earthderived dust however how theepithelium integrates these microbial and nonmicrobial signals into a finetuned response is incompletelyunderstoodin order to transmit signals from the environment epithelial cells use sophisticated communicationsystems with other lung cell types the airway epithelium and that of gut and skin plays key roles intransmitting signals from microanisms and environmental stimulito instruct antigenpresentingdendritic cells to direct immunity towards inflammation or tolerance epithelial cells interact not onlywith other immune cells such as macrophages neutrophils innate lymphoid cells and tcells but alsowith structural cells such as fibroblasts airway smooth muscle cells and endothelial cells via a plethora ofdifferent mechanisms figure [ ] this array of interactions with environmental triggersmicroanisms and lung cells allows epithelial cells to orchestrate host defence and immunity and tomaintain epithelial integrity and mediate pathologic airway remodelling figure establishment and maintenance of the lung microbiotathe establishment of the lung microbiota likely occurs in the first days and weeks of life although earlyhighprofile reports suggested the presence of a placental microbiome that could influence prepartumlung development subsequent wellcontrolled studies have failed to detect bacterial signals distinctfrom contaminating dna present in negative controls the lung microbiota of newborn mice isbelow the limit of detection via quantitative pcr and increase in total burden in the following days andweeks [ ] in human infants the composition of the respiratory microbiome seems to mature in apredictable wellcharacterised pattern during the first year oflife besides the special nichecharacteristics of the lung earlylife respiratory microbiota are influenced by mode of delivery vaginalversus caesarean method of feeding breast versus bottle and exposures siblings daycare attendance provocatively earlylife respiratory microbiota may predict subsequent susceptibility to respiratorytract infections [ ] suggesting roles of the local microbes in immune homeostasis and resistance topathogens in contrast being exposed to an increased microbial diversity during childhood promotes thedevelopment of balanced immunity and is protective against inflammatory responses to allergens andasthma development [ ] this protection is partly associated with distinct farm dust which in vitroincreases epithelial barrier function and antiviral defences once established the composition of the lung microbiome is determined by three ecological factorsimmigration elimination and relative growth rates of community members figure in health lungcommunities are sparse and dynamic largely determined by the equilibrium between immigration viamicroaspiration and elimination via cough mucociliary clearance and immune defences littleevidence supports the presence of resident lung bacteria in health that reproduce and survive selectivepressures [ ] nevertheless the transient and dynamic communities detected in health are largelyviable and exert a detectable effect on the immune constitution of the lower respiratory tract [ ]the establishment of a diverse respiratory microbial flora is influenced by many different factors there isa finetuned balance between tolerance of commensal or beneficial bacteria at the epithelial surface andthe development of active immune responses to pathogens notably this balance is regulated by both hostand microbederived signals sudden shifts in this tightly controlled equilibrium such as outgrowth ofspecific pathogens or for example virally induced damage to the airway epithelium destroying the barrierand inducing local immune responses can have detrimental effects on both the host and the microbiomeand might therefore contribute to the pathogenesis of respiratory diseasesrole of the environment in shaping the lung microbiomeenvironmental influences play pivotal roles in the development of lung diseases this might be due to directeffects on the host epithelial barrier and immune responses but most known risk factors such as cigarettesmoke air pollution [ ] viral infections and di so directly impact the microbiota [ ]thus a combination of both might be critical for disease developmentfor example one of the most studied inhaled toxins cigarette smoke supposedly has a detrimental effecton both the host and microbial communities prolonged cigarette smoke exposure contributes to increasedbaseline inflammation in the airways and epithelial remodelling towards goblet cell hyperplasia and areduction in cilia and ciliary activity and can reduce the antimicrobial defence provided by the airway10118313993003023202019 0crespiratory basic science r gosens mucociliaryclearingmicroaspirationairway regionbacterial metabolitesouter membrane vesiclesquorumsensing moleculesalveolar regionantimicrobial peptidesciliary beatingmucuscytokineschemokinesgrowth factorsextracellular vesiclescritically altered in chronic lung diseasebacteriarespiratory virusclub cellciliated cell goblet cell alveolar type cell alveolar type cellfibroblastmucus layer basal celldendritic cell macrophagesmooth muscle cellcapillaryfigure hostmicrobiome crosstalk in the lung microenvironment the lung microenvironment consists of different cell types depending onthe location in the proximaltodistal airway tree the epithelial layer in larger airways is constantly exposed to a variety of different microbes ofthe local microbiota while the composition of the latter is influenced by host factors such as elimination via mucociliary clearance it alsodepends on the competition among the microbial inhabitants it is now evident that there is a complex crosstalk between host and microbes inthis environment accordingly bacterial metabolites or outer membrane vesicles can influence the host status while antimicrobial peptides orcytokines can shape the composition of the microbiota as virtually all of these single factors are altered in chronic respiratory disease it is ofutmost importance to appreciate and assess the complexity of this system in future studiesepithelium in patients with copd it has been shown to be associated with reduced lung bacilli andincreased haemophilus influenzae and streptococcus pneumoniae but the largest study to date inhealthy smokers has not found a significant effect of cigarette smoke on the lung microbiome thus it is intriguing to speculate that cigarette smoke primarily affects the host system which over timeand upon disease copd development in susceptible individuals may also affect the microbiomeanother example of environmental influence on respiratory disease is diet which has profound effects onboth microbiota and health even in the short term the intake of dietary fibre induces similarbeneficial microbiota changes in the lung and gut while lowfibre diets change the gut microbialcommunity over multiple generations in mice highfibre diets have beneficial effects in pregnant miceand suppress allergic airway disease aad in mothers and their offspring this also highlights theimportance of crosstalk between the gut and lung highfibre diets induce the production of shortchainfatty acids by gut bacteria which are transported systemically to the lung where they exertantiinflammatory actions and ameliorate aad in mice in contrast a lipidrich di ters themicrobiota and promotes metabolic inflammation and has been associated with premetastatic nichedevelopment in lung cancer however the detailed mechanisms of action remain unclearalong with bacteria common major respiratory viruses including respiratory syncytial virus rhinoviruscoronaviruses influenza and adenoviruses are part of the respiratory microbiome and contribute tothe pathogenesis of chronic respiratory diseases this may result from complex interactions of viruses withthe hosts immune system and the microbiota including other pathogens these interactions can influencethe prevalence of bacterial pathogens by increasing the expression of adhesion molecules on therespiratory epithelium damaging respiratory epithelial cells compromising barrier functionimpairingmucociliary clearance and altering host immunity and the lung microenvironment [] interestinglyit has been suggested that viruses and bacteriophages induce consistent and reproducible changes inrespiratory microbiomes in chronic disease but not the healthy state and have been shown to increasemicrobial diversity in the nasopharynx [ ] furthermore fungi such as aspergillus spp contributeto the pathogenesis of chronic respiratory diseases as pathogens on their own but also by activation of theimmuneinteractions with microbiota particularly withnontuberculous mycobacteria [ ]through theirsystemand probablyimportantly the absence of certain members from the microbiome can have detrimental influences on lunghealth this is illustrated by the relative disappearance of parasitic worms which have been a constant partof our gut microbiome and have even been found in fossils from the period of jawed fish in fact our10118313993003023202019 0crespiratory basic science r gosens modern immune system developed in the continuous presence of helminths which may explaintheir important regulatory role in immunity typically worms induce type2 immune responses which areconsidered instrumentalin host defence against these parasitic infections however worm infectionsinduce regulatory responses that are aimed to suppress immune responses directed at worm antigenswhich allows worms to live in their host for years additionally this regulatory response has a bystandereffect by promoting allergen tolerance this is illustrated by studies in mouse models in whichvarious helminth infections have been shown to provide protection against the development of allergicasthma the range of helminthinduced protective mechanisms includes the inhibition of interleukinil33 in the airways and the induction of regulatory tcells [ ] bcells [ ] andmacrophages intriguingly the presence of helminths also affects the composition of the bacterialcommunity which might be important for the protection against allergic airway disease in mice niche and microbiome alterations in respiratory diseasein disease the ecology of the lower respiratory tract changes dramatically the airways and alveolinormally inhospitable to bacterial reproduction are radically altered by the influx of nutrientrichoedema and mucus establishment of stark oxygen gradients surge of bacterial growthpromotinginflammatory responses [ ] and impairment of local host defences [ ] thus it is unsurprisingthat crosssectional studies have identified altered lung microbiomes in established lung diseases aschanges in ecological niches supposedly result in different microbial communities due to selective pressurethe composition of the lung microbiome then becomes increasingly determined by the relative growthrates of its constituentswhile there are some studies on the role of the microbiome in ipf and lung cancer mostknowledge is based on studies of chronic inflammatory respiratory diseases such as asthma copd andcystic fibrosis generally those patients have increased susceptibility to infections and exacerbations thatagain affect the microbiome [ ] h influenzae moraxella catarrhalis and s pneumoniae areassociated with the development of severe asthma and copd they modify the lung microbiome anddrive inflammation oxidative stress symptoms and exacerbations [ ] which may form a viciouscircle perpetuating the disease m catarrhalis and hinfluenzae in particular induce neutrophilicinflammation more severe disease and steroid resistance [] furthermorein asthma alteredrespiratory microbiome profiles are associated with asthma phenotype and severity responses toallergens and treatment in cystic fibrosis and bronchiectasis haemophilus and pseudomonas spp increasingly dominate the lungmicrobiota [ ] genetic association studies in phe508del cystic fibrosisaffected homozygousindividuals have shown associations of single nucleotide polymorphisms with important disease featuresrelated to bacterial colonisation for example gene variants of human leukocyte antigen class ii genesimplicating a role for tcell and bcell immunity associate with age of onset of persistent p aeruginosainfection indicating the role of the hosts immune system in selecting a distinct microbiota like the bacterial composition ofthe lung microbiome antiviral responses are altered in chronicrespiratory diseases in asthma where airway epithelial repair in response to viral infection is aberrant dueto changes in genes regulating epithelial barrier function and repair the airway epithelium hasreduced innate immunity to common viruses such as rhinovirus [ ] this is due to reduced type iand iii interferon and increased mirna levels [ ] transforming growth factor tgfβ a cytokinecommonly upregulated in asthma suppresses airway epithelialinnate immune responses throughsuppressor of cytokine signalling socs1 and socs3 contributing to impaired antiviral immunity this impaired antiviral immunity may also alter the bacterial microbiome and contribute to coinfectionsby bacteria rhinovirus infection can also impair the phagocytosis of bacteria by alveolarmacrophages which can lead to bacterial outgrowth in copd murine studies showed thatinfluenzainfected epithelial progenitors of the distal lung exhibited severely impaired renewal capacity dueto virusinduced blockade of βcatenindependent fgfr2b signalling this could contribute toimpaired repair of the distal lung in copd although its involvement in human disease is still unclearthus viral and bacterial infections and the inability to resolve them may aggravate impaired airway andalveolar repair combined with a heightened airway andor alveolar immune tone aberrant repairmechanisms following pathogen exposures in vulnerable individuals might represent a mechanism for howhostmicrobiome interactions contribute to disease progression it is currently unclear if alterations in thelung microbiome can also precede lung structural changes and inflammation and thereby contribute toonset of diseaseas discussed earlier the relationship between lung dysbiosis and lung inflammation is bidirectionaldisordered lung communities trigger epithelial and luminal inflammation further altering growth conditionsof the lung microenvironment chronic inflammation and perpetuating dysbiosis [ ] a common10118313993003023202019 0crespiratory basic science r gosens isthe outgrowth oftheinflammatory lung conditionsfinding acrossinflammationassociatedproteobacteria phylum and in mice the lung microbiome has been shown to be associated withpulmonary levels of the innate cytokine il1α additionally alterations in the microbiome of the gutmight influence the immune tone of the lung which is shown by the development of more severeexperimental asthma in germfree mice [ ] or mice with disturbed microbiomes due to earlylifeantibiotic treatment [ ] in contrast the absence of microbiota in germfree mice or due toantibiotic treatment has recently been shown to protect mice with kras mutations and p53 loss from lungcancer development according to the authors this effect was due to the induction of il1β andil23 through the microbiota that led to il17 production by γδt cells and tumorigenesisin humans copd patients are two to three times more likely to have crohns colitis and of peoplewith inflammatory bowel disease also have pulmonary inflammation [] the gastrointestinal tracthas by far the highest microbial content and thus an interaction of gut microbiota and their metaboliteswith the gastric mucosa affects systemic immunity which may in turn impactthe lung consequently alterations in gut microbiota and physiology might contribute to respiratory disease andvice versa possibly via the gutlung axis however it is important to note that germfree breeding and tosome extent antibiotic treatment will affect both the gut and the lung microbiome so it is still notcompletely clear whether these effects can fully be explained by gutlung crosstalk or if they areinfluenced by aberrant local microbiotaimmune crosstalk along this line in mice the lung immune tonehas been suggested to be more correlated to lung bacteria than gut bacteria in contrast in mice colitisinduced pulmonary pathology was associated with increased inflammation andgramnegative bacterial endotoxins in the lung that probably emanate from the gut furthermorecigarette smokeinduced experimental copd models [] indicate that reduced gas exchange andhypoxia in the lung is associated with hypoxia in the gut causing colon remodelling cell deathinflammation and impaired barrier function additionally activation of nodlike receptors bybacteria in the gut increase production of reactive oxygen species by alveolar macrophages suggesting thatthe gastrointestinal microbiome contributes to oxidative stress in the lung conversely oralapplication of beneficial probiotic bacteria bifidobacterium and lactobacillusbased reduced airwayinflammation and emphysema in cigarette smokeexposed mice a large canadian study reported that four bacterial genera lachnospira veillonella faecalibacterium androthia were reduced in the faeces of human infants that subsequently developed asthma and inoculating ahuman faecal microbiome supplemented with these four taxa into germfree mice partially protected theirf1 progeny from experimentally induced aad thus there are encouraging studies in mousemodels highlighting cause and effect of the gutlung axis in respiratory disease however findings firstneed to be validated in humans before proposing this crosstalk as a treatable trait for respiratory diseasein summary at the hostmicrobiome interface disordered communities are probably both cause andeffects of host inflammation however given the close reciprocal interactions between the microbiome andhost at all times it might be impossible to determine the real cause of the very first changes in diseasedevelopment as host and microbiome factors are coinciding and closely intertwinedimplications for the development of novel therapiesdue to the strong and dynamic interdependency between host and microbiome in local niches it isunsurprising that most drugs used in clinical practice that were designed to target the host also affect themicrobiome accordingly inhaled corticosteroids and proton pump inhibitors affect the lung microbiota[] as well as subsequent pneumonia risk [ ] macrolide antibiotics broadly effective acrosschronic lung conditions such as copd affect both lung microbiota and host immunity perhapsmost provocatively baseline differences in lung microbiota appear to predict patient responsiveness totherapies such as inhaled corticosteroids suggesting that variation in lung microbiota may representan untapped phenotype of precision medicine in the lung this opens new possibilities to exploit thisimportant crosstalk in therapeutic interventions but in order to do so we first need to improve ourunderstanding of the molecular mechanismsincreasing evidence of the importance of the microbiome raises the concept of restoring diseasedmicrobiomes to prevent or treat diseases using microbiotadirected therapies or hosttargeted therapiessuch as probiotics metabolites lung microbiota transplantation or vitamin d therapy which we discusshere in more detailmany clinical studies have investigated the efficacy of probiotic bacteria which are supposedly beneficialfor the host to prevent chronic diseases such as asthma or allergic rhinitis however these studies havelargely produced contradictory outcomes which might be due to the fact that the used probioticswere not selected based on potential mechanistic effects and may not be ideal the microbiome is a10118313993003023202019 0crespiratory basic science r gosens complex ecosystem comprised of a variety of different inhabitants and influenced by many externalhostderived factors thus the addition of single strains may not make a profound difference thus thetransfer of whole microbiomes via faecal microbiota transplantation fmt could be a more promisingapproach the introduction of healthy microbiota into diseased hosts has restored immunity andphysiology demonstrating that intestinal microbiota and their products can modulate host immunitylocally and systemically and that fmt can replace diseaserelated microbiomes with healthy ones fmt is remarkably successfulin treating antibioticresistant clostridium difficileinducedcolitis and is now being used as treatment in selected patients [ ] the new microbiomeengrafts quickly and lasts for at least a month indicating a potential difficulty in inducing longtermbeneficial changes in the microbiome via only targeting the microbial side while there are encouragingdata questions remain whether fmt may also affectlung health and whether lung microbiotatransplantation is feasible furthermore recently severe complications of fmt have been reported due totransfer of drugresistant bacteria thus it is essential to determine whether such approaches that sofar only transiently change the microbiome can be used for the required longterm treatments of chroniclung diseases that coincide with a variety of structural changes aberrant mucociliary clearance and manymore such as[]specific microbial moleculeslipopolysaccharide lps andalong with living bacteriapeptidoglycan can induce or modulate inflammatory responsesin addition culturesupernatants of probiotic bacteria display antiinflammatory effects which have been ascribed to thepresence of secreted immunemodulatory metabolites for example culture supernatants of certainprobiotic bifidobacterium species decreased the secretion of type cytokines from immune cell lines andthe expression of costimulatory molecules on primary dendritic cells a likely mechanism is quorumsensing quorum sensing is a means of communication among bacteria of the same species to coordinateeffector functions such as biofilm formation sporulation or toxin secretion the bestdescribed quorumsensing molecules are acyl homoserine lactones ahls several ahls are targeted by the host tointerfere with growth of pathogens and are in turn exploited by bacteria to regulate host geneexpression for their benefit some ahls can bind to distinct bitter taste receptors expressed on the airwayepithelium and innate and adaptive immune cells [ ] thereby modulating barrier andimmune functions the moststudied ahl 3oc12hsl can activate phagocytesto increasephagocytosis expression of adhesion receptors and chemotaxis [ ] but is itself cleaved andinactivated by airway epithelial cells another example of bacterialderived modulators of the hosts immune responses are outer membranevesicles omvs omvs are spherical bilayered membrane vesicles released from the surface of bothgramnegative and grampositive bacteria and contain much of the biological material from the parentbacterium but in a nonreplicative form [ ] evidence suggests that the release of omvs providesbacteria with competitive advantages when exposed to acute and chronic hostassociated stressors innate and adaptive immune responses [] antimicrobialthey may protect bacteria againstpeptides and antibiotics [ ] moreover omvs contain factors eg siderophores aiding in theacquisition of nutrients in an environment devoid of crucial elements such as iron besidessupporting the survival of the parent bacteria omvs may also play role in the progression of pulmonarydiseases bacteria frequently associated with copd exacerbations are known to release omvs furthermore macrophages stimulated with omvs derived from prominent airway pathogens such asp aeruginosa h influenzae or m catarrhalis release higher amounts of tumour necrosis factorα and il6 legionelladerived omvs significantly enhanced bacterial replication in macrophages andbacteriafree p aeruginosa omvs have been shown to potently induce pulmonary inflammation in mice strengthening the idea that omvs exert diseasepromoting activities in addition omvs have beenshown to induce tolerance and hyporesponsivenessthereby facilitating bacterial adherence to andinternalisation by macrophages which may contribute to clearance of the infection [ ] thusdespite our increasing knowledge on omvs and their potential role in interkingdom communicationthere is a need for further research to better understand their pathogenic properties and possibletherapeutic or prophylactic implications eg novel vaccinesan interesting alternative to fmt or probiotic bacteria might be to use immunemodulatory microbialmetabolites or beneficial omvs such chemically defined bacterial substances could be produced at largescale under controlled conditions applied in defined effective doses both systemically or locally and mayhave fewer adverse sideeffects ie in immunocompromised patients compared to live bacteria several studies have already used defined bacterial metabolites to treat aad in mice lps from escherichiacoli o111 bacterial polysaccharide a oligodeoxynucleotides with bacterial cpg motifs flagellin b shortchain fatty acids dtryptophan and the neutro | Colon_Cancer |
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