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"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 |
"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 |
"colorectal cancer crc remains the third most prevalent cancer type and leading cause of cancerrelated deaths with million cases and deaths worldwide during the occurrence and progression of crc result from a wide array of cellular transformation processes which include genetic and epigenetic mutations that drive uncontrolled cell proliferation and escape from apoptosis2 chemotherapy and surgery remain the major therapeutic treatment for crc patients5 fluoropyrimidinebased chemotherapy eg 5fluorouracil has been used as the firstline systemic chemotherapy of treating advanced crc for over a half century6 however most patients receiving chemotherapy finally develop drug resistance which is considered to be the major reason for crc therapy failure7 furthermore even though chemotherapy has significant antitumor activity the side effects can affect the quality of a patient's life which makes the new therapeutic approaches urgentdrug design development and therapy sun this work is published and licensed by dove medical press limited the full terms of this license are available at wwwdovepresscomtermsphp and incorporate the creative commons attribution non commercial unported v30 license httpcreativecommonslicensesbync30 by accessing the work you hereby accept the terms noncommercial uses of the work are permitted without any further permission from dove medical press limited provided the work is properly attributed for permission for commercial use of this work please see paragraphs and of our terms wwwdovepresscomtermsphp 0csun dovepresstraditional chinese medicines such as dendrobium have been shown to exert anticancer activity in many kinds of cancers89 erianin 2methoxy5[2345trimethoxy phenylethyl]phenol figure 1a a natural compound derived from dendrobium candidum shows various pharmacological activities and therapeutic potential to inhibit multiple cancers in vivo and in vitro10 li demonstrated that erianin inhibited the proliferation of acute promyelocytic leukemia hl60 cells by regulating the expression of bcl2 and bax10 in addition erianin caused moderate growth delay in xenografted human hepatoma bel7402 and melanoma a37511 furthermore erianin induced cell cycle g2mphase arrest and apoptosis via the jnk signalling pathway in osteosarcoma and bladder cancer1213 erianin can also inhibit cell invasion metastasis and angiogenesis in lung cancer and breast cancer by the figure erianin inhibited crc cells growth a chemical structure of erianin b and c sw480 and hct116 cells were treated with indicated concentration b and time c of erianin cell viability was assessed by cck8 assay p Ë p Ë d and e ncm460 cells were treated with indicated concentration d and time e of erianin cell viability was assessed by cck8 assay f sw480 and hct116 cells were performed colony formation assay after being treated with indicated concentration of erianinsubmit your manuscript wwwdovepresscom dovepress drug design development and therapy 0cdovepress sun regulation of ido mpp and timp expressions1415 interestingly besides the function on cell growth apoptosis and migration erianin was found to strongly affect the serum levels of cytokines and immune response in liver cancer16 more importantly in addition to the anticancer effects previous a study also suggested that erianin had no major anrelated toxicities12however to the best of our knowledge neither the mechanism nor the effect of erianin on colorectal cancer has been reported hence in this study we evaluate the antitumor potential and molecular mechanisms of erianin in human colorectal cancer sw480 and hct116 cells and provide a theoretical basis of erianin application for colorectal cancer therapymaterials and methodsmaterialsantibodies against cleaved parp cat bak cat bax cat bcl2 cat bclxl cat catenin cat cyclin d1 cat cmyc cat hdac2 cat and gapdh cat were purchased from cell signaling technologies danvers ma usa antibody against αtubulin cat t6199 was purchased from sigma aldrich co st louis mo usaerianin was purchased from shanghai yuanye bio technology co ltd china and dissolved in dmso wntcatenin signaling inhibitor wnt974 was purchased from medchemexpress monmouth junction nj usa and dissolved in dmsocell culturethe human colorectal cancer cell lines sw480 and hct116 were purchased from american type culture collection atcc manassas va usa cells were maintained in rpmi1640 medium supplemented with fbs thermo fisher scientific waltham ma usa uml penicillin and µgml streptomycin thermo fisher scientific and cells were cultured at °c with co2cell viability and colony formation assaycell viability was assessed with the cell counting kit cck8 dojindo japan according to the manufactorers instructionsfor the colony formation assay crc cells cells well were seeded in a sixwell plate and maintained in medium for days subsequently the colonies were fixed with paraformaldehyde and stained with crystal violet and the number of clones was counted using an inverted microscopekit quantitative realtime pcr qrtpcrtotal rna from crc cells was isolated using rna isolation kit omega norcross ga usa according to the manufacturers protocol total rna µg was used as the template for cdna synthesis by using iscripttm reverse transcription super mix biorad laboratories inc hercules ca usa before the samples were analyzed using sybr green master mix on a realtime pcr system biorad laboratories inc the primer sequences used were as follows cmyc forward 5ʹ aaacacaaacttgaaca gctac3ʹ reverse 5ʹ atttgaggcagtttacatt atgg3ʹ cyclin d1 forward 5ʹaggcggatgagaac aagcaga3ʹ reverse 5ʹcaggcttgactccagaag gg3ʹ cd47 forward 5ʹggcaatgacgaaggaggt ta3ʹ reverse 5ʹatccggtggtatggatgaga3ʹ and gapdh forward 5ʹcacccactcctccacctttg3ʹ and reverse 5ʹccaccaccctgttgctgtag3ʹ the 2δδcq method was used to calculate the relative expression levelswestern blottingfor western blotting μg cellular protein extracts were separated in sdspage gel and were then transferred to nitrocellulose membranes emd millipore burlington ma usa the membrane was blocked with nonfat milk and incubated with primary antibodies overnight at ° then the membranes were incubated with secondary antibody and the proteins were visualized using super signal west pico chemiluminescent substrate thermo fisher scientifictransit transfectionplasmid pegfpn1betacatenin was purchased from addgene watertown ma usa lipofectamine thermo fisher scientific carlsbad ca usa was used for transit transfection according to the instructionscatenin sirna was purchased from sigmaaldrich co lipofectamine rnaimax thermo fisher scientific was used for transfection according to the instructiondrug design development and therapy submit your manuscript wwwdovepresscom dovepress 0csun dovepresscell cycle analysisafter treated with vehicle or indicated drugs crc cells were harvested by trypsinization fixed with ethanol and retained at °c overnight after cells were centrifuged and washed with pbs they were resuspended in propidium iodide pi solution containing rnase μgml in the dark at room temperature for min and then studied in a flow cytometercaspase37 activity assayapoone¢ homogeneous caspase37 assay promega corporation madison wi usa was used to measure caspase37 activity briefly apoone® homogeneous caspase37 reagent μlwell was added to a 96well plate and the plate was then placed on a shaker for five minutes rpm before incubating for h at room temperature the reading of each well was measured by spectrofluorometerapoptosis assay by annexin vannexin vfitc staining was used to detect the extent of apoptosis induced by erianin briefly crc cells were treated with erianin for h and were then collected and resuspended in μl annexin vbinding buffer and μl pi for minat room temperature in the dark then the cells were finally analyzed by the flow cytometry bd facs calibur with an emission filter of nm for pi red and nm for fitc greenapoptosis assay by dapithe effect of erianin on apoptosis induction was evaluated by dapi staining assay crc cells à were seeded in a 96well plate after treatment the cells were washed three times with pbs and paraformaldehyde was added to each well for fixation after permeabilization with triton x100 solution dapi solution was added the cells with condensed and fragmented chromatin were analyzed by echo fluorescence microscopycellular thermal shift assayfor cellular thermal shift assay crc cells were pretreated with μm mg132 for one hour and then incubated with erianin for four hours after washing with icecold pbs cells were aliquot into pcr tubes μl each and incubated at different temperatures for four minutes after being frozen and thawed twice using liquid nitrogen cells were centrifuged and proteins were analyzed by western blottingtopfop luciferase reporter assaythe transcriptional activity of catenin was assessed using the topfop dualluciferase reporter system dual glo¢ luciferase assay system promega the renilla luciferase plasmid prltk promega which controls for transfection efficiency was cotransfected with catenin responsive firefly luciferase reporter plasmid topflash emd millipore or the negative control fopflash emd millipore using the lipofectamine thermo fisher scientific cells were harvested after h in culture and the luciferase activity was determined by the luciferase assay system promega using a microplate luminometer berthold bad wildbad germanyflow cytometry analysiserianin treated crc cells were washed and resuspended in μl facs buffer and stained with fitcconjugated anticd47 bd biosciences san jose ca usa antibodies all samples were incubated for minutes at °c and then washed twice with facs buffer flow cytometry analyses were performed on bd facs canto iiin vitro phagocytosis assayfor phagocytosis assay thp1 derived macrophages were seeded in a sixwell tissue culture plate erianintreated crc cells were washed and labeled with μm of carboxyfluorescein succinimidyl ester cfse thermo fisher scientific after incubating macrophages in serum free medium for two hours cfselabeled crc cells were added to the macrophages for another two hours at °c macrophages were then washed and imaged with an inverted microscope the phagocytosis efficiency was calculated as the number of macrophages containing cfse labeled crc cells per macrophageschromatin immunoprecipitation chip assaychip assays were performed using the simplechip® enzymatic chromatin ip kit cell signaling technologies according to manufacturer's instructions using the antibodies against h3k27ac immunoprecipitated dna was analyzed by qrtpcr using the following primers cd47 promoter fragment f ²aggatgaatgatgtggcctgt3² and r ² caaacaggcattagcagcgt3² fragment f submit your manuscript wwwdovepresscom dovepress drug design development and therapy 0cdovepress sun ²ggggatgtgttggatacgct3² and r ² ctctg cgttcggctcgtcta3² fragment f ²agggaag agcagagcgagta3² and r ² ttgctttcactcc caccctc3² fragment f ²agagagaggacag tggggc3² and r ² ccagtcgcaggctccaga3² fragment f ² gccgcgtcaacagca3² and r ² aaaggcatcattcttggaaattgt3²with ¨° sw480 cells per mouse suspended in vivo xenograftnodscid shanghai slac laboratory animal co ltd china mice were injected subcutaneously in right flank in µl pbs and mixed with an equal volume of matrigel animals with tumors volume mm3 were divided into two groups n6 and treated with either placebo or mgkg erianin for continuously three weeks by intraperitoneal injection tumor size were measured at the indicated times all the animalrelated procedures were approved by the animal care and use committee of the changchun university of chinese medicine all animal experiments were conducted according to the nih guide for the care and use of laboratory animalsstatistical analysisdata were presented as mean ±sd from three independent experiments p value was determined using paired students ttest and a p value Ë was deemed to indicate statistical significanceresultserianin inhibited crc cell growthfigure 1a illustrates the chemical structure of erianin to investigate the inhibitory effect of erianin on crc cell viability we treated two crc cell lines sw480 and hct116 with different concentrations of erianin and nm for and h as shown in figure 1b and c erianin treatment significantly inhibited the viability of crc cells in a dose and timedependent manner importantly erianin did not show cytotoxic effects on normal human colon mucosal epithelial cell line ncm460 figure 1d and e in addition consistent with the shortterm growth assay our colony forming unit assay also showed that erianin inhibited the colony formation ability of sw480 and hct116 cells figure 1ferianin elevated cell cycle arrest and apoptosisto verify the causal relation of cell viability inhibition the cell cycle distribution was analyzed erianin increased cell number at g2m phase but decreased cell number at s and g0g1 phases after 24h incubation with indicated concentration in sw480 and hct116 cells figure 2a and b to explore the effect of erianin on apoptosis we examined the activity of caspase the protein level of cleaved parp bax bak bcl and bclxl as shown in figure 2ce the activity of caspase protein level of cleaved parp bak and bax pro apoptosis increased as the concentration of erianin increased in contrast the protein level of bcl2 and bclxl anti apoptotic decreased after erianin treatment figure 2e annexin v flow cytometry and dapi staining further confirmed that erianin could induce cell apoptosis figure 2f and gerianin inhibited catenin translocationincreasing evidence revealed that the wntcatenin pathway plays critical role in colorectal cancer tumorigenesis we hypothesized that erianin might have effect in modulating the wntcatenin pathway first we investigated the effect of erianin on catenin phosphorylation as shown in figure 3a no obvious change was observed on catenin phosphorylation level we then evaluated the effect of erianin on catenin translocation as shown in figure 3be catenin expression in cytoplasm was increased whereas expression in the nucleus was decreased with the treatment of erianin in a dose and timedependent manner to further explore the effect of erianin on catenin transcription activity we performed topfop dual luciferase assay we found that topfop relative luciferase activity was significantly decreased after erianin treatment both in sw480 and hct116 cells figure 3f and gerianin bound catenin directlysince erianin inhibited catenin translocation to the nuclear without changing its phosphorylation level we hypothesized that erianin might bind catenin directly to determine whether erianin physically binds catenin we performed a cellular thermal shift assay the results from this experiment indicated that erianin treatment increased the thermal stability of catenin when cells were pretreated with the proteasome inhibitor mg132 for one hour figure 4a and b in contrast erianin treatment had no effect on the thermal stability of gapdh a loading control figure 4a and b these results strongly suggested a specific physical interaction between erianin and catenindrug design development and therapy submit your manuscript wwwdovepresscom dovepress 0csun dovepressfigure erianin elevated cell cycle arrest and apoptosis a and b sw480 and hct116 cells were treated with erianin for h and then analyzed by pi staining to determine cell cycle phase distribution c sw480 and hct116 cells were treated with erianin for h the relative caspase37 activity was measured using apoone¢ homogenous caspase37 assay p Ë p Ë d and e the protein level of cleaved parp1 bak bax bcl2 and bclxl were analyzed by western blotting after treated with indicated concentration of erianin f and g sw480 and hct116 cells were treated with erianin for h apoptosis was assessed using annexinv flow cytometry analysis f or dapi staining gsubmit your manuscript wwwdovepresscom dovepress drug design development and therapy 0cdovepress sun figure erianin inhibited catenin translocation a the protein level of indicated proteins was analyzed by western blotting after being treated with indicated concentration of erianin for h be the protein level of catenin in cytosol and nucleus was analyzed by western blotting after treated with erianin for indicated concentration b and c and time d and e f and g sw480 and hct116 cells were treated with erianin for indicated concentration f and time g the transcriptional activity of catenin was assessed by topfop luciferase reporter assay p Ë p Ëerianin inhibited the expression of cmyc and cyclin d1as cmyc and cyclin d1 are the direct targets of the wnt catenin pathway we then evaluated the mrna and protein level of cmyc and cyclin d1 unsurprisingly both mrna and protein level of these two proteins were significantly decreased after erianin figure 5ac interestingly no synergetic effect was observed when combining erianin with wntcatenin signaling inhibitor wnt974 which indicated that erianin regulates cmyc treatment drug design development and therapy submit your manuscript wwwdovepresscom dovepress 0csun dovepressfigure erianin interacted with catenin a and b sw480 a and hct116 b cells were treated with μm mg132 for one hour followed by four hours incubation with nm erianin before performing thermal shift assay the lower panel shows the charts of percentages of nondenatured protein fractionand cyclin d1 via wntcatenin signaling figure 5d furthermore the inhibitory effect of erianin on cmyc and cyclin d1 expression and cell viability could be reversed by catenin overexpression figure 5e and f which indicated that erianin regulates crc cell growth via catenincd47 mediated phagocytosis we used an in vitro assay by coculturing thp1 derived macrophage with crc cell lines sw480 or hct116 as shown in figure 6g and h treatment of erianin markedly promote colorectal cancer cell phagocytosis by macrophages these results suggest that erianin treatment can attenuate cd47 expression and ultimately promote phagocytosis of crc cellserianin decreased cd47 expression and increased phagocytosisthe immune checkpoint protein cd47 is included in the list of wntcatenin target molecules with a role in immunity escape17 since catenin depletion by sirna inhibited the expression of cd47 figure 6a we then sought to know whether erianin regulates the expression of cd47 first we explored the effects of erianin on cd47 mrna protein and cell surface level in both sw480 and hct116 cells erianin treatment significantly decreased the mrna protein and cell surface level of cd47 figure 6bd promoter analysis by ucsc genome browser demonstrates that h3k27 acetyl marks are enriched in cd47 promoter regions figure 6e next our chip assay demonstrated that h3k27ac enrichment specifically near promoter region f3f5 was significantly decreased with erianin treatment figure 6f to investigate the effect of erianin on erianin inhibited tumor growth in vivoto investigate the possibility of erianin as a potential therapy in crc we tested the function of erianin on tumor growth in a mouse model the mouse model was established by s c injection of sw480 cells into nodscid mice after three weeks treatment we analyzed the tumor size and weight as shown in figure 7ac the tumor size and weight from the erianin treatment group were significantly lower than that from the control group in addition after days of bearing tumor the weight of the mice had no significant change figure 7dto examine the impact of therapy on catenin and its downstream signaling localization of catenin protein level of cd47 cmyc bcl2 and bax three representative tumors from each group were analyzed using western blotting as shown in figure 7e and f catenin expression in cytoplasm was increased whereas expression in nucleus was decreased with the treatment of erianin the submit your manuscript wwwdovepresscom dovepress drug design development and therapy 0cdovepress sun figure erianin inhibited the expression of cmyc and cyclin d1 ac after treated with indicated concentration and time of erianin mrna and protein level of cmyc and cyclin d1 were analyzed by qrtpcr and western blotting p Ë d sw480 cells were treated with erianin orand wnt974 for h protein levels of cmyc and cyclin d1 were analyzed by western blotting e and f sw480 cells were treated with erianin for h followed by overexpression with catenin plasmid for h protein levels of cmyc and cyclin d1 were analyzed by western blotting e and cell viability was assessed by cck8 assay f p Ëprotein level of cd47 cmyc and bcl2 decreased while bax increased after erianin treatment these data indicated that erianin inhibited tumor growth via catenin in vivodiscussioncrc is one of the most malignant and commonly diagnosed solid tumors all around the world18 although crc incidence rates have declined somewhat chemotherapies are inefficient in most crc patients due to resistance2122 thus the development of acquired therapeutic drugs researching novel and safe treatment strategies is essential for improving the prognosis of crc patients in recent years natural medicinal plants are receiving more and more attention and considered to be important sources of treatment23 novel dendrobium is considered as one of the most important herbs in the orchidaceae family and shows diverse pharmacological functions including anticancer neuroprotective antidiabetic and immunemodulating activities24 erianin derived from dendrobium is one of the most for cancer drug design development and therapy submit your manuscript wwwdovepresscom dovepress 0csun dovepressfigure erianin decreased cd47 expression and increased phagocytosis a sw480 cells were transfected with nontarget nt or catenin sirna for h protein levels of indicated protein weres measured by western blotting bd sw480 and hct116 cells were treated with erianin for indicated dose the mrna level b protein level c and cell surface cd47 d were detected by qrtpcr and flow e the ucsc genome browser revealed the enrichment of h3k27ac on cd47 promoter f the enrichment of h3k27ac on cd47 promoter f1f6 was detected by chip assay g and h sw480 and hct116 were treated with indicated concentration of erianin for h representative images showed the effect of erianin on phagocytosis g and bar graphs showed quantitative analysis of phagocytosis h p Ë p Ësubmit your manuscript wwwdovepresscom dovepress drug design development and therapy 0cdovepress sun figure erianin inhibited tumor growth in vivo a typical photos of tumors from the control and erianin treated groups b and c erianin decreased tumor volume and weight p Ë d mice body weight of control and erianin treated groups was measure at indicated time e the protein level of catenin in cytosol and nucleus in three representative tumors from mouse to mouse of each group were analyzed by western blotting f the protein level of indicated protein in three representative tumors from mouse to mouse of each group were analyzed by western blottingdrug design development and therapy submit your manuscript wwwdovepresscom dovepress 0csun dovepressnoteworthy constituents that have been used as an antipyretic and an analgesic in traditional chinese medicine25 recently several studies have proved that erianin shows significant antitumour activity in a variety of human cancer cells10 consistent with literature in this study we found that erianin had a significant antiproliferative effect against crc cells the inhibitory effect caused by erianin may result from induction of apoptosis and arrest of cell cycle at g2m since the effect of erianin on crc cells has never been studied before we further confirm its antitumor activity in a mouse model which indicated that erianin significantly inhibited tumor growth in vivoseveral signaling pathways including egfrmapk pi3kakt or wntcatenin have been linked to crc genesis and progression26 as the aberrant activation is present in almost all crc cases wntcatenin signaling is prominent among these pathways27 inactivated mutations in the apc gene leads to stabilization and ensuing nuclear translocation of catenin to facilitate tcflef dependent transcription of wntcatenin signaling target genes such as cmyc and cyclin d1 to drive cell proliferation survival and metastasis28 to understand the mechanisms of action of erianin we assessed the effect of erianin on wntcatenin pathway interestingly we found that erianin treatment had no effect on catenin phosphorylation but inhibited the translocation of catenin in the nucleus which suggested to us that erianin physically interacts with catenin our cellular thermal shift assay confirmed this hypothesis the thermal stability of catenin increased after erianin treatment as catenin downstream targets the expression level of cmyc and cyclin d1 significantly decreased after erianin treatmentcd47 a transmembrane glycoprotein expresses ubiquitously and mediates a selfdonoteatme signal on normal cells however cd47 is often upregulated in tumor cells to evade innate immunity31 anticd47 antibodies which block cd47 sirpα interactions and promote macrophage mediated phagocytosis of tumor cells has shown promise in several solid tumors31 in colorectal cancer cd47 promotes colon cancer cell migration and metastasis34 in addition upregulated immuneescape pathways such as cd47 sirpα are responsible for immune escape and survival in circulating tumor cells of colorectal cancer35 myc an oncogene identified as a wntcatenin target gene was reported to control cd47 transcription therefore mutations in components of the wntcatenin signaling pathway which induced | Colon_Cancer |
"colorectal cancer crc remains the third most prevalent cancer type and leading cause of cancerrelated deaths with million cases and deaths worldwide during the occurrence and progression of crc result from a wide array of cellular transformation processes which include genetic and epigenetic mutations that drive uncontrolled cell proliferation and escape from apoptosis2 chemotherapy and surgery remain the major therapeutic treatment for crc patients5 fluoropyrimidinebased chemotherapy eg 5fluorouracil has been used as the firstline systemic chemotherapy of treating advanced crc for over a half century6 however most patients receiving chemotherapy finally develop drug resistance which is considered to be the major reason for crc therapy failure7 furthermore even though chemotherapy has significant antitumor activity the side effects can affect the quality of a patient's life which makes the new therapeutic approaches urgentdrug design development and therapy sun this work is published and licensed by dove medical press limited the full terms of this license are available at wwwdovepresscomtermsphp and incorporate the creative commons attribution non commercial unported v30 license httpcreativecommonslicensesbync30 by accessing the work you hereby accept the terms noncommercial uses of the work are permitted without any further permission from dove medical press limited provided the work is properly attributed for permission for commercial use of this work please see paragraphs and of our terms wwwdovepresscomtermsphp 0csun dovepresstraditional chinese medicines such as dendrobium have been shown to exert anticancer activity in many kinds of cancers89 erianin 2methoxy5[2345trimethoxy phenylethyl]phenol figure 1a a natural compound derived from dendrobium candidum shows various pharmacological activities and therapeutic potential to inhibit multiple cancers in vivo and in vitro10 li demonstrated that erianin inhibited the proliferation of acute promyelocytic leukemia hl60 cells by regulating the expression of bcl2 and bax10 in addition erianin caused moderate growth delay in xenografted human hepatoma bel7402 and melanoma a37511 furthermore erianin induced cell cycle g2mphase arrest and apoptosis via the jnk signalling pathway in osteosarcoma and bladder cancer1213 erianin can also inhibit cell invasion metastasis and angiogenesis in lung cancer and breast cancer by the figure erianin inhibited crc cells growth a chemical structure of erianin b and c sw480 and hct116 cells were treated with indicated concentration b and time c of erianin cell viability was assessed by cck8 assay p Ë p Ë d and e ncm460 cells were treated with indicated concentration d and time e of erianin cell viability was assessed by cck8 assay f sw480 and hct116 cells were performed colony formation assay after being treated with indicated concentration of erianinsubmit your manuscript wwwdovepresscom dovepress drug design development and therapy 0cdovepress sun regulation of ido mpp and timp expressions1415 interestingly besides the function on cell growth apoptosis and migration erianin was found to strongly affect the serum levels of cytokines and immune response in liver cancer16 more importantly in addition to the anticancer effects previous a study also suggested that erianin had no major anrelated toxicities12however to the best of our knowledge neither the mechanism nor the effect of erianin on colorectal cancer has been reported hence in this study we evaluate the antitumor potential and molecular mechanisms of erianin in human colorectal cancer sw480 and hct116 cells and provide a theoretical basis of erianin application for colorectal cancer therapymaterials and methodsmaterialsantibodies against cleaved parp cat bak cat bax cat bcl2 cat bclxl cat catenin cat cyclin d1 cat cmyc cat hdac2 cat and gapdh cat were purchased from cell signaling technologies danvers ma usa antibody against αtubulin cat t6199 was purchased from sigma aldrich co st louis mo usaerianin was purchased from shanghai yuanye bio technology co ltd china and dissolved in dmso wntcatenin signaling inhibitor wnt974 was purchased from medchemexpress monmouth junction nj usa and dissolved in dmsocell culturethe human colorectal cancer cell lines sw480 and hct116 were purchased from american type culture collection atcc manassas va usa cells were maintained in rpmi1640 medium supplemented with fbs thermo fisher scientific waltham ma usa uml penicillin and µgml streptomycin thermo fisher scientific and cells were cultured at °c with co2cell viability and colony formation assaycell viability was assessed with the cell counting kit cck8 dojindo japan according to the manufactorers instructionsfor the colony formation assay crc cells cells well were seeded in a sixwell plate and maintained in medium for days subsequently the colonies were fixed with paraformaldehyde and stained with crystal violet and the number of clones was counted using an inverted microscopekit quantitative realtime pcr qrtpcrtotal rna from crc cells was isolated using rna isolation kit omega norcross ga usa according to the manufacturers protocol total rna µg was used as the template for cdna synthesis by using iscripttm reverse transcription super mix biorad laboratories inc hercules ca usa before the samples were analyzed using sybr green master mix on a realtime pcr system biorad laboratories inc the primer sequences used were as follows cmyc forward 5ʹ aaacacaaacttgaaca gctac3ʹ reverse 5ʹ atttgaggcagtttacatt atgg3ʹ cyclin d1 forward 5ʹaggcggatgagaac aagcaga3ʹ reverse 5ʹcaggcttgactccagaag gg3ʹ cd47 forward 5ʹggcaatgacgaaggaggt ta3ʹ reverse 5ʹatccggtggtatggatgaga3ʹ and gapdh forward 5ʹcacccactcctccacctttg3ʹ and reverse 5ʹccaccaccctgttgctgtag3ʹ the 2δδcq method was used to calculate the relative expression levelswestern blottingfor western blotting μg cellular protein extracts were separated in sdspage gel and were then transferred to nitrocellulose membranes emd millipore burlington ma usa the membrane was blocked with nonfat milk and incubated with primary antibodies overnight at ° then the membranes were incubated with secondary antibody and the proteins were visualized using super signal west pico chemiluminescent substrate thermo fisher scientifictransit transfectionplasmid pegfpn1betacatenin was purchased from addgene watertown ma usa lipofectamine thermo fisher scientific carlsbad ca usa was used for transit transfection according to the instructionscatenin sirna was purchased from sigmaaldrich co lipofectamine rnaimax thermo fisher scientific was used for transfection according to the instructiondrug design development and therapy submit your manuscript wwwdovepresscom dovepress 0csun dovepresscell cycle analysisafter treated with vehicle or indicated drugs crc cells were harvested by trypsinization fixed with ethanol and retained at °c overnight after cells were centrifuged and washed with pbs they were resuspended in propidium iodide pi solution containing rnase μgml in the dark at room temperature for min and then studied in a flow cytometercaspase37 activity assayapoone¢ homogeneous caspase37 assay promega corporation madison wi usa was used to measure caspase37 activity briefly apoone® homogeneous caspase37 reagent μlwell was added to a 96well plate and the plate was then placed on a shaker for five minutes rpm before incubating for h at room temperature the reading of each well was measured by spectrofluorometerapoptosis assay by annexin vannexin vfitc staining was used to detect the extent of apoptosis induced by erianin briefly crc cells were treated with erianin for h and were then collected and resuspended in μl annexin vbinding buffer and μl pi for minat room temperature in the dark then the cells were finally analyzed by the flow cytometry bd facs calibur with an emission filter of nm for pi red and nm for fitc greenapoptosis assay by dapithe effect of erianin on apoptosis induction was evaluated by dapi staining assay crc cells à were seeded in a 96well plate after treatment the cells were washed three times with pbs and paraformaldehyde was added to each well for fixation after permeabilization with triton x100 solution dapi solution was added the cells with condensed and fragmented chromatin were analyzed by echo fluorescence microscopycellular thermal shift assayfor cellular thermal shift assay crc cells were pretreated with μm mg132 for one hour and then incubated with erianin for four hours after washing with icecold pbs cells were aliquot into pcr tubes μl each and incubated at different temperatures for four minutes after being frozen and thawed twice using liquid nitrogen cells were centrifuged and proteins were analyzed by western blottingtopfop luciferase reporter assaythe transcriptional activity of catenin was assessed using the topfop dualluciferase reporter system dual glo¢ luciferase assay system promega the renilla luciferase plasmid prltk promega which controls for transfection efficiency was cotransfected with catenin responsive firefly luciferase reporter plasmid topflash emd millipore or the negative control fopflash emd millipore using the lipofectamine thermo fisher scientific cells were harvested after h in culture and the luciferase activity was determined by the luciferase assay system promega using a microplate luminometer berthold bad wildbad germanyflow cytometry analysiserianin treated crc cells were washed and resuspended in μl facs buffer and stained with fitcconjugated anticd47 bd biosciences san jose ca usa antibodies all samples were incubated for minutes at °c and then washed twice with facs buffer flow cytometry analyses were performed on bd facs canto iiin vitro phagocytosis assayfor phagocytosis assay thp1 derived macrophages were seeded in a sixwell tissue culture plate erianintreated crc cells were washed and labeled with μm of carboxyfluorescein succinimidyl ester cfse thermo fisher scientific after incubating macrophages in serum free medium for two hours cfselabeled crc cells were added to the macrophages for another two hours at °c macrophages were then washed and imaged with an inverted microscope the phagocytosis efficiency was calculated as the number of macrophages containing cfse labeled crc cells per macrophageschromatin immunoprecipitation chip assaychip assays were performed using the simplechip® enzymatic chromatin ip kit cell signaling technologies according to manufacturer's instructions using the antibodies against h3k27ac immunoprecipitated dna was analyzed by qrtpcr using the following primers cd47 promoter fragment f ²aggatgaatgatgtggcctgt3² and r ² caaacaggcattagcagcgt3² fragment f submit your manuscript wwwdovepresscom dovepress drug design development and therapy 0cdovepress sun ²ggggatgtgttggatacgct3² and r ² ctctg cgttcggctcgtcta3² fragment f ²agggaag agcagagcgagta3² and r ² ttgctttcactcc caccctc3² fragment f ²agagagaggacag tggggc3² and r ² ccagtcgcaggctccaga3² fragment f ² gccgcgtcaacagca3² and r ² aaaggcatcattcttggaaattgt3²with ¨° sw480 cells per mouse suspended in vivo xenograftnodscid shanghai slac laboratory animal co ltd china mice were injected subcutaneously in right flank in µl pbs and mixed with an equal volume of matrigel animals with tumors volume mm3 were divided into two groups n6 and treated with either placebo or mgkg erianin for continuously three weeks by intraperitoneal injection tumor size were measured at the indicated times all the animalrelated procedures were approved by the animal care and use committee of the changchun university of chinese medicine all animal experiments were conducted according to the nih guide for the care and use of laboratory animalsstatistical analysisdata were presented as mean ±sd from three independent experiments p value was determined using paired students ttest and a p value Ë was deemed to indicate statistical significanceresultserianin inhibited crc cell growthfigure 1a illustrates the chemical structure of erianin to investigate the inhibitory effect of erianin on crc cell viability we treated two crc cell lines sw480 and hct116 with different concentrations of erianin and nm for and h as shown in figure 1b and c erianin treatment significantly inhibited the viability of crc cells in a dose and timedependent manner importantly erianin did not show cytotoxic effects on normal human colon mucosal epithelial cell line ncm460 figure 1d and e in addition consistent with the shortterm growth assay our colony forming unit assay also showed that erianin inhibited the colony formation ability of sw480 and hct116 cells figure 1ferianin elevated cell cycle arrest and apoptosisto verify the causal relation of cell viability inhibition the cell cycle distribution was analyzed erianin increased cell number at g2m phase but decreased cell number at s and g0g1 phases after 24h incubation with indicated concentration in sw480 and hct116 cells figure 2a and b to explore the effect of erianin on apoptosis we examined the activity of caspase the protein level of cleaved parp bax bak bcl and bclxl as shown in figure 2ce the activity of caspase protein level of cleaved parp bak and bax pro apoptosis increased as the concentration of erianin increased in contrast the protein level of bcl2 and bclxl anti apoptotic decreased after erianin treatment figure 2e annexin v flow cytometry and dapi staining further confirmed that erianin could induce cell apoptosis figure 2f and gerianin inhibited catenin translocationincreasing evidence revealed that the wntcatenin pathway plays critical role in colorectal cancer tumorigenesis we hypothesized that erianin might have effect in modulating the wntcatenin pathway first we investigated the effect of erianin on catenin phosphorylation as shown in figure 3a no obvious change was observed on catenin phosphorylation level we then evaluated the effect of erianin on catenin translocation as shown in figure 3be catenin expression in cytoplasm was increased whereas expression in the nucleus was decreased with the treatment of erianin in a dose and timedependent manner to further explore the effect of erianin on catenin transcription activity we performed topfop dual luciferase assay we found that topfop relative luciferase activity was significantly decreased after erianin treatment both in sw480 and hct116 cells figure 3f and gerianin bound catenin directlysince erianin inhibited catenin translocation to the nuclear without changing its phosphorylation level we hypothesized that erianin might bind catenin directly to determine whether erianin physically binds catenin we performed a cellular thermal shift assay the results from this experiment indicated that erianin treatment increased the thermal stability of catenin when cells were pretreated with the proteasome inhibitor mg132 for one hour figure 4a and b in contrast erianin treatment had no effect on the thermal stability of gapdh a loading control figure 4a and b these results strongly suggested a specific physical interaction between erianin and catenindrug design development and therapy submit your manuscript wwwdovepresscom dovepress 0csun dovepressfigure erianin elevated cell cycle arrest and apoptosis a and b sw480 and hct116 cells were treated with erianin for h and then analyzed by pi staining to determine cell cycle phase distribution c sw480 and hct116 cells were treated with erianin for h the relative caspase37 activity was measured using apoone¢ homogenous caspase37 assay p Ë p Ë d and e the protein level of cleaved parp1 bak bax bcl2 and bclxl were analyzed by western blotting after treated with indicated concentration of erianin f and g sw480 and hct116 cells were treated with erianin for h apoptosis was assessed using annexinv flow cytometry analysis f or dapi staining gsubmit your manuscript wwwdovepresscom dovepress drug design development and therapy 0cdovepress sun figure erianin inhibited catenin translocation a the protein level of indicated proteins was analyzed by western blotting after being treated with indicated concentration of erianin for h be the protein level of catenin in cytosol and nucleus was analyzed by western blotting after treated with erianin for indicated concentration b and c and time d and e f and g sw480 and hct116 cells were treated with erianin for indicated concentration f and time g the transcriptional activity of catenin was assessed by topfop luciferase reporter assay p Ë p Ëerianin inhibited the expression of cmyc and cyclin d1as cmyc and cyclin d1 are the direct targets of the wnt catenin pathway we then evaluated the mrna and protein level of cmyc and cyclin d1 unsurprisingly both mrna and protein level of these two proteins were significantly decreased after erianin figure 5ac interestingly no synergetic effect was observed when combining erianin with wntcatenin signaling inhibitor wnt974 which indicated that erianin regulates cmyc treatment drug design development and therapy submit your manuscript wwwdovepresscom dovepress 0csun dovepressfigure erianin interacted with catenin a and b sw480 a and hct116 b cells were treated with μm mg132 for one hour followed by four hours incubation with nm erianin before performing thermal shift assay the lower panel shows the charts of percentages of nondenatured protein fractionand cyclin d1 via wntcatenin signaling figure 5d furthermore the inhibitory effect of erianin on cmyc and cyclin d1 expression and cell viability could be reversed by catenin overexpression figure 5e and f which indicated that erianin regulates crc cell growth via catenincd47 mediated phagocytosis we used an in vitro assay by coculturing thp1 derived macrophage with crc cell lines sw480 or hct116 as shown in figure 6g and h treatment of erianin markedly promote colorectal cancer cell phagocytosis by macrophages these results suggest that erianin treatment can attenuate cd47 expression and ultimately promote phagocytosis of crc cellserianin decreased cd47 expression and increased phagocytosisthe immune checkpoint protein cd47 is included in the list of wntcatenin target molecules with a role in immunity escape17 since catenin depletion by sirna inhibited the expression of cd47 figure 6a we then sought to know whether erianin regulates the expression of cd47 first we explored the effects of erianin on cd47 mrna protein and cell surface level in both sw480 and hct116 cells erianin treatment significantly decreased the mrna protein and cell surface level of cd47 figure 6bd promoter analysis by ucsc genome browser demonstrates that h3k27 acetyl marks are enriched in cd47 promoter regions figure 6e next our chip assay demonstrated that h3k27ac enrichment specifically near promoter region f3f5 was significantly decreased with erianin treatment figure 6f to investigate the effect of erianin on erianin inhibited tumor growth in vivoto investigate the possibility of erianin as a potential therapy in crc we tested the function of erianin on tumor growth in a mouse model the mouse model was established by s c injection of sw480 cells into nodscid mice after three weeks treatment we analyzed the tumor size and weight as shown in figure 7ac the tumor size and weight from the erianin treatment group were significantly lower than that from the control group in addition after days of bearing tumor the weight of the mice had no significant change figure 7dto examine the impact of therapy on catenin and its downstream signaling localization of catenin protein level of cd47 cmyc bcl2 and bax three representative tumors from each group were analyzed using western blotting as shown in figure 7e and f catenin expression in cytoplasm was increased whereas expression in nucleus was decreased with the treatment of erianin the submit your manuscript wwwdovepresscom dovepress drug design development and therapy 0cdovepress sun figure erianin inhibited the expression of cmyc and cyclin d1 ac after treated with indicated concentration and time of erianin mrna and protein level of cmyc and cyclin d1 were analyzed by qrtpcr and western blotting p Ë d sw480 cells were treated with erianin orand wnt974 for h protein levels of cmyc and cyclin d1 were analyzed by western blotting e and f sw480 cells were treated with erianin for h followed by overexpression with catenin plasmid for h protein levels of cmyc and cyclin d1 were analyzed by western blotting e and cell viability was assessed by cck8 assay f p Ëprotein level of cd47 cmyc and bcl2 decreased while bax increased after erianin treatment these data indicated that erianin inhibited tumor growth via catenin in vivodiscussioncrc is one of the most malignant and commonly diagnosed solid tumors all around the world18 although crc incidence rates have declined somewhat chemotherapies are inefficient in most crc patients due to resistance2122 thus the development of acquired therapeutic drugs researching novel and safe treatment strategies is essential for improving the prognosis of crc patients in recent years natural medicinal plants are receiving more and more attention and considered to be important sources of treatment23 novel dendrobium is considered as one of the most important herbs in the orchidaceae family and shows diverse pharmacological functions including anticancer neuroprotective antidiabetic and immunemodulating activities24 erianin derived from dendrobium is one of the most for cancer drug design development and therapy submit your manuscript wwwdovepresscom dovepress 0csun dovepressfigure erianin decreased cd47 expression and increased phagocytosis a sw480 cells were transfected with nontarget nt or catenin sirna for h protein levels of indicated protein weres measured by western blotting bd sw480 and hct116 cells were treated with erianin for indicated dose the mrna level b protein level c and cell surface cd47 d were detected by qrtpcr and flow e the ucsc genome browser revealed the enrichment of h3k27ac on cd47 promoter f the enrichment of h3k27ac on cd47 promoter f1f6 was detected by chip assay g and h sw480 and hct116 were treated with indicated concentration of erianin for h representative images showed the effect of erianin on phagocytosis g and bar graphs showed quantitative analysis of phagocytosis h p Ë p Ësubmit your manuscript wwwdovepresscom dovepress drug design development and therapy 0cdovepress sun figure erianin inhibited tumor growth in vivo a typical photos of tumors from the control and erianin treated groups b and c erianin decreased tumor volume and weight p Ë d mice body weight of control and erianin treated groups was measure at indicated time e the protein level of catenin in cytosol and nucleus in three representative tumors from mouse to mouse of each group were analyzed by western blotting f the protein level of indicated protein in three representative tumors from mouse to mouse of each group were analyzed by western blottingdrug design development and therapy submit your manuscript wwwdovepresscom dovepress 0csun dovepressnoteworthy constituents that have been used as an antipyretic and an analgesic in traditional chinese medicine25 recently several studies have proved that erianin shows significant antitumour activity in a variety of human cancer cells10 consistent with literature in this study we found that erianin had a significant antiproliferative effect against crc cells the inhibitory effect caused by erianin may result from induction of apoptosis and arrest of cell cycle at g2m since the effect of erianin on crc cells has never been studied before we further confirm its antitumor activity in a mouse model which indicated that erianin significantly inhibited tumor growth in vivoseveral signaling pathways including egfrmapk pi3kakt or wntcatenin have been linked to crc genesis and progression26 as the aberrant activation is present in almost all crc cases wntcatenin signaling is prominent among these pathways27 inactivated mutations in the apc gene leads to stabilization and ensuing nuclear translocation of catenin to facilitate tcflef dependent transcription of wntcatenin signaling target genes such as cmyc and cyclin d1 to drive cell proliferation survival and metastasis28 to understand the mechanisms of action of erianin we assessed the effect of erianin on wntcatenin pathway interestingly we found that erianin treatment had no effect on catenin phosphorylation but inhibited the translocation of catenin in the nucleus which suggested to us that erianin physically interacts with catenin our cellular thermal shift assay confirmed this hypothesis the thermal stability of catenin increased after erianin treatment as catenin downstream targets the expression level of cmyc and cyclin d1 significantly decreased after erianin treatmentcd47 a transmembrane glycoprotein expresses ubiquitously and mediates a selfdonoteatme signal on normal cells however cd47 is often upregulated in tumor cells to evade innate immunity31 anticd47 antibodies which block cd47 sirpα interactions and promote macrophage mediated phagocytosis of tumor cells has shown promise in several solid tumors31 in colorectal cancer cd47 promotes colon cancer cell migration and metastasis34 in addition upregulated immuneescape pathways such as cd47 sirpα are responsible for immune escape and survival in circulating tumor cells of colorectal cancer35 myc an oncogene identified as a wntcatenin target gene was reported to control cd47 transcription therefore mutations in components of the wntcatenin signaling pathway which induced | Colon_Cancer |
recently extensive evidence has clarified the crucial role of circular rnas circrnas as a protumor or anticancer participant in human malignancies a new circrna derived from oxysterol binding protein like osbpl10 circosbpl10 has not been researched in cervical cancer cc yetmethods the expression of molecules was analyzed by rtqpcr or western blot several functional assays were applied to explore the biological influence of circosbpl10 on cc the interaction between rnas was estimated via luciferase reporter rna immunoprecipitation and rna pulldown assaysresults circosbpl10 characterized with cyclic structure was revealed to possess elevated expression in cc cells circosbpl10 downregulation elicited suppressive impacts on cc cell proliferation and migration interestingly circosbpl10 regulated cc progression by interacting with microrna1179 mir1179 moreover ubiquitin conjugating enzyme e2 q1 ube2q1 targeted by mir1179 was positively regulated by circosbpl10 in cc furthermore enhanced ube2q1 expression or suppressed mir1179 level countervailed the repressive effect of circosbpl10 depletion on the malignant phenotypes of cc cells moreover forkhead box a1 foxa1 was confirmed to induce circosbpl10 expression in cc cellss foxa1induced circosbpl10 facilitates cc progression through mir1179ube2q1 axis highlighting a strong potential for circosbpl10 to serve as a promising therapeutic target in cckeywords circosbpl10 mir1179 ube2q1 foxa1 cc as a frequent type of human gynecological malignancies worldwide cervical cancer cc is depicted as one of the dominating causes contributing to cancerassociated death in women [ ] it is estimated that that nearly correspondence luo0700777337163com lushun1982livecn department of radiotherapy sichuan cancer hospital institute sichuan cancer center school of medicine university of electronic science and technology of china no renmin south road chengdu sichuan china pharmacy department sichuan jinxin women and childrens hospital no jingxiu road jinjiang district chengdu sichuan chinafull list of author information is available at the end of the new cases are diagnosed with cc annually in china cc is also regarded as one of the most prevalent lethal tumors over the past few years in spite of the application of human papillomavirus hpv vaccine in treatment cc is still a major stumbling block for female health [ ] the majority of patients at an early stage of cc are likely to be cured through surgery whereas no or limited efficient therapeutic approaches for those at the advanced stages in order to make advances in the treatment of cc researchers have focused on exploring and developing tumorparticular biomarkers for cc for example melatonin was identified as a new adjuvant agent in treating patients with cc so was curcumin 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 0cyang a0et a0al cancer cell int page of however more efforts should be made in developing new effective therapeutic strategies for cc hence it is imperative to make indepth exploration of the underlying molecular mechanisms in ccnoncoding rnas ncrnas have been commonly considered as the potential key modulators in gene regulation to impact on tumor development [ ] including genetic and epigenetic manners in recent years ncrnas have been indicated as clinical biomarkers in diverse diseases [ ] including cancer [ ] as a member of ncrnas circular rnas circrnas own a covalently closed structure which are tolerant to rnase rmediated degradation [ ] increasing analyses have suggested the abnormal expression of circrnas in various cancers [ ] including cc [ ] emerging researches have testified the implication of circrnas in tumorigenesis and progression via regulation of different biological processes which includes cell proliferation migration and invasion [] emerged as a new circrna circosbpl10 circbase id hsa_circ_0064669 is derived from backsplicing of osbpl10 mrna messenger rna to our knowledge the critical regulatory mechanism of circosbpl10 has not been investigated in cc yetin this study the main purpose was to decipher the potential regulatory role of circosbpl10 in cc data from a series of assays uncovered that foxa1induced upregulation of circosbpl10 contributes to cc progression via mir1179ube2q1 axis revealing that circosbpl10 might be a hopeful biomarker for ccmethodscell culturehuman normal cervical epithelial cells h8 and human cc cells c33a caski hela and siha were bought from chinese academy of sciences shanghai china cells were cultured with dulbeccos modified eagle medium dmem invitrogen carlsbad ca usa adding fetal bovine serum fbs invitrogen penicillinstreptomycin sigmaaldrich milan italy and then incubated in an incubator of co2 at a0°ccell transfectionhela and siha cells were transfected with specific short hairpin rnas shrnas against circosbpl10 shcircosbpl1012 foxa1 shfoxa112 and their corresponding negative control nc shnc as well as pcdna31circosbpl10 pcdna31ube2q1 pcdna31foxa1 and empty pcdna31 ± circrna mini vector empty pcdna31 vector severally the mir mimics mir1179 inhibitor nc mimics and nc inhibitor were synthesized by genepharma shanghai china transfection experiments were executed by lipofectamine invitrogenrealtime quantitative polymerase chain reaction rtqpcrtotal rna of cells was isolated using trizol reagent followed by cdna complementary dna synthesis with reverse transcription kit invitrogen rtqpcr was measured by sybrgreen realtime pcr kit takara tokyo japan operated on biorad cfx96 system takara relative expression level was calculated utilizing δδct method with normalization to glyceraldehyde3phosphate dehydrogenase gapdh or u6 the sequences of primers were presented in additional file a0 table a0s1cell counting kit cckin short cells were plated into a 96well plate after incubation for specific times a0h cells were processed with a0 μl of cck8 reagent for additional a0 h absorbance at a0 nm was measured via a microplate reader olympus tokyo japancolony formation assaytransfected cells were first coated into 6well plates after a0weeks of incubation cells were rinsed with phosphate buffer saline pbs sigmaaldrich san francisco usa fixed in methanol sigmaaldrich and dyed using crystal violet sigmaaldrich the visible colonies were counted manuallyactinomycind actd and a0rnase r treatmentto block transcription a0mgml actinomycin d actd sigmaaldrich or dimethylsulphoxide dmso sigmaaldrich was added into culture medium total rna was cultivated with or without a0uμg of rnase r epicentre technologies madison wi usa for a0min after treatment with actd or rnase r rtqpcr was applied for determining the expression levels of circosbpl10 and osbpl10 mrnanucleic acid electrophoresisconvergent primers and divergent primers were designed to amplify osbpl10 mrna or circosbpl10 the level of circosbpl10 in pcr products from cdna and genomic dna was examined by agarose gels with te trisethylene diamine tetraacetic acid buffer from thermo scientific waltham ma usaterminal deoxynucleotidyl transferasemediated dutp nickend labeling tunel assayapoptosis transfected siha and hela cells were assessed utilizing tunel apoptosis kit invitrogen ²6diamidino2phenylindole dapi sigmaaldrich was employed 0cyang a0et a0al cancer cell int page of to dye above cells the percentage of positive stained cells was observed by fluorescence microscopy olympus and then analyzedflow cytometry analysiscell apoptosis analysis was performed via cell apoptosis analysis kit takara after incubation in 6well plates siha and hela cells were rinsed with pbs and resuspended in binding buffer followed by fixation with icecold ethanol sigmaaldrich cells were doublestained by annexin vfluorescein isothiocyanate and propidium iodide last cell apoptosis rate was detected by flow cytometer bectondickinson ma usamigration assaycell migration abilities were examined using transwell chambers corning ny usa transfected cells with serumfree medium were placed into top compartment while medium with fbs was added into the lower compartment a0h later cells in the lower chamber were immobilized and dyed in methanol and crystal violet separately then migratory cells were then counted in five random chosen fields under a microscope olympuswound healingsiha and hela cells were added in 6well plates for cultivation when cell confluence was scratches were produced in cell layer using sterile pipette tip afterward cells were cleaned using pbs and incubated in a culture medium for a0h images of migrating cells were detected at lastwestern blottotal protein was reaped in radio immunoprecipitation assay ripa lysis buffer beyotime shanghai china bicinchoninic acid bca kit beyotime determined protein concentrations proteins were separated through sodium dodecyl sulfatepolyacrylamide gel electrophoresis sdspage and moved onto polyvinylidene fluoride pvdf membranes the membranes were sealed with skimmed milk and cultivated with primary antibodies against ube2q1 orb77618 biorbyt san francisco california usa and gapdh ab8245 abcam cambridge uk secondary antibody was added for incubating for a0h gapdh was an internal control proteins quantities were evaluated via chemiluminescence detection systemluciferase reporter assaythe wildtype wt and mutant mut binding sites of mir1179 in circosbpl10 or ube2q1 ²utr was subcloned into pmirglo dualluciferase vector to construct circosbpl10wtmut or ube2q1wtmut and plasmids were cotransfected with mir1179 mimics or nc mimics into siha and hela cells respectively the pgl3osbpl10 promoter was cotransfected with pcdna31foxa1 or pcdna31 into cells dualluciferase reporter assay system promega usa detected the luciferase activitysubcellular fractionationusing neper¢ nuclear and cytoplasmic extraction fractions of cytoplasmic and nuclear were separated reagents invitrogen and gathered by rneasy midi kit qiagen hilden germany to determine the cellular localization of circosbpl10 rtqpcr was used to examine the levels of circosbpl10 u6 nuclear control and gapdh cytoplasmic controlrna pulldownbriefly cell lysates were incubated with biotinylated rna including biomir1179wt biomir1179mut and bionc moreover m280 streptavidin magnetic beads sigmaaldrich were added to coculture for a0h the relative enrichment of rnas pulled down in each group were assayed by rtqpcrrna immunoprecipitation riprip assays were progressed with magna riptm rnabinding protein immunoprecipitation kit millipore bedford usa siha and hela cells were lysed with rip lysis buffer followed by incubation with magnetic beads conjugated with antiago2 millipore or antiigg millipore the rtqpcr was performed to evaluate the expression levels of circosbpl10 mir1179 and ube2q1 in the precipitateschromatin immunoprecipitation chipvia magna chip kit millipore chip experiment was achieved dna in cell lysates was interrupted into 300bp chromatin fragments by ultrasound after that lysates were subjected to immunoprecipitation with antifoxa1 or antiigg negative control group the precipitated dna fragments were detected by rtqpcrstatistical analysisexperimental data from at least three independent experiments were shown as mean ± standard deviation sd statistical analysis was performed using graphpad prism software graph pad la jolla ca usa significance in differences between or more groups was analyzed via students t test or oneway analysis of variance anova p had statistical significance in requirements 0cyang a0et a0al cancer cell int page of resultscircosbpl10 is a0highly expressed in a0cc and a0its depletion impedes cc cell proliferation and a0migrationto study the cellular function of circosbpl10 in cc we first applied rtqpcr analysis and unveiled a marked elevation of circosbpl10 expression in cc cell lines compared with a0h8 cells fig a01a then nucleic acid electrophoresis manifested that in siha and hela cells divergent primers could produce circosbpl10 from cdna but not from genomic dna gdna while convergent primers amplified linear osbpl10 from both cdna and gdna fig a0 1b besides osbpl10 mrna was greatly degraded by actd whereas circosbpl10 exhibited as resistant to actd fig a0 1c additionally osbpl10 expression was dramatically reduced whereas circosbpl10 expression demonstrated no evident change after siha and hela cells were treated with rnase r fig a01d then we verified that circosbpl10 expression was lowered in two cc cells after transfection with shcircosbpl1012 while those with shcircosbpl101 showed higher silencing efficiency fig a0 1e subsequently cell proliferation assays depicted a notably weakened proliferation ability of siha and hela cells under circosbpl10 silence fig a0 1f g moreover cell apoptosis capability was proved to be facilitated after silencing circosbpl10 in siha and hela cells fig a01h i furthermore it was uncovered that circosbpl10 deficiency gave rise to attenuated migration ability of siha and hela cells fig a01j k taken together circosbpl10 is expressed at high levels in cc and knockdown of it impairs malignant behaviors in cc cellscircosbpl10 sponges mir in a0ccfor the purpose of investigating the molecular mechanism of circosbpl10 in regulating cc we first detected its cellular sublocalization in siha and hela cells via subcellular fractionation as illustrated in fig a0 2a circosbpl10 was majorly distributed in cytoplasm thus we speculated that circosbpl10 might affect cc via serving as a sponge of specific mirna after searching starbase httpstarb asesysueducn with certain condition clip data strict stringency ¥ degradome data low stringency ¥ three mirnas mir1179 mir27a3p and mir27b3p were revealed to have binding potentials with circosbpl10 fig a02b then we discovered a significant downregulation of mir1179 whereas no apparent changes on the levels of mir27a3p and mir27b3p in cc cell lines compared to normal h8 cells fig a02c therefore mir1179 was chosen for further analysis subsequently circosbpl10 and mir1179 were presented to be conspicuously concentrated in antiago2 group fig a02d afterwards two binding sites between circosbpl10 and mir1179 were predicted via starbase fig a02e moreover we validated that mir1179 bound with circosbpl10 at site fig a02f to further test whether circosbpl10 promoted cc progression via its interaction with mir1179 we mutated the sequence of circosbpl10 recognized by mir1179 as displayed in fig a02g the expression of circosbpl10 was observably elevated in c33a and caski cells after overexpressing circosbpl10 and circosbpl10 mut interestingly it seemed that cell proliferation and migration in c33a and caski cells could be fortified by overexpression of fulllength circosbpl10 but not by upregulation of circosbpl10 with mutated mir1179 binding sites fig a0 2h i indicating that the function of circosbpl10 in cc depended on its binding to mir1179 in circosbpl10 interacts with mir1179 to drive cc progressioncircosbpl10 upregulates ube2q1 level in a0cc by a0sequestering mirto further investigate the downstream mechanism of circosbpl10 in cc starbase was utilized as predicted under certain circumstances clip data strict stringency ¥ degradome data low stringency ¥ four candidates ube2q1 prpf38b cacul1 and luc7l3 were found as the targets of mir1179 fig a03a the following rna pulldown assay demonstrated the distinct enrichment of ube2q1 whereas limited harvest of other three mrnas in biomir1179wt group fig a0 3b more importantly ube2q1 level was cut down in siha and hela cells by circosbpl10 knockdown as well as by augmented mir1179 expression fig a03c and additional file a0 besides rtqpcr obtained a conspicuous elevation on ube2q1 expression in cc cell lines relative to h8 cells at both mrna and protein levels fig a0 3e and see figure on next pagefig circular rna circosbpl10 was highly expressed in cc and knockdown of it suppressed cc progression a circosbpl10 expression was detected by rtqpcr in cc cell lines h8 cells b it was delineated by nucleic acid electrophoresis analysis that divergent primers amplified circosbpl10 from cdna but not from gdna gapdh was a negative control c the resistance of circosbpl10 and osbpl10 mrna to actd in siha and hela cells was analyzed by rtqpcr d rtqpcr assay was conducted to determine the abundance of circosbpl10 and linear osbpl10 mrna in siha and hela cells treated with rnase r normalized to mock treatment e rtqpcr was utilized to analyze the efficacy of circosbpl10 knockdown in siha and hela cells f g the proliferation ability of siha and hela cells transfected with shcircosbpl101 or shnc was evaluated via cck8 and colony formation h i cell apoptosis ability in transfected cells was measured by tunel assay and flow cytometry analysis j k transwell and wound healing assays were conducted to analyze the migration of transfected cells p p 0cyang a0et a0al cancer cell int page of 0cyang a0et a0al cancer cell int page of 0cyang a0et a0al cancer cell int page of see figure on next pagefig circosbpl10 sponged mir1179 in cc a subcellular fractionation was applied to detect the cellular sublocalization of circosbpl10 in siha and hela cells b mir1179 mir27a3p and mir27b3p were predicted via starbase to have the binding capacity with circosbpl10 c mir1179 expression in cc cell lines and h8 cells was detected via rtqpcr d rip assay unveiled the significant enrichment of circosbpl10 and mir1179 in antiago2 group e two binding sites between circosbpl10 and mir1179 were predicted via starbase f luciferase reporter assay validated the interaction between circosbpl10 and mir1179 g the expression of circosbpl10 in siha and hela cells transfected with pcdna31 pcdna31circosbpl10 or pcdna31circosbpl10 mut was analyzed through rtqpcr h i cell proliferation and migration in different groups was analyzed via cck8 and transwell assays respectively p p additional file a0 afterwards the binding sites between ube2q1 and mir1179 were obtained through starbase prediction fig a0 3f importantly we observed declined luciferase activity of ube2q1wt owing to mir1179 upregulation through luciferase reporter assay however no overt changes on that of ube2q1mut were noted between two groups fig a03g more importantly a significant enrichment of circosbpl10 mir1179 and ube2q1 in antiago2 groups was observed via rip analysis fig a03h further ube2q1 expression was visibly upregulated by circosbpl10 overexpression whereas enhanced expression of mutated circosbpl10 had no such influence on ube2q1 expression in these two cc cells fig a03i and additional file a0 in sum circosbpl10 facilitates ube2q1 expression by sponging mir1179 in cccircosbpl10 accelerates cc progression via a0mirube2q1 axisgiven that circosbpl10 sponged mir1179 to upregulate ube2q1 expression in cc we intended to detect whether this mechanism contributed to cc progression herein the efficacy of elevating ube2q1 expression or lowering mir1179 level was analyzed at first by rtqpcr and the outcome appeared to be satisfactory in siha cells fig a04a afterwards we testified that ube2q1 upregulation or mir1179 inhibition could offset circosbpl10 depletionmediated suppressive effect on cell proliferation fig a04b c in addition cell apoptosis facilitated by circosbpl10 knockdown was counteracted by ube2q1 overexpression or mir1179 suppression fig a0 4d e moreover the attenuated cell migration capability in circosbpl10depleted cells was restored by upregulation of ube2q1 or inhibition of mir1179 fig a04f g to conclude circosbpl10 promotes cc progression via mir1179ube2q1 axisfoxa1 activates circosbpl10 expression in a0ccsince the downstream signaling responsible for the regulation of circosbpl10 in cc had been studied we subsequently focused on figuring out its possible upstream mechanism through utilizing ucsc university of california santa cruz httpgenom eucscedu foxa1 seemed to be a probable transcription factor of osbpl10 the host gene of circosbpl10 prior to testify the influence of foxa1 on circosbpl10 expression in cc we first silenced or overexpressed foxa1 with satisfactory efficacies in siha and hela cells fig a0 5a of interest we discovered that the expression of circosbpl10 was distinctly declined by foxa1 depletion whereas overtly augmented by foxa1 upregulation fig a0 5b later on we employed jaspar database httpjaspa rgener egnet and obtained the dna motif of foxa1 fig a05c seen from fig a05d the sequences of osbpl10 promoter were fragmented into five parts p15 interestingly it was verified by chip assay that foxa1 mainly bound to osbpl10 promoter at p4 region fig a0 5e moreover when overexpressing foxa1 in siha and hela cells the luciferase activity of osbpl10 promoterwt was observably increased while there were no evident changes on that of osbpl10 promotermut with mutated foxa1 binding sites predicted in p4 region fig a05f in a word foxa1 activates osbpl10 transcription and thereby facilitates circosbpl10 expression in ccdiscussionmounting evidence has manifested that circrnas serve vital parts in cc initiation and progression for instance circrna smarca5 regulates cc progression by sponging mir620 increased expression of circ_0067934 accelerates cc development by targeting mir545eif3c see figure on next pagefig circosbpl10 upregulated ube2q1 expression by competitively binding with mir1179 in cc a venn diagram showed the overlaps of potential mir1179 targets predicted by pita microt pictar mirmap b the binding capacity between mir1179 and four mrnas was verified through rna pulldown assay c d the expression of ube2q1 in siha and hela cells transfected with different plasmids was detected via rtqpcr and western blot e ube2q1 expression in cc cell lines and h8 cells was detected via rtqpcr f the binding sites between ube2q1 and mir1179 obtained from starbase were displayed g the interaction between ube2q1 and mir1179 was validated by luciferase reporter assay h rip analysis revealed that circosbpl10 mir1179 and ube2q1 coexisted in riscs rnainduced silencing complexes i rtqpcr and western blot were utilized to detect the expression of ube2q1 in indicated cells p 0cyang a0et a0al cancer cell int page of 0cyang a0et a0al cancer cell int page of fig circosbpl10 accelerated cc progression via mir1179ube2q1 axis a the efficacy of ube2q1 overexpression and mir1179 inhibition in siha cells was analyzed by rtqpcr b c the proliferation ability of siha cells under diverse conditions were measured by cck8 and colony formation assays d e the apoptosis ability of transfected cells were measured via tunel assay and flow cytometry analysis f g transwell and wound healing assays were carried out to analyze cell migration under different contexts p 0cyang a0et a0al cancer cell int page of fig foxa1 activated circosbpl10 expression in cc a the efficacy of foxa1 knockdown and overexpression in siha and hela cells was obtained through rtqpcr analysis b the expression of circosbpl10 in different groups was detected by rtqpcr c the dna motif of foxa1 was obtained from jaspar database d fulllength of osbpl10 promoter was fragmented into pieces p15 and jaspar predicted a potential binding site for foxa1 to osbpl10 promoter at p4 region e chip assay proved that the p4 region of osbpl10 promoter was recognized by foxa1 in both siha and hela cells f the interaction between osbpl10 promoter and foxa1 at above predicted sites was testified by luciferase reporter assay p axis circrna hsa_circ_0023404 plays an oncogenic part in cc circosbpl10 derived from backsplicing of osbpl10 mrna is a circular rna that has not been studied in cc but is worth exploring in this research circosbpl10 was evidenced to have a circular structure and possess high levels in cc additionally silenced circosbpl10 exerted suppressive impacts on cc cell proliferation and migrationincreasing researches have elucidated that circrnas might serve as a molecular sponge of specific mirnas which has been suggested to be of significant value in cc [ ] so as to regulate the tumorigenesis and 0cyang a0et a0al cancer cell int page of development of numerous cancers including cc [ ] in current study on the basis of bioinformatics prediction and molecular mechanism experiments mir1179 that was unveiled as an antitumor regulator in some cancers [ ] was screened out and validated to be implicated in circosbpl10 regulated cellular processes in ccpreviously ube2q1 has been depicted as a critical participator in tumor progression [ ] in present study ube2q1 was verified capable of binding with mir1179 and its expression was boosted by circosbpl10 in cc through mir1179 sequestration more importantly the followup rescue experiments indicated that ube2q1 upregulation or mir1179 inhibition could rescue circosbpl10 depletionmediated suppressive effects on malignant phenotypes in ccincreasing researches have indicated that foxa1 expresses at high levels in many cancers such as lung cancer glioma and prostate cancer besides a previous study indicated that foxa1 could directly bind with plod2 promoter and activate plod2 transcription in nsclc similarly we revealed that foxa1 activates osbpl10 transcription and thereby facilitates circosbpl10 expression in cc in this studyin foxa1induced circosbpl10 promotes cc progression by targeting mir1179ube2q1 axis providing novel insights into exploring more effective treatment of cc however the biggest regret is that no clinical data are included in this work and the findings in our study need to be further testified by clinical samples in the futuresupplementary informationsupplementary information accompanies this paper at https doi101186s1293 additional file a0 table a0s1 list of the sequences of primers used in rtqpcr additional file a0 figure s1 the size of marker for western blot gelsabbreviationscircrna circular rna cc cervical cancer ube2q1 ubiquitin conjugating enzyme e2 q1 foxa1 forkhead box a1 ncrnas noncoding rnas fbs fetal bovine serum actd actinomycin d gdna genomic dnaacknowledgementswe are very grateful to all individuals and groups involved in this study authors contributionssy yj xr and df designed this study sy lz fz yj and xr devoted to experiment data curation and interpretation sy yj xr df dh sh and lz were in charge of investigation experiment record figures and writing sl edited the manuscript all authors read and approved the final manuscript fundingthis study was supported by the national natural science foundation of china the national cancer center climbing foundation of chinancc201808b016 department of science and technology of sichuan province20yyjc3815 department of science and technology of sichuan province20gjhz0088 availability of data and materialsresearch data and material are not sharedethics approval and consent to participatenot applicableconsent for publicationnot applicablecompeting intereststhe authors declare that they have no competing interestsauthor details department of gynecological radiotherapy harbin medical university cancer hospital no haping road nangang district harbin heilongjiang china department of radiotherapy sichuan cancer hospital institute sichuan cancer center school of medicine university of electronic science and technology of china no renmin south road chengdu sichuan china pharmacy department sichuan jinxin women and childrens hospital no jingxiu road jinjiang district chengdu sichuan china received september accepted june references jemal a bray f center mm ferlay j ward e forman d global cancer statistics ca torre la bray f siegel rl ferlay j lortettieulent j jemal a global cancer statistics ca tewari ks sill mw long hj 3rd penson rt huang h ramondetta lm landrum lm oaknin a reid tj leitao mm et al improved survival with bevacizumab in advanced cervical cancer new engl j med chen w zheng r zeng h zhang s he j annual report on status of cancer in china chinese j cancer res testing for cervical cancer new recommendations from the american cancer society american society for colposcopy and cervical pathology and american society for clinical pathology ca burki tk cervical cancer screening and risk with age lancet oncol 2014153e107 noordhuis mg fehrmann rs wisman gb nijhuis er van zanden jj moerland pd ver loren van themaat e volders hh kok m ten hoor ka et al involvement of the tgfbeta and betacatenin pathways in pelvic lymph node metastasis in earlystage cervical cancer clin cancer res shafabakhsh r reiter rj mirzaei h teymoordash sn asemi z melatonin a new inhibitor agent for cervical cancer treatment j cell physiol ghasemi f shafiee m banikazemi z pourhanifeh mh khanbabaei h shamshirian a amiri moghadam s arefnezhad r sahebkar a avan a et al curcumin inhibits nfkb and wntβcatenin pathways in cervical cancer cells pathol res pract perez ds hoage tr pritchett jr ducharmesmith al halling ml ganapathiraju sc streng ps smith di long abundantly expressed noncoding transcripts are altered in cancer hum mol genet guttman m donaghey j carey bw garber m grenier jk munson g young g lucas ab ach r bruhn l et al lincrnas act in the circuitry controlling pluripotency and differentiation nature 0cyang a0et a0al cancer cell int page of khani p nasri f khani chamani f saeidi f sadri nahand j tabibkhooei a mirzaei h genetic and epigenetic contribution to astrocytic gliomas pathogenesis j neurochem mirzaei h stroke in women risk factors and clinical biomarkers j cell biochem saeedi borujeni mj esfandiary e baradaran a valiani a ghanadian m codo±erfranch p basirat r alonsoiglesias e mirzaei h yazdani a molecular aspects of pancreatic βcell dysfunction oxidative stress microrna and long noncoding rna j cell physiol asma v zahra s ahmad m soheila m sima f hamid rm afshin n amir s hamed m long noncoding rnas as epigenetic regulators in cancer curr pharm des mirzaei h yazdi f salehi r mirzaei h sirna and epigenetic aberrations in ovarian cancer j cancer res ther qu s zhong y shang r zhang x song w kjems j li h the emerging landscape of circular rna in life processes rna biol qu s yang x li x wang j gao y shang r sun w dou k li h circular rna a new star of noncoding rnas cancer lett shabaninejad z vafadar a movahedpour a ghasemi y namdar a fathizadeh h pourhanifeh mh savardashtaki a mirzaei h circular rnas in cancer new insights into functions and implications in ovarian cancer j ovarian res naeli p pourhanifeh mh karimzadeh | Colon_Cancer |
" gastric neoplasms containing neuroendocrine carcinoma nec components are rare malignancieswith highly aggressive behavior and a poor prognosis and include pure nec and mixed tumors containing neccomponents we aimed to investigate whether there is a distinct difference in overall survival os between gastricneoplasms containing nec components and gastric adenocarcinomamethods surgically resected gastric neoplasms containing nec components n and gastricadenocarcinomas n from january to december at peking university cancer hospital wereretrospectively analysed patients were categorized into a surgical group and a neoadjuvant group and adjustedusing propensity score matching in the two groups gastric neoplasms containing nec components were dividedinto pure nec and mixed tumors with less than ghminen between and ghminen andmore than ghminen neuroendocrine carcinoma components os was compared between thesegroups and the gastric adenocarcinoma groupresults the os of gastric neoplasms containing neuroendocrine nec components was poorer than that of gastricadenocarcinomas in the surgical group regardless of whether the percentage of neuroendocrine cancercomponents was less than between and more than or cox multivariable regressionanalysis suggested that tumor category neoplasms containing nec components or gastric adenocarcinoma wasan independent risk factor for prognosis interestingly among patients receiving neoadjuvant therapy thedifference was not significantcontinued on next page correspondence buzhaodecjcrcn jijiafuhscpkueducn jiahui chen anqiang wang and ke ji contributed equally to this workdepartment of gastrointestinal surgery key laboratory of carcinogenesisand translational research ministry of education peking university cancerhospital institute no fucheng road haidian district beijing china 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 0cchen bmc cancer page of continued from previous pages gastric neoplasms containing any proportion of nec components had poorer overall survival thangastric adenocarcinoma in patients treated with surgery directly indicating that these neoplasms are moremalignant than gastric adenocarcinoma among the patients receiving neoadjuvant therapy the difference inoverall survival was not significant which was in sharp contrast with the results of the surgery group suggestingthat neoadjuvant therapy may have a good effect in the treatment of these neoplasmskeywords neuroendocrine carcinoma gastric adenocarcinoma overall survival gastric neoplasms containing neuroendocrine carcinomanec components are a heterogeneous subgroup ofgastric cancer with highly aggressive behavior and poorprognosis and include pure necs and mixed tumorscontaining nec components every yearthere areapproximately million new cases of gastric cancerworldwide and gastric neoplasms containing nec components account for approximately of thesecases [ ] given the low incidence there is little comprehensive basic and clinical research to systematicallyguide the treatment of these gastric neoplasms makingthe prognosis of these tumors unsatisfactory []according to the world health anizationwho digestive neuroendocrine tumor classificationneuroendocrine neoplasm nen can be divided intothree categories based on ki67 levels and mitotic counts hpf grade g1 ki67 mitoses grade g2 ki67 mitoses grade g3ki67 mitoses meanwhile the americanjoint committee on cancer ajcc defines highly differentiated nen as a neuroendocrine tumor net and thepoorly differentiated nen as a neuroendocrine carcinoma nec based on the degree of tumor cell differentiation generally g1 g2 and rare welldifferentiated g3nens belong to the nets while poorly differentiatedg3 nens belong to necs[ ] gastric mixedneuroendocrinenonneuroendocrineneoplasm gminen is a special type of gastric nen that is definedas containing more than of both neuroendocrineand nonneuroendocrine components accountingfor approximately of all gnens and of gastricneuroendocrine carcinomas gnecs [] for thosemixed tumors with less than or more than neuroendocrine carcinoma components there is no uniform definition consideringthe heterogeneity ofminen and the malignancy degree of the different components in the tumor la rosa [ ] proposeddividing minen into three categories highgradeintermediategrade and lowgrade highgrade minenconsists of nec and carcinomaadenoma intermediategrade mimen consists of net and carcinoma and lowgrade minen consists of net and adenoma thereforein this study gastric highgrade mixed neuroendocrinenonneuroendocrine neoplasm ghminen was defined as gastric cancer containing more than of bothneuroendocrineadenocarcinomacomponentscarcinomaandgenerally the prognosis of mixed tumors is largely determined by the most malignant component kim found that gnec has shorter progressionfree survival pfs than gastric adenocarcinoma huang found that the prognosis of patients with more than of neuroendocrine cancer components is significantly poorer than that of patients with less than components all of these studies provide evidence thattumors containing neuroendocrine cancer componentsmay contribute to a worse prognosis therefore wehypothesized that a mixed tumor containing neuroendocrine carcinoma components would have a worse prognosis than pure adenocarcinoma alone we sought tofind studies on the overall survival os comparison between ghminen and gastric adenocarcinoma butfailed thus we think that a study of the comparison ofthe os of ghminen and gastric adenocarcinoma willprovide a valuable supplement to current research on ghminen to overcome the bias caused by the differences between the covariates in the comparison we usedpropensity score matching psm to match importantfactors such as age gender tumor location tumor sizepathological staging and adjuvant chemotherapy between the two groups making the research results morereliablemethodspatient selectionwe retrospectively collected patients diagnosed withgastric nens and underwent radical resection at pekinguniversity cancer hospital beijing from january to december the inclusion criteria were as follows pathologically confirmed pure nec or tumorcontaining nec components no other tumors werediagnosed before the operation complete clinicopathological information and survival information thatcould be obtained through followup patients diagnosedwith cm1 or ct4b before surgery or died from perioperative complications were excluded from the study 0cchen bmc cancer page of patients with gastric adenocarcinomas undergoing radical surgery were randomly selected for psm analysesperformed the chisquared test and mannwhitney utest were used to further verify the matching resultsfollowupwe followed the patients at least twice a year serumtumor markers test gastroscope and computed tomography ct scans were used to reexamine patients aftersurgery depending on the patients status magneticresonance imaging mri and positron emission tomography computed tomography petct were alsoconsidered for patients who cannot regularly visit ourcenter for postoperative examination we use telephonefollowup to obtain survival informationdiagnosis and classificationwe reevaluated the diagnosis and classification of ghminen mixed tumors with less than or morethan neuroendocrine carcinoma components werealso included in this study which were defined as ghminen and ghminenrespectively atumor consisting of nec is defined as pure necall neuroendocrine tumors were identified diagnosedand classified by two independent pathologists in accordance with the who classification of tumors neuroendocrine components were identified byhistological features and immunohistochemical specificity marks such as synaptophysin syn chromogranina cga and neuro cell adhesion molecule cd56 orncam the tumor staging described in the study wasbased on the ajcc 8th edition tnm staging guidelines all possible disagreements were discussed in ourstudy groupdefinition of variables and groupsin this study patients were divided into a surgical groupand a neoadjuvant group based on whether they had received neoadjuvant therapy before surgery patients inthe surgery group were assessed by the ptnm stagingsystem while patients in the neoadjuvanttreatmentgroup were assessed by the yptnm staging system osrefers to the time from surgery to the last followup thetime of death or the end ofloss offollowup or other cause of deathfollowup egpropensity score matchingto accurately compare the prognosis of ghminenand gastric adenocarcinoma we employed psm to balance the differences between the two groups psm wasperformed through the pamatching plugin in spss software logistic regression models were used toestimate propensity scores based on gender age tumorlocation tumor size and pathological staging given a caliper width nearest neighbor matching wasstatistical analysisall statistical analyses were performed using spss statisticalsoftware ibm united states the chisquared test and mannwhitney u test were used forstatistical analysis of categorical variables and continuous variables respectively kaplanmeier method wasused for the comparison of os the logrank test wasused to compare survival rates multivariable cox proportional hazards models were used to identify predictors of survival outcome p was regarded as thethreshold of significanceresultspatient selection and psm resultsbetween and among the patients treated atthe gastrointestinal cancer center of peking universitycancer hospital a total of patients with gastric neoplasms containing nec components met the inclusioncriteria for the study including cases of pure necand cases of mixedtype of these patients a total of patients received neoadjuvant therapy nec ghminen ghminen ghminen while the remaining patients receivedsurgery directly nec ghminen ghminen ghminen there were aninsufficient number of patients in group ghminen group to conduct effective statistical analysisso we combined the ghminen group with thenec group for further analysis we also randomly selected patients with gastric adenocarcinoma whounderwent radical surgery among them patientsreceived neoadjuvant therapy and the remaining patients were treated with surgery directly fig immunohistochemical specificity markers were utilizedto identify the neuroendocrine components fig 2asyn was expressed in almost all neoplasms containingnec components while the positive rates ofcga and cd56 were much lower and respectively no significant difference in the positiverate of syn and cga was observed between pure nec ghminen ghminen and ghminenfig 2b c only the positive rate of cd56 was found tobe higher in the pure nec group than that in the ghminen group fig 2dtherefore priorto os comparison psm wasperformed to ensure that there were no significant differences in patient gender age tumor location tumorsize pathological staging and adjuvant chemotherapybetween the two groups 0cchen bmc cancer page of fig flow chart of patient enrolmentcomparison of os between all patients with neccomponents and patients with gastric adenocarcinoma inthe surgical group and neoadjuvant groupbefore psm we compared the survival curves between all patients with nec components and patientswith gastric adenocarcinoma by the kaplanmeiermethod fig apparently patients with nec components had a poorer os than those with gastricadenocarcinoma fig 3a p in the surgicalgroup in contrast no significant difference was observed between the patientsreceiving neoadjuvanttherapy fig 3b p according to the proportion of nec components patients were classifiedinto pure nec ghminen ghminenand ghminen the os was also comparedbetween patients with adenocarcinomaand thesegroups and the results were similar to the overallcomparison fig 3c dfig illustrations of immunohistochemical staining patterns in gastric neoplasms containing nec components a an overview of the expressionof syn cga and cd56 in tumors containing nec components b syn expression in different nec component groups c cga expression indifferent nec component groups d cd56 expression in different nec component groups cd56 neuro cell adhesion molecule cgachromogranin a nec neuroendocrine carcinoma syn synaptophysin pvalue 0cchen bmc cancer page of fig see legend on next page 0cchen bmc cancer page of see figure on previous pagefig comparison of os between gastric neoplasms containing nec components and gastric adenocarcinoma a os comparison betweengastric neoplasms containing nec components and gastric adenocarcinoma before psm in the surgical group b os comparison between gastricneoplasms containing nec components and gastric adenocarcinoma before psm in the neoadjuvant group c os comparison between differentnec content groups pure nec ghminen ghminen and ghminen and gastric adenocarcinoma before psm in the surgicalgroup d os comparison between the different nec content groups and gastric adenocarcinoma before psm in the neoadjuvant group e oscomparison for patients in the surgical group after psm f os comparison for patients in the neoadjuvant group after psm nec neuroendocrinecarcinoma os overall survival psm propensity score matchingbefore psm significant differences between the baseline characteristics were observed in the surgical groupand the neoadjuvant group table table to balance the clinicopathological differences between the twogroups psm was performed to ensure that there wereno significant differences in patient gender age tumorlocation tumor size pathological staging and adjuvantchemotherapy between the two groups the detailedclinicopathological characteristics before and after psmare shown in table and table as a result patients with nec components and patients with gastric adenocarcinoma were matchedin the surgical group table patients with nec components also had a poorer os than those with gastricadenocarcinoma fig 3e p multivariable analysis showed that adjuvant therapy tumor category andtnm stage werefactorstable independent prognosticto investigate whether neoadjuvant therapy had an effect on os patients with nec components and patients with gastric adenocarcinoma were matched inthe neoadjuvant group table interestingly kaplanmeier analysis showed that among patients receivingneoadjuvant therapy there was still no significant difference in os between the two groups fig 3f p comparison of os between patients with differentproportions of nec components and patients with gastricadenocarcinomato investigate whether the level of nec componentshad an effect on os in the surgical group ghminen ghminen pure nec and pure nec plus ghminen were compared with gastric adenocarcinoma after psm the results showed that even thegroup with the lowest proportion of nec componentsthe ghminen group had a poorer os thanadenocarcinoma fig 4a p as expected theghminen pure nec and pure nec plus ghminen groups each with relatively high proportionsof nec components had worse os than the gastricadenocarcinoma group fig 4bd p detailed clinical information after matching isshown in additional file tables s1s4psm was also performed in the neoadjuvant group incontrast to the results of the surgery group in the purenec group containing the highest proportion ofnec componentstill no significantdifference in os from gastric adenocarcinoma fig5d the other three groups with lower nec contentwere also notfrom gastricadenocarcinoma in terms of os fig 5ac detailedclinicopathologicaland afterpsm are shown in additional file tables s5s8characteristics beforethere wassignificantly differentdiscussionamong gastric neuroendocrine neoplasms the tumorcontaining nec components is a special type includingpure nec and mixed tumor containing nec components the incidence of these tumors is extremely lowbut they are more invasive and have a poorer prognosisthan welldifferentiated gnens [ ]received neoadjuvantin previous study kim found that in patientschemotherapywho had notprogressionfree survivalpfs of pure gnec waspoorer than that of gastric adenocarcinoma while thepfs of mixedtype tumors was not significantly differentin kimsfrom that of gastric adenocarcinoma study the mixed type was defined as net mixed withgastric cancer rather than nec net is much less malignant than nec [ ] this may be the reason whythere was no significant difference in os between mixedtype and gastric adenocarcinomas in addition mixed tumors with less than or more than of nec components were not included in that study which webelieve was a deficit of the study pfs is an important indicator for evaluating prognosis in many cases it can reflect the trend of os based on kims research resultswe regarded tumors containing nec components as awhole and found that the os of these tumors was poorerthan that of adenocarcinoma in the surgical group inthe comparison of os between mixed tumors with different proportions of nec components and gastricadenocarcinoma the results for pure nec cases wassimilar to kims while the os of mixed tumors was alsopoorer than that of gastric adenocarcinoma whether theproportion of neuroendocrine cancer components wasless than between and or more than which was not mentioned in kims study cox multivariable regression analysis showed thattumor categoryneoplasm with nec component or adenocarcinoma 0cchen bmc cancer page of table comparison of clinicopathological characteristics before and after psm in surgical grouppatient characteristicsunmatched comparisonpatients with neccomponents n p valuematched comparisonpatients with neccomponents n age year mean ± sdgender malefemalebmi mean ± sdadjuvant therapyyesnotumor locationupper thirdmiddle thirdlower thirdentiretumor size cm¥ cmtype of gastrectomytotal gastrectomydistal gastrectomy ± ± proximal gastrectomy surgical procedureopenlaparoscopict staget1t2t3t4n stagen0n1n2n3m stagem0m1ptnm stageiiiiiiiv gastricadenocarcinoman ± ± ± ± p value gastricadenocarcinoman ± ± bmi body mass index minen mixed neuroendocrinenonneuroendocrine neoplasm nec neuroendocrine carcinoma psm propensity score matchingpatients with nec components nec high grade minen high grade minen and high grade minen 0cchen bmc cancer table comparison of clinicopathological characteristics before and after psm in neoadjuvant groupmatched comparisonpatient characteristicsunmatched comparisonpatients with neccomponents n age year mean ± sdgender malefemalebmi mean ± sdadjuvant therapyyesnotumor locationupper thirdmiddle thirdlower thirdentiretumor size cm¥ cmtype of gastrectomytotal gastrectomydistal gastrectomyproximal gastrectomysurgical procedureopenlaparoscopict staget0t1t2t3t4n stagen0n1n2n3m stagem0m1yptnm stageiiiiiiiv ± ± gastricadenocarcinoman ± ± p valuepatients with neccomponents n ± ± page of p valuegastricadenocarcinoman ± ± bmi body mass index minen mixed neuroendocrinenonneuroendocrine neoplasm nec neuroendocrine carcinoma psm propensity score matchingpatients with nec components nec high grade minen high grade minen and high grade minen 0cchen bmc cancer page of table univariate and multivariate analyses of survival after psm in surgical grouppatient characteristicsunivariate analysishr cimultivariate analysishr cip valueage yeargendermale vs femalebmiadjuvant therapyyes vs notumor size¥ cm vs cmtumor categorycarcinoma with nec component vsgastric adenocarcinoma vstype of gastrectomytotal gastrectomydistal gastrectomyproximal gastrectomysurgical procedurelaparoscopic vs opentnm stageiiiiiiivp value tumor size and tnm staging were independent risk factors for prognosis this suggests that the prognosis ofgastric neoplasms with nec components is substantiallydifferent from that of gastric adenocarcinoma and evena small percentage of nec components can alsoimpair prognosis which challenges the current cutoffvalue of the proportion of each component that must theoretically be greater than was set in andsince who has also adopted this standard to define minen this largely avoids the overdiagnosisof minen in tumors with only focal neuroendocrinemarker expression and no corresponding morphologicalchanges in additionit also prevents clinicians fromdealing with these rare neoplasms too often withoutguidelines nevertheless it is now being questionedby an increasing number of scholars the componentsin mixed tumors are not evenly distributed for large tumorsthe randomness of biopsy and postoperativepathological sampling causes the proportion of eachcomponent to fluctuate greatly making it difficult to describe the proportion of each component precisely park compared the os between tumors with morethan nec components and gastric adenocarcinomawith or without less than nec and they found thattumors with an nec composition of more than hada worse prognosis this suggests that even a small proportion of malignant components can affect prognosis while in parks study for unknown reasons the authors did not compare the prognosis of mixed tumorswith nec components less than with gastricadenocarcinomas directly nor did they compare allneccontaining tumors as a whole with gastric adenocarcinoma which we believe was a deficit of the studyin our study we regarded tumors containing neccomponents as a whole and found that the os of thesetumors was poorer than that of adenocarcinoma in thesurgical group in addition we also found that the os ofmixed tumors with less than between and more than nec components or pure nec wasworse than that of gastric adenocarcinoma analysis ofimmunohistochemical markers show that there was nosignificant difference in the positive rate of syn and cgabetween different nec content groups only the positiverate of cd56 was found to be higher in the pure necgroup than that in the ghminen group therole of cd56 in the diagnosis of nec is still controversial however syn and cga are two wellrecognized 0cchen bmc cancer page of fig comparison of os between gastric neoplasm with different proportions of nec and gastric adenocarcinoma in the surgical group aoverall survival comparison between ghminen and gastric adenocarcinoma b overall survival comparison between ghminen andgastric adenocarcinoma c overall survival comparison between ghminen plus pure nec and gastric adenocarcinoma d overall survivalcomparison between pure nec alone and gastric adenocarcinomamarkers therefore from the results of immunohistochemistry we believed that there was no significantlydifference in tumors containing nec componentsstudies on the molecular mechanism of pathogenesisshow that nec components and adenocarcinoma components have similar genomic abnormalities similarlosses of heterozygosity loh and mutations at multiple loci and key oncogenes such as tp53 apc and rbgenes all these results imply that the two componentsin the mixed tumor may have a common origin and acquire biphenotypic differentiation during carcinogenesis[] moreoverin the who definition of mixedneuroendocrine and nonneuroendocrine neoplasms ofother ans ie lung and thyroid no minimumpercentage for either ingredient is established thereforewe believe that mixed tumors containing nec components are actually of the same origin have similar biological characteristics and are differentfrom gastricadenocarcinoma we propose considering mixed tumorscontaining nec components as a whole rather than defining them based on the definition for both tumorcomponents which has not been raised by other studiespreviously many studies have confirmed the efficacyof neoadjuvant chemotherapy in gastric adenocarcinoma[ ] in a retrospective study involving patientsma found that neoadjuvant chemotherapy improves the survival of patients with nec and hminenof the stomach van der veen reported that 0cchen bmc cancer page of fig comparison of os between gastric neoplasm with different proportions of nec components and gastric adenocarcinoma in theneoadjuvant group a overall survival comparison between ghminen and gastric adenocarcinoma b overall survival comparisonbetween ghminen and gastric adenocarcinoma c overall survival comparison between ghminen plus pure nec and gastricadenocarcinoma d overall survival comparison between pure nec and gastric adenocarcinomaneoadjuvant chemotherapy could not benefit the survivalof patients with mixed tumors containing nec components however because only eight patients wereincluded in the neoadjuvant group vans results arequestionable in our study among patients receivingneoadjuvanttherapy no significant difference in osbetween mixed tumor and gastric adenocarcinoma wasobserved even for the pure nec group with the highestnec contentthere was no significant differencesuggesting that neoadjuvant therapy may have a positiveeffect on these neoplasmsalthough this is only a singlecenter retrospectivestudy the sample we reported is considerable for thisrare disease which can provide new ideas for clinicaland basic research in addition we proposed treatingall gastric neoplasms containing nec components asa whole and found that neoadjuvanttherapy mayhave a good effect on these neoplasms in the futurewe will conduct more genomics studies to confirmour ideas this study also has its limitations due tothe lack of recurrence and detailed chemotherapy information we were unable to compare progressionfree survival and analyse the effects of differentchemotherapy regimens as a retrospective study despite our performing psm in advance selection biascannot be completely avoided in addition since theexact proportion of each componentin the mixedtumor could not be obtained we could not determine 0cchen bmc cancer page of whether there is a cutoff value for the diagnosis ofthe mixed tumor with nec componentless than so we could only treat all mixed tumors withnec component as a wholesour study demonstrated that gastric neoplasms withnec components regardless of the proportion of components have poorer overall survival than gastric adenocarcinomaindicating a higher degree of malignancythan gastric adenocarcinoma among the patients receiving neoadjuvant therapy the difference in overallsurvival was not significant which was in sharp contrastwith the results of the surgery group suggesting thatneoadjuvant therapy may have a good effect on theprognosis of these malignancies therefore for this typeof malignancy we should adopt more aggressive andpowerful treatments than those used for gastric adenocarcinoma to improve the prognosis of patients neoadjuvant chemotherapy may be a good way to improve theefficacy offor these tumors at advancedstagestreatmentsupplementary informationsupplementary information accompanies this paper at httpsdoi101186s12885020072817additional file table s1 comparison of clinicopathologicalcharacteristics before and after psm of 30ghminen patients insurgical group table s2 comparison of clinicopathologicalcharacteristics before and after psm of ghminen patients in surgicalgroup table s3 comparison of clinicopathological characteristics beforeand after psm of 70ghminen plus pure nec patients in surgicalgroup table s4 comparison of clinicopathological characteristics beforeand after psm of pure nec patients in surgical group table s5 comparison of clinicopathological characteristics before and after psm of 30ghminen patients in neoadjuvant group table s6 comparison ofclinicopathological characteristics before and after psm of ghminen patients in neoadjuvant group table s7 comparison of clinicopathologicalcharacteristics before and after psm of 70ghminen plus pure necpatients in neoadjuvant group table s8 comparison of clinicopathological characteristics before and after psm of pure nec patients in neoadjuvant groupabbreviationsajcc american joint committee on cancer ct computed tomography ghminen gastric highgrade mixed neuroendocrinenonneuroendocrineneoplasm gnec gastric neuroendocrine carcinoma hpf high power fieldminen mixed neuroendocrinenonneuroendocrine neoplasmnec neuroendocrine carcinoma nen neuroendocrine neoplasmnet neuroendocrine tumor mri magnetic resonance imaging os overallsurvival petct positron emission tomography computed tomographypfs progressionfree survival psm propensity score matching who worldhealth anizationacknowledgmentsthanks to dr zhongwu li of the department of pathology peking universitycancer hospital and his colleagues for their assistance in pathologicaldiagnosis and review thanks to all colleagues in the department ofgastrointestinal surgery of peking university cancer hospital and dr jianghong from the statistics department for their assistance in this studyauthors contributionsall authors contributed to the study conception and design jc performeddata collection and wrote the manuscript aw wrote and t revised hemanuscript kj helped with statistical analysis and prepared the illustrationszb edited the manuscript jj conceived the study and reviewed themanuscript all authors read and approved the final manuscriptfundingthis work was supported by the national science foundation for youngscientists of china beijing youth talent plan qml20191101 andscience foundation of peking university cancer hospital thefunders had no role in study design data collection and analysis decision topublish or preparation of the manuscriptavailability of data and materialsthe datasets used andor analysed during the current study are availablefrom the corresponding author on reasonable requestethics approval and consent to participatethe study was approved by the ethics committee of peking universitycancer hospital and the patients written consent was also obtained writteninformed consent for publication was obtained and stored in pekinguniversity cancer hospitalconsent for publicationnot applicablecompeting intereststhe authors declare that they have no competing interestsreceived may accepted august referencesbray f ferlay j soerjomataram i siegel rl torre la jemal a global cancerstatistics globocan estimates of incidence and mortality worldwidefor cancers in countries ca cancer j clin matsubayashi h takagaki s otsubo t iiri t kobayashi y yokota t advanced gastric glandularendocrine cell carcinoma with 1year survivalafter gastrectomy gastric cancer park jy ryu mh park ys park hj ryoo by kim mg prognosticsignificance of neuroendocrine components in gastric carcinomas eur jcancer la rosa s inzani f vanoli a klersy c dainese l rindi g histologiccharacterization and improved prognostic evaluation of gastricneuroendocrine neoplasms hum pathol ishida m sekine s fukagawa t ohashi m morita s taniguchi h neuroendocrine carcinoma of the stomach morphologic andimmunohistochemical characteristics and prognosis am j surg patholrayhan n sano t qian zr obari ak hirokawa m histological andimmunohistochemical study of composite neuroendocrineexocrinecarcinomas of the stomach j med investig jiang sx mikami t umezawa a saegusa m kameya t okayasu i gastriclarge cell neuroendocrine carcinomas a distinct clinicopathologic entityam j surg pathol ohike nan la rosa s who classification of tumours of endocrineans 4th ed lyon iarc press amin mb edge sb ajcc cancer s | Colon_Cancer |
thyroid carcinoma is presently the malignancy with the most rapidly increasing incidence in the worldand is the most widely recognized endocrine carcinoma in the western world thyroid cancers derivedfrom follicular thyroid cells can be sorted into papillary thyroid carcinoma ptc follicular thyroid carcinoma ftc and anaplastic thyroid carcinoma atc according to the histological subtype clinicalresults vary across these subtypesthe annual rate of thyroid cancer has more than doubled within the past two decades with the vast majority of this increase being ascribed to ptc which accounts for of all thyroid carcinomas inaddition patients with ptc suffer from cervical lymph nodes metastasis or remote metastasis which leadsto unfavorable results and approximately of cases may progress to a potentially fatal recurrentailment due to these reasons uncovering the causes of ptc and its fundamental mechanisms andfinding molecular biomarkers for early diagnosis and customized treatment are significant and important the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commons attributionlicense cc by 0cbioscience reports bsr20201555101042bsr20201555taskswith the advancement and continuous improvement of gene sequencing and geneediting technology it is nowconvenient to recognize the hub biomarkers related to neoplasm metastasis and survival status using a large amountof information available by applying bioinformatics currently there are no effective sensitive biomarkers for earlydiagnosis treatment and prevention of lymph node metastasis of ptc an examination of differentially expressedgenes degs between tumor and paracarcinoma tissue may help identify critical biomarkers of papillary thyroidcarcinoma as a form of molecular marker mrna containing the most abundant genetic information is necessaryfor protein translation and it is separate from the pathological process of cancer at various stages some studiesutilized public databases such as the cancer genome atlas tcga and the gene expression omnibus geo toidentify significant biomarkers of papillary thyroid carcinoma however these investigations were only founded onsingle datasets with constrained sample sizes or just based on online databases used to screen out the degsin the present study we analyzed the degs in ptc tissues versus the matched adjacent tissues by rnaseq andbioinformatics methods to obtain the degs then we screened out the key modules and extracted the key genes inthose modules by constructing degs interaction network then the possible role of differentially expressed geneswas analyzed using go annotation and kegg pathway enrichment analysis the expression validation survivalanalysis and functional enrichment analysis of key genes were conducted by using relevant databases finally wefound that the three genes adora1 apoe and lpar5 were highly expressed in ptc and were associated withptc methylation tnm staging and immune infiltrationmethodstissue samplesthirty pairs of ptc and adjacent tissues were collected from january to july at the first affiliated hospitalof hebei north university this experiment was approved by the ethical committee of the first affiliated hospitaland all patients provided informed consent all tissues were frozen in liquid nitrogen after surgical resectionrna library construction and sequencingtotal rna was isolated from four adjacent normal and cancerous thyroid samples utilizing trizol reagent qiagenvalencia ca usa as indicated by the manufacturers guidelines rnas of ptc tissues and paracancerous tissuessample numbers 1c 1p 2c 2p 3c 3p 4c 4p the number represents different samples the c indicates a cancersample and the p represents a matched paracancerous tissue sample were used six libraries were built utilizingan illumina standard kit as indicated by the manufacturers protocol all sequencing was carried out on an illuminahiseq lc bio chinadifferentially expressed genes screeningthe level of expression of mrnas was evaluated using stringtie by calculating fpkm the degs between ptcand paracancerous tissue were screened with log2 fold change1 and p005 was regarded as statistically significant the analyses were conducted using the r package ballgown functional enrichment analysis and pathway analysisto reveal the functional roles of the degs the annotation visualization and integrated discovery function annotation tool david httpdavidabccncifcrfgovhomejsp was used to perform gene ontology go enrichmentanalysis and kyoto encyclopedia of genes and genomes kegg pathway enrichment analysis p values less than were considered as cutoff criteriappi network construction and identiï¬cation of hub genesppi networks were constructed successively using string database tringdb the interactions ofdegs with a combined score were set as significant and cytoscape software version was utilized tovisualize and analyze the results of the string database to find key hub genes in this ppi network the significantmodule was analyzed by using the plugin mcode of cytoscape software the criteria for selection were set to thedefault the key genes were chosen with degrees ¥ subsequently genes in that module were used to analyse theirfunctional roles with funrich software the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commons attributionlicense cc by 0cbioscience reports bsr20201555101042bsr20201555table pcr primersgene symboladora1actinapoelpar5bp base pair f forward primer r reverse primerprimer sequencef5cid3ccacagacctacttccacacc3cid3r5cid3taccggagagggatcttgacc3cid3f5cid3cactcttccagccttccttcc3cid3r5cid3aggtctttgcggatgtccac3cid3f5cid3 gttgctggtcacattcctgg 3cid3r5cid3 gcaggtaatcccaaaagcgaccid3f5cid3 cacttggtggtctacagcttg3cid3r5cid3 gcgtagtaggagagacgaacg3cid3data validation and analysisto verify the accuracy of our rnaseq results we used the gene expression profiling interactive analysis databaseto verify the expression of key genes in ptc and adjacent tissues the overall survival and diseasefree survivalanalyses were performed by kaplanmeier plots for these ptcrelated hub genes genetic alterations of hub genesin ptc and their correlations with other genes were analyzed utilizing the cbioportal for cancer genomics hub genesrelated to clinicopathological features were analyzed using the online database ualcan httpualcanpathuabedu the correlation of adora1 apoe and lpar5 expression with the immune infiltration level in ptc and theexpression of these three genes in different kinds of cancers was performed using the tumor immune estimationresource database for qrtpcr analysis total rna was isolated from normal and cancerous papillary thyroid samples utilizingtrizol reagent qiagen valencia ca usa cdna was synthesized with rna reverse transcription kit tiangenbiotech beijing china qrtpcr was performed with an abi realtime pcr system applied biosystems life technologies usa the expression of the genes of interest was normalized to actin the primers foradora1 apoe lpar5 and actin are shown in table for western blot ripa buffer was used to extract protein from four pairs of tissue from ptc patients and theprotein concentrations were measured via bca methods briefly the sdspage gel was used for electrophoresis andpdvf membrane was used for transmembrane transfer pdvf membrane was blocked and then incubated with primary antiadora1 antibodies dilution bioss bs6649r apoe dilution bioss bs4892r lpar5antibodies dilution bioss bs15366r and actin dilution bioss bs0061r at ¦c overnight followed by incubation with secondary antibodies zhongshanjinqiao dilution at ¦c for h the signal wasdetected using ecl methodstatistical analysisall the data were analyzed by r and spss spss inc usa kaplanmeier method was used to estimate thesignificant difference in survival between the overexpression group and the lowexpression group of key genes inpapillary thyroid carcinoma the statistical difference was set at p resultsdifferentially expressed genes screening based on rnaseqto screen out the genes or modules that may play a role in promoting cancer in papillary thyroid carcinoma weperformed rnaseq experiments on four pairs of thyroid cancer tissues and their matched paracancerous tissues toobtain differentially expressed genes after rnaseq we acquire million reads for each sample the fold changesbetween ptc cancer tissues and matched paracancerous samples were calculated setting the cutoff criterion as pvalue and a fold change there were upregulated and downregulated genes these degswere considered to be candidate genes for subsequent study figure 1a showed the expression of the top genes inptc versus matched paracancerous tissuesfunctional enrichment analysis and pathway analysisconsidering that there were many falsepositive genes among these degs we verified our results one by onethrough the tcga database we found that only genes in our data were consistent with the gene expression of the the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commons attributionlicense cc by 0cbioscience reports bsr20201555101042bsr20201555figure identiï¬cation of degs by rnaseqthe heat map a and ppi network of the degs b c the volcano plots of the degs d the most significant module was selectedby mcode in cytoscape red represents the upregulated genes and blue represents the downregulated genesfigure go and kegg pathway enrichment analysis of degs through rnaseqa bubble plot of gene ontology enrichment analysis of degs b bubble plot of kyoto encyclopaedia of genes and genomespathway enrichment analysis of degstcga database to investigate the potential function of these degs in ptc genes functional enrichment was conducted by using go and kegg pathway analyses for the biological process category the degs were significantly involved in the regulation of axonogenesis regulation of cell morphogenesis extracellular structure anization extracellular matrix anization synapse anization cellsubstrate adhesion and urogenital system development thecellular component category results showed ptcrelated degs were enriched in collagencontaining extracellularmatrix synaptic membrane cellcell junction glutamatergic synapse neurontoneuron synapse postsynaptic membrane basolateral plasma membrane degs in molecular function were mainly involved in cell adhesion moleculebinding passive transmembrane transporter activity extracellular matrix structural constituent glycosaminoglycanbinding growth factor binding transmembrane receptor protein kinase activity and transmembrane receptor proteintyrosine kinase activity figure 2aas figure 2b showed the kegg pathway results showed degs were enriched in cytokinecytokine receptorinteraction mapk signaling pathway proteoglycans in cancer rap1 signaling pathway axon guidance cushing the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commons attributionlicense cc by 0cbioscience reports bsr20201555101042bsr20201555figure go enrichment analysis and kegg analysis for the key genesa top elements involved in biological processes b top elements involved in molecular function c top elementsinvolved in cellular components d top pathways related to the key genes through kegg analysissyndrome parathyroid hormone synthesis secretion and action agerage signaling pathway in diabetic complications growth hormone synthesis secretion and action salivary secretion circadian entrainment cholinergic synapse p53 signaling pathway ecmreceptor interaction arrhythmogenic right ventricular cardiomyopathyarvc endocrine resistance renin secretion type ii diabetes mellitus bladder cancer nicotinate and nicotinamidemetabolism and apoptosismultiple speciesppi network construction and module analysisppi networks were constructed successively by the database by loading the ptc related dges into the stringdatabase figure 1bc using cytoscape we analyzed the most significant module in the ppi network figure 1dthe ppi network consisted of nodes and edges following the use of mcode in cytoscape the significantmodule was selected the top hub genes adcy8 adora1 adra2c apoe c5ar1 ccl13 ccl20 cdh2chgb cxcl12 eva1a fam20a fn1 gnai1 gpc3 grm4 lpar5 meltf or mfi2 mfge8 nmu oprm1serpina1 sstr3 timp1 and tnc were evaluated by degree in the ppi network figure 1d the resultsshowed that the functions of the key genes were mainly concentrated in signal transduction cell communicationgprotein coupled receptor activity cell adhesion molecule activity and gpcr ligand binding figure data analysis and validationafter the key genes were selected the expression of key genes in ptc and its adjacent tissues were verified by thegepia database figure adora1 apoe eva1a lpar5 mfge8 oprm1 serpina1 sstr3 and timp1were positively related to the overall survival analysis of ptc patients while c5ar1 and gnai1 were negativelyrelated figure adcy8 adora1 chgb fn1 lpar5 nmu and tnc showed positive associations withdiseasefree survival analysis of ptc patients but not apoe figure next we analyzed the alterations of the key genes by using the cbioportal database figure the key geneswere changed in of queried samples figure 7b figure 7a showed the frequency of alterations of eachptc related key gene sstr3 fn1 and adora1 were altered the most and respectively figure 7dshowed the network of the genes and their altered neighbouring genes in ptc patients out of a total of among these genes only adora1 apoe and lpar5 genes simultaneously showed statistical significance foroverall survival analysis and diseasefree survival analysis of ptc patients the qpcr experiments and western blotdata verified that these three survivalrelated genes were all overexpressed in ptc figure then based on ual the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commons attributionlicense cc by 0cbioscience reports bsr20201555101042bsr20201555figure validation of the key degs in the gepia databaseadora1 apoe ccl13 cdh2 cxcl12 eva1a fam20a fn1 gnai1 lpar5 mfge8 nmu serpina1 timp1 and tnc areoverexpressed in ptc tissues compared with paracancerous tissue while gnai and gpc3 are downregulated the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commons attributionlicense cc by 0cbioscience reports bsr20201555101042bsr20201555figure overall survival analysis of key genes in ptc using kaplanmeier plotsexpression levels of adora1 apoe c5ar1 eva1a fam20a gnai1 lpar5 mfge8 oprm1 serpina1 sstr3 and timp1are related to the overall survival of patients with ptccan the clinical features and degree of methylation of these three genes were analyzed the transcription levels ofadora1 apoe and lpar5 were significantly higher in ptc patients than normal tissues according to subgroupsof sample types individual stages and nodal metastasis status figure in addition ador1 and lpar5 exhibiteda hypomethylation state in the cancer group but apoe showed a hypermethylation state in ptc samples figure10ato further clarify the role of these genes we conducted an analysis of immune infiltration the ador1 expression level was positively corelated with infiltrating levels of b cells r0111 p151e2 cd8 t cells r0246p396e neutrophils r0162 p331e and dcs r0232 p232e the expression of apoe was the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commons attributionlicense cc by 0cbioscience reports bsr20201555101042bsr20201555figure diseasefree survival analysis of key genes in ptc using kaplanmeier plotsexpression levels of adcy8 adora1 apoe chgb fn1 lpar5 nmu and tnc are significantly related to the diseasefreesurvival of patients with ptcpositively associated with b cells r0228 p439e cd8 t cells partialcor p930e neutrophils r0197 p114e and dcs partialcor p358e lpar5 expression level was positively related to b cells r0259 p815e cd4 t cells r0238 p103e macrophages r0175p105e neutrophils r027 p142e and dcs r0256 p104e and negatively related to purity r p294e and cd8 t cells r p618e figure 10b these findings stronglysuggested that lpar5 ador1 and apoe may play specific roles in immune infiltration in ptc especially those ofdcs finally we examined the expression of these three genes in common cancer tissues and adjacent tissues and wefound that these three genes were highly expressed in most cancer tissues figure the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commons attributionlicense cc by 0cbioscience reports bsr20201555101042bsr20201555figure the key genes expression and mutation analysis in ptc by the cbioportal for cancer genomicsa the genetic alterations of key genes of ptc samples queried genes are altered in of queried patientssamplesb the expression heatmap of key genes c the alteration frequency of key genes in ptc d network of key genesmutations and their frequently altered neighboring genes in ptc the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commons attributionlicense cc by 0cbioscience reports bsr20201555101042bsr20201555figure the mrna and protein expressions of adora1 apoe and lpar5 in ptc tissuesac validation of expression levels of adora1 apoe and lpar5 by rtqpcr in cases of ptc and matched adjacent tissuesd adora1 apoe and lpar5 protein levels are increased in four cases of ptc and matched adjacent tissues as measured bywestern blot p0001discussionptc is a common cancer with great heterogeneity in morphological features and prognosis although most papillary thyroid carcinomas exhibit low biological activity there are still a few patients with higher invasive and metastaticclinical features activation of oncogene expression and loss of function of tumor suppressor genes may lead tothe development or progression of ptc to better clarify the molecular mechanism of ptc occurrence development and metastasis we identified key genes related to ptc progression through comprehensive bioinformaticsmethods and we screened three of the ptc prognosisrelated genes for a comprehensive analysisin the present study we identified differentially expressed genes by rnaseq with go enrichment analysis showing that the degs were enriched in the regulation of the axonogenesis regulation of cell morphogenesis extracellular structure anization extracellular matrix anization synapse anization cellsubstrate adhesion urogenital system development collagencontaining extracellular matrix synaptic membrane cellcell junction glutamatergic synapse neuron to neuron synapse postsynaptic membrane basolateral plasma membranecell adhesion molecule binding passive transmembrane transporter activity extracellular matrix structural constituent glycosaminoglycan binding growth factor binding transmembrane receptor protein kinase activity andtransmembrane receptor protein tyrosine kinase activity and kegg pathway results showed degs were enrichedin cytokinecytokine receptor interaction mapk signaling pathway proteoglycans in cancer rap1 signaling pathway axon guidance cushing syndrome parathyroid hormone synthesis secretion and action agerage signalingpathway in diabetic complications growth hormone synthesis secretion and action salivary secretion circadian entrainment cholinergic synapse p53 signaling pathway ecmreceptor interaction arrhythmogenic right ventricularcardiomyopathy arvc endocrine resistance renin secretion type ii diabetes mellitus bladder cancer nicotinateand nicotinamide metabolism and apoptosismultiple speciesto further explore the interrelationship of differentially expressed genes in papillary thyroid carcinoma we constructed a ppi regulatory network a total of degs with nodes greater than were screened out in the networkthe key genes were adcy8 adora1 adra2c apoe c5ar1 ccl13 ccl20 cdh2 chgb cxcl12 eva1afam20a fn1 gnai1 gpc3 grm4 lpar5 meltf mfge8 nmu oprm1 serpina1 sstr3 timp1 andtnc biological process and molecular function analyses of these key degs indicated that they were significantly the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commons attributionlicense cc by 0cbioscience reports bsr20201555101042bsr20201555figure relative expression of adora1 apoe and lpar5 in normal thyroid tissues and ptc tissues individual cancerstages and nodal metastasis status respectively ualcanp0001involved in cancer regulation processes such as adjustment of cell growth or maintenance cell immune response celladhesion molecular activity and extracellular matrix structural constituentto verify the credibility of the experiments and data the degs screened were verified by the gepia databaseamong the selected genes genes showed expression differences consistent with our rnaseq data among the the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commons attributionlicense cc by 0cbioscience reports bsr20201555101042bsr20201555figure methylation level and immune inï¬ltration level of adora1 apoe and lpar5a relative methylation level of adora1 apoe and lpar5 based on normal thyroid tissues and ptc tissues individual cancerstages and nodal metastasis status respectively ualcan b the correlation between the three genes and tiics timer tiicstumor infiltrating immune cells genes adora1 apoe ccl13 cdh2 eva1a fam20a fn1 lpar5 mfge8 nmu serpina1 timp1and tnc levels were overexpressed in ptc tissues while gpc3 and gnai1 were downregulatedadora1 belongs to the gprotein coupled receptor family and protects human tissues and cells under physiological conditions lin et al suggested that adora1 may promote the proliferation of breast cancer cellsby positively regulating oestrogen receptoralpha in breast cancer cells similarly jayakar indicated that knockdown of apoe expression can reduce the level of mmps by regulating the ap1 signaling pathway and thus reducethe invasion and metastasis of oral squamous cell carcinoma bioinformatics predictions were that apoe mrnashows a significant increase in ptc ccl13 is a coding gene involved in immune regulation and inflammatoryresponses and it has been reported that ccl13 has a role in promoting the proliferation of tumorforming volumein nude mice cdh2 is overexpressed in various cancers some research results indicate that overexpression ofcdh2 can increase the invasive ability and induce emt in lung cancer cells qiu et al confirmed cdh2 actsas an oncogene in papillary thyroid carcinoma which is consistent with our findings eva1a acts as a regulatorof programmed cell death and shen et al indicates that eva1a can inhibit the proliferation of gbm cells by inducing autophagy and apoptosis via inactivating the mtorrps6kb1 signaling pathway fam20a may play a keyrole in haematopoiesis there are few reports on the relationship between fam20a and cancer and our experimentfound that fam20a is more highly expressed in papillary thyroid carcinoma than in other cancers fn1 is involvedin regulating cell adhesion cell movement wound healing and maintaining cell morphology some researchersindicated that fn1 participates in regulating many types of cancer progression such as gastric cancer skin squamous cell carcinoma and papillary thyroid carcinoma it has been shown that lpar5 is related tothe pathogenesis of pancreatic cancer consistent with our study zhang et al believes that lpar5 may be involved in the development of papillary thyroid carcinoma according to previous reports mfge8 is involvedin the progression of various malignancies such as breast cancer melanoma bladder tumors and ovarian cancer the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commons attributionlicense cc by 0cbioscience reports bsr20201555101042bsr20201555figure the expression of adora1 apoe and lpar5 in thyroid cancer tissues and normal thyroid tissuesthe three genes expression were analyzed in different kind of cancer tissues and normal tissues via the timer database p005p001 p001 the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commons attributionlicense cc by 0cbioscience reports bsr20201555101042bsr20201555[] mfge8 is considered to be a potential therapeutic target for ovarian cancer owing to its carcinogenic effect consistent with our data zhang et al indicate that nmu is one of the degs of papillary thyroid carcinoma recently a researcher has shown that abnormal expression of nmu is associated with a variety of cancers for serpina1 there are currently six s pointing out that serpina1 may be a key gene for ptc consistentwith our results [] clinical studies have shown that high expression of timp1 is positively correlated witha poor prognosis of colon brain prostate breast lung and several other cancers tnc is a component of theextracellular matrix ecm and is closely related to the malignant biological behavior of cancer in particular tncoverexpression is positively associated with liver cancer oral squamous cell carcinoma and lymph node metastasisof breast cancer gpc3 belongs to the glypicans family it has been reported that overexpression of gpc3can promote the metastasis of hepatocellular carcinoma but we found that it is expressed at low levels in ptcsimilar to gpc3 some scholars believe that gnai1 is a tumorpromoting gene and reported upregulated gnai1mrna in human glioma which is inconsistent with our data only the adora1 apoe and lpar5 genes simultaneously showed statistical significance for overall survivaland diseasefree survival of ptc patients considering that the occurrence and metastasis of cancer is a complexand multiregulated process we further analyzed the regulatory mechanisms of these three genes we found thatthe mrna and methylation levels of these three genes were significantly correlated with tnm staging in additionadora1 apoe and lpar5 were all related to immune infiltration especially to dendritic cells finally we foundthat these three genes were more highly expressed in cancer tissues than matched adjacent tissueshowever our research has certain limitations first only four pairs of cancer and adjacent tissues were analyzedusing rnaseq in this experiment so further research requires a larger sample size second further experiments areneeded to validate the specific mechanisms of these key genesdata availabilitythe data used to support the findings of this study are available from the corresponding author upon requestcompeting intereststhe authors declare that there are no competing interests associated with the manuscriptfundingthis study was supported by grants from the hebei provincial department of finance specialist capacity building and specialistleadership program [grant number ] the hebei provincial natural science foundation project [grant number h201840505]and the hebei north university basic research business expenses project [grant number jyt2019015]author contributionxu lin conducted the bioinformatics analysis xu lin and gang xue contributed as first authors xu lin wrote the manuscriptjingfang wu and gang xue critically revised the gang xue and da pei obtained clinical specimens and the others contributed to verification of the rnaseq resultsabbreviationsatc anaplastic thyroid carcinoma ecm extracellular matrix ftc follicular thyroid carcinoma ptc papillary thyroid carcinomareferences kitahara cm and sosa ja the changing incidence of thyroid cancer nat rev endocrinol 101038nrendo2016110 aschebrookkilfoy b ward mh sabra mm and devesa ss thyroid cancer incidence patterns in the united states by histologic type thyroid 101089thy20100021 pourseiraï¬ s shishehgar m ashraf mj and faramarzi m papillary carcinoma of thyroid with nasal cavity metastases a case report iranj med sci ullmann tm gray kd moore md zarnegar r and fahey iii tj current controversies and future directions in the diagnosis andmanagement of differentiated thyroid cancers gland surg 1021037gs20170908 jin x deng b ye k et al comprehensive expression proï¬les and bioinformatics analysis reveal special circular rna expression and potentialpredictability in the peripheral blood of humans with idiopathic membranous nephropathy mol med rep 103892mmr201910671 rapisuwon s vietsch ee and wellstein a circulating 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bcl9 and pygo are bcatenin cofactors that enhance the transcription of wnt targetgenes they have been proposed as therapeutic targets to diminish wnt signaling output inintestinal malignancies here we find that in colorectal cancer cells and in developing mouseforelimbs bcl9 proteins sustain the action of bcatenin in a largely pygoindependent mannerour genetic analyses implied that bcl9 necessitates other interaction partners in mediating itstranscriptional output we identified the transcription factor tbx3 as a candidate tissuespecificmember of the bcatenin transcriptional complex in developing forelimbs both tbx3 and bcl9occupy a large number of wntresponsive regulatory elements genomewide moreover mutationsin bcl9 affect the expression of tbx3 targets in vivo and modulation of tbx3 abundance impactson wnt target genes transcription in a bcatenin and tcflefdependent manner finally tbx3overexpression exacerbates the metastatic potential of wntdependent human colorectal cancercells our work implicates tbx3 as contextdependent component of the wntbcatenindependenttranscriptional complexintroductionthe wnt pathway is an evolutionarily conserved cell signaling cascade that acts as major drivingforce of several developmental processes as well as for the maintenance of the stem cell populations within adult tissues nusse and clevers deregulation of this signaling pathway resultsin a spectrum of consequences ranging from lethal developmental abnormalities to several forms ofaggressive cancer nusse and clevers most prominently colorectal cancer crc is initiatedby genetic mutations that constitutively activate wnt signaling kahn secreted wnt ligands trigger an intracellular biochemical cascade in the receiving cells that culminates in the calibrated expression of target genes mosimann this transcriptionalresponse is orchestrated by nuclear bcatenin that acts as a scaffold to buttress a host of cofactorsto cisregulatory elements occupied by the tcflef transcription factors valenta among the cofactors the two paralogs bcl9 and bcl9l referred to as bcl99l and pygo12proteins reside within the wntbcatenin transcriptional complex and their concerted action isrequired to efficiently activate wnttarget gene expression kramps parker for correspondencekonradbaslerimlsuzhch kbandreasmoorbsseethzchaemclaudiocantuliuse cc these authors contributedequally to this workpresent address ¡division ofmolecular pathology thenetherlands cancer instituteamsterdam netherlandscompeting interests theauthors declare that nocompeting interests existfunding see page received april accepted august published august reviewing editor roel nussestanford university unitedstatescopyright zimmerli this is distributed under theterms of the creative commonsattribution license whichpermits unrestricted use andredistribution provided that theoriginal author and source arecreditedzimmerli elife 20209e58123 107554elife58123 of 0cshort reportdevelopmental biologyfigure the intestinal epitheliumspecific recombination of pygo12 does not recapitulate the effects of deleting bcl99l a schematic representationof the wntbcatenin transcriptional complex with emphasis on the socalled chain of adaptors components bcatenin bcl99l and pygo wntfigure continued on next pagezimmerli elife 20209e58123 107554elife58123 of 0cshort reportfigure continueddevelopmental biologyresponsive element wre the homology domains hd13 of bcl99l are shown b epithelialspecific pygo12 deletion via vilcreert2 pygo12ko does not lead to any obvious histological or functional defect neither in the small intestine nor in the colon as seen by hematoxylin and eosinstaining left panels the proliferative compartment detected via ki67 right panels seems also unaffected also refer to the count in figure figuresupplement 1de c quantitative rtpcr detecting lgr5 mrna extracted from colonic epithelium of control black bcl99l blue or pygo12 redconditional mutants ko d weekold male mice were treated with five tamoxifen tam injections ip mgday for five consecutive days days later mice were treated with dextran sodium sulfate dss ad libitum in drinking water for days while of control mice n wereseverely affected or died due to the dss treatment red lines of conditional bcl99lko n mice performed poorly in this test deletion ofbcl99l increased significantly the death rate after dss treatment pvalue000013 in fishers exact test no difference between pygo12ko andcontrol mice could be measured of control mice n and of pygo12ko n were affected upon dss treatment pvalue05626 infishers exact test e weekold female mice were exposed to a single dose of the carcinogenic agent azoxymethane aom followed by daysof dss administration in the drinking water this regimen results in the emergence of dysplastic adenomas that are collected for rna extraction andanalysis of the indicated targets via rtpcr wnt target genes and genes expressed during epithelialtomesenchymal transition emt associated withcancer metastasisthe online version of this includes the following figure supplements for figure figure supplement efficient epithelialspecific pygo12 deletion does not lead to obvious defectsfigure supplement intestinal epitheliumspecific recombination of pygo12 does not recapitulate the effects of deletingbcl99l van tienen figure 1a during vertebrate development their requirement in thebcateninmediated transcription appears to be contextdependent cantu li and they also have evolved bcateninindependentfunctions cantu cantu curiously however bcl9 and pygo always seem to act as a duetkennedy importantly bcl99l and pygo proteins were found to significantly contribute to the malignanttraits typical of wntinduced crcs deka gay jiang mani mieszczanek moor talla and brembeck theseobservations provided impetus to consider the bcl9pygo axis as relevant targetable unit in crclyou mieszczanek talla and brembeck zimmerli however here we noticed an apparent divergence between the roles of bcl99l and pygo proteins we found that genetic abrogation of bcl99l in mouse crc cells results in broader consequences than pygo12 deletion suggesting that bcl9 function does not entirely depend on pygo12among the putative bcateninbcl9 interactors we identified the developmental transcription factortbx3 intriguingly we show that also during forelimb development bcl99l possess a pygoindependent role in this in vivo context tbx3 occupies bcateninbcl9 target loci genomewide andmutations in bcl99l affect the expression of tbx3 targets finally tbx3 modulates the expression ofwnt target genes in a bcatenin and tcflefdependent manner and increases the metastaticpotential of human crc cells when overexpressed we conclude that tbx3 can assist the wntbcatenin mediated transcription in selected developmental contexts and that this partnership could beaberrantly reactivated in some forms of wntdriven crcsresults and discussionwe induced intestinal epitheliumspecific recombination of pygo12 loxp alleles pygo12ko thatefficiently deleted these genes in the whole epithelium including the stem cells compartment figure figure supplement 1a and b consistently with recent reports mieszczanek talla and brembeck and similarly to deletion of bcl99l deka mani moor pygo12ko displayed no overt phenotypic defects figure 1b figure figure supplement 1ce we were surprised in noticing that the expression of lgr5 themost important intestinal stem cell marker and wnt target gene barker was heavilydownregulated upon loss of bcl99l but unaffected in pygo12ko figure 1c to address the functionality of the stem cell compartment in these two conditions we subjected both bcl99l andpygo12 compound mutants koregeneration by dss treatmentkim figure 1d while bcl99lko mice showed a defect in regeneration after insultdeka pygo12ko proved indifferent when compared to controllittermatesfigure 1d while we cannot exclude that pygo12 also contributes to the wntbcateninto a model ofintestinalzimmerli elife 20209e58123 107554elife58123 of 0cshort reportdevelopmental biologydependent transcriptional regulation our results highlight that the bcl99l function in the intestinalepithelium homeostasis and regeneration does not entirely depend on pygo12 this was surprising since bcl99l proteins were thought to act as mere bridge proteins that tethered pygo tothe bcatenin transcriptional complex figure 1a fiedler mosimann both bcl9 and pygo proteins have been implicated in colorectal carcinogenesis gay jiang mieszczanek talla and brembeck we tested if the consequence of the deletion of bcl99l and pygo12 genes was also different in the context of carcinogenesis specifically we looked at the contribution to gene expression in chemicallyinduced aomdsscolorectal tumors figure 1e as previously observed bcl99lko tumors exhibit a massive decreasein wnt target gene expression epithelialtomesenchymal transition emt and stemness traitsdeka moor which was not observed in pygo12ko tumors figure 1efigure figure supplement 2a and b this phenotypic difference is consistent with a recentstudy in which bcl99l but not pygo12 loss reduced the activation of wnt target genes induced byapc lossoffunction mieszczanek we interpret this as an independent validation ofour observation all these experiments open up the question of how bcl99l imposes its functionindependently of pygosurprisingly the intestinespecific deletion of the homology domain hd1 of bcl99lfigure 2a that was previously annotated to interact only with pygo12 cantu kramps i suppressed the metastatic phenotype of the aomdss tumors while deletion of pygo12 did not figure 2b and ii induced a strong downregulation of wnt target emt andstemness genes figure 2c figure figure supplement the discrepancy between the geneexpression changes induced by recombining pygo12 or deleting the hd1 domain of bcl99l impliesthat currently unknown proteins assist bcl99l function we set out to identify new candidate bcl9partners that might be responsible for the different phenotypes to this aim we performed a pulldown of tumor proteins expressing either a fulllength or a hd1deleted variant of bcl9 followedby mass spectrometry figure 2d among the proteins differentially pulled down by control but notby mutant bcl9 we detected tbx3 figure 2d and e and selected it for further validation the invivo deletion of the hd1 domain in bcl99ldhd1 embryos leads to severe forelimb malformationswhile pygo12ko embryonic forelimbs are unaffected figure 2falso see schwab limb development thus represents another context where bcl99l appear to act independently ofpygo of note tbx3 plays a fundamental role in the development of this structure frank we confirmed cytological vicinity between transfected tagged versions of bcl9 and tbx3 byproximity ligation assay pla figure figure supplement 2ab however overexpressionbasedin vitro coimmunoprecipitation experiments could not detect any stable interaction between thesetwo proteins suggesting absence of direct binding or a significantly lower affinity than that betweenbcl9 and pygo figure figure supplement 2c hence we aimed at testing the functional association between tbx3 and bcl9 in a more relevant in vivo context to this aim we collected ca forelimbs from dpc wildtype mouse embryos and subjected the crosslinked chromatin toimmunoprecipitation using antibodies against bcl9 salazar or tbx3 followed bydeepsequencing of the purified dna chipseq figure 3a by using stringent statistical parameters and filtering with irreproducible discovery rate idr we extracted a list of high confidencebcl9 and tbx3 peaks figure 3b and c surprisingly we discovered that bcl9 occupies a largefraction ca 23rd of the tbx3bound regions figure 3d suggestive of a role for tbx3 within thewntdependent transcriptional apparatus motif analysis of the common tbx3bcl9 target loci identified statistical prevalence for tcflef and homeobox transcription factor consensus sequencesbut not for any tbx transcription factor figure 3e this suggests that tbx3 interacts with the dnain these locations via affinity to the wntbcatenin cofactors rather than via direct contact with dnaaccordingly tbxspecific motifs were detected within the group of tbx3 exclusive peaks which donot display bcl9 binding figure figure supplement notably tbx3 and bcl9 occupancywas detected at virtually all previously described wntresponsiveelements wre within known wnttarget genes figure 3fto test whether the in vivo abrogation of the simultaneous interactions mediated by bcl99lwould influence the expression of genes associated with tbx3 peaks we set out to mutate the bcl99l interaction domains while leaving tbx3 protein unaffected we combined different bcl99l allelesin which the hd2 bcatenininteracting and hd1 pygonew cofactorinteracting motifs arezimmerli elife 20209e58123 107554elife58123 of 0cshort reportdevelopmental biologyfigure identification of tbx3 as a putative bcl9 cofactor a the deletion of the hd1 pygointeracting domain of bcl9 and bcl9l induces avariation in the chain of adaptors causing the loss of pygo association with the wntbcatenin transcriptional complex cantu bimmunofluorescence staining of tumors collected from control or conditional pygo12ko and bcl99lko mice prox1 red and dapi blue are shownin in the top panels vimentin green and laminin red in the bottom panels c quantitative rtpcr of selected groups of targets compare it withfigure continued on next pagezimmerli elife 20209e58123 107554elife58123 of 0cshort reportfigure continueddevelopmental biologythe same analysis of pygo12ko in figure 1e of rna extracted from control or bcl99ldhd1 tumors d experimental outline of the tumor proteinspulldown and massspectrometry tbx3 was identified among the proteins potentially interacting with bcl9 but not with bcl9dhd1 e the ipproteins analyzed by mass spectrometry were in parallel subjected to sds page electrophoresis and probed with an antitbx3 antibody upper panelthe expression of tbx3 in control compared to bcl99ldhd1 tumors n was evaluated via qrtpcr bottom panel to exclude that differentialpulldown was due to lost expression in mutant tumors f dpc bcl99ldhd1 embryos display forelimb malformations and absence of digitsemphasized by dashed white lines a characteristic tbx3mutant phenotype upper panels the limb defect is absent in pygo12ko embryosbottom panels underscoring that bcl99l act in this context independently of pygo12the online version of this includes the following figure supplements for figure figure supplement qrtpcr of target genes associated with intestinal stem cell functionfigure supplement cytological proximity of bcl9 and tbx3deleted cantu double heterozygous animals for the hd1 bcl9dhd1 bcl9ldhd1 orthe hd2 bcl9dhd2 bcl9ldhd2 deletions are viable and fertile the cross between them leads to atransheterozygous genetic configuration in which both domain deletions are present bcl9dhd1dhd2bcl9ldhd1dhd2 referred to as bcl99ld1d2 figure 1a as in these mice bcl99l retain both thehd1 and the hd2 domains in heterozygosity this allelic combination is a way of testing the consequences of abrogating the tripartite complex mediated by the two interacting motifs of bcl99lwithout causing a full lossoffunction of these proteins bcl99ld1d2 embryos also display forelimbmalformations the cause of which cannot be due to pygo figure 2f but must be caused by thefailure of recruiting the new hd1interacting partner by bcl99l onto the bcatenin transcriptionalcomplex we collected forelimbs from control and bcl99ld1d2 mutant embryos at and measured gene expression via rnaseq figure 3g we found a significant enrichment hypergeometrictest p14e6 of tbx3 targets among the genes differentially expressed in bcl99ld1d2 mutantsfigure 3h the enrichment was particularly significant when considering downregulated genes inbcl99ld1d2 mutants indicating that the bcl9tbx3 partnership sustains transcriptional activationfigure 3h of note the design of our experiment directly implicates that these tbx3 transcriptional targets are also bcatenindependent the overlap list includes several regulators of limbdevelopment such as meis2 capdevila irx3 li and eya2 grifone figure 3h heat map on the right despite being of correlative nature this analysis supportsa model in which bcl99l and tbx3 cooperate to the activation of target genesso far we have presented genetic evidence that bcl9 proteins require additional cofactors andthat tbx3 associates with the bcateninbcl9 bound regions on the genome possibly influencing theexpression of target genes however the similarity of genomic binding profiles between tbx3 andbcl9 might be due to their binding in different cells and the decreased expression of genes withnearby enhancers bound by bcl9 and tbx3 might imply a requirement for bcl99l but not necessarily for tbx3 we reasoned that our hypothesis in which bcl9 functionally tethers tbx3 to the bcatenin transcriptional complex raises several testable predictions that will be addressed belowfirst our model implies that tbx3 could impact on wnt target gene expression and its activityshould be dependent on the main constituents of the wntbcatenin transcriptional complex second if tbx3 is tethered by bcl9 to its targets mutations in bcl99l should influence the ability oftbx3 to physically associate with wres finally as for bcl9 tbx3 should be capable of enhancingthe metastatic potential of colorectal cancer cellsto test our first prediction implying a potential role of tbx3 in the transcription of wnt targetgenes we overexpressed it in hek293t cells and monitored the activation status of wnt signalingusing the transcriptional reporter supertopflash stf consistent with its role as repressor tbx3led to a moderate but significant transcriptional downregulation that was importantly specific tothe stf but not the control reporter plasmid figure 4a upon wnt signaling activation achievedvia gsk3 inhibition tbx3overexpressing cells exhibited a markedly increased reporter activity whencompared to control cells in particular at nonsaturating pathway stimulating conditions figure 4aleft panel importantly tbx3 proved transcriptionally incompetent on the stf if the cells carriedmutations in tcflef d4tcf or ctnnb1 encoding for bcatenin dbcat doumpas strongly supporting the notion of its cellautonomous involvement in the activation of canonical wnttarget gene transcription figure 4a central and right panels respectively endogenous wnt targets showed a similar expression behaviour to that of stf upon tbx3 overexpression figure zimmerli elife 20209e58123 107554elife58123 of 0cshort reportdevelopmental biologyfigure tbx3 and bcl9 occupy wnt responsive elements wre in vivo a artistic representation of the chipseq experimental outline bc barplots showing the genomic distribution of highconfidence bcl9 peaks b total and tbx3 peaks c total d overlap of the highconfidence peak groups between bcl9 and tbx3 e selected result entries from motif analysis performed on the bcl9tbx3 overlapping highconfidence peaks significant enrichment was found for tcflef and homeobox motifs no tbx consensus sequence was detected in this analysis ffigure continued on next pagezimmerli elife 20209e58123 107554elife58123 of 0cshort reportfigure continueddevelopmental biologyselect genomic tracks demonstrating occupancy of bcl9 and tbx3 within the wnt responsive element wre of known wnttarget genes axin2ccnd1 nkd1 and lef1 and genes important in limb morphogenesis hand1 and hand2 the scale of peak enrichment is indicated in the topleftcorner of each group of tracks in light blue the bcl9 salazar and in orange the tbx3 replicates and in green the control track igggenomic tracks are adapted for this figure upon visualization with igv integrative genomic viewer igv two independent replicates forbcl9 and tbx3 chipseq experiments are shown g volcano plot displays all the differentially expressed genes degs in developing forelimbs uponmutation of bcl99l bcl99ld1d2 vs ctrl degs were a total of p005 with upregulated and downregulated n of individualmouse embryos for each condition were used for this analysis h a significant portion of degs exhibited overlap with tbx3 chipseq peaksthe overlap with tbx3 chipseq peaks appeared statistically significant in particular when the downregulated genes were considered hierarchicalclustering of samples ctrl versus bcl99ld1d2 right panel based on genes overlapping between degs and genes annotated for tbx3 chipseqpeaks normalized rnaseq read counts wards clustering method euclidian distance annotation added for genes associated by gene ontology townt signaling fgf10 ptk7 kremen1 zfp703 bmp2 and gli3 and genes known as regulators of limb development meis2 irx3 and eya2the online version of this includes the following figure supplements for figure figure supplement overlap of the highconfidence bcl9 and tbx3 peaks in developing murine limbs reveals the existence of bcl9 exclusive oneexample displayed in the genomic tracks on the left and tbx3 exclusive peaks one example in the genomic track on the rightfigure supplement while our experiments show that tbx3 can influence the expression of wnttarget genes the mechanisms by which this occurs remain to be elucidatedwe then addressed our second hypothesis in which bcl99l are required for tethering tbx3onto wres we performed chip of tbx3 in hek293t cells followed by quantitative pcr to detectenrichment on the wre present in the axin2 promoter jho consistent with an effecton transcription in the absence as well as in the presence of chir99021 chir figure 4a tbx3 wasbound to this region both in off and in on conditions figure 4b we then exploited ahek293t clone devoid of both bcl9 and bcl9l db99l van tienen and tested iftbx3 was capable of physical association with the wre of note enrichment of tbx3 in db99 l cellswas dramatically reduced to background levels figure 4b while we cannot exclude that tbx3might act independently of bcl99l on several of its targets this observation supports the notionthat bcl99l are responsible for tbx3 recruitment on classical wres figure 4c this also suggeststhat the previously identified targets of both bcl9 and tbx3 figure 3df must display simultaneous cooccupancy of these two factors in agreement with the notion that bcl99l are themselvesrecruited by the tcfbcatenin axis the physical association of tbx3 with the axin2 promoter wasalso lost in d4tcf and dbcat cells figure 4bfinally we evaluated the effects of tbx3 overexpression oe on growth and metastatic potentialof hct116 human colorectal tumor cells a representative model of crc driven by activating mutations in ctnnb1 mouradov using a in vivo zebrafish xenograft model rouhi approximately labelled control or tbx3oe hct116 cells were implanted in theperivitelline space of hours postfertilization hpf zebrafish embryos figure 4d three days afterinjection tbx3oe cells displayed a marked increase in number in the caudal hematopoietic plexusfigure 4ef the main metastatic site for cells migrating from the perivitelline space rouhi of note tbx3oe hct116 cells maintained consistently high expression of tbx3 within fishembryos throughout the experiment and this was accompanied by increased wntbcatenindependent transcription as measured by axin2 expression figure 4g while this experiment does notallow to exclude that tbx3 might also act independently of bcl9bcatenin in this context it showsthat increased expression of tbx3 enhances proliferation and migratory capability of human crccells bearing constitutively active wnt signaling and this is associated with simultaneous enhancement of the wntbcatenindependent transcription figure 4gtaken together our experiments show that in specific developmental and disease contexts thetranscription factor tbx3 can take active part in the direct regulation of wnt target genes by functional interplay with the bcateninbcl9dependent transcriptional complex our study suggests anew paradigm in which tissuespecific cofactors might be the key to understand the spectrum ofpossible transcriptional outputs observed downstream of wntbcatenin signaling nakamura moreover tbx3 has been linked to different cancer types willmer our observations suggest that tbx3 or its downstream effectors could be considered as new relevant targetsto dampen crc progressionzimmerli elife 20209e58123 107554elife58123 of 0cshort reportdevelopmental biologyfigure tbx3 controls the expression of wnt target genes a bcatenintcf luciferase reporter stf assay in parental left bcatenin knockout dbcat center and tcf knockout d4tcf right hek293t cells cells were treated with the indicated concentration of chir or dmso overnightoverexpression of tbx3 oe black bars compared to control ev empty vector white bars showed that tbx3 acts as a repressor on a wnttcfpathway reporter but switches to activator upon pathway induction only significant pvalues p005 are indicated three independent experimentsfigure continued on next pagezimmerli elife 20209e58123 107554elife58123 of 0cshort reportfigure continueddevelopmental biologyn are shown note that the logarithmic scale on the yaxis of the histogram on the left is different from the linear scale of the central and middlepanels b chip followed by qpcr in hek293t cells treated with dmso wntoff or chir wnton enrichment was identified on axin2promoter and the downstream enhancer note that the enrichment on the enhancer is only present upon pathway stimulation we interpret this asevidence for the enhancer looping onto the promoter occurring when the wntdependent transcriptional regulation is active the data are normalizedto immunoprecipitation performed in cells transfected with an empty vector ev and presented as the mean ± standard deviation of independentexperiments the fold enrichment of tbx3flag on axin2 promoter and enhancer n is lost upon mutations in bcl99l db99l n cnttb1dbcat n and tcflef d4tcf n c schematic representation of the axin2 locus indicates the position of the primers used black arrowsto test the binding of tbx3 despite the apparent absence of direct physical interaction between tbx3 and bcl99l the data support a model of tbx3recruitment by bcl99l onto the bcatenintcf transcriptional complex d schematic diagram of the human crc zebrafish xenografts model parentaland tbx3 overexpressing hct116 colorectal tumor cells were harvested and labeled with dii dye red the stained cells were injected into theperivitelline space of day old zebrafish embryos zebrafish were visualized with fluorescent microscopy at day post injection dpi and three dpi andprimary tumor cell invasion and metastasis were counted e representative images of hct116 tumor invasion and dissemination at and dpi inzebrafish xenografts for both control and tbx3 overexpressing cells the red asterisks indicate the position of the primary tumor red arrowheadspoint at clusters of disseminatingmetastatic cells f scatter plot representing the quantification of primary tumor growth and metastasis after hct116xenograft horizontal bars represent the mean value only significant pvalues p005 are displayed g quantitative rtpcr confirmed continuedincreased expression of tbx3 while hct116 disseminate through zebrafish tissue and that this is accompanied by enhanced wntbcatenintranscription as seen by axin2 expression each datapoint represents the extraction of total rna from pools of zebrafish embryos figure legendof figure supplementsthe online version of this includes the following figure supplements for figure figure supplement axin2 and nkd1 are here considered as representative wnt transcriptional targetsmaterials and methodstreatment of mice and histological analyseshomeostasis weekold male and female mice bcl9floxfloxbcl9lfloxflox vilcreert2 and bcl9floxfloxbcl9lfloxflox no cre littermates pygo1floxfloxpygo2floxflox vilcreert2 and bcl9floxfloxbcl9lfloxflox no cre were treated with five tamoxifen injections ip mgday sigma for five consecutivedays and the small intestine and colon were analyzed at different time points thereafter mouseexperiments were performed in accordance with swiss guidelines and approved by the veterinarianoffice of vaud switzerlandinduction of dss colitis weekold male mice were treated with five tamoxifen injections ip mgday for five consecutive days days later dss mg mp biomedicals catno was administered ad libitum in the drinking water for daysinduction of tumors weekold female mice were treated with five tamoxifen injections ip mgday for five consecutive days days later they were injected ip with mgkg body weightdmh 2hcl nn dimethylhydrazine dihydrochloride after another days later dss was administered ad libitum in the drinking water for daysmice were monitored clinically for rectal bleeding prolapse and general signs of morbidityincluding hunched posture apathetic behavior and failure to groomthe relative body weight in was calculated as follows x weight at a certain dayweight atthe first day of dss treatment epithelial damage of dss treated mice was defined as percentage ofdistal colon devoid of epitheliumto determine proliferation rates mice were injected ip with mgkg brdu sigma hr priorto sacrifice small intestines and colons divided into three equal segments to be named proximalmiddle and distal colon were dissected flushed with cold pbs cut open longitudinally and fixed in paraformaldehyde for hr at rt and paraffin embedded sections mm were cut and used forhematoxylineosin and alcian blue staining and for immunohistochemistry the primary antibodiesused were rabbit antisynptophysin dako rabbit antilysozyme dako mouse antiki67 novocastra mouse antibrdu sigma antibcatenin bd pharmigenantiactive caspase cell signalingthe peroxidaseconjugated secondary antibodies used were mouse or rabbit envision dakoor mouse antirat hrp biosourcezimmerli elife 20209e58123 107554elife58123 of 0cshort reportdevelopmental biologyrealtime pcr genotypingto determine the deletion rate the intestinal epithelium was separated from the underlining musclethe intestine was dissected flushed with pbs cut open longitudinally and incubated in mm ethylenediamine tetraacetic acid edta and mm dithiothreitol dtt in pbs for hr at rt on arotor the tubes were shaken vigorously the muscle removed and the epithelium centrifuged andused for genomic dna extraction sybr green realtime pcr assays were performed on each sample analyzedchromatin immunoprecipitationforelimb buds were manually dissected from ca rjorlswiss outbred dpc mouse embryoschromatin immunoprecipitation was performed as previously described cantu brieflythe tissue was dissociated to a single cell suspension with collagenase 1mgml in pbs for hr at Ëc washed and crosslinked in ml pbs for min with the addition of mm ethylene glycolbissuccinimidyl succinatethermo scientific waltham ma usa for proteinprotein crosslinkingsalazar and formaldehyde for the last min of incubation to preserve dnaprotein interactions the reaction was blocked with glycine and the cells were subsequently lysed in ml hepes buffer sds tritonx m nacl mm edta mm egta mmhepes chromatin was sheared using covaris s2 covaris woburn ma usa for min with the following set up duty cycle max intensity max cyclesburst max mode power tracking the sonicated chromatin was diluted to sds and incubated overnight at Ëc with mg of antibcl9abcam ab37305 or antitbx3 santacruz sc17871 or igg and ml of protein ag magneticbeads upstate the beads were washed at Ëc with wash buffer sds deoxycholate triton x100 m nacl mm edta mm egta mm hepes wash buffer sds sodium deoxycholate triton x100 m nacl mm edta mm egta mmhepes wash buffer m licl sodium deoxycholate np40 mm edta mmegta mm hepes and finally twice with tris edta buffer the chromatin was eluted with sds m nahco3 decrosslinked by incubation at Ëc for hr with mm nacl extractedwith phenolchloroform and ethanol precipitated the immunoprecipitated dna was used as inputmaterial for dna deep sequencing the pull downs | Colon_Cancer 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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 |
coronaviruses covs belong to a family of envelopedviruses with a positive sense singlestranded rna genomecovs cause illness ranging from upper respiratory tractinfections urtis resembling the common cold to lowerrespiratory tract infections lrtis such as bronchitis pneumonia and even severe acute respiratory syndrome sarswith most serious disease outcomes in the elderly immunocompromised patients and infants [ ] hcovoc43oc43 hcov229e 229e hcovnl63 nl63 andhcovhku1 hku1 were the ï¬rst documented humancovs hcovs which usually cause urtis and less frequently are associated with ltri diseases in the lastdecades two human coronaviruses created great concernfor the world medical community due to significant diseaseand mortality [ ] in severe acute respiratorysyndromecoronavirus sarscov was characterized byacute atypical pneumonia and diï¬use alveolar damagedad in roughly patients and with almost deathsrepresenting a nearly mortality rate more recentlyin a new human coronavirus designated as middle eastrespiratory syndromecoronavirus merscov was identiï¬ed and the global ongoing outbreak of mers with over oï¬cial cases and deaths represented approximately case fatality rate to date in humans over the last few months a new strain of human coronavirus sarscov2 also known as 2019ncov hascaught the worlds seven continents attention with its rapidglobal spread aï¬ecting at least countries and territoriesinfecting more than and claiming more than lives worldwide the coronavirus pandemichas promoted isolation and uncertainly fear and panicworldwide in additionlikely lead to changes inpolitical and economic power in ways that can be determined only later it willit is important to note that there are many similaritiesamong diï¬erent coronavirus species but not in all aspects 0coxidative medicine and cellular longevitydepending on the molecular mechanism of viral inhibitionpromoted by an antiviral agent the analysis of the data andcomparison between animal and human covs must be donevery carefully in fact it is important to note that there arediï¬erences between human and animal cov receptorswhich will likely result in diï¬erent aï¬nities or unlikely interactions of an antiviral agent with the diï¬erent cov receptors howeverif the antiviral agent interferes with thereplication andor assembly of the covs there is a higherprobability of obtaining similar antiviral activity results inhuman cov tests [ ] following this line our searchin specialized literature was focused mainly on studies thatinvestigated the anticoronavirus eï¬ects of natural antioxidants by inhibiting proteases for viral replication materials and methodsthe present study was carried out based on a search of the literature of natural antioxidants and coronavirus the searchperformed in the pubmed database included studies published until march and used the following keywordscoronavirusstressmerscov sarscov 229e nl63 oc43 hku1merscov virus infection and middle east respiratorysyndrome virus the scientiï¬c publications were selectedfrom studies published in the english languageantioxidants ï¬avonoids oxidative pathogenic mechanism of coronavirusinduced cell damagethe high mortality rate associated with the three pathogenichcovs has been mainly attributed to the development ofdigestive and respiratory tract injuries observed followinginfection acute atypical pneumonia and diï¬use alveolardamage that progress to deposition of ï¬brous tissue denudedairways haemorrhage and elevated macrophage ltrationare sometimes accompanied by watery diarrhoea dehydration and vomiting [ ]despite the molecular mechanisms of coronavirusinduced intestine and lung pathogenesis not fully elucidatedand still unclear studies have suggested that lateterm diseaseprogression is unrelated to viremia it is now believed morelikely to be associated with the immunopathological mechanism [ ] viral clearance and subsequent recovery frominfection require activation of an eï¬ective host immuneresponse however many immune eï¬ector cells may alsocause injury to host tissues together with ammatoryand immune response signaling the presence of oxidativecompounds such as reactive oxygen species ros playsimportant roles in the pathogenic mechanism of cell damageinduced by covs through oxidative stress oxidative stress is deï¬ned as an interruption andorderegulation of the signaling and redox system that can becaused by an imbalance in the production of oxidant andantioxidant species among the main oxidant agentsros and reactive nitrogen species rns stand out in orderto counterbalance the oxidant species there is an antioxidantsystem formed by enzymes and nonenzymatic molecules however during pathological events such as viral infections there may be an increase in the production of oxidantspecies not neutralized by the antioxidant system resultingin oxidative stress that promotes cellular damage throughprotein denaturation changes in the functions of nucleicacids lipid peroxidation and cell death []in addition during viral infection oxidative stress contributes to viral pathogenesis through stimulating ammation loss of immune function and increased viral replicationthat may occur due to the activation of the nuclear factorkappa b nfκb transcription pathway [] currentevidence suggests that cytokine dysregulationalso calledcytokine stormcontributes to severe disease caused by thepathogenic covs [ ] the exact mechanisms are notclear yet but research on uenza a virus shows that infection causes a rapid ux of ammatory cells this isfollowed by an increase in reactive oxygen species productionand cytokine expression and release which ultimately leadsto acute lung injury in general rna viruses promotechanges in the bodys antioxidant defense system aï¬ectingenzymes such as superoxide dismutase sod and catalasecat in addition to reducing the levels of antioxidantmolecules such as ascorbic acid carotenoids and reducedglutathione gsh [] wu reported that glucose6phosphate dehydrogenasean important antioxidantenzyme that produces nadph knockdown cells were moresusceptible to infection by hcov229e than normal cells interestingly ye and colleagues have reported that theinhibition of ros production alleviates ammation causedby uenza a virus infections in an experimental model of sarsinduced acute lunginjury in mice it was noted that phospholipid oxidationdue to oxidative stress is one of the main triggering factorsof acute lung injury this happens through the activation ofthe innate immune response culminating in the activationof pulmonary macrophages via tlr4triftraf6nfκbsignaling furthermore hypoxia caused by acute lunginjury can cause myocardial injury due to the production ofros aggravating infections caused by coronavirus disease covid19 mitochondria have an essential function in energy generation and for this reason their function and integrity arestrictly regulated in order to respond to varying energyrequirements and environmental conditions mitochondria are known to function as the control point in apoptoticpathways releasing proapoptotic factors mainly ros whichfunction as a signaling molecule that may result in cell death[ ] some studies have shown a relationship betweencoronavirus infection and dysfunctional or damaged mitochondria leading to the release of ros and other proapoptotic substances [ ] in a recent study xu reportedthat ros and p53 play key roles in regulating many kindsof the cell process during coronavirus infection in vero cellsaccording to the authors coronavirus infection appears toinduce a timedependent ros accumulation which in turnis linked to regulatory mechanisms of p53 activation andapoptosis in infected cells antioxidant substances promote improvement in casesof disease caused by coronaviruses such as apolipoproteinda lipocalin that promoted a neuroprotective eï¬ect against 0coxidative medicine and cellular longevityencephalitis induced by human coronavirus oc43in micethis protective eï¬ect occurred through the reduction of oxidative stress cerebral lipid peroxidation and regulation ofammation [ ] also the treatment with antioxidantssuch as pyrrolidine dithiocarbamate or nacetylcysteine significantly inhibits moreover melatonin promotes downregulation of acute lungoxidative injury due to its antiammatory and antioxidantactions making it a possible compound in the treatment ofcovid19 based on these studies compounds thathave antioxidant actions can be helpful in the treatment ofinfections promoted by coronaviruscoronavirusinduced apoptosisin general antioxidant properties of polyphenolic compounds such as some ï¬avonoids have been associated withthe presence of aromatic phenolic rings that promote theelectron donation and hydrogen atom transfer to free radicals acting as free radical scavengers reducing agents andquenchers of single oxygen formation thus the aimof this study was to investigate the antioxidant capacity andantiviral activity of natural antioxidants against coronavirusthe compounds are illustrated in figure occurrence and antioxidant properties ofanticoronavirus compoundsquercetin can be found in plants such as rubus fruticosus land lagerstroemia speciosa l pers [ ] also quercetinshows antioxidant activity at a concentration of μmoll inhepg2 cells inhibiting oxidative stress promoted by h2o2 promotes an increase in sod cat and glutathioneperoxidase gpx and reduces lipid peroxidation in rats withchronic prostatitischronic pelvic pain syndrome moreover quercetin improves sepsisinduced acute lung injury inrats by reducing lipid peroxidation and ammation andincreasing sod and cat levels in addition quercetin glycosides with antioxidant activity such as quercetin 3βglucoside have already been isolated from plants such as passiï¬ora subpeltata ortega andchamomilla suaveolens pursh rydb [ ] the administration of quercetin 3βglucoside mgkg poinstreptozotocininduced diabetic rats promotes an increasein the levels of antioxidant enzymes sod cat andgpx and nonenzymatic antioxidants vitamins c and eand gsh and a reduction of lipid peroxidation quercetin 3βgalactoside hyperoside is found mainly in plantsof the hypericum genus such as hypericum perforatum l[ ] moreover it showed cardioprotective activity inhigh glucoseinduced injury of myocardial cells throughdecreased apoptosis and ros production and increasedsod levels quercetin 7ramnoside is also found inplants of the hypericum genus such as hypericum japonicum thunb ex murray this ï¬avonoid shows hepatoprotective activity against carbon tetrachloride in mice bydecreasing lipid peroxidation and increasing cat andgsh levels in addition to presenting values of μmand22diphenyl1picrylhydrazyldpph and ²azinobis3ethylbenzthiazoline6sulphonic acid abts assays respectively μm intheepigallocatechin gallate is present in parkia roxburghii gdon and is one of the main metabolites found in green teaand liubao tea camellia sinensis lo kuntze also gallocatechin gallate can be found in this plant [] literaturedata reveal that the administration ip of mg100 g ofepigallocatechin gallate in rats with streptozotocininduceddiabetes mellitus promotes a reduction in oxidative stressthrough reductions in parameters such as indirect nitricoxide synthesis and status total oxidative as well as anincrease in levels of cat and total antioxidant capacity ofplasma furthermore it promotes cardioprotection byantioxidant mechanisms green tea has a high antioxidant capacity due to the highlevels of catechins present he and collaborators compared the antioxidant activities of catechins and reported thatepigallocatechin gallate has greater antioxidant activity viaradical scavenging activity μm with values of ± ± and ± compared to its epimergallocatechin gallate with values of ± ± and ± in the dpph abts and ferric reducingantioxidant power frap respectively amentoï¬avone is a biï¬avonoid present in leaves ofginkgo biloba l garcinia brasiliensis l and nandinadomestica l [] this biï¬avonoid has a high antioxidantcapacity demonstrated in scavenging tests ofdpph abts superoxide and hydroxyl radicals moreover amentoï¬avone prevents acute lung injury induced bysepsis in rats by decreasing thiobarbituric acid reactive substance tbars levels and by increasing levels of sod andgsh apigenin is mainly present in ï¬owers and leaves beingabundantly found in apium graveolens l petroselinum crispum mill fuss and matricaria chamomilla l sánchezmarzo and collaborators evaluated the antioxidantcapacity of apigenin using the trolox equivalent antioxidantcapacity teac oxygen radical absorbance capacityorac and frap assays the results show that apigeninhas good antioxidant activity with values of ± μmol teammol ± μmol teammol and ± μmol fe2mmol respectively in addition oraladministration of apigenin mgkgday for days in anexperimental model of cardiotoxicity induced by doxorubicin in rats promoted cardioprotection by reducing levels ofmalondialdehyde mda increasing sod levels and preventing cardiomyocyte apoptosis luteolin is present in foods such as carrot cabbage teaand apple and is found in ugni molinae turcz [ ] datashow that luteolin μgml increases the levels of gsh theexpression of gsh synthetase and the activity of sod andcat in human colon cancer cells ht29 furthermoreluteolin attenuates the sepsisinduced acute lunginjury in mice by reducing lipid peroxidation and increasingsod and cat activity in addition to suppressing the nfκbpathway herbacetin is ubiquitous in plants of the genus rhodiolasuch as rhodiola rosea l herbacetin glycosides are alsopresent in the roots of r sachalinensis a bor and show antioxidant activity veeramani reported that theadministration of herbacetin mgkg po in mice with 0coxidative medicine and cellular longevityohohohohhoohooohohhooohoh oquercetinohohoooohohohquercetin 3ð½galactosideohohooohoohoohohhoohohohoh oquercetin 7ramnosideohooohohoohoohoohohohquercetin 3ð½glucosidehooohooohohohohohohepigallocatechin gallateohgallocatechin gallateohhooohoh ohooluteolinhooohoooohoohohoohrhoifolinhohohoohohohohohohoohoohooh oamentoflavoneohhooohohohoherbacetinhohoooohooohohohoapigeninooohooohhopectolinarinooopsoralidinhooohohohohcatechinohhoohoooh ohelichrysetinohohomyricetinohohohhohoohoohoisobavachalconeohhooohscutellareinohresveratrolfigure chemical structures of bioactive antioxidants against coronavirusobesityassociated insulin resistance promotes an increasein the activity of the enzyme glucose6phosphate dehydrogenase which is directly related to the production ofnadph pectolinarin is present in plants of the genus cirsiumsuch as cirsium setidens nakai and cirsium japonicum dcthe administration of pectolinarin and mgkg pofor two weeks in rats promotes antioxidant eï¬ects in hepatic 0coxidative medicine and cellular longevitytable antioxidant properties of natural inhibitors of coronaviruscompoundtestedassaysexperimentalconcentration doseantioxidant eï¬ectreferencetype of cells μmoll mgkg po mgkg poinhibiting oxidative stress promoted byh2o2promoted an increase in sod cat andgpx and reduced lipid peroxidationreduces lipid peroxidation and increasessod and cat levelsincreases levels of sod cat gpx mgkg povitamins c and e and gsh and reduces nmoll mgkgic50 μm dpphec50 μm abtsrats with streptozotocininduced diabetes mellitus mg100 g iprats with streptozotocinnicotinamideinduceddiabetes mellitus mgkg po μm mgkg μgml ± μmol teammol ± μmol teammol and ± μmol fe2mmolrespectively mgkg pomodelshepg2 cellsrats with chronicprostatitischronic pelvicpain syndromesepsisinduced acute lunginjury in ratsstreptozotocininduceddiabetic ratshigh glucoseinducedinjury of myocardial cellsccl4induced liver damagemodel in micedpphabtsquercetinquercetin 3βglucosidequercetin 3βgalactosidequercetin ramnosideepigallocatechingallateepigallocatechingallatedpphabtsfrapgallocatechingallateamentoï¬avoneacute lung injury inducedby sepsis in ratsdpph abts superoxideand hydroxyl radicalsteacoracfrapcardiotoxicity induced bydoxorubicin in ratshuman colon cancer cellsht29acute lung injury inducedby sepsis in micemice with obesityassociated insulinresistance inductionhepatic injury induced bydgalactosamine in ratsoracapigeninluteolinherbacetinpectolinarinrhoifolincatechinlipid peroxidationdecreases apoptosis and ros productionand increases sod levelsdecreases lipid peroxidation and increasescat and gsh levelsscavenging of free radicalsreduces indirect nitric oxide synthesis andtotal oxidative statusincreased levels of cat and totalantioxidant capacity of plasmaincreased levels of cat sod and gshreduced levels of superoxide and proteincarbonyl pco and prevented dnadamage ± ± and ± ± and ± respectively ± respectivelydecreases tbars levels and increaseslevels of sod and gshscavenging of free radicalsscavenging of free radicalsreduces levels of mda increases sodlevels and prevents cardiomyocyteapoptosis μgmlincreases levels of gsh expression of gshsynthetase and the activity of sod and mgkg ip mgkg po and mgkg poapproximately troloxequivalents μmcatreduces lipid peroxidation increases theactivity of sod and cat and suppressesthe nfκb pathwayincreases the activity of glucose6phosphate dehydrogenaseincreases levels of sod gsh glutathionereductase and glutathione stransferasescavenging of free radicalsscavenging of free radicals 0coxidative medicine and cellular longevitytable continuedtype of cellscompoundtestedassaysexperimentalconcentration doseantioxidant eï¬ectreferencemodelsabtsfrap ± mol troloxequivalentsmol ± mol trolox equivalentsmoldihydrorhodamine oxidation assayandic50 ± μmisobavachalconedpph sc50frapabts sc50psoralidinelectron spin resonancemyricetinhelichrysetinscutellareinresveratroldpphchinese hamster lungï¬broblast cells v794treated with h2o2oracdpphabtssuperoxide radicalsdpph sc50r2rats with obstructive lungdiseasehypercholesterolemicapoeko mouserespectively μm ± mmic50 μmequivalent to feso4 mm respectively·7h2o and μgml and μgml μgml ± trolox equivalents ± μm ± μm and ± μm respectivelyscavenging of free radicalsscavenging of free radicalsscavenging of free radicals and respectivelyprevents dna damage and lipidperoxidationincreases the activity of sod cat andgpxscavenging of free radicalsscavenging of free radicals μmoldmscavenging of free radicals mgkg mgkgincreases sod activity and reduces mdalevelsinhibits the activity and expression ofnadph oxidasesincreases sod gpx and cat levels injury induced by dgalactosamine by increasing levels ofsod gsh glutathione reductase and glutathione stransferase [ ]rhoifolin is found in citrus fruits such as citrus limettarisso studies have indicated that its radical peroxyl scavenging capacity is higher than trolox in orac assays approximately trolox equivalents μm [ ]meanwhile the catechin is a ï¬avonoid present inleaves of green tea wine and fruits [ ] grzesik investigated the antioxidant action of catechinthrough the abts scavenging activity and frap teststhe results show values of ± mol troloxequivalentsmol and ± mol trolox equivalentsmol respectively in addition catechin shows greaterprotective properties in the dihydrorhodamine oxida ± μm than gsh and ascorbiction assay ic50acid and μm respectively psoralidin is a prenylated coumestan which is found inplants of the fabaceae family such as psoralea corylifolia lxiao and collaborators investigating the antioxidant potential of compounds isolated from p corylifolia observed thatpsoralidin shows the best antioxidant activity by the methodof electron spin resonance spectroscopy with an ic50 value of μm the compound isobavachalcone has been isolated fromplants of the fabaceae and moraceae families [ ] isobavachalcone shows a strong antioxidant activity in dpphsc50 frap and abts sc50 assays with values of μm ± mm equivalent to feso4·7h2o and mm respectively in addition the compound has beenreported to inhibit the nfκb pathway in sephadexinducedlung injury in rats [ ]helichrysetin is a chalcone that is found in plants of thehelichrysum genus such as helichrysum odoratissimum l in a study investigating the antioxidant activity of natural and prenylated chalcones vogel found that helichrysetin is the substance that shows the highest antioxidantactivity in the orac test with values of ± troloxequivalents myricetin is widely found in the plant families myricaceae and anacardiaceae and is widely used as health foodsupplement due to its antioxidant properties [ ]bennett demonstrated that myricetin reacts withoxygencentered galvinoxyl radicals more than timeshigher than vitamin e dalphatocopherol furthermoremyricetin was able to scavenge and on the dpphassay μgml and μgml respectively interestinglythe compound prevents dna damage by lipid 0coxidative medicine and cellular longevity2h2o2 2gsh2o2h2h2o2vit evit cgshnadphnonenzymatic antioxidantsgpxsodcatsemyzne tnadixoitna2h2o gssgo2 h2o2o2 h2omdapcotbarsbio m arkers of oxidative stressgeneration of rosoxidative stresscell damagenaturalantioxidants sesadixohpdan2o2 nadphnadp 2o2 hfigure the main antioxidant mechanisms of natural compounds reported in this review dashed line inhibition full line activationperoxidation and increasing the activity of sod cat andgpx in chinese hamster lung ï¬broblast cells v794treated with h2o2 scutellarein is found in scutellaria barbata d don andpolygonum viscosum buchham [ ] liu investigated the antioxidant activity of scutellarein through thedpph abts and superoxide scavenging assays they notedthat the compound shows good antioxidant activity withvalues of ± μm ± μm and ± μmrespectively while the trolox a standard antioxidant compound presented ± μm ± μm and ± μm respectively resveratrol is found in grapes peanuts and blueberriesand can be isolated from veratrum grandiï¬orum o loes literature shows that resveratrol has good antioxidantvalues of μmoldmactivity with dpph sc50r2moreover it is able to reduce the production of ros by inhibiting the activity and expression of nadph oxidases byeliminating oxidant agentsincluding radical hydroxylsuperoxide hydrogen peroxide and peroxynitrite the treatment of resveratrol mgkg po in ratsreduces oxidative stress in obstructive lung disease byincreasing sod activity and reducing mda levels indicatinga decrease in lipid peroxidation table shows themain actions of natural antioxidants discussed in this studyand figure illustrates these activities effect of natural antioxidants incoronavirus infectionsthis review focused on studies reporting on the anticoronavirus activity of natural antioxidants based on exclusioncriteria data from nineteen compounds were discussedthe oxidative stress pathway could potentially be a keyelement in coronavirusinduced apoptosis and pathogenesis for this reason it is interesting to investigate the useof antioxidants as potential therapeutic toolseither as analternative or as an adjuvant to conventional therapiesinthe treatment of coronavirus infections among the antioxidant compounds evaluated as for coronavirus infectionsare the ï¬avonoids which are compounds widely found infruits vegetables and certain beverages in fact researchgroups have reported that antioxidant ï¬avonoids includingcatechin luteolin apigenin quercetin and quercetin rhamnosideinhibit ros accumulation and apoptosis ofcells infected with diï¬erent coronavirus including porcineepidemic diarrhoea coronavirus pedv and transmissiblegastroenteritis coronavirus tgev []as shown with the recent covid19 pandemic thesearch for alternative or new antiviral therapies for theremainstreatment of coronavirus diseasesimportantbased on the literature antioxidanttherapies oï¬er anattractive option 0coxidative medicine and cellular longevitytable natural antioxidants tested in in vitro coronavirus infection models and their main results and mechanism of actionantioxidanttype of cells testedconcentrationic50antiviral eï¬ectmechanism of actionreferencecatechintgevinfected st cellscatechin μminhibition of tgevinduced apoptosissuppression of the tgevinducedbcl2 reduction baxredistribution cytochrome crelease and caspase3 activation resveratrolmersinfected vero e6cellsresveratrol μmquercetinepigallocatechingallategallocatechingallate gcgquercetin rhamnosideq7rrecombinant 3clprowas expressed in pichiapastoris gs115quercetin μmepigallocatechingallate μmgallocatechingallate μmpedvinfectedvero cellsq7r μminhibition ofmersinducedreduction of the expression ofinfectionapoptosis andnucleocapsid n protein essential prolonged cellular survivalfor merscov replicationafter virus infectioninhibition of coronavirusreplicationreduction of the formationof a visible cytopathiceï¬ect cpe without dnafragmentationgcg displayed a binding energyof kcal mol1 to the active siteof 3clpro and the galloyl moietyat 3oh position was required for3clpro inhibition activitynot speciï¬city amentoï¬avoneapigeninluteolinquercetinquercetin3βgalactosideherbacetinrhoifolinpectolinarinsarscov 3clproinhibition usingï¬uorescence resonanceenergy transfer analysismolecular dockingsprfretbasedbioassays andmutagenesistryptophanbasedï¬uorescence methodherbacetinisobavachalconequercetin3βdglucosidehelichrysetintryptophanbasedï¬uorescence methodamentoï¬avone3βgalactoside μmapigenin μmluteolin μmquercetin μmquercetin μmherbacetin μmrhoifolin μmpectolinarin μmherbacetin μmisobavachalcone μmquercetin3βdglucoside μmhelichrysetin μminhibition of sarscovreplicationflavonoids exhibited sarscov3clpro inhibitory activity[ ]inhibition of merscovreplicationflavonoids exhibited merscov3clpro inhibitory activity isobavachalconepsoralidinlineweaverburk anddixon plotsmyricetinscutellareinsprfretbasedbioassaysisobavachalcone μmpsoralidin μmmyricetin μmscutellarein μminhibition of sarscovreplicationisobavachalcone and psoralidinexhibited sarscov papainlikeprotease inhibitory activitymyricetin and scutellareininhibition of sarscovpotently inhibit the sarscovreplicationhelicase protein in vitro byaï¬ecting the atpase activity the high number of deaths and clinical complicationsobserved in sars and merscov epidemics motivatedthe search for eï¬ective therapeutic agents this was necessitated when many of the tested conventional drugs andtherapies proved ineï¬ective in treating sarsantiviralcov infections for exampletreatment ofthe initial 0coxidative medicine and cellular longevitycell culture coronavirusnaturalantioxidants suppress bcl2 reductionsuppress bax redistributionsuppress cytochorome c releasesuppress caspase3 activationreduce formation of a visiblecytopathic effect without dnafragmentationreduce nucleocapsid n protein expressioninhibit 3clike proteaseinhibit 3clike proteaseinhibit papainlike proteaseinhibit helicase protein by affectedatpase activitytgevpedvmerscovsarscovfigure inhibitory actions of natural antioxidants against coronavirussarscov with antiviral agents such as ribavirin and corticosteroids did not achieve very satisfactory resultsmainly because corticosteroids exertimmunosuppressoreï¬ects on the humoral and cellular immune systems[ ] other drugs such as pentoxifylline were considered for the treatment of sars due to its interestingtherapeutic propertiesantiammatoryantiviral immunomodulatory and bronchodilatory eï¬ectshowever it too was not successful in the clinical treatment of sarscov infection includethatinhibition ofmany antioxidant compounds show antiviral activityagainst sarscov the antiviral activity has been mainlyattributed to thethe 3clike protease3clpro of sarscov a vital enzyme for sarscov replication as an example multiples studies havereported that quercetin and quercetinderived compoundssuch as quercetin 3βgalactoside display potent 3clproinhibitory e5ï¬ect and consequent reduction of sarscovreplication other antioxidants such as epigallocatechin gallate gallocatechin gallate amentoï¬avone apigeninluteolin herbacetin rhoifolin and pectolinarin are alsofound to eï¬ciently block the enzymatic activity of sarscov 3clpro [ ]moreover some natural antioxidants exhibit promisingantiviral activity against sarscov infection by interferingwith diï¬erent targets involved in sarscov replication inparticular the sarscov papainlike protease plpro andsarscov helicase protein kim reported that isobavachalcone and psoralidin inhibit plpro in a dosedependentmanner with ic50 ranging between and μm previously yu reported that myricetin and scutellareinpotently inhibit the sarscov helicase protein in vitro byaï¬ecting the atpase activity merscov is another zoonotic coronavirus transmitted between animals and human beings that causes severemorbidity and mortality no antiviral medicines with satisfactory eï¬cacy for the treatment of merscovinfectedpatients have been identiï¬ed to date similar to sarscov natural antioxidant libraries have been probed forpotentialinhibitory compounds against merscov 3clike protease jo showed that herbacetin isobavachalcone quercetin 3βdglucoside and helichrysetinfourcompounds with recognized antioxidant activity can blockthe enzymatic activity of merscov 3clpro using atryptophanbased ï¬uorescence method furthermore theexperimental and computational studies show that ï¬avonoland chalcone are favourite scaï¬olds to bind with the catalytic site of merscov 3clpro in a study performed by lin the antiviral activitiesof resveratrol were investigated in mersinfected vero e6cells the authors reported a significantinhibition ofmerscov infection and prolonged host cell survival aftervirus infection which they speculate was promoted by resveratrol in addition they also found that the expression ofthe nucleocapsid n protein which is essential for merscov replicationis decreased after resveratrol treatment it is important to mention that in vitro models of coronavirus infection also show antiviral activity of ï¬avonoidsextracted from ï¬owering cherry cultivars and black tea[ ] finally antioxidants such as resveratrol alsoare able to block infection produced by herpesvirus the discovery of antiviral compounds from a bioactivecompound against other viruses is an interesting strategy forobtaining new antiviral drugs table shows the mainactions of the natural antioxidants against the coronavirusand figure summarizes these activities 0c conclusionsin conclusion this review shows that antioxidant compounds prominently ï¬avonoids exhibit antiviral action inmodels of coronavirus infections in general the antiviralactivity might be attributed at least in part to the inhibitoryeï¬ect on the enzymatic activity of targets involved in coronavirus replication including sarscov 3clpro sarscovpapainlike protease plpro sarscov helicase proteinand merscov 3clpro in addition some studies provideevidence that the reduction of ros accumulation retardsthe coronavirusactivated apoptotic signaling thereforethe mechanisms of oxidative stress could be the key elementto be studied in coronavirus infectionsincluding thoserelated to ammatory processes arising from the action ofthis virus obviously further investigations are needed toelucidate other pharmacological mechanisms by which natural antioxidants play an antiviral eï¬ect despite the ï¬ndingsreported in this reviewthey cannot be generalized tocovid19 however the data provided support to the investigation of natural antioxidants as a potential therapeuticapproach in the treatment for covid19 and its severe clinical complications either as an alternative or as an adjuvantto conventional therapies and contribute to the search fornew prototypes in the development of drugs against coronavirus infectionsabbreviations229e3clproabtshuman coronavirus229e3clike protease²azinobis3ethylbenzthiazoline6sulphonic acidcoronavirusescatalasediï¬use alveolar damage22diphenyl1picrylhydrazylferric reducing antioxidant powerreduced glutathioneglutathione peroxidasehuman coronaviruseshuman coronavirushku1lower respiratory tract infectionsmalondialdehydecovscovid19 coronavirus disease catdaddpphfrapgshgpxhcovshku1lrtismdamerscov middle east respiratory syndromecoronavirusnfκbnuclear factor kappa bhuman coronavirusnl63nl63human coronavirusoc43oc43oxygen radical absorbance capacityoracprotein carbonylpcoporcine epidemic diarrhoea coronaviruspedvplpropapainlike proteasereactive nitrogen speciesrnsreactive oxygenated speciesrossarssevere acute respiratory syndromesarscov severe acute respirator | Colon_Cancer |
as one of the most common gynecological malignant tumors cervical cancer is the fourth leadingcause of cancerrelated death among women worldwide although eï¬orts including periodiccancer screening prompt surgical treatment chemotherapy and radiotherapy have been madeto decrease the mortality of cervical cancer the prognosis of patients is still poor and cervicalcancer remains an important public health issue the pathogenesis of cervical cancer has notbeen clearly illustrated but it is conï¬rmed that the activation of tumorpromoting genes and theinactivation of tumor suppressor genes participate in the progression of cervical cancer toscreen for novel abnormally expressed genes functioning in cervical cancer may provide potentialprognostic markers and therapeutic targets for treatmentedited byihab youniscarnegie mellon university inqatar qatarreviewed byweifeng dingnantong university chinamassimo brogginimario negri pharmacologicalresearch institute irccs italycorrespondencelin xuxulin83njmueducnemei lulem13705179888sinacnbinhui renrobbishren163com these authors have contributedequally to this workspecialty sectionthis was submitted tocancer geneticsa section of the frontiers in oncologyreceived april accepted june published august citationzhu b wu y luo j zhang qhuang j li q xu l lu e and ren b mnx1 promotes malignantprogression of cervical cancer viarepressing the transcription ofp21cip1 front oncol 103389fonc202001307frontiers in oncology wwwfrontiersinaugust volume 0czhu mnx1 enhances progression of cervical cancermnx1 motor neuron and pancreas homeobox also knownas hb9 hlxb9 is a member of homeobox gene family andencodes a nuclear protein the homeobox genes are agroup of genes containing homeobox a base pairs longdna sequence and encode homeodomain proteins that actas transcription factors many homeobox genes have beenproved to be implicated in various human cancers acting asoncogenes or tumor suppressors mnx1 was ï¬rstly foundto be expressed in lymphoid and pancreatic tissues and deï¬nedas a novel human homeobox gene in early studiesshowed that mnx1 was involved invertebrate and pancreaticdevelopment and motor neuronal diï¬erentiation defects in this gene result in currarino syndrome an autosomicdominant congenital malformation in followup studymnx1 was found to be abnormally expressed in several cancertypes including prostate cancer hepatocellular carcinoma andacute myeloid leukemia furthermore recent studiesconï¬rmed that mnx1 played oncogenic roles in colorectalcancer breast cancer and bladder cancer the aim of this study is to identify the expression andfunction of mnx1 in cervical cancer our results revealedthat mnx1 was significantly upregulated in cervical cancerand correlated with poorer prognosis the knockdown oroverexpressed mnx1 inhibited or promoted aggressiveness ofcervical cancer including proliferation migration and invasioncapacities by enhancing or repressing the transcription of p21cip1thus regulating the g2m cell cycle transition these ï¬ndingssuggested that mnx1 might be a potential diagnostic marker andtherapeutic target for cervical cancermaterials and methodsbioinformaticsthe tcga dataset termed tcga_cesc_exp_hiseqv22015 was downloaded from the ucsc cancer browser genomecancerucscedu to evaluate the expression ofmnx1 in cervical cancer and adjacent normal tissues gepiagene expression proï¬ling interactive analysis httpgepiacancerpku cnindexhtml was used to analyze the expressionof mnx1 with disease free survival dfs of cervical cancerpatients the cbioportal website httpwww cbioportal was utilized to obtain highly coexpressed genes with mnx1totally genes highly correlated with mnx1 pearson score table s1 were submitted to david bioinformaticsresources httpdavidabccncifcrfgov for geneontology go kyoto encyclopedia of genes and genomeskegg and reactome pathway analysis and we analyzed thebinding site of mnx1 and p21cip promoters through the jaspardatabase httpjaspardevgeneregnet human cervical cancer cell linesthe human normal cervical celllines hacat and cervicalcancer cell lines hela siha caski and c33a were purchasedfrom american type culture collection atcc usa helasiha c33a and hacat cells were incubated in dmem mediumkeygen nanjing china and caski cells were cultured inrpmi1640 keygen nanjing china medium containing fetal bovine serum gibcobrl invitrogen carlsbad causa and cultured at ¦c in a humidiï¬ed incubator containing co2human cervical cancer tissuesthe pairs of cervical cancer tissues and adjacent tissues wereselected from the aï¬liated cancer hospital of nanjing medicaluniversity and informed consent was obtained from all subjectsall tumors and paired nontumor tissues were conï¬rmed byexperienced pathologists and no patients received chemotherapyor radiotherapy before surgery the mrna expression ofmnx1 and p21cip1 in cervical cancer tissues was detected byqrtpcr collection of human tissue samples was conductedin accordance with the international ethical guidelines forbiomedical research involving human subjects cioms thisstudy was approved by the ethics committee of the nanjingmedical university aï¬liated cancer hospitaltissue microarrayspaired cervical cancer tissue microarrays were obtained fromshanghai outdo biotech co ltd cat no odctrputr03 and odctrputr03006 totally pairs of paraï¬nembedded human cervical cancer sections were analyzed formnx1 expression all tissues were examined by two experiencedpathologists and the tnm stage was conï¬rmed in each patientwith blinded methods the sections were incubated with an antimnx1 primary antibody abcam ab79541 the ihcscores were calculated according to intensity and percentage ofpositive cells the staining intensity was evaluated as the basis offour grades negative staining 1weak staining moderatestaining or strong staining the product percentage ofpositive cells and respective intensity scores was used as the ï¬nalstaining scores a minimum value of and a maximum valueof rna preparation reverse transcriptionand qrtpcrtrizol reagent invitrogen carlsbad ca usa was used toextract total rna from tissue samples or cultured cells accordingto the manufacturers protocol a reverse transcription kittakara cat rr036a keygen was utilized to generate cdnaqrtpcr was performed with sybr select master mix appliedbiosystemscat keygen nanjing china and primersare shown in table s2western blottinglysis buï¬er ripa keygen containing protease inhibitorspmsf keygen was used to extract protein of cells andtissues and protein concentration was detected with a bcakit keygen protein samples µg were loaded into sodium dodecyl sulfate polyacrylamide electrophoresissdspage gels and transferred onto a pvdf membraneafter electrophoresis the membrane was blocked with nonfatmilk for h and incubated overnight with antibodies againstrespective antibodies mnx1 abcam ab79541 p21cip1cell signaling technology pthr161cdk1 cellsignaling technology cdk1 cell signalingfrontiers in oncology wwwfrontiersinaugust volume 0czhu mnx1 enhances progression of cervical cancertechnology p27kip1 cell signaling technology cyclinb1 abcam ab72 cycline1abcam ab3927 cycline1 abcam ab3927 cyclind1santa cruz biotechnology sc246 actinabcam ab15265 sirna and plasmid transfectionthe sirnas targeting mnx1 and p21cip1 were conductedand purchased from ribobio guangzhou china all sirnasequences are shown in table s3 the fulllength cdnaof human mnx1 were pcrampliï¬ed and cloned intothe expression vector pgpu6gfpneo vigene biosciencesshandong china the sirnas and overexpression plasmidswere transfected into cervical cancer cells according to thelipofectamine reagent invitrogen carlsbad ca usaprotocol nonsense rnai sinc and empty plasmids oencwas used as negative controls²analysiscell proliferation assaythe cell proliferation assays were performed h aftertransfection for real timexcelligencesystemrtca cells100 µl were seeded in eplates and theplates were locked into the rtca dp device in the incubator tocalculate the cell index value in colony formation assay a totalof cells were placed in afresh 6wellplate and the cells werestained with crystal violet solution after days for5ethynyl2deoxyuridine edu assay keyfluor488 clickitedu kit ribobio guangzhou china the transfected cells wereplaced in 96wellplates cellswell overnight in a co2incubator then cells were incubated with µlwell of µmedu for h at ¦c and ï¬xed with µl paraformaldehydecontaining pbs for min at room temperature subsequentlythe cells were cultured for min with µl of mgmlglycine and then washed with µl bsa in pbs afterpermeabilization with triton x100 for min the cellswere cultured with à clickit reaction solution for minat room temperature in dark conditions after that cells wereincubated with µlwell of à hoechst solutionsfor min at room temperature in the dark after washing with µl of pbs the cells were then imaged using ï¬uorescencemicroscopy and proliferation cell ratios were counted fromï¬ve random ï¬elds in every well each experiment was repeatedthree times a total of cells in a fresh sixwellplates weremaintained in medium containing fbs the medium wasreplaced every or days after weeks the cells were ï¬xedwith paraformaldehyde and stained with crystal violeteach experiment was repeated three timesmigration and invasion assayfor wound healing assay cells were growing on the 6wellplate then artiï¬cial scratch on a conï¬uent monolayer of cellswas created with a µl pipette tip the medium wasreplaced with the serumfree and cells imaged h later fortranswell and matrigel assay totally transfected cells wereadded to the upper chamber of transwell assay inserts µmpet 24well millicell or a matrigel coated membrane bdbiosciences containing µl serumfree dmem media thelower chambers were ï¬lled with µl dmem media containing fbs after a 24h migration assay or 48h invasionassay incubation the cells were ï¬xed with polyformaldehydestained with crystal violet and imaged migration and invasionwere assessed by counting cell nuclei from ï¬ve random ï¬elds onevery ï¬lter each experiment was repeated three times rtcawas also used to evaluated the ability of migration and invasioncimplates installation and baseline measurement was carriedout according to the instructions add µl of mixed serumfree cell suspension à cells to the upper chamber in cimplates and the plates were locked into the rtca dp device in theincubator to calculate the cell index valuecell cycle analysiscells were digested with trypsinedta and ï¬xed with ethanol for h at ¦c the ethanolsuspended cells werecentrifuged and stained with pi staining solution for minin the dark at ¦c a facscalibur ï¬ow cytometer was usedto detect cell cycle distribution the percentage of the cells ing0g1 s and g2m were counted and comparedchromatin immunoprecipitation chipcells were crosslinked in paraformaldehyde and the reactionwas quenched with glycine after two washes with cold pbscells were added with precooling pbs containing cocktailhalttm protease inhibitor cocktail thermo scientiï¬c and scraped into a centrifuge tube the cells were centrifugedfor min at g at ¦c then added with µl celllysis buï¬er containing µl cocktail and incubated onice for min cells were then centrifuged for min at à g ¦c and cell precipitates were resuspended in µlnucleus lysis buï¬er containing µl cocktail the cellswere sonicated amplitude on ice for min and solublechromatin was obtained by centrifuging for min at g at¦c five micrograms of antimnx1 antibody sigmaaldrichsab2101494 coupled to magnetic beads resin m2 sigmashanghai china was used to immunoprecipitate the dnaprotein complex and the igg antibody was used as a negativecontrol the immunoprecipitation products were washed with µl low salt buï¬er high salt buï¬er lici buï¬er and tebuï¬er successively all for min at ¦c the chip elution buï¬ercontaining proteinase k was used for dna puriï¬cation thebeads were wiped out on a magnetic frame and the dna waseluted with elution buï¬er c from adsorption column chipdna samples were subjected to pcr ampliï¬cation with primersspeciï¬c to p21cip1 promoter region pcr products were then usedfor agarose gel electrophoresis the sequence of primers used areshown in table s4 and gapdh was used as a controlluciferase reporter assaythe p21cip1 cdkn1a promoter region bp wasampliï¬ed and cloned into luciferase reporter plasmid pgl3basic the p21cip1 promoter wildtype plasmids or mutanttype plasmids were cotransfected with cmvmnx1 expressionplasmids in hek293t cells and cmvempty vectors were usedas a negative control relative luciferase activity was corrected forfrontiers in oncology wwwfrontiersinaugust volume 0czhu mnx1 enhances progression of cervical cancerrenilla luciferase activity of pgl3basic and normalized to theactivity of the controlxenograft modelall animal studies were conducted in accordance with nihanimal use guidelines and protocols were approved by nanjingmedical university animal care committee sixteen femalenude mice weeks old were purchased from nanjingmedical university school of medicines accredited animalfacility the mice were randomly divided into two groupsusing random number generator in each group à exponentially growing cervical cancer cells were injected inaxilla subcutaneously before tumor transplantation cells weretransfected with shrnas or overexpression plasmids thetransfection was performed by transient transfection accordingto the speciï¬cation of lipofectamine invitrogen carlsbadca usa the shnc and empty vector pcdna31 were usedas controls and totally µg plasmid vectors were transfectedinto cells for each group the sequences of shrnas are shown intable s5 tumors were harvested at weeks after injection theweight of tumor was measured on the scale and tumor volumewas estimated using calipers [length à width2] and tissueswere immunohistochemically stained with mnx1 abcamab79541 ki67 abcam ab79541 and p21cip1abcam ab109520 western blotting was performed aspreviously described no blinding was done in the animal studiesstatistical analysisresults are presented as the mean ± standard deviation sdstatistical analyses were performed using spss statistics version chicago ill and graphpad prism software graphpadsoftware inc la jolla ca usa p was consideredstatistically significantresultsoverexpression of mnx1 correlates withpoorer prognosis and more aggressiveclinical featuresanalysis of tcga dataset revealed that the mrna expressionof mnx1 was remarkably upregulated in cervical cancer tissuescompared with paratumor tissues p figure 1a ingepia gene expression proï¬ling interactive analysis websitepatients with higher expression of mnx1 bore a worse diseasefree survival nhigh nlow p figure 1b theexpression of mnx1 in cervical cancer tissues were significantlyhigher than adjacent tissues in of cervicalcancer patients p figures 1cd ihc assays based ontissue microarrays tmas were performed to detect the proteinexpression of mnx1 in paired human cervical cancer tissuesand paratumor tissues and results showed that staining scoresof mnx1 were higher in cancer tissues p figure 1ecombined with the patients clinical information the expressionof mnx1 was higher in patients with more advanced tnm stagestage iii vs iiiiv p figure 1f t stage t1 vst2t3 p figure 1g and n stage n0 vs n1 p figure 1h moreover mnx1 staining scores were linkedto higher pathological grade level ii vs iii p figure 1iand larger tumor maximum diameter d vs ¥ cm p figure 1j and ihc images of two patients with diï¬erentclinical stages were presented figure 1kknockdown of mnx1 inhibited progressionof cervical cancer in vitroto evaluate the expression of mnx1 in cell lines qrtpcr andwestern blotting were performed and results showed that mnx1was generally upregulated in cervical cancer cell lines comparedwith normal human cervical cell lines hacat figures 2ab tofurther investigate the biological function of mnx1 in cervicalcancer two speciï¬c sirnas targeting mnx1 were transfectedinto hela and siha cells both two sirnas showed favorablesuppression eï¬ciency in hela figures 2cd and siha cellsfigures 2ef the rtca proliferation assay figure 2g eduassay figure 2h and colony formation assay figure 2ishowed that knockdown of mnx1 inhibited the proliferationability of cervical cancer in hela and siha cells moreover rtcamigration assay figure 2j transwell assay and matrigel assayfigure 2k and wound healing assay figure 2l revealed thatsilencing mnx1 inhibited the ability of cervical cancer cells tomigrate and invade these results suggest that mnx1 plays a vitalrole in the malignant phenotype of cervical cancerectopic expression of mnx1 enhancedaggressiveness of cervical cancer in vitroto further verify the biological role of mnx1 in cervical cancer apcdna31 plasmid to overexpress mnx1 was constructed andtransfected into c33a and hela cells the plasmid eï¬ectivelyupregulated the expression of mnx1 conï¬rmed by qrtpcrand western blotting figures 3ab consistently our resultsshowed that ectopic expression of mnx1 promotes proliferationmigration and invasion figures 3cg of cervical cancer cellssimnx1 induced g2m cell cycle arrestand upregulated the expression of p21cip1two hundred and eight genes highly correlated with mnx1were used for go kegg and reactome pathway analysisresults showed that mnx1 may participate in transcriptionand metabolism pathway figure 4a cell cycle detectionshowed that knockdown of mnx1 induced g2m cell cyclearrest in hela and siha cells figure 4b furthermore weexamined the eï¬ect of mnx1 on the expression of cell cyclekeyrelated genes including p15ink4b p16ink4a p21cip1 p27kip1cdk1 cdk2 cdk4 cyclinb1 cyclind1 and cycline1 bothin hela and siha cells knockdown of mnx1 upregulated theexpression of p21cip1 which has been conï¬rmed as a tumorsuppressor gene in multiple cancers figure 4c and westernblotting results suggested that knockdown of mnx1 increasedthe expression of p21cip1 while decreased the expression ofphosphorylated cdk1 pthr161cdk1 a downstream eï¬ectorof p21cip1 figure 4d consistently with these results ectopicexpression of mnx1 decreased the expression of p21cip1 whileincreased the expression of pthr161cdk1 in c33a and helacells figures 4ef it suggested that mnx1 might exerted itsbiological function via modulating the expression of p21cip1frontiers in oncology wwwfrontiersinaugust volume 0czhu mnx1 enhances progression of cervical cancerfigure mnx1 is upregulated in cc tissues and positively correlates with aggressive clinical characteristics a mnx1 is upregulated in cc tissues compared withadjacent normal tissues in tcga dataset p b patients with high expression of mnx1 have poor disease free survival dfs in cc p cd themrna expression of mnx1 in cervical cancer tissues was significantly higher than that in adjacent normal tissues in patients p e the mnx1staining score was upregulated compared with that in adjacent normal tissues p f the mnx1 staining score was positively correlated with tnm stage p g t stage p h lymph node metastasis p i tumor differentiation p and j local primary tumor diameter p incc patients k representative ihc staining images in tmas were shown error bars represent the mean ± sd values ns no signiï¬cance represents p frontiers in oncology wwwfrontiersinaugust volume 0czhu mnx1 enhances progression of cervical cancerfigure knockdown of mnx1 suppressed the proliferation migration and invasion in cc cells ab mnx1 mrna and protein level are upregulated in cc celllines cf two speciï¬c sirna si1 and si2 of mnx1 were designed and the transfection efï¬ciencies of sirnas in hela and siha cells were analyzed by qrtpcrand western blot gi the proliferation abilities were evaluated by xcelligence system assay edu incorporation assay and colony formation assay were inhibitedafter knockdown of mnx1 in hela and siha cells j the xcelligence system assay k transwell and matrigel assay and l wound healing assay indicated thatmigration and invasion capacities were suppressed after simnx1 in hela and siha cells error bars represent the mean ± sd values of three independentexperiments p p p ns no signiï¬cancefrontiers in oncology wwwfrontiersinaugust volume 0czhu mnx1 enhances progression of cervical cancerfigure ectopic expression of mnx1 enhanced aggressive abilities in c33a and hela cells ab the pcdna31mnx1 was synthesize and the transfectionefï¬ciencies were analyzed by qrtpcr and western blot the proliferation functions were measured by c the xcelligence system assay d colony formationassays and e edu incorporation assays were elevated in oemnx1 c33a and hela cells f the transwell assay and matrigel invasion assay g wound healingassay also showed that oemnx1 strengthened migration and invasion capacities error bars represent the mean ± sd values of three independent experiments p p p ns no signiï¬cancemnx1 suppressed the expression ofp21cip1 via binding to its promoter regionour previous results showed that knockdown or ectopicexpression of mnx1 altered the expression of p21cip1 to furtherverify the mechanism we analyzed the correlation betweenmnx1 and p21cip1 in cases of cc samples and the resultswere shown that mnx1 and p21cip1 had a negative correlationn p figure 5a as transcription factors usuallybind to sequencespeciï¬c dna to regulate transcription weutilized jaspar database to predict the binding site betweenmnx1 and the promoter region upstream bp of codingregion of cdkn1a the gene symbol of p21cip1 it turnedout that mnx1 was predicted to have four binding sites withthe promoter region of cdkn1a of which bpfrontiers in oncology wwwfrontiersinaugust volume 0czhu mnx1 enhances progression of cervical cancerfigure knockdown of mnx1 expression induced g2m phage arrest by regulating the p21cip1 expression a many genes were enriched in regulation oftranscription by go analysis most of the genes were enriched in the metabolic pathways by kegg and reactome pathway analysis b knockdown of mnx1generated g2m stage arrest in hela and siha cells were measured by ï¬ow cytometry cf the p21cip1 mrna levels were upregulated or downregulated after si oroemnx1 in cc cell lines de the protein level of p21cip1 was upregulated or downregulated while the expression of pthr161cdk1 was decreased or increased afterknockdown or ectopic mnx1 of cc cells the expression of cdk1 ccne1 ccnd1 and ccnb1 had no obvious changes error bars represent the mean ± sdvalues of three independent experiments p p p ns no signiï¬canceaacaataaat and bp gcccattaat showedhigher combination scores figure 5b accordingly the wildcdkn1a promoter region and mutant types 226mt and1371mt were generated and cloned into luciferase reportervector pgl3basic figure 5c and in luciferase reporterassay overexpression of mnx1 inhibited the transcriptionalactivity of the wild cdkn1a promoter but not mutant typefigure 5d moreover chip assay also revealed that mnx1bound to the p21cip1 promoter region in hela and sihacells figures 5effrontiers in oncology wwwfrontiersinaugust volume 0czhu mnx1 enhances progression of cervical cancerfigure mnx1 bounds to the p21cip1 promoter region and suppresses p21cip1 transcription a the expression of mnx1 and p21cip1 is negatively correlated in cervical cancer tissues p b the jarspar database indicates that mnx1 has several binding sites with the promoter region of p21cip1 c schematicdiagram shows that the two sites with the highest score of mnx1 on p21cip1 promoter and the mutant p21cip1 promoter were selected d overexpression of mnx1remarkably decreased wild type but not mutant p21cip1 promoter luciferase activity p21cip1226 p p21cip11371 p e chromatinimmunoprecipitation chip assays using normal igg or antimnx1 demonstrated that mnx1 directly binding to p21cip1 promoter region f the results of chippcrproduct electrophoresis were showed that a clear band was observed in the antimnx1 group while almost no band was detected in the igg control groupp p frontiers in oncology wwwfrontiersinaugust volume 0czhu mnx1 enhances progression of cervical cancerfigure downregulation of p21cip1 partially recovered the malignant phenotypes of simnx1 cells a the transfection efï¬ciency of p21cip1 was determined byqrtpcr and si1p21cip1 was chosen to further experiments bd the proliferative abilities were partially rescued after knockdown p21cip1 in simnx1 hela cellswere measured by the xcelligence system assay colony formation assay and edu incorporation assay ef the invasion and migration capacities have also beensignificantly improved after knockdown p21cip1 in simnx1 cells compared with simnx1 alone cells g the protein level of p21cip1 and pthr161cdk1 were partiallyreversed when knockdown of p21cip1 in simnx1 compared with simnx1 alone error bars represent the mean ± sd values of three independent experiments p p p ns no signiï¬cancefrontiers in oncology wwwfrontiersinaugust volume 0czhu mnx1 enhances progression of cervical cancerfigure knockdown or overexpression of mnx1 inhibited or promoted tumor growth in vivo ab the transfection efï¬ciency of shmnx1 was measured byqrtpcr and western blot c a total of eight nude female mice were sacriï¬ced and xenograft tumors were collected after injection with shmnx1 cells weeksde tumor volume and weight were reduced in the shmnx1 group compared with those in the shnc group f the expression of mnx1 and ki67 wasdownregulated and p21cip1 was upregulated in shmnx1 xenograft tumors analyzing by ihc staining g the protein level of mnx1 pthr161cdk1 weredownregulated and p21cip1 was upregulated in shmnx1 mouse xenograft tumors analyzed by western blot h a total of eight nude female mice were sacriï¬ced andxenograft tumors were collected after injection with oemnx1 cells weeks jk tumor volume and weight was increased in the oemnx1 group compared withthose in the oenc group i the expression of mnx1 and ki67 was upregulated and p21cip1 was downregulated in oemnx1 xenograft tumors analyzing by ihcstaining error bars represent the mean ± sd values p p p ns no signiï¬cancesilencing p21cip1 rescued the function ofsimnx1to determine whether the function of mnx1 was relied onp21cip1 we designed three sirnas table s3 to knockdownthe expression of p21cip1in hela cells the si1p21cip1showed the best transfection eï¬ciency figure 6a and it wasused for the following experiment rtca proliferation assaycolony formation assay edu assay transwell assay matrigelassay and would healing assay revealed that silencing p21cip1partially rescued the decreased proliferation migration andinvasion ability of hela cells caused by knockdown of mnx1figures 6bf and western blotting showed that the proteinlevel of p21cip1 and pthr161cdk1 were partially reversed bysilencing p21cip1 figure 6gmnx1 promoted tumor growth of cervicalcancer in vivothe xenograft models were used to explore the function ofmnx1 in vivo the shrnamnx1 shrnanc as control wastransfected into hela cells and the knockdown eï¬ciency wasconï¬rmed by qrtpcr and western blotting figures 7abresults showed that knockdown of mnx1 inhibited tumrowth measured by tumor weight and volume in vivofigures 7ce ihc staining and western blotting of harvestedtumors revealed that knockdown of mnx1 upregulated theprotein level of p21cip1 and downregulated ki67 and pthr161cdk1 in vivo figures 7fg moreover ectopic expression ofmnx1 promoted tumor growth and altered the expression ofp21cip1 and ki67 in vivo figures 7hkfrontiers in oncology wwwfrontiersinaugust volume 0czhu discussionin this study we identiï¬ed mnx1 a transcription factor ofhomeobox family was significantly upregulated and involvedin the progression of cervical cancer the overexpression ofmnx1 correlated with advanced clinical stages and poorerprognosis of cervical cancer patients furthermore mnx1exerted its oncogenic role via modulating the expression ofp21cip1 especially by targeting the promoter region of p21cip1thus to repress its transcriptionin accordance with ourï¬ndings a recent showed that mnx1 had a role in theprogression of cervical cancer partially through upregulating cellcycle regulators ccne1 and ccne2 and mnxas1 theantisense lncrna of mnx1 was also reported to promote theinvasion and metastasis of gastric cancer through repression ofcdkn1a all this results indicated that mnx1 played acritical role in cancer growth and cell cycle progression andmnx1 might serve as a useful diagnostic and treatment targetfor cervical cancermnx1is a member of homeobox gene family which allcontain a homeobox a dna sequence around basepairs long and encode homeodomain protein products astranscription factors this cluster of genes has beenidentiï¬ed to participate in the regulation of development andmorphogenesis in animals fungi and plants for examplecdx1 which is stably expressed in the human intestine playsan important role in embryonic epicardial development and the protagonist of our study mnx1 participates inmotor neuron development and neurodegenerative processesof zebraï¬sh and moreover controls cell fate choice inthe developing endocrine pancreas in recent years moreand more researches uncovered the role of developmentrelatedhomeobox genes in carcinogenesis and these genes show greatapplication prospect in tumor diagnosis and prevention asthe role of carcinoembryonic antigen cea in gastroenterictumors and alpha fetal protein afp in liver cancer for instance pdx1 is a key regulator in pancreatic developmentand cell function and meanwhile dynamically regulatespancreatic ductal adenocarcinoma initiation and maintenance hoxc13 a highly conserved transcription factor involvedin morphogenesis of all multicellular anisms is aberrantlyexpressed and associated with cancer progression in esophagealcancer lung adenocarcinoma and liposarcomas likewise mnx1 has been reported to promote sustainedproliferation in bladder cancer by upregulating ccne12 and to act as a novel oncogene in prostate cancer and in ourstudy mnx1 was also conï¬rmed to be upregulated in cervicalcancer and enhance the progression of cervical cancerin terms of mechanism we found that mnx1 promotedtumor growth of cervical cancer via accelerating the progressionof the cell cycle especially by modulating the expression ofp21cip1 cell cycle is a vital process by which a cellleadsto duplication and disorders of the cell cycle regulation maylead to tumor formation the cell cycle progress isdetermined by two types of regulatory factors cyclins and cyclindependent kinases cdks active cyclincdk complexesphosphorylate proteins to elevate the expression levels of cyclinsmnx1 enhances progression of cervical cancerand enzymes required for dna replication converselythe cell cycle progression can be prevented by inhibitors bybinding to and thus inactivating cyclincdk complexes suchas p21cip1 p27kip1 p16ink4a and so on the p21cip1also known as cyclindependent kinase inhibitor cdkn1ahas been identiï¬ed as a regulator of cell cycle and a tumorsuppressor in multiple kinds of cancers our results provedthat mnx1 repressed the transcription of p21cip1 by directlytargeting its promoter region and furthermore promoted thephosphorylation of downstream cdk1 the mnx1p21cip1pthr161cdk1 axis played crucial roles in the progression ofcervical cancer and meanwhile provided new evidence forthe pathogenesis of cervical cancer moreover the associationbetween cervical cancer and hpv has long been identiï¬ed as asexually transmitted agent hpv are involved in transformationand maintaining of transformed status many studies havereported that hpv can also alter the expression of p21 thus we searched the geo dataset to seek for information aboutthe relationship between mnx1 and hpv viral infection weanalyzed the gse dataset and found that there were nosignificant changes in the expression of mnx1 nm_005515 inhacat cells infected with hpv11e6 or hpv18e6 in gse3292gds1667 hpv positive or negative head and neck squamouscell carcinoma hnscc showed no expression diï¬erences ofmnx1 figure s1 this information suggests that mnx1 mightnot be directly involved in hpv carcinogenesis and furtherinvestigations are needed in the future in addition cervicalcancer i | Colon_Cancer |
" mutations in the wilms tumor gene cause a spectrum of podocytopathy ranging from diffusemesangial sclerosis to focal segmental glomerulosclerosis in a considerable fraction of patients with wilms tumor mutations the distinctive histology of immunecomplextype glomerulonephritis has been reported however theclinical relevance and etiologic mechanisms remain unknowncase presentation a 5yearold child presented with steroidresistant nephrotic range proteinuria initial renalbiopsy revealed predominant diffuse mesangial proliferation with a doublecontour and coexisting milder changesof focal segmental glomerulosclerosis immunofluorescence and electron microscopy revealed a fullhousepatterndeposition of immune complexes in the subendothelial and paramesangial areas serial biopsies at and years ofage revealed that more remarkable changes of focal segmental glomerulosclerosis had developed on top of theinitial proliferative glomerulonephritis identification of a de novo wilms tumor splice donorsite mutation in intron nm_0244266c1447 4c t and 46xygonadal dysgenesis led to the diagnosis of frasier syndromes our findings together with those of others point to the importance of heterogeneity inclinicopathological phenotypes caused by wilms tumor mutations and suggest that immunecomplexmediatedmembranoproliferative glomerulopathy should be considered as a histological variantkeywords focal segmental glomerulosclerosis frasier syndrome membranoproliferative glomerulonephritis wilmstumor mutations in wilms tumor wt1 gene cause severaldiseases characterized by renal and or genital anomaliessuch as denysdrash syndrome dds frasier syndromefs and isolated focal segmental glomerulosclerosisfsgs dds patients typically present earlyonset diffuse correspondence tsukaguhhirakatakmuacjp daisuke matsuoka and shunsuke noda contributed equally to this work7second department of internal medicine division of nephrology kansaimedical university shinmachi hirakata osaka japanfull list of author information is available at the end of the mesangial sclerosis dms a 46xy disorder of sex differentiation and wilms tumor wt fs patients tend toexhibit milder phenotypes with an onset at adolescenceincluding fsgs maletofemale sex reversal and gonadoblastoma but usually lack wt given the high incidence of wt and gonadoblastoma in dds and fsprophylactic gonadectomy and nephrectomies are recommended over of dds patients carry missensemutations in exons and whereas fs is commonlycaused by a splicedonor site mutation in intron 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 0cmatsuoka bmc nephrology page of wt1related nephropathy is generally ascribed to developmental defects in glomerular podocytes [ ] several patients with dds or fs display membranoproliferativeglomerulonephritis mpgn that is mainly characterizedby subendothelial immune deposits [] suggesting thatrenal pathologies resulting from wt1 mutations are complex and affected by multiple factors possible pathogenicmechanisms are discussed by reviewing the current literature here we present a case of a child with fs to thebest of our knowledge this is the first case report whererenal histological changes have been followed up for years before and after immunosuppressive therapy ourcase report should alert clinicians to consider the possibleexistence or wt1 mutations behind seemingly immunologic forms of mpgncase presentationa child was first diagnosed with proteinuria during aregular checkup at years of age but remained untreated until the age of years old when proteinuriareached the nephrotic range since the proteinuria didnot resolve after weeks of oral prednisolone mgkgday the child was referred to our hospital for furtherevaluationpregnancy and perinatal periods were uneventful withno family history of kidney disease physical examinationproteinuria ggrevealed no edema rash or arthralgia and the child hadnormal female external genitalia laboratory studies indicated nephrotic syndrome hypoalbuminemia gdl hyperlipidemia total cholesterol mgdl andmassivecreatinine withouthematuria bloodcell count renal function and serumcomplement c3 and c4 levels were all normal serological tests for hepatitis b and c and antinuclear antibodies were negative in the first renal biopsy at age see additional files and of glomerulidisplayed global mildtosevere mesangial proliferationwhereas others showed fsgs fig 1a in some glomerular tufts the capillary wall was irregularly thickenedwith a doublecontour configuration fig 1b foamcells had infiltrated focally around the tubular interstitium immunofluorescence revealed a coarsely granularfullhouse deposition pattern of positive for iggigm iga c3 and c1q along with trace c4 labelingin the mesangial and peripheral capillary loops electronmicroscopy revealed electrondense deposits in the subendothelial and paramesangial areas fig 1c the glomerular basement membranes gbm exhibited normalthickness and contour while focally showing subendothelial widening these histologic features were consistent withicglomerulonephritis with partial mpgn pattern whereasimmunecomplexendocapillaryfig representative light and electron microscopy images of the first and second biopsies a mesangial proliferation with segmental sclerosisarrowheads periodic acidschiff staining original magnification b doublecontour formation arrows periodic acid methenamine silverstaining original magnification c subendothelial asterisks and paramesangial arrowheads deposits electron micrograph originalmagnification ac images from the first biopsy at age d irregularities of the gbm asterisks with mesangial interposition asteriskselectron micrograph original magnification d image from the second biopsy at age gbm glomerular basement membrane 0cmatsuoka bmc nephrology page of minor fsgs changes were occasionally observed basedon pathological assessment intravenous methylprednisolone pulse intravenous cyclophosphamide cyclosporinea and mycophenolate mofetil were administered however the proteinuria was unresponsivethe second biopsy at age see additional files and following intravenous methylprednisolone pulseand cyclophosphamide showed remarkable attenuationof iga c3 and c1q depositions in mesangiocapillaryareas relative to the first biopsy irregular gbm thickening was more apparent with a doublecontour patterndue to mesangial interposition fig 1d along with astepwise increase in cyclosporine a dosage proteinuriagradually declined gg creatinine thereby achievingpartial remission the third biopsy at age see additional files and revealed coarse granular deposits ofigm and c31 at the capillary periphery suggesting macromolecule entrapment in sclerosing glomeruliand we observed significantly fewer foam cells aroundthe tubular interstitium throughout the clinical courseic depositions had ameliorated in response to immunosuppressive therapy however as the child aged glomerular capillary remodeling and podocyte injuries hadprogressed thereby causing the development of moreremarkable fsgs features onto the initial proliferativeglomerulopathybecause of the steroidresistant nephrotic syndromegenetic testing was recommended targeted sequencingfor known renal disease genes additional files detected a splicedonor site mutation in wt1 intron nm_0244266 c1447 4c t segregation analysis offamily members confirmed a de novo mutation fig subsequent gband analysis revealed a 46xy karyotypebilateral streak gonads were observed by magnetic resonance imaging confirming fs diagnosis the patientunderwent gonadectomy at age was diagnosed withgonadoblastoma and is currently treated with cyclosporine a and an angiotensin iireceptor blocker the proteinuria is now in the nephrotic range butrenalfunction remains normaldiscussion and sherein we presented a case of a child with fs withsteroidresistant nephrotic syndrome whose renal histology initially showed a predominant proliferative glomerulonephritis that later progressed into fsgs the firstrenal histology at age was consistent with mesangialproliferative glomerulonephritis with mpgn patternfig pedigree and sequencing analyses sanger sequencing of the wt1 exon and intron boundary in the affected individual and familymembers the affected child ii1 proband shown by arrow harbored a single nucleotide substitution in the canonic donor kts splice site of wt1intron ivs9 c1447 4c t refseq nm_0244266wt1 isoform d clinvar000003500 dbsnprs587776577 which was absent in family membersindicating a de novo mutation this variant has been reported elsewhere under alternate variant designations eg 4c t or ivs9 4c t wt1 wilms tumor suppressor gene 0cmatsuoka bmc nephrology page of diagnosis based on mesangial proliferation doublecontour gbm and fullhouse granular ic depositsalong the glomerular capillary loops as well as the paramesangial region the pathologic findings were indistinguishable from those commonly seen in lupus nephritisalthough our patient lacked serological abnormalitiesthe second and third biopsies following immunosuppressive therapy at ages and respectively revealedthat fsgs features ie focal segmental capillary obsolescence and tubule interstitialinfiltrationwere superimposed on the ic glomerulonephritis andhad become more apparent despite a partial responsepersistent steroidresistant nephrotic proteinuria incentivized us to conduct genetic testing thereby allowingthe diagnosis of fs caused by a typical splicedonor mutation in intron of wt1foam cellwt1 mutations can cause a broad spectrum of clinicaldiseases affecting urogenital development and sexual differentiation at variable severity and combinations mutational survey of wt1 in steroidresistant nephroticsyndrome cohorts [ ] revealed that an intron splicemutation typically causes fsgs with a gonadal tumorwhereas missense and truncating mutations result indms with nephroblastoma however morphologic abnormalities considerably vary in histologic appearanceamong individuals with ddsfs [] detailed analysisof the renal histology of dds individuals revealed complex glomerular changesincluding endotheliosislikeendothelial injuries footprocess fusion and gbm alterations previous studies report a significant fractionof ddsfs individuals including original and some familial cases display mpgn with ic deposition inaddition to fsgs or dms a histopathology commonlyseen in wt1related glomerulopathy [] tables and out of six dds cases four patients harbored aparg467trp nm_0244266c1399c t variant []the most common substitution present in of ddspatients and two harbored a nonsense parg463ternm_0244266c1387c t variant [ ] manifesting inan mpgn pattern moreover nine fs cases have beenreported including two monozygous twins harboring adonor splice site mutation in intron and initially presenting with mpgn [ ] in these cases ics comprisedof either fullhouse or combined igg c3 patterns weredeposited along glomerular capillaries glomerulopathyin dds usually manifests earlier and progresses fasterinto endstage renal disease relative to fs notablythere are dds cases initially presenting with thromboticmicroangiopathy or atypical hemolytic uremia syndromehus [] table it is not clear how the wt variant could facilitate huslike severe endothelial injuries cooccurrence of atypical hus with other glomerular diseaseseg fsgs has been reported suggesting that complement activation and podocytedysfunction may be related mechanistically however toaddress this hypothesis we need the description of further case reports as well as additional data collectionfrom experimental studies as noris suggested we should also take into account the possibility thatanother genetic abnormality or triggering environmentalfactor may be the main mpgn etiology in this case overthe wt1 glomerulopathy the mechanisms by which wt1 mutations causempgn have not been defined three factors might beimplicated in the pathogenesis of icmediated glomerulonephritis involving wt1 mutations the wtderivedprecipitating antigen promoting ic formation alteredimmune responses and increased vulnerability to endothelial injuries in structurally maldeveloped glomerular capillaries first mpgn is occasionally associated withmalignancy typically in lymphoproliferative disorders butalso in solid tumors ie lung colon and renal carcinoma[ ] in this context it is plausible that ics might formthrough aberrant immune responses against oncofetalandor nonautologous tumor antigens and trigger endothelial injuries of glomerular capillaries in wt patients clinicallyin most dds cases glomerulonephritisprecedes or manifests simultaneously with wt diagnosis[ ] tables and moreover nephrotic syndrome can persist even after complete excision of tumorswith no evidence of recurrence and metastasis [ ]however the existence of wtspecific circulating antibodies has not been well defined these observations indicate no convincing biological evidence linking wt tompgn thereby warranting further studysecond dysfunctional wt1 might be associated withleading to ic formationaberrant immune responses based on its role in the transcriptional regulation ofmultiple genesimplicated in the differentiation ofhematopoietic stem cells and apoptosis consistentwith our case clinical studies report the effectiveness ofcyclosporin in wt1 glomerulopathy with no reportof mpgn recurrence after renal transplant in patientsbearing wt1 mutations except for one dds patient which represents an unusual case of mpgn recurrencein the allografted kidney even after wt resection andsubsequent thorough immunosuppressive therapyan mpgn pattern resembling a glomerular morphology distinctive from classic podocytopathy fsgsdms is recognizable in some ddsfsgs cases by thedisrupted glomerular capillary integrity and as of yetundetermined predisposing factors for ic deposition indepth evaluation of glomerular histology in dds patientsand mouse models harboring parg467trp nm_0244266c1399c t demonstratescomplex disturbances in podocytes and endothelial cells as well asgbm maturation [ ] several studies of fs patientssuggest gbm alterations as the first histological changes 0cmatsuoka bmc nephrology page of 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liacgrusretfasemoctuolanernoisicxeryegatadrselatnedcniiryegatadrseyspotuanotwtayspotuanotwlatnedcniimegaymotcerhpenthgriangpmrosmdcggingpmngpmymotcerhpenthgriesuohllufngpmretfadehsinavcidetsisrepiairunetorpgnitaucriclymotcerhpenlaitraptfellatotthgriymotcerhpendetsisrepiairunetorpnoitadariavdrseavymotcerhpenthgrienummicinicymonitcaaenitsircnvivesaesidlaneregatsdnedrsengpmngpmsgsfyparehtromutlygootsihlaneriairunetorprosntwmtesnomtesnoepytonehpepytoyrakerutaretilromutsmliwhtiwemordnyshsardsynednisnoitatsefinamlanerfoyrammuselbatsitirhpenouremogllevitarefilorponarbmemngpmsisorelcsliagnasemesuffidsmdidenmretedtondnemordnyscitorhpensnromutsmliwtwirtadepjlatesgraepsybldetauaveersawesactneitapsihtrofsisorelcsouremoglllatnemgeslacofsgsflygootsihalxepmocsesacdetsilehtnidenifedtonerewsnoitatumtwnoiteedlpdetcepsusbailatinegleamefletepmocniailatinegsuougbmaiyxdn][ahsardailatinegsuougbmaiyx][sgraepsailatinegsuougbmaiailatinegsuougbmaileamefyxyxdn][mtttarrab][efyoccm][prenrohtiadirinabyx][eryllucs 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tsericnegorhpenraboartnliserutaefromutepytonehpmegataymotcedanogdnaymotcerhpenthbgxthrotcafltnemepmochfcemordnyscimerulcityomehlacipytasuhasuhcnorhcingpmmyteicosnoitairavemonegnamuhehtmorferutalcnemontnairavfonoitadnemmocerehtdna_mntwfotpircsnartdmrofositsegnolehtfoecneuqesecnereferehtnodesabnwohserastnairavtwehtlamotsabodanogbgemordnyscimerulcityomehsuhsitirhpenouremogllevitarefilorponarbmemngpmidenmretedtondnnoisnetrepyhthtnemtaertsfrhtpsadetroperylliangirosawsfrhtpehtprtgraptctccqesnoitangisedtnairavetanretlarehtodnaprtgrapdetroperylliangirosawtnairavprtgrapehtlebacilppatonanemordnyshsardsynedsddesaesidlaneregatsdnedrseemordnyscitorhpensnenegrosserppusromutsmliwtwiyhtapognaorcmcitobmorhtiroemordnysicmerulcityomehliacpytanagnitneserplyhtapouremoglldetaertwfoyrammuselbatserutaefrehtopuwollofegasisongadilaitinieelllahhfcthxtretfaecnerruceronxtretfaecnerruceronsuhasuhasuhalygootsihlanerdrseegaryryantesnomsnnoitatumtwepytonehpepytoyrakprtgrapxxleamefsfrhtpyxleamprtgrapyxleamdnyxleamsisongadisddsddsddsddacniillc][rjeitobrehs][ljegalerutaretil][cjlievnam 0cmatsuoka bmc nephrology page of that precede overt features of mpgn ic deposits andfsgs interstitial foam cells [ ]this case adds to the evidence of endothelial injuriesas essential components in the pathogenesis of wt1related glomerulopathy in addition to dms or fsgs pathologies icmediated mpgn should be considered as ahistological variant in patients harboring wt1 mutations early recognition of wt1 mutations allows forpersonalized choices ofimmunosuppressive reagentsand prevention of tumorigenesissupplementary informationsupplementary information accompanies this paper at httpsdoi101186s12882020020070additional file fig s1 renal histology of the first biopsy at age a b representative images of the first renal biopsy at age mostglomeruli appear grossly normal b arrows indicate foam cells in theinterstitium scale bar μm c higher magnification b showingfoam cell aggregation with a striped appearance arrowheads andsegmental sclerosis arrow there were no inflammatory or scleroticlesions in the interlobular artery asterisk scale bar d erepresentative images of glomeruli show mild mesangial proliferationwith gbm thickening arrows and d foam cells in the periglomerulartubular interstitium arrowhead e some glomeruli showed tuftadhesion and segmental sclerosis arrows scale bar μm periodicacidschiff stainingadditional file fig s2 immunofluorescence images of the first renalbiopsy at age immunofluorescence images of renal biopsy at age immunoglobulins igg igm and iga and complement proteins c3 andc1q were diffusely deposited along the capillary wall and expressed atsimilar levelsadditional file fig s3 electron micrographs of the first biopsy atage a representative images of electrondense deposits in the paramesangial area asterisks thickness and contour of the gbm appear generally normal while there was partial scalloping in the paramesangialregion scale bar μm b enlarged view of the boxed area in a someportion of the gbm was slightly thickened showing double layers ofdense matrix arrowheads scale bar μm c electrondense deposits inthe subendothelial and paramesangial spaces asterisks with occasionalthickening of the adjacent gbm in podocytes there were numerouscytoplasmic vacuoles and deformities including footprocess effacementand microvilli formation scale bar μm d enlarged view of the boxedarea in c the gbm appeared abnormally thickened with granular subendothelial electrondense deposits asterisks scale bar μmadditional file table s1 summary of serial immunofluorescencestudiesadditional file fig s4 immunofluorescence images of the secondrenal biopsy at age representative immunofluorescence images of thesecond renal biopsy at age dense igm deposition localized in themesangial area as well as in the periphery of glomerular capillariesforming a fringelike pattern arrowheads igg codeposited to alesser degree than iga c3 overall immunocomplex deposition wassignificantly lower than that found in the first biopsy indicating successful removal of ics by immunosuppressive therapyadditional file fig s5 histology of the second renal biopsy at age a b representative images of the second renal biopsy at age mostglomeruli showed an increase in mesangial matrices whereas somedisplayed segmental sclerosis arrows foam cells accumulated primarilyin the interstitium arrowheads a scale bar μm b scale bar μm c increases in mesangial matrix were more pronounced inperihilar regions arrow tubular atrophy and dilatation asterisk wereobserved in the interstitium adjacent to the sclerosing glomerulihistology resembled fsgs more closely despite the coexistence of somempgn characteristics scale bar μm d representative image ofglomeruli with global mesangial proliferation and aggregation of foamcells within the capillary lumen and bowmans space arrowheads someglomeruli exhibited tuft adhesion and segmental sclerosis arrow scalebar μm periodic acidschiff staining ac periodic acid methenaminesilver stainingadditional file fig s6 electron micrographs of the second biopsy atage ultrastructure of glomeruli after immunosuppressive therapy athe amount of capillary deposition decreased relative to that of the firstbiopsy however some deposits remained in the subendothelial arrowand subepithelial regions arrowheads double arrows indicate depositfree capillary wall scale bar μm b scalloping and irregular thickeningof the gbm observed along with electronlucent matrix expansion asterisks podocytes were deformed with cytoplasmic vacuolization footprocess effacement and microvilli formation scale bar μm c mesangial matrices and fragmented electrondense depositions asterisks increased in the paramesangial regions scale bar μm d the gbm wasabnormally thickened and partially split due to an accumulation of finegranular deposits in the subendothelial and subepithelial regions as wellas mesangial interposition scale bar μmadditional file fig s7 immunofluorescence images of the thirdrenal biopsy at age dense and diffuse igm deposition observedpredominantly along the glomerular capillary complement proteins c3c1q and c4 codeposited with igm in the tufts suggesting nonspecificentrapping of macromolecules due to a gradual loss of glomerular structural integrityadditional file fig s8 renal histology of the third biopsy at age representative images of the third renal biopsy at age an increasingfraction of glomeruli showed segmentaltoglobal sclerosis arrows andexpansion of interstitial fibrosis asterisks suggesting fsgs progressionfoam cells focally aggregated in the interstitium arrowheads howeveroverall cell density was significantly lower than in previous biopsies ascale bar μm b scale bar μm c arrows indicate the doublecontour in the glomerular capillary and the arrowhead indicates segmental luminal dilation and foam cell accumulation scale bar μm d eglomerulus with increased mesangial matrix and tuft adhesion arrowalong with foam cell accumulation in bowmans space and the capillarylumen arrowheads e double arrowheads indicate hyaline nodules inthe vascular pole scale bar μm the arteriole asterisk appeared normal ac periodic acid methenamine silver staining d e periodic acidschiff stainingadditional file table s2 a list of the genes included in the nextgeneration sequencing panel screeningadditional file care checklistabbreviationsdds denysdrash syndrome fs frasier syndrome fsgs focal segmentalglomerulosclerosis gbm glomerular basement membrane ic immunecomplex mpgn membranoproliferative glomerulonephritis wt wilmstumor wt1 wilms tumor acknowledgementsnot applicableauthors contributionsdm and sn made substantial contributions to the conception of this reportand clinical data collection dm drafted the manuscript hs evaluated thehistopathology kn ki and ht performed genetic studies and evaluated themutants dm sn mk yh yy and tm were actively involved in the clinicalcare of the patients ht reviewed the draft and made critical modificationsall authors read and approved the final manuscriptfundingnoneavailability of data and materialsall data generated or analyzed during this study are included in thispublished and its additional files 0cmatsuoka bmc nephrology page of membranoproliferative glomerulonephritis and secondary focal segmentalglomerulosclerosis arch pathol lab med no authors listed scully re case records of the massachusettsgeneral hospital weekly clinicopathological exercises case a yearold boy with aniridia and proteinuria years after nephrectomy for awilms' tumour n engl j med alge jl wenderfer se hicks j bekheirnia mr schady da kain js hemolytic uremic syndrome as the presenting manifestation of wt1mutation and denysdrash syndrome a case report bmc nephrol sherbotie jr van heyningen v axton r williamson k finn ls kaplan bshemolytic uremic syndrome associated with denysdrash syndrome pediatrnephrol manivel jc sibley rk dehner lp complete and incomplete drashsyndrome clinicopathologic study of five cases of a dysontogeneticneoplastic complex hum pathol noris m mele c remuzzi g podocyte dysfunction in atypical haemolyticuraemic syndrome nat rev nephrol sethi s fervenza fc membranoproliferative glomerulonephritisa new lookat an old entity n engl j med cambier jf ronco p onconephrology glomerular diseases with cancerclin j am soc nephrol wilm b mu±ozchapuli r the role of wt1 in embryonic development andnormal an homeostasis methods mol biol gellermann j stefanidis cj mitsioni a querfeld u successful treatment ofsteroidresistant nephrotic syndrome associated with wt1 mutationspediatr nephrol ratelade j arrondel c hamard g garbay s harvey s biebuyck n amurine model of denysdrash syndrome reveals novel transcriptionaltargets of wt1 in podocytes hum mol genet publishers notespringer nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliationsethics approval and consent to participatenot applicableconsent for publicationwritten informed consent was obtained from a family of the patient for thepublication of this case reportcompeting intereststhe authors declare that they have no competing interestsauthor details1department of pediatrics shinshu university school of medicinematsumoto japan 2department of pediatrics nagano red cross hospitalnagano japan 3center for medical genetics shinshu university hospitalmatsumoto japan 4department of pathology aizawa hospital matsumotojapan 5department of obstetrics and gynecology shinshu university schoolof medicine matsumoto japan 6department of pediatrics kobe universitygraduate school of medicine kobe japan 7second department of internalmedicine division of nephrology kansai medical university shinmachihirakata osaka japanreceived april accepted august referencesniaudet p gubler mc wt1 and glomerular diseases pediatr nephrol ruf rg schultheiss m lichtenberger a karle sm zalewski i mucha b prevalence of wt1 mutations in a large cohort of patients withsteroidresistant and steroidsensitive nephrotic syndrome kidney intchernin g vegawarner v schoeb ds heeringa sf ovunc b saisawat p genotypephenotype correlation in nephrotic syndrome caused bywt1 mutations clin j am soc nephrol neuhaus tj arnold w gaspert a hopfer h fischer a recurrence ofmembranoproliferative glomerulonephritis after renal transplantation indenysdrash pediatr nephrol karmila ab yap yc appadurai m oh l fazarina m abd ghani f focal segmental membranoproliferative glomerulonephritis a histologicalvariant of denysdrash syndrome fetal pediatr pathol schumacher va jeruschke s eitner f becker ju pitschke g ince y impaired glomerular maturation and lack of vegf165b in denysdrashsyndrome j am soc nephrol bockenhauer d van't hoff w chernin g heeringa sf sebire njmembranoproliferative glomerulonephritis associated with a mutation inwilms' tumor suppressor gene pediatr nephrol ito s hataya h ikeda m takata a kikuchi h hata j alport syndromelike basement membrane changes in frasier syndrome an electronmicroscopy study am j kidney dis aucella f bisceglia l de bonis p gigante m caridi g barbano g wt1mutations in nephrotic syndrome revisited high prevalence in young girlsassociations and renal phenotypes pediatr nephrol klamt b koziell a poulat f wieacker p scambler p berta p frasiersyndrome is caused by defective alternative splicing of wt1 leading to analtered ratio of wt1 kts splice isoforms hum mol genet frasier sd bashore ra mosier hd gonadoblastoma associated with puregonadal dysgenesis in monozygous twins j pediatr drash a sherman f hartmann wh blizzard rm a syndrome ofpseudohermaphroditism wilms' tumor hypertension and degenerativerenal disease j pediatr spear gs hyde tp gruppo ra slusser r pseudohermaphroditismglomerulonephritis with the nephrotic syndrome and wilms' tumor ininfancy j pediatr no authors listed barratt tm two children with kidney diseasedemonstrated at the royal college of physicians of london br med j mccoy fe jr franklin wa aronson aj spargo bh glomerulonephritisassociated with male pseudohermaphroditism and nephroblastoma am jsurg pathol thorner p mcgraw m weitzman s balfe jw klein m baumal r wilms'tumour and glomerular disease occurrence with features of 0c" | Colon_Cancer |
cancer stem cells cscs are important factors contributing to tumorigenesiswe examined whether cscs isolated from colorectal cancer crc cells possessmetastatic properties that can be transferred to noncscs via the deliveryof mir200c enclosed in extracellular vesicles evs the inhibitory effectof atractylenolide i atl1 a traditional chinese medicinal compound onmir200c activity and metastatic transfer was investigated evs were isolatedfrom colorectal cscs the expression of mir200c was evaluated in cscs andcscderived evs and horizontal transfer of metastatic properties via evs tononcscs was investigated in terms of cell behavior and phosphatidylinositol45bisphosphate 3kinase pi3kprotein kinase b aktmammalian target ofrapamycin mtor signaling cscs isolated from metastatic crc cells exhibitedhigher levels of mir200c than those in nonmetastatic crc cells overexpressionof mir200c in cscs enhanced metastatic potential by promoting proliferationand inhibiting apoptosis in turn leading to the release of evs carrying an excessof mir200c noncscs cocultured with mir200ccontaining evs exhibitedenhanced invasion and stemness maintenance associated with pi3kaktmtoractivation demonstrating successful metastatic transfer via ev delivery furthermore atl1 impaired the evmediated transfer of metastatic propertiesby suppressing mir200c activity and disrupting ev uptake by noncscsevs are critical signal transducers that facilitate intercellular communicationand exchange of metastatic properties which can be controlled by atl1 theabbreviations akt protein kinase b anova analysis of variance atl atractylenolide crc colorectal cancer csc cancer stem cell evextracellular vesicle fbs fetal bovine serum fitc fluorescein isothiocyanate mir microrna mtor mammalian target of rapamycin mtt345dimethylthiazol2yl25diphenyltetrazolium bromide oct4 octamerbinding transcription factor pbs phosphatebuffered saline pi3kphosphatidylinositol45bisphosphate 3kinase sd standard deviation sox9 sexdetermining region ybox tem transmission electron microscopythis is an open access under the terms of the creative commons attribution license which permits use distribution and reproduction in any medium provided theoriginal work is properly cited the authors clinical and translational medicine published by john wiley sons australia ltd on behalf of shanghai institute of clinical bioinformaticsclin transl med 202010e139101002ctm2139wileyonlinelibrarycom ctm2 of 0c of tang findings are useful in the development of micrornabased anticancer strategiesby targeting evmediated activity especially using natural compoundsk e y wo r d satractylenolide i extracellular vesicles metastasis pi3kaktmtor stemnessintroductioncolorectal cancer crc is one of the most prevalent cancers worldwide with an estimated new cases and deaths in in the united states alone1 mostcrcrelated deaths are caused by metastasis of malignant tumours2 and research on crc treatment has thusfocused on controlling the occurrence of metastasis thefactors that affect crc metastasis are manifold and it ischallenging to gain a comprehensive view of the mechanisms involved within micrornas mirs have beena recent highlight in diverse areas of medical researchincluding targeted cancer therapy because of their effectson a variety of biological responses such as oncogenesis and tumor metastasis the identification of specificmirs as oncomirs or tumor suppressors has advancedour knowledge of cancer development and contributedto the emergence of mirbased therapeutic schemes forexample targeting the oncogenic mir155 delayed tumrowth3 and a deliverable therapeutic formulation againstlung cancer has been developed based on the tumor suppressor mir344 interestingly mir200c has been identified as both an oncomir5 and a tumor suppressor6 in crcwith these contrasting reports it is necessary to furtherinvestigate the role of mir200c in crc development andmetastasisthe metastatic properties of mir200c have beendemonstrated to be transferrable via extracellular vesicles evs as carriers between tumor cells with differentmetastatic abilities7 evs are spherical ps that arecategorized based on their size and origin for examplethose with diameters of nm are known as exosomesand originate from endosomes whereas those with diameters of nm to µm are known as microvesicles oroncosomes in cancer cells originating from the plasmamembrane8 evs derived from cancer stem cells cscsreportedly mediate the crosstalk between cells via the horizontal transfer of tumorigenic factors between cells9 suchas oncogenes and proteins although cscs form only asubset of the cancer cell population they are believed tobe a key determinant of tumorigenesis and play important roles in regulating the tumor environment and metastasis the maintenance of stemlike properties in cscsis critical in promoting disease onset and thus studieshave focused on therapeutic means of disrupting or impairing the maintenance of stemness1011 whether the protumorigenic potential of cscs such as metastatic andstemlike traits can be conferred to nonmetastatic crccells via the transfer of mircontaining evs remains thusfar unknownaside from conventional anticancer schemes such assurgical intervention and chemotherapy natural herbaland plantbased compounds have gained attention as adjuvant or complementary therapies in cancer treatmentbaizhu or atractylodes macrocephala koidz is a dryperennial rhizome in the asteraceae family and has beenapplied as a typical component of many traditional chinese medical formulations1213 among the components ofbaizhu three forms of atractylenolides atls i ii andiii have been identified to exert pharmacological properties and atl1 is the main active ingredient that has exhibited therapeutic effects against crc1417 despite this studies on atl1 in contemporary cancer treatment are scarceas are those on its effects on cscs and the dynamics ofmircarrying evswe are interested in investigating whetherthemetastatic potential of crc cells can be amplifiedwhen they are cultured with cscderived evs carryingmir200c which is presumed to be oncogenic we alsoexamined whether atl1 possesses the ability to suppresscrc metastasis by exploring its effect on the transferof mir200c by evs and provide a speculation on themechanisms involved in its antitumorigenic actionmaterials and methodscell culture transfection andtreatmenttwo human crc cell lines highmetastasis lovo cellsand lowmetastasis ht29 cells were obtained from thecell bank of the chinese academy of sciences shanghai branch shanghai china lovo cells were culturedin f12k medium gibco waltham ma containing fetal bovine serum fbs gibcoand ht29 cells were cultured in mccoys 5a medium gibco containing fbs at ¦c in an 0ctang of atmosphere containing co2 to isolate cscs from lovoor ht29 cells denoted as lovocscs and ht29cscsrespectively four eppendorf tubes were prepared and µl of cell suspension cellsml were added to eachtube then µl of cd44 antibody ebioscience waltham ma was added to tube µl of epithelial cell adhesion molecule epcam antibody ebioscience was added to tube cd44 and epcam antibody µl of each were added to tube and no antibodywas added to tube empty label after min of incubation at ¦c the cells were subjected to flow cytometricsorting the isolated cscs were stored in sterile eppendorftubes until use to overexpress or silence mir200c mimics and inhibitors genepharma shanghai china weretransfected into crc cells or colorectal cscs transfectionwas performed by incubating cells with mir200c mimicsor inhibitors at nm for h for experiments involving atl1 treatment mg of atl1 purity ¥ hplcbatch no dst170606014 dsiter chengdu china wasprepared in µl of dimethyl sulfoxide to prepare a stocksolution with a concentration of approximately mmthe stock solution was then diluted in culture medium andadministered at µm for h phosphatebuffered salinepbs was used as a controlcscderived evsisolation and characterization ofevs were isolated from nontreated or transfected lovocscs or ht29cscs using the exoquicktc exosome precipitation solution for culture media spinal fluid andurine exotc50a1sbi system biosciences palo altoca the cells were cultured in fbsfree lowglucose dulbeccos modified eagle medium for h after which themedium was collected and centrifuged at g for min the supernatant was collected and centrifuged at g for min and the supernatant was collectedagain and ultracentrifuged at g for min theprecipitate was resuspended in ml of pbs and ultracentrifuged at g for min after which the precipitate was resuspended in pbs at a ratio of the mixturewas centrifuged at g for min and the supernatantwas subjected to sucrose density gradient purification ofevs after the gradient was ultracentrifuged at g for min the ev fraction ¼ sucrose was carefullycollected using a long pipette tip the collected fraction wasultracentrifuged at g for min and the resultingprecipitate containing isolated evs was collected all subsequent experiments involving coculture with evs exceptfor pkh labeling were performed with µgml evs for htransmission electron microscopytransmission electron microscopy tem was performedto identify the isolated evs the evs were fixed with glutaraldehyde in m pbs ph and the fixed evswere added dropwise to a treated nickel mesh for minafter the mesh was washed with pbs glutaraldehydewas added dropwise and incubated for min after whichthe mesh was washed several times with doubledistilledwater then filtered uranyl acetate was added to thesample dropwise and incubated for min excess liquidwas blotted with filter paper and the sample was dried themorphology of the evs was observed using tem345dimethylthiazol2yl25diphenyltetrazolium bromideassaycrc cells or colorectal cscs in the logarithmic growthphase were collected for 345dimethylthiazol2yl25diphenyltetrazolium bromide mtt assay the cells wereseeded in 96well plates at cellswell and culturedovernight at ¦c the cells were subjected to transfectionor atl1 treatment as described in section if applicable after or h of culture µl of mgmlmtt reagent pab180013 bioswamp wuhan china wasadded to each well and the cells were further cultured for h then the mtt solution was removed and µl ofdimethyl sulfoxide was added to each well the plate wasgently shaken for min and the absorbance of the wellswas measured using a plate reader at nmand invasiontranswell assay of cell migrationtranswell chambers corning inc corning ny wereplaced in the wells of a 24well plate and immersed inpbs for min before the experiment after cells were subjected to µgml ev andor µm atl1 treatmentfor h they were cultured in fbsfree medium for hfor the migration assay18 the cells were trypsinised resuspended in fbs and seeded into the upper transwellchambers at cellsml mlwell in the bottomtranswell chambers ml of medium containing fbs was added in each well the plates were incubatedat ¦c for h and the cells were fixed with ml of paraformaldehyde in each well for min at room temperature the fixative was removed and the cells were washedonce with pbs then ml of crystal violet solutionpab180004 bioswamp was added to each well and after 0c of tang min of staining the cells were washed three times withpbs cells that did not migrate were removed using a cotton swab and migrated cells were observed and countedat using an inverted microscope dm il led leicamicrosystems wetzlar germany the invasion assay wasperformed following the same procedure except that eachchamber was coated with µl of matrigel bdbiosciences franklin lakes nj at ¦c for min priorto cell seedingcell markers and apoptosisflow cytometry detection of stemthe purity of isolated cscs was evaluated by flow cytometry using cd44 and epcam19 cscs weresuspended in µl of flow cytometry buffer in aneppendorf tube and µl of fluorescein isothiocyanatefitcconjugated cd44 or phycoerythrinconjugatedepcam antibody was added to each tube the tubes wereincubated at ¦c for in the absence of light then thecells were washed with ml of buffer and centrifuged at g at ¦c for min and the supernatant was removedthe cells were resuspended in µl of buffer and subjected to flow cytometry using a cytoflex s apparatusbeckman coulter brea ca data were analyzed usingnovoexpress software acea biosciences inc san diegoca for apoptosis flow cytometry was performed usinga fitcannexinv apoptosis detection kit bdbiosciences h after crc cells or colorectal cscs weretreated with µm atl1 or subjected to transfectiontrypsinised cells were centrifuged at g for minand the supernatant was removed approximately cells were resuspended in pbs and centrifuged at g at ¦c for min the previous step was performed threetimes and the cells were resuspended in µl of bindingbuffer thereafter µl of annexin vfitc and µl ofpropidium iodide were added to the cells after min ofincubation at ¦c in the dark µl of binding bufferwas added and the cells were subjected to flow cytometrythe data were analyzed using novoexpress softwarepolymerase chain reactionquantitative reverse transcriptionstemloop quantitative reverse transcription polymerasechain reaction was performed to quantify the expressionof mir200c in crc cells colorectal cscs and cscderived evs after h of culture with µgml evsandor µm atl1 rna was extracted using trizol ambion inc foster city ca according to themanufacturers instructions and reverse transcriptionta b l e polymerase chain reactionprimer namemir200c stemloopmir200c forwardmir200c reverseu6 forwardu6 reverselist of primers for quantitative reverse transcriptionsequencectcaactggtgtcgtggagtcggcaattcagttgagtccatcatgggtaatactgccgggtaaactggtgtcgtggagtcggcctcgcttcggcagcacaaacgcttcacgaatttgcgtof the extracted rna into cdna was carried out usingthe advantage rtforpcr kit takara dalianchina quantitative polymerase chain reaction was conducted using the sybr green pcr reagent kit km4101kapabiosystems with the primers listed in table pcrproceeded as follows initial denaturation for min at¦c cycles of denaturation for s at ¦c annealingfor s at ¦c and extension for s at ¦c and finalextension for s at ¦c and s at ¦c data wereacquired using qbase plus software and analyzed by theδδct method western blotphosphatidylinositol45bisphosphateto confirm that ev isolation was carried out successfully western blot of exosomal markers cd63 cd81and tsg101 was performed in cscs cscderivedevs and the lysates of cells from which the evs wererelated to stemness maintenanceisolated proteinsand3kinasepi3kprotein kinase b aktmammalian target ofrapamycin mtor signaling were also evaluated bywestern blot after h of culture with µgml evsandor µm atl1 proteins were extracted fromcells or evs radioimmunoprecipitation assay bufferpab180006 bioswamp containing protease and phosphatase inhibitors was added to lyse the cells at ¦c thelysates were transferred to an eppendorf tube heatedfor min at ¦c and centrifuged at g for min the protein content in the supernatant was quantified using a bicinchoninic acid assay kit pab180007bioswamp for western blot µg of protein was subjected to sodium dodecyl sulfatepolyacrylamide gel electrophoresis the electrophoresed proteins were transferredto a polyvinylidene fluoride membrane ipvh00010millipore burlington ma and blocked using skimmilk for h at room temperature thereafter the membranes were incubated overnight at ¦c with rabbitprimary antibodies againstthe following proteinsoctamerbinding transcription factor oct4 0ctang of ab18976 abcam cambridge uksexdeterminingregion ybox sox9 ab185966 abcam nanog ab106465 abcam cd63 pab33929bioswamp cd81 pab33928 bioswamp tsg101 pab32949 bioswamp pi3k ab191606abcam phosphopi3k ppi3k ab182651abcam akt ab8805 abcam pakt ab38449 abcam mtor ab32028 abcampmtor ab84400 abcam and gapdh pab36264 bioswamp after three washes withpbstween for min each the membranes wereincubated with goat antirabbit igg secondary antibody sab43711 bioswamp for h at room temperature and washed again three times with pbs for mineach for visualization of the proteins the membraneswere incubated with an enhanced chemiluminescenceagent wbkls0010 millipore in the dark and proteinband gray values were analyzed using an automaticscanner tanon5200 tanon shanghai china the dataare presented as the ratio to the gray value of the controlgroup taken as a value of in arbitrary units aufluorescence labelingevaluation of ev uptake via pkh67evs were labeled following the instructions of the pkh67green fluorescent cell linker midi kit midi671ktsigmaaldrich st louis mo isolated evs were mixedwith ml of diluent and µl of pkh67 solution both provided in the kit and incubated for min then ml of bovine serum albumin in pbs was added for quenching the labeled evs were ultracentrifuged twice at g at ¦c for min and resuspended in µl of pbs toexamine ev uptake lovo or ht29 cells were seeded in a24well plate at cellswell and incubated at ¦cfor h in an atmosphere containing co2 then µg of pkh67labeled evs were added to each well and theplate was further incubated at ¦c in co2 for or h the cells were fixed for min with paraformaldehyde and the nuclei were stained with hoechst afterwhich they were observed under a leica dm il led fluorescence microscope using a objective lens for evuptake the microscopy imaging parameters were set atinitial acquisition and were kept constant between acquisitions green fluorescence represents pkh67 labelingof evsstatistical analysisstatistical analysis was performed using originpro thedata are expressed as the mean ± standard deviation sdof three technical replicates n differences betweentwo groups were compared using onesample ttest oneway analysis of variance anova followed by tukeyspost hoc test was conducted for comparisons between multiple groups more than two p indicates statisticalsignificanceresultsproperties of colorectal cscseffect of atl1 on the behavior andcolorectal cscs were successfully isolated from lovo andht29 cells as evidenced by the flow cytometric profilesof cd44 and epcam figures 1a and s1 which aremarkers of cscs15 we first evaluated the influence of µm atl1 on the behavior of colorectal cscs figures 1band 1c demonstrate that in cscs isolated from both lovohigh metastatic potential and ht29 low metastaticpotential cells proliferation was inhibited by atl1 forup to h atl1 also disrupted stemness maintenance inboth lovocscs figure 1d and ht29cscs figure 1eas revealed by the decreased protein expression of stemness indicators oct4 sox9 and nanog next themigration and invasion of cscs was investigated usingtranswell assays ht29cscs exhibited low metastaticpotential and their limited migratory and invasive abilitiesresulted in unsuccessful trials of transwell assay for thisreason only results for lovocscs are shown evidentlyatl1 at µm significantly decreased the migrationfigure 1f and invasion figure 1g of lovocscs theseresults are complemented by flow cytometry observations showing that the administration of atl1 inducedremarkable apoptosis in cscs compared to that in nontreated cells figure 1h corresponding flow cytometricprofiles of cell cycle progression in cscs are provided infigure s2relationship between mir200c andthe metastatic potential of colorectal cscsto elucidate whether there is a correlation betweenmir200c and the metastatic potential of crc cells andcolorectal cscs we detected the expression of mir200cin lovo and ht29 cells we anticipated that the highlymetastatic lovo cells would exhibit higher expression ofmir200c than ht29 cells and the results confirmed ourspeculation figure 2a the same trend was observedin the cscs derived from the respective crc cells wethen proceeded to compare the expression of mir200cin colorectal cscs with that in the crc cell line from 0c of tang f i g u r e characterization of colorectal cscs isolated from lovo and ht29 cells a flow cytometric sorting of cscs using cd44 andepcam as markers the percentage of parental lovo and ht29 cells expressing both cd44 and epcam was approximately representingthe proportion of cscs cscs isolated from parental lovo and ht29 cells denoted as lovocscs and ht29cscs respectively exhibited highexpression of both markers relative proliferation of b lovocscs and c ht29cscs was inhibited by atl1 at µm for up to h compared to that of control pbs cscs the protein expression of stemness markers oct4 sox9 and nanog relative to gapdh 0ctang of which they were isolated comparing the same numberof lovo or ht29 cells and lovocscs or ht29cscsthe cscs showed a clear increase in mir200c expressionfigure 2b administration of µm atl1 to cscssignificantly attenuated the expression of mir200c compared to that in nontreated cscs figure 2c from theseresults it is speculated that the high metastatic potentialof lovo cells may be associated with the expression ofmir200c in the csc subpopulationwe further investigated the role of mir200c in regulating crc cell behavior in particular proliferation andapoptosis lovo and ht29 cells were treated with mir200c mimics mir200cmim or inhibitors mir200cinh the transfection efficiency was excellent in lovocells wherein mir200cmim and mir200cinh significantly upregulated and downregulated the expressionof mir200c respectively however in ht29 cells theinhibitor demonstrated high efficiency whereas the efficiency of the mimic was suboptimal figure 2d aftermimic or inhibitor treatment crc apoptosis and cell proliferation were examined using flow cytometry and mttassay respectively in terms of apoptosis mir200cinhinduced an increase in the percentage of apoptotic lovoand ht29 cells figure 2e corresponding flow cytometric analysis of cell cycle progression is demonstrated in figure s3 in addition in both lovo figure 2f and ht29figure 2g cells proliferation was enhanced by mir200cmim but suppressed by mir200cinh together with theprevious results these findings suggest that mir200c confers crc cells with metastatic traits by promoting cellproliferation and inhibiting apoptosis subsequently weinvestigated whether mir200c overexpression and interference exert similar effects in colorectal cscs as theydid in crc cells lovocscs and ht29cscs were transfected with mir200cmimics or inhibitors and the transfection efficiency was validated figure 3a similar tothe crc cells flow cytometric analysis showed a markedincrease in apoptosis in both types of cscs transfected withmir200cinh figure 3b corresponding flow cytometric analysis of cell cycle progression in transfected cscsis demonstrated in figure s4 this was supported by themtt assay which revealed that the proliferation of bothtypes of cscs was enhanced by mir200cmim but inhibited by mir200cinh over a period of h figures 3cand 3d collectively the results demonstrate that transfection of mir200c mimics and inhibitors had a directeffect on csc behavior thus the effects of mir200cobserved previously in crc cells may be a result of changesin the properties of the colorectal csc subpopulationfound withinfrom mir200ctransfected colorectal cscscharacterization of evs derivedwe then explored whether the properties conferred bymir200c can be horizontally transferred between cellsvia a carrier agent to do this we isolated evs fromlovocscs and ht29cscs lovocscsevs and ht29cscsevs respectively western blot of exosomal markers cd63 cd81 and tsg10120 confirmed successful evisolation as these markers were exclusively expressed inevs and were almost nonexistent in the lysates of cscsfrom which the evs were derived figures 4a and 4bevs were also identified by tem figure 4c next weexamined whether the cscderived evs acted as a carrier for mirnas in particular mir200c we first confirmed that the basal level of mir200c was lower in ht29cscsevs than that in lovocscsevs figure 4d thencscs isolated from lovo or ht29 cells were transfectedwith mir200c mimics or inhibitors as described and evswere isolated from the transfected cscs evs from nontransfected cscs acted as controls we observed that mir200cmimtransfected lovocscs and ht29cscs produced evs that exhibited significantly elevated mir200cexpression than did the nontransfected cscs contrarilytransfection of mir200cinh in cscs resulted in the isolation of evs with attenuated mir200c expression compared to that in control evs figure 4e to determinewhether evs carrying mir200c mediated the horizontal transfer of metastatic traits we cultured lovo cellswith evs derived from either lovocscs or ht29cscstransfected with mir200c mimics or inhibitors lovo cellsshowed higher expression of mir200c when mir200coverexpressing evs were added whereas correspondinglyevs from mir200cinhtransfected cscs led to lowermir200c expression figure 4f the migratory and invasive capabilities of the cultured lovo cells were thenassessed using a transwell assay with evs from nontransfected cscs used as controls in the case of both lovocscs and ht29cscs the transfection of mir200cmimin the cscs resulted in evs that greatly enhanced thein d lovocscs and e ht29cscs was downregulated by atl1 at µm after h of culture compared to that of control pbscscs transwell assay of the f migration and g invasion of lovocscs demonstrated decreased cell counts in both cases when atl1 wasadministered at µm compared to those of control pbs cscs h the percentage of apoptotic lovocscs and ht29cscs was elevatedin the presence of atl1 at µm compared to that of control pbs cscs the data represent the mean ± sd of three independent technicalreplicates ttest p p at h 0c of tang f i g u r e mir200c expression in parental crc cells and colorectal cscs a relative basal expression of mir200c in lovo cells highmetastatic potential and ht29 cells low metastatic potential as well as isolated cscs lovocscs and ht29cscs demonstrates a possiblerelationship between mir200c and metastatic property b mir200c expression in lovo and ht29 cells relative to that of their correspondingcscs the same number of colorectal cscs clearly exhibited higher mir200c expression than did crc cells c mir200c expression wasattenuated by atl1 at µm in both lovocscs and ht29cscs d transfection efficiency of mir200c mimics mir200cmim inhibitorsmir200cinh and their corresponding negative controls mir200cmimnc and mir200cinhnc in lovo and ht29 cells mir200cmimand mir200cinh respectively induced significant upregulation and downregulation of mir200c in lovo and ht29 cells e the percentageof apoptotic lovo and ht29 cells was increased by mir200cinh but remained unchanged in the case of mir200cmim relative proliferationof f lovo and g ht29 cells subjected to transfection of mir200c mimics or inhibitors or their corresponding nc mir200cmim andmir200cinh respectively enhanced and inhibited the proliferation of both types of parental crc cells the data represent the mean ± sd ofthree independent technical replicates ttest or anova p p at hmigratory and invasive capabilities of lovo cells interference of mir200c on the other hand produced cscderived evs that limited the migration and invasion oflovo cells figures 4g and 4h we also examined whether µm atl1 had an inhibitory effect on migration andinvasion as it did on cell proliferation demonstrated previously as anticipated atl1 impaired the migration andinvasion of lovo cells in the presence of evs as confirmed by the significant decrease in cell count in thecase of transfected and nontransfected cscs figures 4gand 4hwith cscderived evscoculture of lovo and ht29 cellswe were interested in the specific effect of cscderivedevs on the stemness maintenance of noncscs and theinvolvement of mir200c therein lovo or ht29 cellswere cultured with evs derived from lovocscs or ht29cscs that were not transfected control or transfectedwith mir200c mimics or inhibitors and the expressionof stemness markers oct4 sox9 and nanog was evaluated compared to control evs from nontransfected 0ctang of f i g u r e transfection of mir200c mimicsinhibitors in colorectal cscs a transfection efficiency of mir200c mimics mir200cmim inhibitors mir200cinh and their corresponding negative controls mir200cmimnc and mir200cinhnc in lovocscs andht29cscs mir200cmim and mir200cinh respectively induced significant upregulation and downregulation of mir200c in both lovocscs and ht29cscs b the percentage of apoptotic lovocscs and ht29cscs was increased by mir200cinh but remained unchangedin the case of mir200cmim relative proliferation of c lovocscs and d ht29cscs subjected to transfection of mir200c mimics orinhibitors or their corresponding nc mir200cmim and mir200cinh respectively enhanced and inhibited the proliferation of both typesof colorectal cscs the data represent the mean ± sd of three independent technical replicates anova p p at hcscsthose isolated from mir200cmimtransfectedlovocscs and ht29cscs induced stronger expressionof stemness markers whereas evs isolated from mir200cinhtransfected cscs suppressed their expressionthis was the general trend observed in lovo cells cocultured with lovocscsevs figure 5a or ht29cscsevs figure 5b as well as in ht29 cells coculturedwith lovocscsevs figure 5c or ht29cscsevs figure 5d additionally we observed that the incorporation of µm atl1 in the coculture of crc cellsand evs had an inhibitory effect on stemness as demonstrated by the decrease in oct4 sox9 and nanog inall caseswe also investigated whether mir200c carried by isolated evs affected the activation of the pi3kaktmtorsignaling pathway in crc cells the same cocultures wereperformed and the expression of phosphorylated pi3kakt and mtor was detected relative to the respectivetotal protein content similar to stemness maintenancepi3kaktmtor activation was promoted in crc cells 0c of tang f i g u r e isolation and characterization of cscderived evs as a carrier of mir200c a expression of exosomal markers cd63 cd81and tsg101 was detected in lovocscs evs isolated from lovocscs and cell lysates after isolation b expression of exosomal markers cd63cd81 and tsg101 was detected in ht29cscs evs isolated from ht29cscs and cell lysates after isolation in both cases the lysates of thecscs showed negligible expression of these markers compared to that in cscs and cscderived evs c transmission electron microscopy ofevs derived from lovocscs scale bar nm d comparison of the relative basal expression of mir200c in evs derived from nontransfected lovocscs and ht29cscs lovocscsevs expressed higher levels of mir200c than did ht29cscsevs e expression of mir200cin evs derived from lovocscs or ht29cscs that were first transfected with mir200c mimics mir200cmim or inhibitors mir200cinhevs derived from nontransfected cscs are denoted as control mir200cmim and mir200cinh respectively induced significant upregulation and downregulation of mir200c in both lovocscsevs and ht29cscsevs relative to control levels f expression of mir200c inlovo cells cocultured with lovocscsevs and ht29cscsevs cscs were subjected to various transfections transfection of cscs withmir200cmim and mir200cinh resulted in the secretion of evs that respectively induced significant upregulation and downregulation ofmir200c in lovo cells relative to control levels g transwell assay of the migration and invasion of lovo cells cocultured with lovocscsevs cscs were subjected to various transfections with or without atl1 administration at µm relative to control levels migration andinvasion were enhanced by evs derived from mir200cmimtransfected cscs but suppressed by those derived from mir200cinhtransfectedcscs in all cases atl1 reduced the degree of migration and invasion scale bar µm h quantification of the data in f by cell countthe data represent the mean ± sd of three independent technical replicates ttest or anova p cocultured with evs from mir200cmimtransfectedcscs but decreased by mir200c interferencethis was the case for lovo cells cocultured with lovocscsevs figure 6a or ht29cscsevs figure 6b aswell as in ht29 cells cocultured with lovocscsevsfigure 6c or ht29cscsevs figure 6d the additionof µm atl1 induced a decrease in the phosphorylation of the abovementioned proteins signifying thatpi3kaktmtor activation was inhibited the resultssuggest that evs derived from cscs carried different 0ctang of f i g u r e effect of ev coculture on stemness maintenance in lovo and ht29 cells lovocscs and ht29cscs were first transfectedwith mir200c mimics mir200cmim or inhibitors mir200cinh lovo cells were cocultured for h with evs isolated from nontran | Colon_Cancer |
objective cannabinoids are able to reduce tumor growth in xenograft models but their therapeutic potential as anticancer drugs in humans is unclear yet in vitro studies of the effect of cannabinoids on cancer cells are often carried out in absence of serum or in low serum concentration ie serum conditions that limit cellular growth and therefore can increase the response of cells to additional challenges such as the presence of cannabinoids however the tumor microenvironment can be teaming with growth factors in this study we assessed the viability and proliferation of cancer cells treated with cannabidiol in presence of a serum concentration that commonly sustains cell growth serumresults the results show that cannabidiol exerts a markedly different effect on the viability of the human ht cancer cell line when cultured in presence of serum in comparison to serum displaying a cytotoxic effect only in the former situation in presence of serum no inhibitory effect of cannabidiol on dna replication of ht cells was detected and a weak inhibition was observed for other cancer cell lines these results indicate that the effect of cannabidiol is cell contextdependent being modulated by the presence of growth factorskeywords paclitaxel colon cancer cannabidiol serumintroductionthe cannabis plant has a therapeutic potential to treat a wide range of diseases including cancer phytocannabinoids are being tested in a0vitro and in a0vivo for the potential to fight different types of cancer cannabis extracts have recently been described to exert a cytotoxic effect on human cancer cell lines however in a0 vitro cancer models present limitations which reduce their predictive validity one of these limitations is to reproduce the nutritional environment of the cells using cell culture media and growth factors many in a0 vitro cancer studies use historical culture media with fetal calf serum fcs however it is usual correspondence albertosainzcgmailcom gh medical barcelona spainfull list of author information is available at the end of the to eliminate or reduce fcs concentrations ie fcs from the media at the moment of drug exposure to avoid confounding effects of growth factors present in serum as in many studies testing the cytotoxic properties of cannabinoids in cancer cells [ ]the deprivation of survival factors from the media can sensitize cells to a subsequent challenge pirkmajer and chibalin showed that the effects of serum starvation in cell cultures are unpredictable according to eastman serum should be kept in cell cultures to avoid both false positive and negative results due to its effects on cell proliferation stipulating the importance of replicating anic conditions to obtain clinically valid resultsin the present study we analyzed the viability response of different cancer cell lines to cannabidiol cbd in presence of a standard concentration of serum in comparison to a low serum concentration 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 0csainzcort a0et a0al bmc res notes page of main textmaterials and a0methodsmaterialscbd was supplied by schibano pharma ag waldsch¶nengrund switzerland mccoys 5a medium alamarblue® ab invitrogen were bought leibovitzs l15 medium l15 and rpmi and from thermofisher scientific barcelona spain paclitaxel ²6diamidino2phenylindole dapi dimethyl sulfoxide lglutamine penicillinstreptomycin and fcs were bought from sigmaaldrich madrid spain cell proliferation reagent wst1 and 5bromo2²deoxyuridine brdu cell proliferation elisa kit were bought from roche sigmaaldrich madrid spain paclitaxel was dissolved in dimethyl sulfoxide and cbd was dissolved in methanol at a0mm and kept at a0°c for a maximum of a0 months when needed paclitaxel and cbd were diluted conveniently in the cell media at the indicated final concentrations cellular controls without cbd or paclitaxel contained cell media without additivescell cultureht29 cells ref htb38 and sw480 cells ref ccl were obtained from american type culture collection ags cells were kindly provided by miguel a pujana catalan institute of oncology idibell barcelona spain and were originally obtained from nuria sala catalan institute of oncology idibell barcelona spain human colon cancer ht29 cells and sw480 cells were maintained in mccoys 5a and l15 media respectively human gastric cancer ags cells kindly provided by francesca mateo catalan institute of oncology bellvitge institute for biomedical research lhospitalet del llobregat spain were maintained in rpmi medium all of the media was supplemented with penicillinstreptomycin and a0nm lglutamine a0h before treatment cells were plated in 96well plates at cellswell a0 h later wells in triplicates received cbd and paclitaxel all assays with sw480 and ags cells included fcs while the assays using ht29 cells included either or fcscell viability and a0proliferation assaysfor the viability and proliferation assay based on resazurin and its redoxmediated reduction we used ab and measured the fluorescence of the wells using a plate readerfor the viability and proliferation assay based on cleavage of tetrazolium salts by mitochondrial dehydrogenase we used wst1for the proliferation based on the measurement of dna synthesis we added brdu to cells and detected its incorporation into dna following manufacturer instructionsto assess cell viability dapi was added to the cell suspension a0 min before the analysis by flow cytometry dapi emits higher fluorescence when bound to dna dapi enters rapidly through altered cell membranes allowing the detection of damaged cells the cell population was selected by gating in a forward scatter vs side scatter dot plot excluding aggregates and cell debris samples were analyzed using a gallios flow cytometerstatistical analysisdata was analysed using ibm spss statistics and real statistics using excelwe used shapirowilk test to assess data normality and nonparametrical independent samples kruskalwallis test to identify significant differences between each experimental condition we used dunn test as a posthoc analysis to identify which groups show statistically significant differencesresultsviability and a0proliferation of a0ht cells with a0serum deprivation fcswhen human colon cancer ht29 cells were incubated in media with serum adding cbd at a0µm reduced cell viability as assessed via the resazurin method which is based on evaluating mitochondrial reductive capacity fig a0 1a interestingly when cbd concentrations were a0 µm cell viability increased during the first a0 h differences between or and a0 µm were statistically significant p and p at a0h the increasing viability with cbd a0 µm disappeared while the blocking effect of a0µm cbd was more pronounced fig a0 1a this suggests that cbd can induce mitochondrial stress as reported by others looking at the morphology of cells the treatment with a0µm cbd led to changes in cell form such as massive cellular detachment cell rounding and presence of wrinkled cells characteristic of dead cells fig a0 1b in fact analyzing the presence of dead cells using dapi dye we found an increased percentage in samples incubated with a0 µm cbd when compared to control cells fig a01c thus the loss of mitochondrial activity observed at cbd a0 µm correlated with cell death of note at longer incubation times ie a0days massive cellular death was also observable at a0µm cbd data not shown in summary a0µm cbd shows cytotoxic activity on ht29 cells cultured in fcs 0csainzcort a0et a0al bmc res notes page of fig a ht cells were incubated with fcs and different concentrations of cbd for and h cell viability was assessed by incubation with ab the mean sd of three assays are shown b morphology of ht cells incubated with or without μm cbd for h representative images are shown bar µm c ht cell viability according to dapi staining see the materials and methods section ht cells were incubated without top or with μm cbd bottom for h stained with dapi and immediately analyzed by flow cytometry the cursor identifies dapipositive cells dead cells showing a higher percentage in cbdtreated cells a representative experiment c is shown p viability and a0proliferation of a0ht cells in a0 fcscontrary to the drop in viability of cells in fcs cbd did not inhibit the viability of ht29 cells even after a0days in media containing fcs fig a02a b an apparent increase in ht29 cell viability was observed at a0µm cbd as assessed by ab or wst1 fig a0 suggesting mitochondrial stress we sought to find whether in these conditions cbd could show additive or synergistic antiproliferative effects with the therapeutic drug paclitaxel paclitaxel partially decreased the viability of ht29 cells according to ab measurement but not wst1 thus cbd at a0µm does not grossly affect the viability of ht29 cells after a0days culture in presence of serumto ascertain whether cbd had any effect on proliferation of ht29 cells we measured the incorporation of brdu into dna no changes in dna synthesis were observed after a0days of incubation of ht29 cells with any concentration of cbd fig a02c although paclitaxel in itself did inhibit dna synthesis cbd did not increase the effect of paclitaxel fig a02c in summary cbd up to a0µm do not decrease the viability nor the proliferation of ht29 cells cultured in fcs none of these results showed statistically significant differencesviability and a0proliferation of a0sw480 and a0ags cellsto know whether other cancer cell lines behaved similarly to ht29 showing little or no response to cbd when cultured in fcs we used sw480 another colon cancer cell line and ags a gastric cancer cell lineags cells did not show changes of viability by incubation with cbd up to a0µm though a0nm paclitaxel did decrease their viability fig a0 3a higher paclitaxel concentrations resulted in a severe decrease of ags cells viability data not shown so we used a0nm paclitaxel to observe potential effects of cbd the viability of sw480 cells with cbd and fcs showed a trend to decline fig a03c surprisingly and contrary to ht29 cells a0µm cbd did actually impair dna replication in ags and sw480 cells fig a03b d in fact the inhibition of dna replication was additive to that produced by paclitaxel the assessment of dna replication in sw480 cells 0csainzcort a0et a0al bmc res notes page of showed significant differences between the control sample and a0µm cbd without paclitaxel p any other statistic analysis did not show significant resultsin summary in presence of fcs and during a0days of culture cbd does not affect the viability of ht29 sw480 and ags cells though cbd at a0µm does impair the proliferation of ags and sw480 cellsdiscussionin this study we investigated the effects of cbd and its combination with paclitaxel on the viability of three different cancer cells ht29 sw480 and ags under two different concentrations of serum a standard appropriate for cell growth for ht29 sw480 and ags and a restrictive one of for ht29 only for ht29 cells cbd only reduces cell viability under low fcs with no effects on viability or dna replication when cells were in fcs however for sw480 and ags dna replication was impaired under a0µm cbd with serum moreover the inhibition of dna replication in sw480 and ags cells by cbd and paclitaxel had an additive effectat low cbd concentrations ht29 cells showed a trend towards increased cell viability though the differences were not significant different concentrations of cbd have previously been shown to have opposing effects on cells thus a0µm cbd induces proliferation of t leukemia cells but at higher concentration kills the cells a low concentration cbd increases mitochondrial ca2 augmenting mitochondrial metabolism and cell growth but at high concentration it leads to fig ht cells were incubated for days with fcs and different concentrations of cbd in absence or presence of nm paclitaxel a the viability was assessed by incubation with ab the mean sd are shown n b the viability was assessed by incubation with wst the mean sd are shown n c before harvesting cells were incubated with brdu for h which incorporated into dna and dna synthesis was quantified the mean sd are indicated n fig ags cells and sw480 cells were incubated for days with different concentrations of cbd in absence or presence of nm paclitaxel ags or nm paclitaxel sw480 a c cell viability was assessed by incubation with ab the mean sd of three ags and six sw480 assays are shown b d before harvesting cells were incubated for h with brdu which incorporated into dna and dna synthesis was quantitated the mean sd of three assays ags and assays sw480 are shown p 0csainzcort a0et a0al bmc res notes page of excessive mitochondrial ca2 mitochondrial dysfunction and cell death appropriate culturing conditions are essential for the survival and growth of cells in many studies cell culture conditions are not sufficiently detailed which is essential for study replication one possible solution to address the potential effect of serum could be using culture media without fcs so the media does not need to be altered during drug exposition in any case neither higher serum concentrations nor lower serum concentrations represent the proper microenvironment of a cancer cell in the human body and both approaches could be valid to test the effects of a drug on cell lines the tumor microenvironment is enriched with metabolites including lactate and adenosine [ ] which increases tumor growth and may modulate the therapeutic effect of a drug in tumors that are highly glycolytic increasing mitochondrial activity as exerted by cbd may add metabolic stress to cells forcing them to decreased growth the effect of a drug on cells can be assessed effectively if the experimental conditions of the treatment are the same as the growing conditions before the treatment once growing conditions and treatment conditions differ from more than one variable drug treatment then the resulting effects cannot be associated only to the treatment but to the combination of variableslimitationsour results did not show statistically significant differences with the exception of the assessment of viability of ht29 cells under cbd treatment and the assessment of dna replication of sw480 under a0µm cbd the lack of statistically significant results could be due to the small sample size n for most of the assays our study was also not able to replicate the strongly inhibitory effect of cbd shown in other studies where cannabinoids were tested against cancer cells cultured with fcs fcs contains many growth factors and nutrients and differences in the fcs source could substantially modify the viability proliferation and differentiation of cultured cells there are also other studies where cancer cells were cultured with fcs and treated with cbd or other synthetic cbdlike molecules the results of these studies showed that cbd a0μgml reduced the viability of cancer cells and also had effects on other survival variables [ ] the cell lines used in these studies being different to the ones used in our study could account for the different results observedabbreviationsab alamarblue brdu bromo²deoxyuridine cbd cannabidiol dapi ²diamidinophenylindole fcs fetal calf serumacknowledgementswe would like to thank manuel reina for his expert adviceauthors contributionsschibano pharma ag participated in the idea of the study as and ee designed the study as and ee acquired analyzed and interpreted the data cm provided technical assistance and carried out some experiments as and ee drafted the work all authors read and approved the final manuscriptfundingthis study was partially funded by schibano pharma ag waldsch¶nengrund switzerland and gh medical barcelona spain the design of the study was prepared by as ee and cm and approved by schibano pharma ag and gh medicalavailability 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 participatenot applicableconsent for publicationnot applicablecompeting interestsas was employee at gh medical while performing this projectauthor details gh medical barcelona spain celltecub department of cell biology physiology and immunology faculty of biology university of barcelona av diagonal barcelona spain received may accepted august references ackermann t tardito s cell culture medium formulation and its implications in cancer metabolism trends cancer https doi101016jtreca n201905004 brand a singer k koehl ge kolitzus m schoenhammer g thiel a matos c bruss c klobuch s peter k kastenberger m bogdan c schleicher u mackensen a ullrich e fichtnerfeigl s kesselring r mack m ritter u schmid m blank c dettmer k oefner pj hoffmann p walenta s geissler ek pouyssegur j villunger a steven a seliger b schreml s haferkamp s kohl e karrer s berneburg m herr w muellerklieser w renner k kreutz m ldhaassociated lactic acid production blunts tumor immunosurveillance by t and nk cells cell metab https doi101016jcmet201608011eastman a improving anticancer drug development begins with cell culture misinformation perpetrated by the misuse of cytotoxicity assays oncotarget https doi1018632 oncot arget estrella v chen t lloyd m wojtkowiak j cornnell hh ibrahimhashim a bailey k balagurunathan y rothberg jm sloane bf johnson j gatenby ra gillies rj acidity generated by the tumor microenvironment drives local invasion can res https doi10115800085472canfantin vr stpierre j leder p attenuation of ldha expression uncovers a link between glycolysis mitochondrial physiology and tumor maintenance cancer cell https doi101016jccr200604023fisher t golan h schiby g prichen s smoum r moshe i peshesyaloz n castiel a waldman d gallily r mechoulam r toren a in vitro and in vivo 0csainzcort a0et a0al bmc res notes page of efficacy of nonpsychoactive cannabidiol in neuroblastoma curr oncol https doi103747co232893jeong s jo mj yun hk kim dy kim br kim jl park sh na yj jeong ya kim bg ashktorab h smoot dt 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k buta c cochrane b dirks wg fu j hickman jj hohensee c kolar r liebsch m pistollato f schulz m thieme d weber t wiest j winkler s gstraunthaler g fetal bovine serum fbs pastpresentfuture altex https doi1014573 altex wu hy huang ch lin yh wang cc jan tr cannabidiol induced apoptosis in human monocytes through mitochondrial permeability transition poremediated ros production free radical biol med https doi101016jfreer adbio med201806023publishers 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 |
colorectal cancer crc is one of the most common malignanttumors in china chen crc is one of the ï¬veleading causes of cancer death and its incidence is graduallyincreasing owing to obesity and lifestyle changes du chen postoperative treatments includingchemotherapy and radiotherapy are important for longer patientsurvival traditional chinese medicine tcm has become anoption for preventing crc metastasis and enhancing the eï¬ectsof chemotherapy shi xu xie tcm is used as an alternative or supplementary treatmentin the united states and europe and has been widely used totreat various diseases in asia especially in china wang tcm has also been widely investigated in asia for eï¬ectiveand lowtoxicity monomer compounds to develop new drugs forcancer therapy and to counteract drug resistance sui zheng xie in china patients usually choose tcm for adjuvant therapyafter curative resection xu the eï¬ectiveness oftcm has been proven in multiple cancers including breastcancer lee hepatocellular carcinoma chen pancreatic cancer kuo and crc shi xu in crc tcm significantlyimproved diseasefree survival in stage ii and iii crc in aretrospective cohort study including patients shi in a multicenter prospective cohort study including patients with stage ii and iii crc postoperative tcmtreatment was associated with better diseasefree survival andoverall survival compared to those of the untreated groupxu certain active ingredients in tcm herbsmay have stronger activity in inhibiting cell proliferation andpromoting cell apoptosis tan huang and hu for example bufalin an active component of the tcmchan su can reverse multidrug resistance by inhibiting theprotein expression and eï¬ux function of abcb1 yuan cinobufagin another cardiotonic steroid isolated fromchan su suppresses tumor neovascularization by disrupting theendothelial mtorhif1α pathway to trigger reactive oxygenspeciesmediated vascular endothelial cell apoptosis li of the frequently used tcm treatments the most eï¬ectivesingle herbs are ginseng radix ren shen hedyotis diï¬usa willdbai hua she she cao scutellaria barbata ban zhi lian andastragali radix huang qi lee wu however the underlying mechanisms of these remedies remainunknown network pharmacology can eï¬ciently and quicklyidentify the interactions between drugs and target proteinsproviding a foundation for tcm application zhang fufang yiliu yin fyy is a tcm formula that has beenused in clinical practice for cancer treatment our previousstudy found that fyy inhibited cell proliferation migration andinvasion and promoted apoptosis in hepatocellular carcinomayang fyy contains eight herbs astragali radixhuang qi ganoderma lucidum ling zhi semen armeniacaeamarum ku xing ren h diï¬usa willd bai hua she she caoaconiti lateralis radix praeparata fu zi glycyrrhiza glabralinne gan cao radix panacis quinquefolii xi yang shenfyy inhibits colorectal cancer progressionand platycodi radix jie geng of these herbs radix panacisquinquefolii ginseng radix h diï¬usa willd and astragaliradix are commonly used in anticancer formulas lee wu g lucidum and platycodi radix alsoreportedly have anticancer eï¬ects radix astragali jung g lucidum dai platycodi radix park andlee and h diï¬usa willd zhang inhibitcancer cell proliferation polysaccharides in g lucidum inhibitthe proliferation of crc cells upregulating the expression of p21protein and blocking cells at the g2m phase na in the current study we investigated the anticancer eï¬ectof fyy on crc cells in vitro and in vivo and a networkpharmacology analysis was performed to explore the potentialmolecular mechanisms the information obtained in this studywill aid in elucidating the previously unavailable mechanismsof action of fyy in crc and developing fyy as an adjuvanttherapy for crcmaterials and methodspreparation of fyy and cell culturethe components of fyy conformed to the provisions stated bythe chinese pharmacopoeia fyy was prepared at the weifanghospital of traditional chinese medicine shandong chinayang fyy mgml was stored at ¦c untiluse and was further diluted to the required concentrations insubsequent cell experiments human crc cell lines hct116and sw480 were purchased from the cell resource center of theshanghai institutes for biological sciences chinese academy ofsciences shanghai china hct116 cells were grown in rpmi medium rpmi1640 hyclone united states and sw480cells were grown in dulbeccos modiï¬ed eagles medium dmemhyclone united states containing fetal bovine serumfbs gibco brl united states and penicillinstreptomycinsigmaaldrich st louis mo united states in co2 at ¦cin a humidiï¬ed incubatorcell viability and colony formationassayscells per well were seeded into 96well plates andincubated overnight at ¦c co2 in a humidiï¬ed incubatorwhen the cells adhered to the wall hct116 and sw480 cellswere treated with or mgml of fyy or pbs asa control for and h cell viability was measured using acell counting kit8 cck8 beyotime institute of biotechnologyinc shanghai china ten microliters of the cck8 solutionwas added to each well and then samples were incubated at ¦cfor h finally the absorbance value at nm was determinedusing a multiskantm fc microplate photometer thermo fisherscientiï¬c inc united stateshct116 and sw480 cells were treated with or mgmlof fyy or pbs as a control for h the cells perwell were then cultured in sixwell plates and the medium waschanged every days for days cell colonies were ï¬xed with paraformaldehyde and then stained with giemsa beyotimeinstitute of biotechnology inc shanghai china for minfrontiers in cell and developmental biology wwwfrontiersinaugust volume 0cdong fyy inhibits colorectal cancer progressiona colony formation assay was performed to count viable colonies cells per colonycell cycle analyseshct116 and sw480 cells were treated with or mgmlfyy or pbs as a control for h the collected cells were ï¬xed in cold ethanol and stored at ¦c overnight the nextday the cells were washed twice with cold pbs then µlrnase a µgml and µl propidium iodide µgmlsigma aldrich st louis mo united states were added to eachsample and incubated for min in the dark measurements weretaken using a ï¬ow cytometer facscan bd biosciences bedfordma united states and the data were analyzed using flowjo software tree star inc ashland or united statescell apoptosis analysescell apoptosis was detected using an apoptosishoechst staining kit beyotime biotechnology shanghai chinasamples were ï¬xed with paraformaldehyde atroomtemperature for min and stained with mgml hoechst at room temperature for min then ï¬uorescencewas detected under an olympus ix50 microscope olympuscorp tokyo japan at magniï¬cation apoptotic cellswere identiï¬ed using an alexa fluor annexin vdead cellapoptosis kit invitrogentmmolecular probes eugene orunited states after centrifugation at g for min the celldensity was counted and diluted in annexinbinding buï¬erto obtain cellsml µl per assay cells were stainedwith µl of annexin vfitc and µl propidium iodide atroom temperature for min in the dark and then µl ofbinding buï¬er was added measurements were taken using a ï¬owcytometer and the data were analyzed using flowjo softwarenetwork pharmacologyactive fyy compounds were screened using the traditionalchinese medicine systems pharmacology database tcmsp1ru with the pharmacokinetic information retrievalï¬lter based on the tcmsp platform the oral bioavailabilityand druglikeness were set to ¥ and ¥ to obtainqualiï¬ed herbal compounds the chemical structures of thecompounds were drawn using chembiooï¬ce kerwin crc targets were predicted and screened using thegenecards database2 stelzer and omim platform3amberger and hamosh venny venny wasused to screen for common targets between fyy and diseaserelated targetsdrug compounddiseasetarget networks were built usingcytoscape v software shannon and themerge function was used to analyze the core compoundsprotein interaction networks of the common fyy and crcrelated targets were built using the string database platform1httptcmspwcomtcmspphp2httpswwwgenecards3httpswwwomim4httpbioinfogpcnbcsicestoolsvennywith medium conï¬dence and rejecting the target proteinindependent of the network szklarczyk gene ontology go analysis and kyoto encyclopediaof genes and genomeskegg pathway analysis wereperformed using metascape zhou enrichedgo terms and relevant pathways with pvalues wereselected for better prediction and veriï¬cation of the biologicalprocess and mechanismwestern blot analysisthe following primary antibodies obtained from cell signalingtechnology inc danvers ma united states were used inthe immunoblotting analysis pi3k p110α akt pan pakt ser473 bcl2 bclxl bax p21 cmyc andgapdh total proteins were extracted fromcells and tissues using ripa lysis buï¬er cwbio beijing chinaequal amounts of protein from each sample were separatedby sdspage electrophoresis and then transferred onto045µm pvdf membranes biorad laboratories herculesca united states subsequently the membranes were blockedwith milk in pbs plus tween pbst for minincubated with primary antibodies overnight at ¦c and thenincubated with goat antirabbit horseradish peroxidases abcamcambridge ma united states or goat antimousehorseradish peroxidases abcam cambridge ma united states for h at room temperature finallythe bandwas detected using an enhanced chemiluminescence reagentand visualized with a fusion fx7 system vilber lourmatfrance imagej software was used to calculate the intensity grayvalue of each protein band and gapdh served as a controlfor normalizationtumor xenografts in nude miceten male balbc nude mice weeks old ± gwere purchased from beijing vital river laboratory animaltechnology co ltd beijing china the mice were housedat ± ¦c under a 12h lightdark cycle with free accessto food and water all animal experiments were completedat the speciï¬cpathogenfree medical animal laboratory of theaï¬liated hospital of qingdao university and approved bythe animal ethics committee of the aï¬liated hospital ofqingdao university ahqu20180310a hct116 cells cells per tumor were subcutaneously injected into the rightarmpit of the nude mice seven days after tumor inoculationthe tumor size was measured using a vernier caliper andthe mice were divided into two groups the fyy treatmentgroup and a control group n mice per group thefyy group was intragastrically administered ml10 g bodyweight daily in a primary concentration of mgml thecontrol group was intragastrically administered an equivalentvolume of pbs tumor sizes were measured every daysand calculated using the following formula tumor volumemm3 length width2 the nude mice werekilled by cervical dislocation on day and the tumorswere excised weighed and photographed finallytumorfrontiers in cell and developmental biology wwwfrontiersinaugust volume 0cdong fyy inhibits colorectal cancer progressiontissue and liver tissue were stored in formalin or at¦c for subsequent immunohistochemistry or western blotanalyses respectivelyimmunohistochemistrytumor and liver tissues of the nude mice were ï¬xed with paraformaldehyde for h and then embedded in paraï¬nembedded paraï¬n sections were dewaxed in xylene andrehydrated in ethanol antigen retrieval was performed in m citrate buï¬er ph using a pressure cooker followed byincubation for min samples were then washed thrice with pbsand ï¬xed in ethanol for min ki67 antibody novuscolorado united states was stained with a streptavidinperoxidase detection kit zsgbbio beijing china accordingto the kit instructionsstatistical analysisdata analysis was performed using graphpad prism softwaresan diego ca united states all experimental data wereexpressed as the mean ± sd the statistical signiï¬cance of theresults was analyzed by oneway analysis of variance anovafor multiple group comparisons and students ttest for two groupcomparisons a value of p was considered statisticallysignificant all experiments were performed in triplicateresultsfyy inhibited proliferation and promotedapoptosis of crc cells in vitrofufang yiliu yin significantly inhibited the growth of hct116and sw480 cells in a dosedependent manner figure 1a thecolony formation assay showed that the number of the coloniesin the fyy group and mgml was lower than that of thecontrol group figure 1bcolony formation ability was significantly inhibited by mgml p and mgml p fyy forhct116 and for sw480 cells respectively the cell cycle analysisshowed no significant diï¬erence in the percentage of cells ins p for mgml and g2m phases p for mgml in hct116 however a significant increase in g0g1phase was found after treatment with increasing concentrationsof fyy p for mgml figure 2a in hct116similar results were obtained for sw480 cells fyy blockedcell cycle at the g0g1 phase in a concentrationdependentmanner fyy inhibited the expression of cmyc p for mgml and promoted the expression of p21 protein p for mgml figure 2b in hct116 similar results wereobserved in sw480 cells this indicated an inhibitory eï¬ect oncell proliferationcell apoptosis as shown by hoechst staining increased afterfyy treatment figure 3a flow cytometry analysis showedthat the early p for mgml and late apoptosisp for mgml of hct116 cells were significantlypromoted figure 3b by fyy treatment similar results wereobtained for sw480 cells figure 3bnetwork pharmacological analysis offyy targeting crca total of compounds from fyy were retrieved oralbioavailability ¥ and drug likeness ¥ from the tcmspdatabase supplementary table a total of genes related tothese compounds and genes related to crc were screenedout using venny figure 4a common targets wereobtained supplementary table data imported into cytoscape to construct compounddiseasetarget networks figure 4a showed that of the fyy compounds may aï¬ect disease targets the top core compounds were screened based on the topologicalproperties of degree as shown in table quercetin kaempferolluteolin betasitosterol isorhamnetin formononetin calycosinjaranol acacetin and naringenin were the top active fyyingredients against crc the other active compoundsare listed in supplementary table two networks wereconstructed for the top core compounds and the remaining active compounds figure 4a the proteinprotein interactionnetwork built using string software used to investigatethe mechanisms of fyy provided common targets aftersetting the conï¬dence level above figure 4b theprioritization of key targets was analyzed according to thedegree of the node exported from the string database andthe top ï¬ve targets were cyclind1 mapk8 egfr myc andesr1 figure 4cbiological function and pathwayenrichment of fyy on crcthe biologicalfunctions and signaling pathways from allcore targets were enriched the top biological enrichmentresults are shown in figure 4d fyy aï¬ected crc throughmultiple go biological processes including apoptotic signalingpathway response to steroid hormone and response to inanicsubstance kegg analysis results included cancer prostatecancer apoptosis and pi3kakt signaling pathwayswe further investigated how the fyy mechanism promotedapoptosis using rtpcr and western blot analysis of hct116and sw480 cells fyy inhibited the relative expression ofpi3k mrna p figure 5a fyy downregulated theexpression of pi3k pakt bcl2 and bclxl and upregulatedthe expression of bax p figures 5bc takentogether these data support the idea that fyy induces crccell apoptosis by modulating the pi3kakt pathway and bcl2family proteinsfyy inhibited tumor growth and cellproliferation in vivothe hct116 cell xenograft model used to investigate theantitumor eï¬ect of fyy showed that fyy significantlyinhibited tumor growth compared to the control figure 6athe average tumor volumesafter days oftreatmentwere ± mm3in the control group and ± mm3 in fyytreated group figure 6b whiletumor weights were ± and ± mgrespectively ki67 significantly decreased in the fyytreatedfrontiers in cell and developmental biology wwwfrontiersinaugust volume 0cdong fyy inhibits colorectal cancer progressionfigure fufang yiliu yin fyy inhibited colorectal cancer cell proliferation a cck8 assay indicated that fyy inhibited the proliferation of hct116 and sw480cells in a dose and timedependent manner after and h of treatment pbs was used for the control treatment n per group b colony formation abilitydecreased after treatment with different concentrations of fyy for both hct116 and sw480 n per group values are shown as the mean ± sd p p and p vs control group the pvalues were obtained using anovafrontiers in cell and developmental biology wwwfrontiersinaugust volume 0cdong fyy inhibits colorectal cancer progressionfigure fufang yiliu yin fyy significantly inhibited the colorectal cancer cell cycle a fyy significantly inhibited the cell cycle progress of hct116 and sw480arresting them at the g2m phase as shown by ï¬ow cytometry assay n per group b the expression of cmyc decreased and p21 increased with fyytreatment n per group values are shown as the mean ± sd p p and p vs control group the pvalues were obtained usinganovafrontiers in cell and developmental biology wwwfrontiersinaugust volume 0cdong fyy inhibits colorectal cancer progressionfigure fufang yiliu yin fyy promoted colorectal cancer cell apoptosis a hoechst staining analysis indicated that fyy promoted apoptosis includingchromatin condensation and nuclear fragmentation in hct116 and sw480 cells magniï¬cation b flow cytometry indicated that fyy promoted the earlyand late apoptosis of hct116 and sw480 cells n per group values are shown as the mean ± sd p p and p vs control groupthe pvalues were obtained using anovafrontiers in cell and developmental biology wwwfrontiersinaugust volume 0cdong fyy inhibits colorectal cancer progressionfigure network pharmacological analysis and biological functional enrichment analysis of fufang yiliu yin fyy a venn diagram showed common targetsof fyy in colorectal cancer crc compounddiseasetarget networks of fyy against crc b proteinprotein interactions identiï¬ed by string software c thepredicted key targets of fyy treatment of crc d go and kegg pathway enrichment analysesfrontiers in cell and developmental biology wwwfrontiersinaugust volume 0cdong fyy inhibits colorectal cancer progressiontable the top bioactive compounds of fufang yiliu yin are listed below according to the degree of similarity of the compounddiseasetarget networkspubchem cidmolecule namequercetinformulac15h10o7ob dlkaempferolc15h10o6luteolinc15h10o6degreestructurebetasitosterolc29h50oisorhamnetinc16h12o7formononetinc16h12o4calycosinc16h12o5jaranolc17h14o6acacetinc16h12o5naringeninc15h12o5glycyrolc21h18o67methoxy2methyl isoï¬avonec17h13no5continuedfrontiers in cell and developmental biology wwwfrontiersinaugust volume 0cdong table continuedpubchem cidmolecule name7omethylisomucronulatolformulac18h20o5ob dllupiwighteonec20h18o5glyasperin fc20h18o6fyy inhibits colorectal cancer progressiondegreestructureob oral bioavailability dl druglikenesscrc tumor xenograft group figure 6b the expression ofpi3k pakt bcl2 and bclxl followed the same trend asthe in vitro study results figure 6cdiscussionboth retrospective and prospective studies have proven theanticancer eï¬ects of tcm on crc shi xu here we reported the anticancer eï¬ect of the fyyformula which contains eight ingredients fyy significantlyinhibited cell proliferation and promoted crc cell apoptosisin vitro fyy also inhibited xenograft tumor growth in vivousing a network pharmacology analysis we found that fyymay act on crc through active compounds targeting crcrelated genes that regulate the apoptosis and pi3kaktsignaling pathwaysto better understand the complementary eï¬ects of fyyformula ingredients we retrieved a total of compounds fromthe tcmsp database supplementary table compounddiseasetarget networks showed that of the compoundsmay aï¬ect crcrelated targets by searching pubmed wethe top compounds table exhibit antifound thatcrc eï¬ects mainly by promoting apoptosis and inhibiting cellproliferation for example quercetin was mostly related toprotective eï¬ects against crc and is found in three of the eightremedies in fyy astragali radix huang qi h diï¬usa willdbai hua she she cao and g glabra linne gan cao quercetininhibits crc progression by promoting cell apoptosis andautophagy as well as inhibiting angiogenesis and inï¬ammationdarband quercetin induces apoptosis by inhibitingdiï¬erent signaling pathways including the mapkerk pi3kaktand nfκb signaling pathways zhang xavier it also inhibits the migration and invasion of crc cells viaregulating the tolllike receptor 4nfκb signaling pathway han further kaempferol induces crc cell apoptosischoi while isorhamnetin formononetin andnaringenin show anticancer eï¬ects by inhibiting cell proliferationli abaza the similarity of theeï¬ects provided by fyy compounds may provide a mutualenhancement eï¬ect but this must be further tested using singleor mixed compoundsfufang yiliu yin induced cell cycle arrest in crc cells at theg0g1 phase and promoted apoptosis in hct116 and sw480cells to explain the mechanism by which fyy inhibits cellproliferation and promotes apoptosis we performed proteinprotein interaction network kegg and go pathway analysesproteinprotein interaction network analysis indicated the topï¬ve targets were cyclind1 mapk8 egfr cmyc and esr1biological functional analysis indicated apoptosis and cancerrelated pathways including the pi3kakt signaling pathwaythen our experimental study conï¬rmed the activation ofthe pi3kakt pathway and bcl2 family proteins as well ascmyc expressiontraditional chinese medicine formulas reportedly inhibitcancer progression by diï¬erent signaling pathways a tcmformula jianpi jiedu inhibits crc tumorigenesis and metastasisvia the mtorhif1αvegf pathway peng another tcm formula huang qin ge gen tang enhances the ï¬uorouracil anticancer eï¬ect by regulating the e2f1ts pathwayliu the zhi zhen fang formula reverses multidrugresistance mediated by the hedgehog pathway in crc sui these formulas as well as fyy all contain astragaliradix huang qi h diï¬usa willd bai hua she she caog glabra linne gan cao and radix panacis quinquefolii xiyang shen however there have been no reports regarding theanticancer eï¬ect of tcm formulas acting through the apoptosisand pi3kakt pathways in crc figure in the current studywe found that fyy decreased the transcription and protein levelof pi3k figure and further inhibited the phosphorylationof akt in both the cells and tumor tissues figures accumulating evidence indicates that the pi3kakt pathwayplays an important role in tumor development pi3k can partiallyactivate akt at the thr308 or ser473 sites by inducing thetranslocation of akt to the cell membrane via phosphoinositidedependent kinase akt inhibition is usually indicated by afrontiers in cell and developmental biology wwwfrontiersinaugust volume 0cdong fyy inhibits colorectal cancer progressionfigure fufang yiliu yin fyy modulated the expression of the pi3kakt signaling pathway and bcl2 family proteins relative pi3k mrna expression wasaltered by fyy treatment in hct116 and sw480 cells a n per group expression levels of pi3k akt pakt bcl2 bclxl and bax were altered by fyytreatment in hct116 b and sw480 cells c n per group values are shown as the mean ± sd p p and p vs control groupthe pvalues were obtained using anovafrontiers in cell and developmental biology wwwfrontiersinaugust volume 0cdong fyy inhibits colorectal cancer progressionfigure fufang yiliu yin fyy inhibited tumor growth in vivo a subcutaneous xenograft tumors after days demonstrated that fyy inhibited xenograft tumrowth n per group b tumor volume was significantly smaller after days of fyy treatment n per group ihc analysis of ki67 expression infyytreated tumor and liver tissues magniï¬cation the pvalues were obtained using anova c protein expression levels of pi3k akt pakt bcl2bclxl and bax in tumor tissues n per group values are shown as the mean ± sd p and p vs control group the pvalues were obtainedusing students ttestdecrease in the pakt ser473 level and is mostly achievedby inhibiting pi3k using pi3kspeciï¬c inhibitors ly294002or wortmannin reener and marti the regulation ofpi3kakttranscription and protein expression by a tcmtreatment has been previously reported tcm interventiondecreased pakt levels following the concentration gradientof the tcm treatment while the total overall akt level wasunchanged gu zhao calycosina component of astragali radix reportedly inhibits crcproliferation through the erβmediated regulation of the igf1rand pi3kakt signaling pathways zhao quercetinkaempferol and rutin in h diï¬usa willd also exhibit anticancereï¬ects in crc by regulating the pi3kakt signaling pathwaycai frontiers in cell and developmental biology wwwfrontiersinaugust volume 0cdong fyy inhibits colorectal cancer progressionfigure schematic representation of the proposed pi3kakt signalinginduced cell cycle arrest and apoptosis triggered by fufang yiliu yin fyy by combiningthe network pharmacological analysis and our results we hypothesized that fyy activates the pi3kakt signaling pathway and modulates the expression of p21cmyc and bcl2 family proteins thereby inducing cell cycle arrest and apoptosiscellapoptosisandinhibited metastasiswe previously found that fyy inhibited cell proliferationofpromotedhepatocellular carcinoma yang fyy may havea similar eï¬ect on diï¬erent types of cancer although wedemonstrated both the anticancer eï¬ects of fyy and the actionmechanism by which it operates limitations of this study includethe following ï¬rst we did not investigate the antimetastaticeï¬ect of fyy on crc a migration and invasion assay andcrc liver metastasis model should be used to investigate thissecondfurther studies should investigate whether mutualenhancement eï¬ects exist between the applications of fyyand regular chemotherapy and also examine its eï¬ect ondrug resistancein conclusion our study ï¬ndings showed that fyy inhibitedproliferation and promoted apoptosis in crc cells by modulatingthe pi3kakt signaling pathway and bcl2 family proteins webelieve that fyy could be a promising adjuvant therapy for crcethics statementthe animal study was reviewed and approved by animalethics committee of the aï¬liated hospital of qingdaouniversity ahqu20180310a written informed consent wasobtained from the ownerstheiranimals in this studyfor the participation ofauthor contributionsbd and cz obtained funding conducted the research andprepared the manuscript zy and qj performed the experimentssz prepared and provided the fyy formula yw and hzperformed the network pharmacology analysis cs designed thestudy and interpreted the data all authors contributed to the and approved the submitted versiondata availability statementall data presented in thissupplementary materialstudy areincluded in thefundingthis work was supported by the china postdoctoral sciencefoundation grant numbers 2016m602098 and 2018m640615the taishan scholars program ofshandong provincefrontiers in cell and developmental biology wwwfrontiersinaugust volume 0cdong fyy inhibits colorectal cancer progressiongrant number the shandong higher educationyoung science and technology support program grant number2020kjl005 the qingdao postdoctoral science foundationgrant number and the national natural sciencefoundation of china grant number supplementary materialthe supplementary material for this can be found onlineat httpswwwfrontiersins103389fcell202000704fullsupplementarymaterialreferencesabaza m s orabi k y alquattan e and alattiyah r j growthinhibitory and chemosensitization eï¬ects of naringenin a natural ï¬avanonepuriï¬ed from thymus vulgaris on human breast and colorectal cancer cancercell int doi 101186s1293501501940amberger j s and hamosh a searching online mendelian inheritance inman omim a knowledgebase of human genes and genetic phenotypes currprotoc bioinformatics doi 101002cpbi27cai q lin j wei l 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tetrastigma hemsleyanum diels et gilg t hemsleyanum mostly known as san ye qing is a kind of folk plant because of its slow growth it usually takes years to meet the requirements of commercial medicinal materials so it is a precious perennial medicinal resource it mainly grows in the eastern central southern and south western provinces of china such as zhejiang jiangsu guangxi fujian and yunnan provinces peng and wang t hemsleyanum is known worldwide as sources of phytotherapeutics which have been used for the treatment of conditions related to inflammatory and immune response and been recorded based on clinical trials or the use of animal models xu as an edible plant the leaves of t hemsleyanum consumed as a functional tea or dietary supplement for its health benefits such as improving the immune system of the body sun while the aerial parts of t hemsleyanum developed as potential new traditional chinese medicine tcm preparations guo corresponding author ningbo research institute of zhejiang university ningbo zhejiang peoples republic of china email address px4142163com x peng 101016jjep2020113247 received may received in revised form july accepted august ofethnopharmacology2642021113247availableonline12august2020037887412020elsevierbvallrightsreserved 0ct ji abbreviations t hemsleyanum tetrastigma hemsleyanum diels et gilg tcm uplcesiqtofmsms ultra high performance liquid traditional chinese medicine chromatography tandem triple quadrupole time of flight mass spectrometry minimum inhibitory concentration glutathione malondialdehyde nuclear factorκb 5hydroxytryptamine norepinephrine dopamine prostaglandin e2 lipopolysaccharide tumor necrosis factoralpha interleukin1 beta interleukin mic gsh mda nfκb 5ht ne da pge2 mapk mitogenactivated protein kinase lps celegans caenorhabditis elegans tnfα il1 il6 il12p40 interleukin subunit p40 stnfr1 soluble tnf receptors il10 il1 il4 inos tlr4 md2 myd88 myeloid differentiation protein jnk gpt got alp sod interleukin interleukin interleukin inducible no synthase tolllike receptor myeloid differentiation factor2 cjun nterminal kinase glutamicpyruvic transaminase glutamicoxalacetic transaminase alkaline phosphatase superoxide dismutase and activities antiinflammatory the root tubers of t hemsleyanum are extensively used either alone or in combination with other herbal medicines in tcm clinics for the treatment of children with fever convulsion pneumonia asthma rheumatism hepatitis menstrual disorders scrofula and pharynx pain sun chen and guo therefore it was called as natural plant antibiotic according to its wide spectrum of prominent bactericidal in february t hemsleyanum was awarded as the new eight famous kinds of tcm in zhejiang province meant that it has become a key object of industrialization development of zhejiangs dominant large varieties of medicinal materials in covid19 broke out and has caused more than deaths in china and infection cases have been reported in more than countries hua shi xuan fei mixture approval number of zhejiang medicine z20200026000 which composed of t hemsleyanum has been approved by zhejiang provincial drug administration for clinical treatment of covid19 futhermore the modern pharmacological studies had shown that t hemsleyanum also had effects of antiinflammatory ji antioxidant hossain antivirus ding antitumor lin antipyretic yang and wang antihepatic injury ma et al immunomodulatory xu antibacterial chen hypoglycemic ru 2018ab etc numerous reports have demonstrated that the biological activities of t hemsleyanum are attributed to its many chemical components fu wang has reported isolated alkaloids from the aerial parts of t hemsleyanum wang ru extracted a novel polysaccharide tdgp3 from is mainly alanine aminotransferase aspartate aminotransferase hyaluronan laminin total bilirubin total protein interferongamma immunoglobulin a secretory immunoglobulin a epithelialmesenchymal transition alt ast ha ln tbili tp ifnÎ iga siga emt mmps matrix metalloproteinase timps matrixmetallo proteinase cytc cat gshpx glutathione peroxidase tregs tgf cox2 foxp3 pdl taoc ccl4 cef hvj vsv a f s1 s2 pef cff eaf baf cytochrome c catalase regulatory t cells transforming growth factor beta cyclooxygenase forkheadwinged helix transcription factor gene population doubling time total antioxidant capacity carbon tetrachloride chicken embryo fibroblast hemagglutinating virus of japan vesicular stomatitis virus alkalicontaining extract of t hemsleyanum ketonecontaining extract of t hemsleyanum crude extract of t hemsleyanum crude extract of t hemsleyanum petroleum ether extractions of t hemsleyanum ethanol extract chloroform extractions of t hemsleyanum ethanol extract ethyl acetate extractions of t hemsleyanum ethanol extract nbutanol extractions of t hemsleyanum ethanol extract t hemsleyanum with a molecular weight of da by enzymolysisultrasonic assisted extraction method ru 2019ab large amounts of flavonoids were found in leaves aerial parts and root tubers of t hemsleyanum xu 2014ab deng yu in addition t hemsleyanum also contains a variety of functional components such as anic acids hu phenolic acids liu minerals fan amino acids fu etc in recent years wild resources of t hemsleyanum have been overexploited and now are on the verge of extinction due to its multiple medicinal values coupled with the strict requirements of the growing environments in it was listed in the preferentially protected crop germplasm resources of zhejiang province based on our teams preliminary research peng peng 2016ab li we comprehensively summarized and analyzed the domestic and overseas research progress on traditional uses the bioactive components of t hemsleyanum pharmacological activities toxicology with the aim of providing guidance for indepth research and reference for its development and utilization materials and methods the available information about the traditional uses phytochemicals and pharmacological properties of t hemsleyanum was searched via web of science google scholar pubmed science direct china national knowledge infrastructure cnki and springer search using chinese or english as the retrieval languages the keywords used include t hemsleyanum root tubers of t hemsleyanum radix tetrastigma ofethnopharmacology26420211132472 0ct ji traditional uses phytochemistry bioactive components pharmacological activities toxicology and other related words all references were from experimental studies and published prior to april were reviewed all chemical structures were drawn using chemdraw pro software heatclearing were botanical characteristics t hemsleyanum is a perennial grass climbing vine with longitudinal ribs glabrous or sparsely pilose it is usually grown in a cool and humid environment and the main soil type is yellow soil or yellow brown soil with rich humus the optimum ph is between and the root tubers are thick spindle shaped or elliptical and single or several are connected into a string of beads generally cm long and cm in diameter fig the epidermis of the root tubers is tan and most of them are smooth a few of them have folds and lenticel like protuberances some of them have depressions in which there are residual tan roots hard and brittle with a flat and rough section the stem of t hemsleyanum is thin and weak with longitudinal rhombus rooting on the lower node palmate compound leaves alternate leaflets are lanceolate oblong or ovate lanceolate the leaflets are cm long and cm wide with a tapered tip and a wedgeshaped or round base the flowers of t hemsleyanum are small yellow green and ovate the flowering stage of t hemsleyanum ranges from april to june and the fruit phase is normally from august to november when the flower withered it will form a small green round fruit with the size of millet when it is mature the fruit will turn from green to red the berries are spherical and soft spherical traditional uses t hemsleyanum belonging to the family vitaceae was firstly recorded in ben cao gang mu ming dynasty ad the aliases of sanyeqing include shi hou zi shi bao zi shi lao shu lan shan hu lei dan zi po shi zhu tu jing wan sou jia feng san ye dui golden wire hanging gourd golden bell golden wire hanging potato etc the root tubers or whole grass of t hemsleyanum traditionally and ethnically used as a medicine for a long time it has been recorded in multiple hemsleyanum ancient books of tcm such as zhi wu ming shi tu kao qing dynasty wu jiangxi herbal medicine common folk herbal medicine in zhejiang all of these ancient works described the effects of toxicityremoving t dyspnearelieving promoting blood circulation and pain relief thus it can be applied to cure febrile convulsion pneumonia bronchitis pharyngitis sore throat acute and chronic hepatitis rheumatic arthralgia viral meningitis bruise eczema insect and snake bite poor joint flexure and extension irregular menstruation of women national compilation team of chinese herbal medicine in the tcm culture the properties of t hemsleyanum was described as bitter and acrid in taste cool in nature which recorded in dictionaries of traditional chinese medicine and zhong hua ben cao shanghai science and technology press the channel tropism was lung heart liver and kidney meridians decocting with water or mashing for external application are the traditional possess methods of t hemsleyanum considering its extensive traditional effects many prescriptions containing t hemsleyanum have been passed down from generation to generation and have been well supported and clarified by modern pharmacological studies excitingly it has reported that jinlian disinfection drink containing san ye qing combined with interferon can treat covid19 he jinqi tablet made up of san ye qing astragalus and ginsenoside was used to treat cases of malignant tumor cases were completely relieved cases were partially relieved the total effective rate was wei moreover zhonggan mixture including san ye qing could improve the quality of life and prolong the survival time of patients with stage iii primary liver cancer jiang and gong in addition it has been used in the treatment of common gynecological diseases such as blood avalanche and leucorrhea gao and it also has a good effect on measles complicated with pneumonia anal fissure chronic bronchitis and mosquito bites ji chemical compounds of themsleyanum the chemical constituents of t hemsleyanum have been widely investigated sun sun zeng xu 2014ab fu fan chen ding 2015a fig the aerial part a root tuber b and raw herb c of t hemsleyanum ofethnopharmacology26420211132473 0ct ji b ding a total of one hundred and fortytwo compounds have been isolated and identified from t hemsleyanum until now the information about compound name molecular weight compound formula detection method analysis sample is summarized in table flavonoids and their glycosides modern phytochemical studies have indicated that flavonoids are the representative and predominated class of constituents isolated from t hemsleyanum lin zhang table to date fiftyone flavonoids and their glycosides have been extracted and identified from t hemsleyanum in this series compounds quercetin orientin vitexin isorhamnetin apigenin and kaempferol are the main types of skeleton some of their analogues can be identified from hydroxy moiety on c3² and c4 on the b ring of flavonoid aglycone at present many modern analytical techniques have been used for qualitative and quantitative analysis of flavonoids among them ultra high performance liquid chromatography tandem triple quadrupole time of flight mass spectrometry uplcesiqtofms has become a powerful tool for identifying the complicated compounds due to its higher mass accuracy and resolution our team used uplcesiqtofms to identify chemical constituents from the aerial part of t hemsleyanum including flavonoids such as isoorientin quercetin kaempferol vitexin isovitexin kaempferol3glucoside etc sun according to the report liu total flavonoids of t hemsleyanum could protect the aged mice from acute lung injury through inhibiting the phosphorylation of mitogenactivated protein kinase mapk and nuclear factorκb nfκb in lung tissue moreover the flavonoids of t hemsleyanum had the activity of antilung cancer wei luteolin a flavonoid found in t hemsleyanum acted as an anticancer agent against various types of human malignancies such as lung breast glioblastoma prostate colon and pancreatic cancers muhammad it is certain that t hemsleyanum flavonoids give a new vision for researchers to explore clinical anticancer drugs polysaccharide saccharide is another important active ingredient extracted from t hemsleyanum shao polysaccharide has great potential in clinical application because of its unique pharmacological activity however due to the complex structure of polysaccharide it is difficult and special to determine and synthesize their structures guo table the prescriptions and traditional uses of t hemsleyanum in china prescriptions name qingteng fengshi qufengshi yaojiu main composition jiu traditional use t hemsleyanum parabarium chunianum tsiang zanthoxylum nitidum roxb dc t hemsleyanum deeringia amaranthoides lam merr blumea aromatica wall dc t hemsleyanum deeringia amaranthoides lam merr zanthoxylum nitidum roxb dc panax notoginseng burk fh chen t hemsleyanum gypsum lonicera japonica thunb houttuynia cordata thunb ophiopogon japonicus linn f kergawl t hemsleyanum t hemsleyanum lysimachia christinae hance imperata cylindrica citrus reticulata blanco t hemsleyanum ginsenoside astragalus propinquus schischkin t hemsleyanum nepeta cataria l lonicera japonica thunb saposhnikovia divaricata trucz schischk huatuo fengtongbao capsule sanyeqing gypsum decoction sanyeqing power zhonggan mixture jinqi tablet hua shi xuan fei mixture extracted the polysaccharides from roots of t hemsleyanum rtp1 rtp2 and rtp3 were successively found by protein precipitation and purification moreover further study indicated rtp31 was high purity polysaccharide with a molecular weight of kda and it is mainly composed of kinds of monosaccharides arabinose galacturonic acid galactose and fructose the proportion is and respectively ru 2018ab extracted a polysaccharide thp from t hemsleyanum with the average molecular weight estimated as kda the results of study on the composition of polysaccharide showed that it was mainly composed of rhamnose arabinose mannose glucose galactose with the molar ratio of in ru 2019ab successfully extracted polysaccharide thdp3 from t hemsleyanum with molecular weight of kda which consists of rhamnose arabinose mannose glucose and galactose with molar ratio of moreover tdgp3 mainly consists of 4αdgalap1 4dgalp1 and 4αdglcp1 residues as backbones and dmanp1 36dmanp1 and αdaraf1residues as branches phenolic acids phenolic acids refer to aromatic carboxylic acids with multiple phenolic groups substituted on one benzene ring as a secondary metabolite phenolic acids are widely found in many natural plants and have antiinflammatory antioxidant and lipid lowering effects twenty three phenolic acids no52 table have been reported in the aerial parts of t hemsleyanum such as caffeic acid chlorogenic acid 1ogalloyldglucose protocatechol glucoside epigallocatechin 1caffeoylquinic acid 3caffeoylquinic acid 4caffeoylquinic acid 5caffeoylquinic acid 1pcoumaroylquinic acid 4pcoumaroylquinic acid and 5pcoumaroylquinic acid there were twentyone phenolic acids in the root tuber of t hemsleyanum some of which were the same as aerial parts alkaloids alkaloids are a group of basic anic compounds containing nitrogen that exist in nature alkaloids are stored in small quantities in t hemsleyanum and the bioactivity investigations of those alkaloids are still rather rare wang fu extracted the aerial parts of t hemsleyanum with ethanol and then isolated ten alkaloids for the first time including seven indole alkaloids an amide a maleimide and treatment of joint pain wind cold dampness arthralgia treatment of arthralgia syndrome rheumarthritis rheumatoid arthritis scapulohumeral periarthritis treatment of arthralgia syndrome rheumarthritis rheumatoid arthritis scapulohumeral periarthritis joint pain muscular constricture treatment of infantile hyperpyretic convulsion treatment of blood avalanche leucorrhea treatment of liver cancer treatment of malignant tumor treatment of covid19 usage oral administration ml once times a day oral administration ml once times a day oral administration capsules once times a day references ministerial standard ministerial standard ministerial standard one dose a day decoct twice in water and take it times after mixing oral administration oral administration ml once times a day oral administration capsules once times a day oral administration ml once times a day xu gao jiang and gong wei zhejiang provincial drug administration ofethnopharmacology26420211132474 0ct ji detection mode analysis parts of sample reference aerial part root tuber aerial part root tuber aerial part root tuber root tuber aerial part root tuber root tuber root tuber aerial part aerial part aerial part aerial part aerial part aerial part aerial part aerial part aerial part aerial part aerial part aerial part root tuber root tuber root tuber aerial part root tuber root tuber aerial part aerial part aerial part aerial part aerial part aerial part aerial part root tuber aerial part root tuber root tuber aerial part root tuber root tuber aerial part root tuber aerial part root tuber aerial part root tuber aerial part aerial part root tuber aerial part root tuber aerial part root tuber root tuber root tuber root tuber root tuber root tuber root tuber aerial part root tuber aerial part root tuber aerial part root tuber aerial part root tuber aerial part root tuber aerial part aerial part aerial part aerial part aerial part aerial part aerial part aerial part aerial part root tuber aerial part aerial part aerial part aerial part root tuber root tuber aerial part root tuber aerial part root tuber aerial part root tuber sun sun zeng sun sun sun zeng zeng sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun zeng sun zeng sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun xu 2014b sun zeng sun zeng sun zeng zeng sun sun sun sun xu 2014b sun xu 2014b sun zeng sun xu 2014b sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun fu sun sun xu 2014b fan xu 2014b fan sun continued on next page uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms table chemical constituents isolated from the different parts of t hemsleyanum name flavonoids and their glycosides quercetin quercitrin quercetin3oglucoside quercetin3orutinoside quercetin3galactoside quercetin3oxylosylglucoside quercetin3oxylosylglucose7orhamnoside orientin orientin2²²orhamnoside isoorientin isoorientin2²²orhamnoside isoorientin cid0 ²²oxyloside vitexin vitexin2²²orhamnoside vitexin2²²oglucoside vitexin2²²oarabinoside isovitexin isovitexin2²²orhamnoside isovitexin2²²oxyloside isorhamnetin isorhamnetin3rutinoside isorhamnetin3pyranoarabinose7glucosylrhamnoside apigenin apigenin7rhamnoside apigenin8cxylosyl6cglucoside apigenin6cαlarabinose8cdglucose eriodictyol eriodictyolohexoside i eriodictyolohexoside ii luteolin luteolin6 8dichexoside catechin catechin glucopyranoside isomer epicatechin kaempferide kaempferol kaempferol3glucoside kaempferol3rutinoside kaempferol3sambubioside kaempferol3oneohesperidin kaempferol3orhamnoside kaempferol7orhamnose3oglucoside kaempferol3robinoside7rhamnoside kaempferol3rutinoside kaempferol3ocarfuran7orhamnosyl glucoside daidzein biochanin a procyanidin dimmer procyanidin b1 procyanidin b2 procyanidin trimer phenolic acids and derivatives gallic acid protocatechuic acid caffeic acid dihydroxybenzoic acid hexoside 1caffeoylquinic acid 3caffeoylquinic acid 4caffeoylquinic acid 5caffeoylquinic acid 1pcoumaroylquinic acid 4pcoumaroylquinic acid 5pcoumaroylquinic acid phydroxybenzaldehyde pcoumaric acid ferulic acid hexoside salicylic acid chlorogenic acid neochlorogenic acid cryptochlorogenic acid protocatechualdehyde uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms 1hnmr13cnmr ms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms ofethnopharmacology26420211132475 0ct ji table continued name salicin2benzoate trihydroxycinnamoylquinic acid isomer protocatechuic acid hexoside apiosylglucosyl 4hydroxybenzoate 1ogalloyldglucose protocatechol glucoside epigallocatechin vanillic acid1ofuran celery glucosyl ester protocatechuic acid1ofuran celery glucosyl ester methoxyphenol1ofuran glycosyloglucoside 2methoxy4methylbenzene1ofuracresyl glucoside oxyresveratrol dicaffeoylquinic acid 4hydroxycinnamic acid alkaloids indole indole3carboxylic acid indole3propanoic acid 5hydroxyindole3carboxaldehyde 5hydroxyindole3carboxylic acid 6hydroxy3 4dihydro1oxocarboline hippophamide 4hydroxycinnamide pyrrole3propanoic acid scid0 trolline fatty acids trihydroxy octadecadienoic acid trihydroxy octadecenoic acid dihydroxy octadecenoic acid 9hydroxy1012octadecadienoic acid 9hydroxy octadecatrienoic acid hydroxyoctadecenoic acid hydroxyoctadecatrienoic acid dihydroxyoctadecatrienoic acid dihydroartemisinin ethyl ether trihydroxy octadecadienoic acid isomer hydroxyoxooctadecatrienoic acid octadecenedioic acid dimeester stearic acid linolenic acid linoleic acid palmitic acid oleic acid anic acids and derivatives malic acid quinic acid citric acid azelaic acid oxalic acid galactonic acid gallic acid succinic acid fumaric acid propanoic acid terpenoids and steroids sitosterol daucosterol campesterol stigmasterol 6obenzoyl daucosterol ergosterol taraxerone taraxerol αamyrine pteroside z ganoderic acid h 3epipapyriferic acid oleanic acid saponins ginsenoside rh1 detection mode uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms 1hnmr lcms nmr uv ms nmr uv ms nmr uv ms nmr uv ms nmr uv ms nmr uv ms nmr uv ms nmr uv ms nmr uv ms nmr uv ms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms gcms tcl hnmr cnmr ms gcms gcms ir hnmr eims ir hnmr ms ir hnmr ms ir hnmr ms ir eims uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms hnmr cnmr ms analysis parts of sample root tuber root tuber root tuber root tuber aerial part aerial part root tuber aerial part root tuber root tuber root tuber root tuber root tuber root tuber root tuber root tuber aerial parts aerial parts aerial parts aerial parts aerial parts aerial parts aerial parts aerial parts aerial parts aerial parts root tuber aerial part root tuber aerial part root tuber root tuber aerial part root tuber aerial part aerial part aerial part aerial part aerial part aerial part aerial part aerial part root tuber root tuber aerial part root tuber aerial part root tuber aerial part root tuber aerial part root tuber aerial part root tuber aerial part root tuber aerial part root tuber aerial part aerial part aerial part aerial part aerial part root tuber aerial part root tuber root tuber root tuber root tuber root tuber root tuber aerial part aerial part aerial part aerial part root tuber root tuber root tuber root tuber reference sun sun sun sun sun sun zeng sun xu 2014b zeng zeng zeng zeng xu 2014b xu 2014b chen fu fu fu fu fu fu fu fu fu fu sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun chen ding sun sun guo ru ru ru ru sun sun sun ding uplcesiqtofmsms root tuber sun continued on next page ofethnopharmacology26420211132476 0ct ji table continued name ginsenoside rh2 vinaginsenoside r1 amino acid and derivatives phenylalanine pyroglutamic acid glutimic acid hexose tryptophan lglutamic acid detection mode uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms analysis parts of sample root tuber root tuber root tuber aerial part aerial part aerial part aerial part aerial part reference sun sun sun sun sun sun sun respectively a carboline by comparing with the spectral data of known compounds the alkaloids were indole3carboxylic acid indole3propanoic acid 5hydroxyindole3carboxaldehyde 5hydroxyindole3carboxylic acid 6hydroxy3 4dihydro1oxocarboline hippophamide 4hydroxycinnamide pyrrole3propanoic acid and scid0 trolline the chemical structures were shown in fig identified as indole anic acids and derivatives the biologically essential anic acids have been isolated and characterized from t hemsleyanum as well ten anic acids and seventeen fatty acids were identified from the aerial parts and root tuber of t hemsleyanum most of which were found in the aerial parts except stearic acid propanoic acid and dihydroxy octadecenoic acid all the anic acids and fatty acids are listed in no112 and no95 of table respectively terpenoids and steroids terpenoids and steroids are other kinds of secondary metabolites of t hemsleyanum thirteen of these compounds have been isolated and identified no122 table liu yang liu isolated and identified αamyrine sitosterol ergosterol taraxerone taraxerol from the aerial part of t hemsleyanum in addition daucosterol campesterol stigmasterol 6obenzoyldaucosterol pteroside z ganoderic acid h 3epipapyriferic acid and oleanic acid were successively separated tuber roots of t hemsleyanum liu and yang from the inanic elements the mineral elements of tcm are indispensable supplements to the bioactive components which are closely related to the efficacy toxicity and side effects of tcm wu wu et al demonstrated that t hemsleyanum contained twentyseven different mineral elements namely li be na mg al k ca v cr mn fe co ni cu zn ga as se rb ag cd cs ba hg ti pb u moreover ca cu ni ba al k have higher loading values which are the characteristic elements of t hemsleyanum wang wang has indicated that the contents of fe mn zn and cu in three populations of t hemsleyanum cultivated in different environments were mg kgcid0 respectively pharmacology the ethnomedical uses of t hemsleyanum have stimulated various pharmacological studies on it the extracts and isolated compounds from t hemsleyanum showed a variety of bioactivities such as antiviral antibacterial antioxidant antipyretic analgesic hepatoprotective immunoregulatory and antitumor activity the detailed pharmacological activities of t hemsleyanum were presented in table and summarized as follows antiviral activity according to yangs literatures yang the nitrogenous alkalicontaining extract a ketonecontaining extract f crude extract s1 and crude extract s2 of t hemsleyanum had different antiviral effect on mice and chicken embryo fibroblast cef infected with hemagglutinating virus of japan hvj influenza virus pr6 vesicular stomatitis virus vsv specifically s2 strongly inhibited the proliferation of influenza virus pr6 with at the concentration of mgml and mgml s1 has obvious antiviral effect on hvj at the concentrations of mgml and mgml both f and s1 displayed a strong suppressive effect on the plaque formation of vsv in vivo a f s1 s2 have different degrees of antiviral activity when the concentration of a was gkg the protective rate was up to and that of s1 gkg was however the author did not give the sample preparation method ding had demonstrated compounds quercetin3orutinoside kaempferol kaempferol3glucoside quercitrin quercetin kaempferol3orutinoside procyanidin dimmer and epicatechin which were isolated from t hemsleyanum were positively related to the activity of t hemsleyanum against h1n1 influenza virus the ethyl acetate extracts of t hemsleyanum have been shown to obviously restrain the secretion of hbsag and hbeag released by hbv with the ic50 values of mgl however the specific mechanism of action needs to be further confirmed yang and wu wang had proved that the nbutanol and ethyl acetate extraction of t hemsleyanum had antiviral activity against rsv and were superior to ribavirin with the ec50 values of mgl wang moreover the t hemsleyanum extracts had different degrees of inhibition to different hiv1 strains the ec50 values were between μgml and μgml and the | Colon_Cancer |
" a critical barrier to improving the quality of endoflife eol cancer care is our lack of understanding of themechanisms underlying variation in eol treatment intensity this study aims to fill this gap by identifying anizational and provider practice norms at major us cancer centers and how these norms influence providerdecision making heuristics and patient expectations for eol care particularly for minority patients with advanced cancermethods this is a multicenter qualitative case study at six national comprehensive cancer network nccn andnational cancer institute nci comprehensive cancer centers we will theoretically sample centers based upon nationalquality forum nqf endorsed eol quality metrics and demographics to ensure heterogeneity in eol intensity andregion a multidisciplinary team of clinician and nonclinician researchers will conduct direct observations semistructuredinterviews and artifact collection participants will include cancer center and clinical service line administrators providers from medical surgical and radiation oncology palliative or supportive care intensive care hospital medicineand emergency medicine who see patients with cancer and have high clinical practice volume or high local influenceprovider interviews and observations and adult patients with metastatic solid tumors and whom the providerwould not be surprised if they died in the next months and their caregivers patient and caregiver interviewsleadership interviews will probe about eol institutional norms and anization we will observe inpatient andoutpatient care for two weeks provider interviews will use vignettes to probe explicit and implicit motivations fortreatment choices semistructured interviews with patients near eol or their family members and caregivers willexplore past current and future decisions related to their cancer care we will import transcribed field notes andinterviews into dedoose software for qualitative data management and analysis and we will develop and apply adeductive and inductive codebook to the datadiscussion this study aims to improve our understanding of anizational and provider practice norms pertinent toeol care in us cancer centers this research will ultimately be used to inform a provideroriented intervention toimprove eol care for racial and ethnic minority patients with advanced cancertrial registration clinicaltrialsgov nct03780816 december keywords endoflife norms heuristics cancer minority health correspondence amberbarnatodartmouthedu2the dartmouth institute for health policy and clinical practice geisel schoolof medicine dartmouth college lebanon nh usa5department of medicine geisel school of medicine hanover nh usafull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cknutzen bmc palliative care page of the national academy of medicine has identified increasingly aggressive burdensome and expensive endoflife eol treatment as a major public health problem the american society of clinical oncology andnational quality forum nqf define aggressive burdensome and expensive eol treatment in cancer as thereceipt of chemotherapy in the last days of life nqf intensive care unit icu admission in the last days of life nqf and non nqf orlate nqf hospice referral such treatmentadversely affects patient quality of life quality of dyingand caregiver bereavement outcomes [] minoritiesare more likely to receive such eol treatment []potentially in disproportion to their preferences [ ]despite attention focused upon integrating early palliative care into advanced cancer treatment [] icuadmission in the last days of life and late hospicereferral have been secularly increasing yet not allanizations are equal cancer centers vary by morethan twofold in these eol intensity measures [ ]these variations cannot be explained by structural characteristics or casemix since centers serving ahigher proportion of minority patients have systematically higher eol intensity than centers serving a higherproportion of white patients these variations inpractice patterns may contribute to racial disparities inburdensome treatment near death moreover despiteefforts to attribute such variation to differences in patient preferences rather regionlevel analyses suggest that the impact of thesepreferences on variation are likely very small [ ]than racial disparitiesin local anizational and providerwe posit that a critical barrier to improving thequality of eol cancer care in the us and amongminorities in particular is our lack of understandingregarding the mechanisms underlying cancer centervariation in eol treatment intensity the overarching hypothesis driving this study is that differencessocialnorms rules about which there is at least some degree of consensus enforced through social sanctions are a key mechanism underlying this variationwe base this hypothesis on our preliminary work attwo us academic medical center hospitals at oppositeextremes of eol treatmentintensity demonstratingmarked differencesicu and lifesustaining treatment decision making these normswere found to directly and indirectly via influencing patient and family treatment expectationsand provider decision making heuristics affect treatment decisions for minority and nonminority patients with advanced cancer norms are fruitfulforstudy because once understood they are potentiallymalleableandleadership effortsthrough explicitin norms ofimplementation of new forms of positive and negativesanctions via social marketing interventions our study aims to study mechanisms underlying cancer center variation in eol treatment intensity amongminority and nonminority patients using a qualitativecase study design and has two objectives first we willidentify the local anizational and provider practicenorms that influence decisions about laterline chemotherapy hospice and icu use among minorities withadvanced cancer at major us cancer centers second wewill assess the influence of these norms on patient andfamily expectations and provider decision making heuristics for laterline chemotherapy hospice and icu useamong minorities with advanced cancer at major uscancer centers below we describe our qualitative studydesign approach to meet our study objectivesrecommendationsmethodsdesignthe design of the studywe chose a qualitative case study design at six sites toidentify local anizational and provider practice normsthat influence variation in eol treatment intensity particularly for minority patients based on medicareclaims data analyses we will recruit of the ncinccn designated cancer centers serving at least african american advanced cancer patients webased our sample size on recent literature related tosample size sufficiencyto reachmultisite data saturation and qualitative research expertise of our study team first our study is guided by atheory based on previous research and uses data frommultiple sources to test and crosscheck for confirmingor disconfirming evidence of our theory a necessarycomponent of ensuring data sufficiency [ ] inaddition conducting qualitative case studies at six siteseach of which will include over interviews plus multiple observations will produce finegrained and rich descriptive analysis to generate and compare theoreticalinsights across sites as well as across stakeholders egproviders patients within sites [] our targetnumbers for interviews and observations are well withinrecommendationsandsaturation given our well defined study aim lastlyresearch suggests that qualitative research expertise including the quality and depth of interview process is animportant criterion to consider for assessing sample size our site visit team has over years of collectiveexperience conducting qualitative research in healthcaretopics and settings ensuring a rigorous and thoroughprocess at each of the six sitesreaching dataadequacyforwe willtarget national comprehensive cancernetwork nccn and national cancer institute ncicomprehensive cancer centers for our study becausethey set national standards for high quality cancer care 0cknutzen bmc palliative care page of we will engage up to six sites serving a high proportionof african american patients ranging in eol care intensity and theoretically sampled to maximize our ability to compare and contrast anizational and providerpractice norms related to eol care we defined highproportion of minority patients as we measuredeol care intensity based upon riskadjusted metrics ofeol quality using medicare feeforservice claimsdata receipt of chemotherapy in the last days of lifenqf intensive care unit icu admission in thelast days of life nqf and non nqf or late nqf hospice referral our approach tocalculating these eol quality metrics has been publishedelsewhere given the multivariable nature of thesemetrics we use data visualization to purposively selectsites for case study that maximize potential heterogeneity in practice patterns following the principles ofpositive deviance sampling[ ] we will samplehigher versus lower eol quality sites in a ratio of we will employ qualitative case study researchmethods in this study including inpatient observationprocedures and provider patient and leadership semistructured interviews that have been previously developed and piloted [ ] we will augment thesemethods with outpatient and tumor board observationprocedures which we developed and tested at a nonstudy ncidesignated cancer center serving a whiterural population we williteratively revise all datacollection procedures based on researcher experiencesand thematic insights following each sampled case studysite visitqualitative data collectionthe study team will collect types of data field notesfrom direct observation of inpatient and outpatient cancer care and cancer tumor boards transcribed audiorecorded semistructured interviews with cancer centerleadership providers and patients family members andcaregivers and artifacts table we will link all datafrom observations using a unique identification idnumber we will use a data collection form for field observations to capture provider and patient demographicinformation as well as location time and individualspresent at the encounter in addition to relevant clinicaldata observers will note sociolinguistic dimensionssuch as turn taking tone affect body positioning andeye contact artifacts collected during the site visit willinclude workflows marketinginformational materialsorientation guidelines quality reporting and communication documents used in the cancer centersemistructured interview guides for site leadershipfocus on institutional norms including resources programs and policies related to eol care and outcomesas well as sitespecific workflows and scheduling logisticsin preparation for site visits cognitive mental modelssemistructured interview guides for providers whichtable data collection rationaledata collection methoddirect observationrationaleto learn about eol care for minority patients with advanced cancer specifically how it is influenced by anizational and provider practice norms provider decision making heuristics patient and family expectationssemistructured interviewsleadershipto probe anizationlevel norms including resources programs and policies sitespecific workflows and scheduling logisticsprovidersto explore individuallevel motivations decision heuristics andor rationalizations unconscious beliefs and assumptions that structure advanced cancer decision making using casevignettes to prime mental modelspatients family memberscaregiversto probe individuallevel preferences for cancer care past current and future decisions related to cancer careartifact collectionto learn how the anization standardizes workflows marketinginformational materials orientation guidelinesquality reporting and communication documents used in the cancer center and how this impacts local anizational and provider practice norms provider decision making heuristics patient and family expectations 0cknutzen bmc palliative care page of include eol case vignettes explore motivations decision heuristics andor rationalizations six eol vignettes were developed bya medical oncologistradiation oncologist and palliative care providers tohighlight key decision points common to outpatient orinpatient providers see table each vignette has oneversion with a photo of an african american patient andone with a photo of a white patient providers will viewone african american case and one white patient caseto facilitate mental models debriefing and uncover unconscious beliefs and assumptions related to race thatstructure advanced cancer decision making semistructured interview guides for patients family membersand caregivers probe past current and future decisionsrelated to their cancer caresite visit teams will consist of researchers we willidentify a sitespecific principal investigator pi at eachsite to help facilitate support from site leadership andidentify and recruit informants for pre sitevisit interviews and providers for sitevisit observation and interviews up to two months in advance of the site visit thestudy team will conduct leadership interviews by phoneincluding physician and nursing leaders outpatient oncology practice managers and key referral service lineleaders from palliativesupportive care hospital medicine and intensive care we will approach other siteleaders for interviews at the suggestion of the site piwhen necessaryleadership interviews will take placeduring and after site visitsone month in advance of the site visit the study teamwill recruit providers for observation in the inpatientand outpatient setting the observation schedule will involve one researcher assigned to each observed providerfor a halfday observation in outpatient clinics morningtable vignette summariessettingvignette number andpatient racespecialtyinpatient african american white african americaninpatient white african americanmedicaloncology white african american whiteradiationoncology african americansurgicaloncology white african american outpatient whitevignette summary and key questionsummary yearold man with metastatic gastric cancer he was living in a skilled nursing facilityafter a long hospitalization for infection he is now hospitalized with recurrent fever respiratorydistress and anxietykey question how to manage anxiety and respiratory distress in a patient with advanced cancerand high risk for shortterm deathsummary yearold woman with recurrent metastatic pancreatic cancer and mild dementia she isscheduled to start palliative chemotherapy next week she presents to the ed with decliningperformance status decreased appetite and abdominal pain her hospital evaluation demonstratespoor kidney function low blood pressure and rapid breathing all worrisome for rapid constitutionaldeclinekey question how to manage a patient with an aggressive cancer presenting to the emergencydepartment with multiple signs of constitutional declinesummary yearold man with advanced metastatic colon cancer he is married and lives at homewith his wife he presents to clinic with pain weight loss and signs of cancer progression he asksdo you think the chemo is workingkey question how to answer patient questions about prognosis and next steps in treatment ofadvanced cancer with limited treatment optionssummary yearold man with a new diagnosis of metastatic renal cell carcinoma he presents withseizures brain metastases and lung metastases he is unmarried and without children his performancestatus is poor and he is not able to make his own health care decisions his eldest brother is his durablepower of attorney and asks doc what would you do if he was your brotherkey question how to approach surrogate decision making about management approach for a patientwith poor prognosissummary yearold man with newly diagnosed nonmetastatic lung cancer he has severe lungdisease and significant vascular disease from heavy smoking he is a poor surgical candidate hementions that the stress of his cancer diagnosis has caused him to drink alcohol more heavily thanusual and he is coughing up about tablespoons of bright red blood dailykey question how to approach a patient with a new diagnosis of a potentially curable cancer whenthere are a number of red flags that the patient may do poorly with surgical treatmentsummary yearold woman with a recent diagnosis of pancreas cancer she has been hospitalizedwith weight loss pain and declining activity her evaluation shows a borderline resectable pancreaticcancer initial treatment would be chemotherapy or chemoradiation if she could tolerate this she hasbeen unable to eat or ambulate for the last five days due to poor appetite and performance statusshe says im a fighter not a quitter and with jesus anything is possible she then asks what comesnextkey question how to approach a patient who has a treatable diagnosis but who does not have theperformance status to tolerate treatment 0cknutzen bmc palliative care page of or afternoon session the emergency department byshift and inpatient setting by timing of daily service orconsult rounds as well as scheduled tumor boards andfamily meetings selection criteria for providers to observe and interview focus on maximizing our ability toassess provider norms for advanced cancer care withinthe particular institutional context that we will exploreduring leadership interviews provider selection criteriaincludes providers who manage patients with metastatic solid tumors ie we excluded leukemia lymphoma and bone marrow transplant providers and haveeither high volumes of patients andor high peerinfluence as perceived by the site pi or other key informants we seek to recruit medical radiation and surgical oncology providers as well as palliativesupportivecare providers who see cancer patients see table fortarget sampling frame we also seek to recruit providersfrom intensive care hospital medicine and emergencymedicine who care for acutely ill cancer patients wepresent an example observation schedule in fig wewill ask all providersrecruited for observation tocomplete an interview interviews with providers willoccur in person during or by phone after the site visitwe will digitally record all interviews and compensateall providers for participating in an interviewat least weeks prior to the site visit we will sendflyers about the study with photos of the study team tothe site pi who will facilitate posting of the flier in publicsettings such as waiting rooms clinic rooms infusionsuites and inpatient units the purpose of this flyer is toalert nonconsented individuals to our study purposeand to provide instructions for opting outduring patient care observation researchers will directly approach patients and their familycaregivers following introduction by the consented providerifpatients or their familycaregiver verbally consent to beinterviewed the study team member will obtain contactinformation to arrange for a phone interview at thepatient family member or caregivers convenience at alater date selection criteria for patient interviews includes adults aged years or older with metastatic solid tumor whom the provider would not besurprised if they died in the next months and seenby a consented provider we will seek to recruit equalnumbers of minority and nonminority patients we willdigitally record all interviews and compensate each participant for participatingtheoretical modelfindingsqualitative data analysiswe will use a qualitative and mixed methods data analysis platform dedoose to manage and analyze all transcribed field notesinterviews and artifacts and linkrelevant data to contextual information eg patient andprovider race site features sociocultural research consultants llc we will develop a codebook first deductively using ourin theliterature and priorresearch and then inductivelythrough an iterative process of close readings and discussion of the data in order to identify additional codesthree qualitative researchers will apply the codebook tothe data two who will divide and code all the data andone who will assess reliability of coding by reviewing asubset ofthree qualitative researchers will discuss differences in coding and resolveby consensus we will repeat the analysis process aftereach site visit to conduct constant comparative analysisregarding similarities and differences between and withinsites in support of study aims and after completionof each site visit the study team will develop a writtensummary of preliminary quantitative and qualitativefindings specific to the site which will then be sent to allparticipants from that site to assess initial validity of thesitespecific findings after completion of all site visitsand analysis of data we will send final study reports toparticipating sitesthe coded data alltable target sampling frame at each sitedata collection settingoutpatientmedical oncologysurgical oncologyradiation oncologysupportivepalliative careemergency medicineinpatienthospital medicineintensive caresupportivepalliative careoncology consultrigor and reproducibilityour research team is also conscious of conducting purposefully informed and respectful research on the cancerexperiences of racial and ethnic minorities and we havetaken steps to ensure scientific rigor of our approachand results through study design development and willcontinue to do so through data collection and data analysis based in a relatively nonracially diverse geographicregion our team n is comprised of racial andethnic minority researchers as such we seek to incorporate greater diversity of racial and ethnic knowledge aswell as disciplinary perspective through an external advisory board with deep topical expertise in cancer carepalliative care racial and ethnic health equity and socialnorms additionally to address potential researcher biasthe entire study team completed implicit bias trainingn 0cknutzen bmc palliative care page of fig mock onsite observation schedule researchers will ideally observe relevant outpatient clinics during week and inpatient servicesduring week researchers will go to tumor boards attended by consented providers as well as other relevant staff meetings eg fellowsmeetings researchers will observe providers during either am or pm blocks using the alternating daily block to dictate field notes and conductinterviews with providers and patients onsitefocused on unconscious biases related to attitudes aboutrace ethnicity cancer cancer treatment death anddying one researcher participating in data collectionwill remain blinded to sites eol treatment intensityclassification until data collection is completefinally we will employ multiple methods of triangulation to assure comprehensiveness and validity of datatwo to three researchers of a multidisciplinary team willparticipate in each site visit and an additional three researchers will conduct qualitative analysisto satisfyinvestigator triangulation method triangulation will include direct observation semistructured interviews andartifact collection we will achieve data triangulation byobserving and interviewing leadership personnel providers and patients family members and caregivers ateach site of various s specialties and diagnoses respectively qualitative analysis will use both deductive and inductive methodsto achieve theorytriangulationethics approval and consent to participatethe study has been approved by the dartmouth collegecommittee forthe protection of human subjectsstudy00031129 and is considered minimal risk allparticipating sites will waive independent irb approval in favor of acknowledging dartmouths irb rely on dartmouths irb via a smart irb reliance or conduct a local ethical review and approval we willobtain a waiver of informed consent for participant observation all providers will provide written electronicconsent for observation and interview and allinterviewed leadership patients and families will provide oralconsent for interview we have obtained a certificate ofconfidentiality from the national institutes of healthnih for this studystaffwe will not record any identifiable or personal information about providers patients family members caregivers orin field notes except demographicinformation a unique id number will link data fromobservations and interviewsincluding demographicdata to consented participants the key linking the idnumber and identifying information of the consentedparticipants will be maintained on a passwordprotectedserver only the research team will have access to thelinkage file all data collected on individuals will belinked to their id number alone we will audiorecordand transcribe all handwritten field notes without anyidentifiable information we will store all original fieldnotes in a locked filing cabinet and all transcripts on apasswordprotected server to which only the researchteam will have access we will give a discreet lapel pinto all providers staff patientsfamily members and 0cknutzen bmc palliative care page of caregivers who do not wish to be observed as advertisedby the informational flyers posted prior to the studyteams arrival at the site we will not document any individual wearing such a pin in field notes nor will weapproach them for an interviewwe will give all individuals participating in interviewsan information sheet prior to the interview and we willobtain informed consent verbally at the time of theinterview we will obtain informed consent verbally asmany of the interviews will be conducted by phone either before or after the site visit the process of obtaining verbal consent has been approved by the dartmouthcollege committee forthe protection of humansubjects we will record all interviews and later professionally transcribe them without any identifiable information we will store all recordings and transcripts on apasswordprotected server to which only the researchteam will have access additionally we consulted theguidelines for endoflife research putforth in themethods of researching end of life care morecareproject while designing the protocol for this study specifically we considered the risks egintervieweedistress and rewards eg potential therapeutic effectthat qualitative interviews may have for patients familymemberstheseprotocolsgivers while designingand carediscussionour study is the first comprehensive qualitative study oflocal anizational and provider norms at minorityserving nccn and ncidesignated comprehensive uscancer centers if the aims of this study are achieved weexpect to identify targets for institutional change at cancer centers with lower eol quality metric performancethe two main deliverables of this research will be knowledge regarding norms and their impact on eoldecision making at participating cancer centers and identification of potential members of a community research advisory board to oversee future institutionlevelinterventions aimed at improving eol care respectiveto the first deliverable participating cancer centers willreceive a customized report of our findings about theirown center following completion of our site visit aftercompletion of all site visits we will work with theamerican cancer society and participating cancercenters to identify local chapters ofthe americancancer society acs and provider medical societieseg county medical and nursing societies at which wecan discuss our findings and their implications for localpatients and providersrespective to the second deliverable we anticipate theopportunity to develop institutionlevelinterventionsaimed at eol care in the future norms are fruitful forstudy because once understood they are potentiallymalleable through explicit leadership efforts and implementation of new forms of positive and negativesanctions specifically social marketing the use ofmarketing principles to influence human behavior to improve health or benefit society is a promisingstrategy for changing norms interventionists have successfully applied the principles of social marketing tochange hiv risk behaviors [ ] and palliative careconsultation use integrating social marketing interventions in cancer centers with high intensity eol carecould have the effect of improving the quality and costof cancer care particularly for racial and ethnic minorities further by studying norms of decision makingamong groups of physicians this project will overcomethe limitation of past research which uniformly hasneglected this important issueabbreviationsnccn national comprehensive cancer network nci national cancerinstitute eol endoflife nqf national quality forum icu intensive careunit id identification pi principal investigator irb institutional reviewboard acs american cancer societyacknowledgementsthank you to inas kayhal for her assistance with the cluster analysis for sitesampling and associated visualizations and to garrett wasp for his input onthe clinical vignettesauthors contributionskek led coordination of the study participated in design of the protocolcontributed to instrument development and led preparation of themanuscript aeb led design and writing of the grant and protocolcontributed to instrument development and participated in preparation ofthe manuscript kes participated in design and writing of the grant andprotocol led instrument development and participated in preparation of themanuscript gfm and rb participated in design of the protocol contributedto instrument development and participated in preparation of themanuscript gab and nsk created the clinical vignettes contributed toinstrument development and participated in design of the protocol ssacontributed to instrument development and participated in design of theprotocol the authors read and approved the final manuscriptfundingthis research is funded by the american cancer society grant number rsg1801701cphps the funding body had no role in the design of the studyand will have no role in the collection analysis and interpretation of data orwriting of the manuscriptavailability of data and materialsendoflife metrics data can be found at httpswwwdartmouthatlasinteractiveappsendoflifecancercare qualitative data will be deidentifiedand made available to researchers through the ninrfunded palliative careresearch cooperative qualitative data repository after analyses in support ofthe primary aims are complete all materials and instruments developed forthis study are available by request of the authorsethics approval and consent to participatethis study has been approved by the dartmouth college committee for theprotection of human subjects study00031129 this study is consideredminimal riskconsent for publicationnot applicable 0cknutzen bmc palliative care page of competing interestsall authors declare no competing interest with respect to the researchauthorship or publication of this author details1department of behavioral social and health education sciences rollinsschool of public health emory university atlanta ga usa 2the dartmouthinstitute for health policy and clinical practice geisel school of medicinedartmouth college lebanon nh usa 3department of general internalmedicine boston medical center boston ma usa 4evidera pharmaceuticalproduct development bethesda md usa 5department of medicine geiselschool of medicine hanover nh usa 6norris cotton cancer center atdartmouthhitchcock medical center lebanon nh usareceived july accepted august refere | Colon_Cancer |
"adipogenesis is the process through which mesenchymalstem cells mscs commit to the adipose lineage and diï¬erentiate into adipocytes during this process preadipocytescease to proliferate begin to accumulate lipid droplets anddevelop morphologic and biochemical characteristics ofmature adipocytes such as hormoneresponsive lipogenesisand lipolytic programs currently there are mainly twomodels of benign adipocyte diï¬erentiation in vitro one isï¬broid pluripotent stem cells which can diï¬erentiate intonot only adipocytes but also muscle cartilage and othercells there are two kinds of ï¬broid pluripotent stem cellsbone marrow and adipose mesenchymal stem cells anothergroup is ï¬broblastic preadipocytes which have a single direction of diï¬erentiation namely lipid diï¬erentiation including3t3l1 and 3t3f422a cells cancer cells with tumorinitiation ability designated as cancer stem cells cscshave the characteristics of tumorigenesis and the expressionof speciï¬c stem cell markers as well as the longterm selfrenewal proliferation capacity and adipose diï¬erentiationpotential in addition to cscs cancer cells undergoing epithelialmesenchymaltransformation emt havebeen reported to be induced to diï¬erentiate into adipocytes[] lung cancer ncih446 cells can be induced to differentiate into neurons adipocytes and bone cells in vitro the adipogenesis diï¬erentiation treatment is promisingin the p53 gene deletion type of ï¬broblastderived cancer cancer cells with homologous recombination defectssuch as ovarian and breast cancer cells with breast cancersusceptibility genes brca mutations can be inducedto diï¬erentiate by poly adpribose polymerase parp 0cstem cells internationalinhibitors the nuclear receptor peroxisome proliferatoractivated receptor Î pparÎ agonist antidiabetic thiazolidinedione drug can induce growth arrest and adipogenicdiï¬erentiation in human mouse and dog osteosarcoma cells thyroid cancer cells expressing the pparÎ fusion proteinppfp can be induced to diï¬erentiate into adipocytes bypioglitazone adipogenesis can be induced in welldiï¬erentiated liposarcoma wdlps and dediï¬erentiatedliposarcoma ddlps cells by dexamethasone indomethacininsulin and 3isobutyl1methyl xanthine ibmx in this review we highlight some of the crucial transcription factors that induce adipogenesis both in mscs and inincluding the wellstudied pparÎ and ccaatcscsenhancerbinding proteins cebps as well as othercell factors that have been recently shown to have an important role in adipocyte diï¬erentiation we focus on understanding the complex regulatory mechanism of adipocytediï¬erentiation that can contribute to the clinical treatmentof human diseases including those caused by obesity andadipocytes dysfunction especially for the malignant tumorwhich can be transdiï¬erentiated into mature adipocytes adipocyte differentiationcell proliferation and diï¬erentiation are two opposingprocesses and there is a transition between these two processes in the early stages of adipocyte diï¬erentiation theinteraction of cell cycle regulators and diï¬erentiation factors produces a cascade of events which ultimately resultsin the expression of adipocyte phenotype adipogenesishas diï¬erent stages each stage has a speciï¬c gene expression pattern in general adipocyte diï¬erentiation ofpluripotent stem cells is divided into two phases the ï¬rstphase known as determination involves the commitment ofpluripotent stem cells to preadipocytes the preadipocytescannot be distinguished morphologically from their precursor cells but also have lost the potential to diï¬erentiate intoother cell types in the second phase which is known asterminal diï¬erentiation the preadipocytes gradually acquirethe characteristics of mature adipocytes and acquire physiological functions including lipid transport and synthesisinsulin sensitivity and the secretion of adipocytespeciï¬cproteins the diï¬erentiation of precursor adipocytes is also dividedinto four stages proliferation mitotic cloning early diï¬erentiation and terminal diï¬erentiation after the precursors are inoculated into the cell culture plates the cellsgrow exponentially until they converge after reaching contact inhibition the growth rate slows and gradually stagnatesand the proliferation of precursor adipocytes stops which isvery necessary for initiating the diï¬erentiation of precursoradipocytes adipocyte precursors exhibit transient mitosiscalled clonal expansion a process that relies on the actionof induced diï¬erentiation factors some preadipocyte cellsmouse cell lines 3t3l1 3t3f442a undergo one or tworounds of cell division prior to diï¬erentiation whereasother cell lines mouse c3h10t12 diï¬erentiating into adipocyte do not undergo mitosis clonal expansion whether mitotic clonal expansion is required for adiposediï¬erentiation remains controversial however it is certainthat some of the checkpoint proteins for mitosis regulateaspects of adipogenesis [ ] when cells enter the terminaldiï¬erentiation stage the de novo synthesis of fatty acidsincreases significantly the transcription factors and adipocyterelated genes work cooperatively to maintain precursor adipocyte diï¬erentiation into mature adipocytes containing largelipid droplets regulatory pathways inpreadipocytes commitmentadipocyte diï¬erentiation is a complex process in which geneexpression is ï¬nely regulated the most basic regulatory network of adipose diï¬erentiation has not been updated inrecent years but some factors and signaling pathways thatdo aï¬ect adipose diï¬erentiation have been continuouslyreported adipocyte diï¬erentiation is the result of the geneexpression that determines the phenotype of adipocyteswhich is a complex and delicate regulatory process figure wnt signal pathway in adipogenesis wnt signaling isimportant for adipocytes proliferation and diï¬erentiationboth in vitro and in vivo the wnt family of secretedglycoproteins functions through paracrine and autocrinemechanisms to uence cell fate and development wntprotein binding to frizzled receptors initiates signalingthrough catenindependent and independent pathways wnt signaling inhibits adipocyte diï¬erentiation in vitroby blocking the expression of pparÎ and cebpα constitutive wnt10b expression inhibits adipogenesis wnt10b isexpressed in preadipocytes and stromal vascular cells butnot in adipocytes in vivo transgenic expression of wnt10bin adipocytes results in a reduction in white adipose tissuemass and absent brown adipose tissue development wnt10a and wnt6 have also been identiï¬ed as determinantsof brown adipocyte development [ ] wnt5b is transiently induced during adipogenesis and promotes diï¬erentiation indicating that preadipocytes integrate inputs fromseveral competing wnt signals the hedgehog hh signaling pathway mechanismthree vertebrate hh ligands including sonic hedgehogshhindian hedgehog ihh and desert hedgehogdhh have been identiï¬ed and initiated a signaling cascademediated by patched ptch1 and ptch2 receptors [ ]hh signaling had an inhibitory eï¬ect on adipogenesis inmurine cells such as c3h10t12 ks483 calvaria mscslines and mouse adiposederived stromal cells thesecells were visualized by decreased cytoplasmic fat accumulation and the expression of adipocyte marker genes after hhsignaling was inhibited although it is generally agreedthat hh expression has an inhibitory eï¬ect on preadipocytediï¬erentiation the mechanisms linking hh signaling andadipogenesis remain poorly deï¬ned erkmapkppar signal pathway extracellularregulated protein kinase erk is required in the proliferativephase of diï¬erentiation erk activity blockade in 3t3l1 0cstem cells internationaldex insulin demxwnt 10band othersshhpbc smotgfð½p smad3 smad3testosteroneð½catentinarirspi3kaktcrebpkapcrebfoxo1a2tcflef gata23cebpð½mapkg3k3ð½p2cebpð½cebpαppará½»bmpssmad1srebpadipocytegenesfigure regulation pathways in preadipocytes commitment bmp and wnt families are mediators of mscs commitment to producepreadipocytes exposure of growtharrested preadipocytes to diï¬erentiation inducers igf1 glucocorticoid and camp triggers dnareplication leading to adipocyte gene expression due to a transcription factor cascade the dotted line indicates an uncertain molecularregulatory mechanismcells and embryonic stem cells can inhibit adipogenesis inthe terminal diï¬erentiation phase erk1 activity leads topparÎ phosphorylation which inhibits adipocyte diï¬erentiation this implies that erk1 activity must be reduced afteradipocyte proliferation so that diï¬erentiation can proceedthis reduction is mediated in part by mitogenactivatedprotein kinase mapk phosphatase1 mkp1 [ ]these extracellular and intracellular regulation factors causeadipocytespeciï¬c gene expression and eventually lead toadipocyte formation adipocyte differentiationregulatory proteins pparÎ and adipocyte diï¬erentiation pparÎ is a member of the nuclearreceptor superfamily and is both necessaryand suï¬cient for adipogenesis forced expression ofpparÎ is suï¬cient to induce adipocyte diï¬erentiation broblasts indeedthe proadipogenic cebps andkrüppellike factors klfs have all been shown to induceat least one of the two pparÎ promoters in contrast antiadipogenic transcription factor gata functioned in part byrepressing pparÎ expression pparÎ itself has twoisomers the relative roles of pparÎ1 and pparÎ2 in adipogenesis remain an open question pparÎ2 is mainlyexpressed in adipose tissue while pparÎ1 is expressed inmany other tissues although both can promote adipocytediï¬erentiation pparÎ2 could do so eï¬ectively at very lowligand concentration compared with pparÎ1 the twoprotein isoforms are generated by alternative splicing andpromoter usage and both are induced during adipogenesispparÎ1 can also be expressed in cell types other than adipocytes ren et al used engineered zincï¬nger proteins tothe expression ofthe endogenous pparÎ1 andinhibitpparÎ2 promoters in 3t3l1 cells ectopic expression ofpparÎ2 promotes adipogenesis whereas that of pparÎ1does not zhang et al reported that pparÎ2 deï¬ciencyimpairs the development of adipose tissue and insulin sensitivity there are transcriptional cascades between adipocytesgenes including pparÎ and cebpα which are the coreadipocyte diï¬erentiation regulators in the early stage of adipocyte diï¬erentiation the expression of cebp and cebpδincrease which upregulates cebpα expressionfurtheractivate pparÎ pparÎ activating cebpα in turn resultsin a positive feedback pparÎ binding with retinoic acid xreceptor rxr forms diï¬erent heterodimers the variousdimmers can combine with the pparÎ response elementppre and initiate the transcription of downstream genesfor diï¬erentiation into adipocytes cebps participate in adipogenesis and several cebpfamily members are expressed in adipocytesincludingcebpα cebp cebpÎ cebpδ and cebphomologous protein chop the temporal expression of thesefactors during adipocyte diï¬erentiation triggers a cascadewhereby early induction of cebp and cebpδ leads tocebpα expression this notion is further supported by thesequential binding of these transcription factors to severaladipocyte promoters duringadipocyte diï¬erentiationcebp is crucial for adipogenesis in immortalized preadipocyte lines cebp and cebpδ promote adipogenesis atleast in part by inducing cebpα and pparÎ cebpαinduces many adipocyte genes directly and plays an important role in adipose tissue development once cebpα isexpressed its expression is maintained through autoactivation despite the importance of cebps in adipogenesis 0cstem cells internationalthese transcription factors clearly cannot function eï¬cientlyin the absence of pparÎ cebp cannot induce cebpαexpression in the absence of pparÎ which is required torelease histone deacetylase1 hdac1 from the cebpαpromoter furthermore ectopic cebpα expressioncannot induce adipogenesis in pparÎï¬broblasts however cebpα also plays an important role in diï¬erentiated adipocytes overexpression of exogenous pparÎ incebpαdeï¬cient cells showed that although cebpα isnot required for lipid accumulation and the expression ofmany adipocyte genes it is necessary for the acquisition ofinsulin sensitivity [ ] figure human ï¬broblasts withthe ability to diï¬erentiate into adipocytes also do not undergomitotic cloning ampliï¬cation however pparÎ exogenousligands need to be added to promote adipocyte diï¬erentiation therefore it can be inferred that mitotic cloning expansion can produce endogenous ligands of pparÎ bmp and transforming growth factor tgf inadipocyte diï¬erentiation a variety of extracellular factorsaï¬ect the preadipocyte commitment of stem cells includingbone morphogenetic protein bmp transforminggrowth factor tgf insulininsulinlike growthfactor igf1 tumor necrosis factor α and interleukin matrix metalloproteinase ï¬broblast growthfactor fgf and fgf2 bmp and tgf have variedeï¬ects on the diï¬erentiation fate of mesenchymal cells the tgf superfamily members bmps and myostatinregulate the diï¬erentiation of many cell types includingadipocytes tgf inhibitor can promote adipose diï¬erentiation of cancer cells with a mesenchymal phenotypein vitro and transgenic overexpression of tgf impairsadipocyte development inhibition of adipogenesis couldbe obtained through blocking of endogenous tgf with adominantnegative tgf receptor or drosophila mothersagainst decapentaplegic protein smad inhibitionsmad3 binds to cebps and inhibits their transcriptionalactivity including their ability to transactivate the pparÎ2promoter [ ] exposure of multipotent mesenchymalcells to bmp4 commits these cells to the adipocyte lineageallowing them to undergo adipose conversion theeï¬ects of bmp2 are more complex and depend on the presence of other signaling molecules bmp2 alone has little eï¬ecton adipogenesis and it interacts with other factors such astgf and insulin to stimulate adipogenesis of embryonicstem cells bmp2 stimulates adipogenesis of multipotentc3h10t12 cells at low concentrations and can contribute tochondrocyte and osteoblast development at higher concentrations klfs in adipocyte diï¬erentiation during adipocyte differentiation some klf family members are overexpressedsuch as klf4 klf5 klf9 and klf15 while klf16 expression is reduced [ ] klf15 is the ï¬rst klf family members which were identiï¬ed to be involved in adipocytediï¬erentiation its expression increased significantly on thesixth day of 3t3l1 adipocyte diï¬erentiation and peakedon the second day of adipocyte induction in mscs andmouse embryonic ï¬broblasts inhibition of klf15 by sirnaor mutation led to a decrease in pparÎ cebpα fatty acidbinding protein fabp4 and glucose transporter glut4 however overexpression of klf15 in nih3t3cells was found to be associated with lipid accumulation aswell as increases in pparÎ and fabp4 mice with complete absence of klf5 showed embryonal lethality and micewith singlechromosome klf5 knockout showed a significant reduction in white fat in adulthood suggesting thatklf5 plays an important role in adipocyte diï¬erentiationklf5 can be activated by cebp or cebpδ which isinvolved in early adipocyte diï¬erentiation klf5 can beactivated by cebp or cebpδ which is involved in earlyadipocyte diï¬erentiation direct binding of klf5 to thepparÎ2 promoter in combination with cebps inducespparÎ2 expression transfection of klf5 dominantnegative mutants in 3t3l1 cells reduced lipid droplet accumulation and inhibited pparÎ and cebpα expressionwhereas overexpression of wild klf5 significantly promotedadipocyte diï¬erentiation even without exogenous hormonestimulation similar to klf5 klf9 knockdown can inhibitthe expression of a series of adipocyte diï¬erentiation genessuch as pparÎ cebpα and fabp4 hence inhibitingadipocyte diï¬erentiation however klf9 overexpressiondid not upregulate the expression of pparÎ and cebpα in addition klf4 can transactivate cebp by bindingto the region of kb upstream of the cebp promoter and promote lipid diï¬erentiation klf6 can forma complex with histone deacetylase3 hdac3 inhibitingpreadipocyte factor1 pref1 expression and promotinglipid diï¬erentiation klf2 is highly expressed in adiposeprogenitors and its expression decreases during the processof lipid diï¬erentiation overexpressed klf2 can bind to thecaccc region of pparÎ2 proximal promoter and inhibitlipid diï¬erentiation as well as the expression of pparÎcebpα and sterolregulated elementbinding proteinssrebp by inhibiting the promoter activity rnasequence analysisshowed that klfl6 expression wasdecreased on the ï¬rst day of adipocyte diï¬erentiation of3t3l1 cells adipocyte diï¬erentiation was promoted byklf16 knockdown but was inhibited by klf16 overexpression via inhibition of pparÎ promoter activity in addition klf3 and klf7 were also found to play a negativeregulatory role in adipocyte diï¬erentiation [ ] signal transducers and activators of transcriptionstats and adipocyte diï¬erentiation the activated statprotein enters the nucleus as a dimer and binds to the targetgene to regulate gene transcription in the adipocyte diï¬erentiation of mouse 3t3l1 cells the expression of stat1 andstat5 was significantly increased while that of stat3and stat6 was not significantly changed in the adipocyte diï¬erentiation of human subcutaneous adipose precursor cells stat1 expression was significantly decreased while the expression of stat3 and stat5 wasincreased and stat6 expression was unchanged therole of stat1 in adipocyte diï¬erentiation is not clearbecause its expression trend in humans and mice diï¬ersduring the adipocyte diï¬erentiation process early adipocytediï¬erentiation of 3t3l1 cells was inhibited by stat1 0cstem cells internationalklf5srebp1cklf15klf2chopcebpá½»krox20ligandcebpð½cebpð¿gata23ppará½»cebpð¼proadipogenicantiadipogenicgenes of terminaladipocytedifferentiationfigure a cascade of transcription factors that regulate adipogenesis pparÎ is one of the key transcription factors in adipogenesis and thecore of the transcriptional cascade that regulates adipogenesis pparÎ expression is regulated by several proadipogenic blue andantiadipogenic red factors cebpα is regulated through a series of inhibitory proteinprotein interactions some transcription factorfamilies include several members that participate in adipogenesis such as the klfs black lines indicate eï¬ects on gene expression violetlines represent eï¬ects on protein activityagonist interferon Î loss of stat1 in 3t3l1 cells can rescue the inhibition of adipocyte diï¬erentiation caused byprostaglandin factor 2α other studies have found thatstat1 is required for adipose diï¬erentiation and stat1overexpression in c3h10t12 cells can prevent the inhibition of lipid diï¬erentiation caused by bcell lymphoma6knockdown there was no abnormal adipose tissuein stat1 knockout mice stat3 not only aï¬ectsthe proliferation of 3t3l1 cells but also coregulates theiradipocyte diï¬erentiation with high mobility group protein the fabp4 promoter was used to speciï¬callyknock out stat3 in the adipose tissue of mice and theresults showed that mice weight significantly increasedand the adipocyte quantity increased compared with thewildtype mice stat5a and stat5b have diï¬erenteï¬ects on adipocyte diï¬erentiation abnormal adipose tissuewas found in the mice with stat5a or stat5b knockout ordouble knockout and the amount of adipose tissue was onlyoneï¬fth of the original adipose tissue in mice withoutknockdown histone modiï¬cation in adipocyte diï¬erentiation histone deacetylase sirtuin sirt plays an important rolein biological processes such as stress tolerance energymetabolism and cell diï¬erentiation during the adipocyte diï¬erentiation of c3h1012 cells sirt1 expressiondecreased overexpression of sirt1 activated thewnt signal which caused the deacetylation of cateninthe accumulation of catenin in the nucleus could inhibitadipocyte diï¬erentiation sirt1 knockdown resulted inincreased acetylation of the histones h3k9 and h4k16 inthe secreted frizzledrelated protein sfrp and sfrp2 promoters thereby promoting transcription of these genes andpromoting lipid diï¬erentiation forkhead box proteino foxo is a member of the transcription factor foxofamily it can recruit cyclic amp response elementbindingprotein cbphistone acetyltransferase p300 to initiate anacetylation the acetylated foxo1 can be phosphorylatedby phosphorylated protein kinase b pkbakt the phosphorylation of foxo1 by akt inhibits the transcriptionalactivation of foxo1 the acetylation of foxo1 lost the ability of dnabinding aï¬nity and promoted its shuttling fromnuclei to cytoplasm sirt1 and sirt2 can deacetylateand active foxo1 activated foxo1 nonphosphorylatednuclear foxo1 in the nucleus binds to the promoters of target genes encoding p21 p27 and pparÎ and initiates subsequent transcriptions sirt2 inhibits the acetylation andphosphorylation of foxo1 thereby induces the accumulation of activated foxo1 in the nucleus activated foxo1could inhibit adipogenesis via pparÎ [] lysinespeciï¬c histone demethylase lsd1 expression increasedduring the adipocyte diï¬erentiation of 3t3l1 cells lsd1could reduce the dimethylation levels of histone h3k9 andh3k4 in the cebpα promoter region thereby promotingadipocyte diï¬erentiation set domaincontaining setd8 catalyzed the monomethylation of h4k20 andpromoted pparÎ expression the activation of pparÎ transcriptional activity leads to the induction of monomethylatedh4k20 and modiï¬cation of pparÎ and its targets therebypromoting adipogenesis enhancer of zeste homolog ezh2 is a methyltransferase and can bind methyl groupsto histone h3k27 which is also necessary for lipid diï¬erentiation the absence of ezh2 in brown fat precursors results inreduced levels of the wnt promoter histone h3k27me3which is also saved by the ectopic ezh2 expression or theuse of a wntcatenin signal inhibitor in addition histone demethylases such as lysinespeciï¬c histone demethylase lsdkdm kdm6 and histone lysine demethylasephf2 are also involved in adipose diï¬erentiation andkdm2b inhibits transcription factor activator protein 2αpromoter via h3k4me3 and h3k36me2 role of microrna and long noncodingrna in adipogenesismicrorna mir can bind and cut target genes or inhibittarget gene translation endogenous sirna can be producedby the action of dicer enzyme and bind to a speciï¬c proteinto change its cellular location many kinds of mirsare involved in regulating adipocyte diï¬erentiation the 0cstem cells internationalexpression of mir143 increased during the diï¬erentiationof adipose progenitor cells overexpression of mir143promoted gene expression involved in adipose diï¬erentiationand triglyceride accumulation inhibition of mir143 prevented the adipose diï¬erentiation of human fat progenitorcells [ ] additionally mir8 promotes adipocyte diï¬erentiation by inhibiting wnt signaling moreover mir mir103 mir21 mir519d mir210 mir30mir204211 and mir375 also play a certain role in promoting adipocyte diï¬erentiation while mir130 mir448and let7y inhibit lipid diï¬erentiation [ ] in additionto mirs long noncoding rna lncrna is a type of noncoding rna and is important during epigenetic regulationand can form a doublestranded rna complex with mrnacauses protein transcription lncu90926 inhibits adipocytediï¬erentiation by inhibiting the transactivation of pparÎ2 as a novel lncrna hoxaas3 expression increasedduring the adipose diï¬erentiation of mscs and hoxaas3 silencing reduced the marker gene of adipose diï¬erentiation and inhibited the adipose diï¬erentiation zhu et al reported that hoxaas3 interacted with ezh2 toregulate lineage commitment of mscs hoxa as3 canregulate the trimethylation level of h3k27 in the runx2promoter region by binding to ezh2 therefore hoxaas3 is considered to be an epigenetic switch regulating mscslineage speciï¬city adipocyte diï¬erentiationassociatedlncrna can act as a competitive endogenous rna of mir in the process of lipid diï¬erentiation thereby promotingthe expression of sirt1 the target gene of mir204 and thusinhibiting lipid diï¬erentiation the lncrna neat1can also regulate adipocyte diï¬erentiation under the uence of mirna140 other lncrna including lncrnablnc1 and plnc are also involved in regulating adipocytediï¬erentiation [ ] other biochemical response involved inadipocyte differentiation unfolded protein responses in adipocyte diï¬erentiationin the endoplasmic reticulum of eukaryotes unfolded protein response involves three proteinsinositolrequiringenzyme 1α doublestranded rnadependent proteinkinaselike er kinase and activating transcription factoratf 6α knockdown of atf6α aï¬ects the expressionof adipocytes genes and inhibits c3h10t12 adipocyte differentiation the inhibitory eï¬ect of berberine on adipocyte diï¬erentiation of 3t3l1 cells is also due to inducedchop and decorin expressions and this inhibitory eï¬ectis ameliorated by chop knockout in the adipocytediï¬erentiation process of 3t3l1 cells increases in pparÎand cebpα as markers of adipocyte diï¬erentiation wereaccompanied by an increase in the corresponding proteinexpressions of phosphorylated eukaryotic translation initiaeif 2α phosphorylated endoribonucleasetion factorire1α atf4 chop and other unfolded protein responsesendoplasmic reticulum stress inducer or hypoxic endoplasmic reticulum stress can inhibit adipocyte diï¬erentiationadditionally eif2α mutation results in continuous activation or overexpression of chop which also inhibits adipocyte diï¬erentiation after the initiation of adiposediï¬erentiation numerous diï¬erentiationassociated proteinsare synthesized exogenous endoplasmic reticulum stressinducers can lead to excessive endoplasmic reticulumresponse which in turn aï¬ects the synthesis of proteinsrelated to diï¬erentiation and inhibits adipocyte formationfigure role of oxidative stress in adipogenesis during thedirectional diï¬erentiation of mscs mitochondrial complexi and iii and nadph oxidase nox4 are the main sourcesof oxygen species ros production currently it is believedthat ros aï¬ects not only the cell cycle and apoptosis but alsodiï¬erentiation through uencing the signaling pathwaysincluding the wnt hh and foxo signaling cascade duringmscs diï¬erentiation the diï¬erentiation ability ofstem cells is determined by the arrangement of perinuclearmitochondria which speciï¬cally manifests as low atpcellcontents and a high rate of oxygen consumption the lackof these characteristics indicates stem cell diï¬erentiation adipocyte diï¬erentiation is a highly dependent rosactivation factor related to mitosis and cell maturation schroder et al found that exogenous h2o2 could stimulate adipocyte diï¬erentiation of mouse 3t3l1 cells andhuman adipocyte progenitor cells in the absence of insulinh2o2 regulates adipocyte diï¬erentiation of 3t3l1 cells ina dosedependent manner high doses of h2o2 and μm promote adipocyte diï¬erentiation [ ] tormos et al found that ros synthesis increased in humanmscs at the early stage of adipose diï¬erentiation and targeted antioxidants could inhibit lipid diï¬erentiation byknocking down rieske ironsulfur protein and ubiquinonebinding protein ros produced by mitochondrial complexiii was found to be necessary in initiating adipose diï¬erentiation however other studies have shown that theexpression levels of adiponectin and pparÎ were decreasedby using h2o2 mm in 3t3l1 cells free radical nitric oxide no also promotes lipid diï¬erentiationbecause treatment with no inducer hydroxylamine or nosynthase nos substrate arginine can significantly induceadipose diï¬erentiation of rat adipose progenitor cells nosinduced adipose diï¬erentiation mainly via enos rather thaninos ros can induce adipose diï¬erentiation primarily by inhibiting wnt foxo and hh signaling pathwaysthat inhibit lipid diï¬erentiation autophagy in adipocyte diï¬erentiation the increase inautophagosomes during lipid diï¬erentiation indicates thatautophagy may play an important role in lipid diï¬erentiation baerga et al conï¬rmed that the adipocyte diï¬erentiation eï¬ciency was significantly inhibited in mouse embryonic ï¬broblasts lacking autophagyrelated gene atg agene encoding an essential protein required for autophagy knockdown of atg5 in 3t3l1 cells promotesproteasomedependent degradation of pparÎ2therebyinhibiting adipocyte diï¬erentiation zhang reportedthat autophagyrelated gene 7atg7 is also crucial for adipose development atg7deï¬cient mice were slim and onlyhad of white fat compared to wildtype mice and the 0cstem cells internationalcebpð½ geneebf1 geneklf4egr2cebpð½cytosolcebpð¿ genecebpð¿klf5geneppará½» geneklf5nr2f2nfkb11433relasrebf1a2rxrappará½»ppará½»rxra heterodimerppará½»rxracorepressor complexfabp4ligands of ppará½»fam120bthrap3ep300ncoa2ncoa3helz2ncoa1crebbpebf1adipoq geneaidrfcebpð¼ geneznf638znf467cebpð¼ncor1hdac3ncor2 slc2a4 geneglut4 genelep genefabp4 genecdk4ccnd3plin1 genepck1 genefabp4cd36 geneppararxracoactivator complexppará½»fatty acidrxramediatorcoactivator complexangptlgeneppargc1amediator complex consensuslpl genenucleoplasmproteins bind to gene promoterstranscription of genes into proteinsacting on proteins compoundingtgfð½1wnt1wnt10btnf77233adipoqglut4slc2a4 tetramerlepfabp4lipid dropletplin1pck1papa pa4xpalmccd36paangptl4lplfigure regulation of adipocyte diï¬erentiation a regulatory loop exists between pparÎ and cebp activation transcription factor coeebf activates cebpα cebpα activates ebf1 and ebf1 activates pparÎ cebp and cebpδ act directly on the pparÎ gene bybinding its promoter and activating transcription cebpα cebp and cebpδ can activate the ebf1 gene and klf5 the ebf1 and klf5proteins in turn bind the promoter of pparÎ which becomes activated other hormones such as insulin can aï¬ect the expression ofpparÎ and other transcription factors such as srebp1c pparÎ can form a heterodimer with the rxrα in the absence of activatingligands the pparÎrxrα complex recruits transcription repressors such as nuclear receptor corepressor ncor ncor1 andhdac3 upon binding with activating ligands pparÎ causes a rearrangement of adjacent factors corepressors such as ncor2 are lostand coactivators such as transcription intermediary factor tif2 cbp and p300 are recruited which can result in the expression of cyclicampresponsive elementbinding protein creb followed by pparÎ pparÎ expression initiates the expression of downstream genesincluding angiopoietinrelated protein pgar perilipin fabp4 cebpα fatty acid transportrelated proteins carbohydrate metabolismrelated proteins and energy homeostasisrelated proteinslipid metabolism and hormoneinduced lipolysis in the adipocytes were altered autophagy related gene atg4b isactivated by cebp in the process of lipid diï¬erentiationand autophagy activation is necessary for the degradationof klf2 and klf3 two negative regulators of lipid diï¬erentiation these results showed that adipose diï¬erentiation andautophagy are mutually complementary in 3t3l1cells autophagy was inhibited by aspartate ammonia or 0cstem cells internationalmethyladenine at diï¬erent lipid induction periods and days and only autophagy inhibition at days hindered the formation of lipid droplets and the expression of lipid marker genes indicating that autophagy wasvery important in the early stage of lipid diï¬erentiation recent studies showed that lc3 is overexpressed in3t3l1 cells further demonstrating the important role ofautophagy in lipid diï¬erentiation role of alternative splicing in adipogenesis selectivesplicing is uenced by splicing regulators which regulateadipocyte diï¬erentiation by regulating the selective splicingof genes speciï¬c to this process lipin1 is an important regulator in the process of adipocyte diï¬erentiation and includestwo isomers lipin1α and lipin1 which have diï¬erenteï¬ects high expression of lipin1α promotes adipocyte differentiation while that of lipin1 promotes lipid droplet formation in sam68deï¬cient mice the ï¬fth intron ofserinethreonineprotein kinase mtor was retained resulting in unstable and rapid mtor degradation and inhibitionof adipocyte diï¬erentiation furthermore there arefour isomers of pref1 pref1a and pr | Colon_Cancer |
" when we encounter patients who present with both a neck mass and nephrotic syndrome bothmalignancy and kimuras disease need to be evaluated as the therapeutic strategies differ vastly between themcase presentation we present the case of a 27yearold male patient with neck mass and nephrotic syndromethe presence of both eosinophilia and elevated immunoglobulin e levels were concerning for kimuras diseasewhich is an allergic syndrome defined by eosinophilic granulomas of neck soft tissue along with peripheraleosinophilia the eventual final diagnosis however was sclerosing mucoepidermoid carcinoma of parotid glandwith both eosinophilia and membranous nephropathy following the surgical resection of the mass the nephroticsyndrome completely resolved detailed histopathological assessments of both the parotid gland and renal tissue were key aspects ofthe diagnosis and management to exclude kimuras diseasekeywords membranous nephropathy nephrotic syndrome sclerosing mucoepidermoid carcinoma with eosinophilia membranous nephropathy can occur in the setting ofmalignant tumors and often recovers following definitivetreatment of malignancy sclerosing mucoepidermoidcarcinoma with eosinophilia is a rare variant of mucoepidermoid carcinoma for which surgical resection isgenerally recommended as a primary treatment thereare currently no published reports of nephrotic syndromeassociated with mucoepidermoid carcinoma kimuras correspondence teimamumedutoyamaacjp1the second department of internal medicine university of toyama sugitani toyama toyama japanfull list of author information is available at the end of the disease which is a benign syndrome accompanied byeosinophilic granulomas of neck soft tissue often found inyoung men of eastasian descent sometimes accompaniesrenal disease and can be treated by steroid therapy [ ]we present here a young male patient who sufferedmucoepidermoid carcinoma of right parotid grand withlocalized spread to lymph nodes and secondary membranous nephropathy both of which had significant eosinophilic infiltration the presence of peripheral eosinophiliaand elevated immunoglobulin e level has a broad differential diagnosis with vastly different treatment pathways 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 0cfujioka bmc nephrology page of case presentationa 27yearold japanese male patient without a history ofany allergic syndromes was admitted to our institutewith bilateral peripheral edema proteinuria and swellingof the right parotid gland cytology of the parotid glandand lymph node biopsy showed no malignancy thougheosinophilic infiltration in the lymph node was observedhe was diagnosed with nephrotic syndrome with gg of creatinine of proteinuria gdl of serum albuminand mgdl of lowdensity lipoprotein mild peripheral eosinophilia μl and elevated immunoglobuline iuml were also present immunoglobulin gwas mgdl soluble interleukin receptor was uml c3 was mgdl c4 was mgdl andtotal complement activity was umlkidney sizes were mm right and mm leftwe performed a kidney biopsy to further investigate themechanism of the observed nephrotic syndrome onlight microscopy the glomerular basement membraneexhibited mild diffuse thickening with spike formationfig 1a eosinophilic interstitialinfiltration was alsoseen fig 1b immunofluorescence staining showed diffusegranular deposits of immunoglobulin g and c3 along theglomerular capillary walls fig 1c immunoglobulin g4 wasnot predominant for immunoglobulin g subclass and kappaand lambda light chains had equal intensity in the immunofluorescence staining immunofluorescence staining for thephospholipase a2 receptor pla2r and the thrombospondin type domaincontaining 7a thsd7a were negativethe electron microscopy showed globalsubepithelialelectrondense deposits and spike formation of the glomerular baseline membrane fig 1d he was diagnosed withstage ii secondary membranous nephropathycomputed tomography showed a mm of tumor inthe right parotid grand accompanied by a mmlymphoid focus fig both of which showed uptake byfluorodeoxyglucoseposition emission tomography weat this point suspected malignant disease instead of analternative benign presentation such as kimuras diseaserepeat biopsies eventually demonstrated carcinoma andlymph node metastasis t3n2m0 stage ivabefore surgical excision steroid pulse therapy methylprednisolone mg days was performed leading tothe partial reduction of proteinuria and eosinophilia atone month following pulse therapy the patient underwentsuccessful right parotidectomy with neck dissection theresected specimen was consistent with the diagnosis ofsclerosing mucoepidermoid carcinoma with eosinophiliaa variant of mucoepidermoid carcinoma pt3n2bfig the proteinuria achieved complete remissionand eosinophilia normalized without steroid therapyduring the 12month followup with concomitantpostoperative radiation therapy gy 30fr he hashad no recurrence of disease over twoyear followupfig histopathological findings in kidney biopsy specimen a diffuse thickness of the glomerular basement membrane with spike formationarrowhead periodic acidmethenaminsilver stain b infiltration of eosinophils in renal interstitium hematoxylineosin stain c diffuse granulardeposits of immunoglobulin g along the glomerular capillary walls immunofluorescence stain for igg d global subepithelial electrondensedeposits and spike formation of the glomerular baseline membrane electron microscopy 0cfujioka bmc nephrology page of fig head computed tomography shows tumor at right parotid gland a and lymphadenopathy of right neck bdiscussion and we diagnosed mucoepidermoid carcinoma with eosinophilia and excluded kimuras disease with successivebiopsies and detailed histopathological assessment thepatient did clinically well following careful exclusion ofkimuras disease for which the correct therapeutic pathway was surgical resection instead of steroid therapyboth kimuras disease and mucoepidermoid carcinomawith eosinophila have several common characteristicsincluding neck mass with lymph node enlargementeosinophilia and high serum immunoglobulin e levelmucoepidermoid carcinoma is the most commonmalignancy among salivary glandorigin tumors andalthough rareit can occur in younger patients fig a tumor of right parotid gland hematoxylineosin stain showing invasion of tumor cells surrounded by sclerotic stroma and numerouseosinophils b normal tissue of right parotid gland hematoxylineosin stain also showing infiltration of eosinophil 0cfujioka bmc nephrology page of mucoepidermoid carcinoma has several variants sclerosing mucoepidermoid carcinoma with eosinophilia is oneof more rarely encountered variants the exact mechanism of why this carcinoma is associated with peripheraleosinophilia and infiltration is uncertain prior studieshave suggested that tumorderived cytokine release mightbe an important pathobiological mechanism []kimuras disease is a rare syndrome accompanied byeosinophilic granulomas of neck soft tissue peripheraleosinophilia and high immunoglobulin e level it is mostoften observed in young eastasian males [ ] this is abenign chronic granulomatous disease characterized byfollicular lymphomas with eosinophilic infiltration thedisease can be treated by steroids or other forms ofsystemic immunosuppression prior reports suggestthat renal disease with nephrotic syndrome may occur inup to of patients with kimuras disease the pathogenesis of kimuras disease remains unknowntrauma infection an immunoglobulin emediated hypersensitivity reaction or an autoimmune process mightstimulate cytokine release that stimulates eosinophil production of note there are no published reports ofkimuras disease in the presence of a confounding malignancy in this case atypical change with eosinophilic interstitial infiltration and negative expression of pla2r andthsd7a on renal biopsy suggested secondary membranous nephropathy instead of a de novo origin to our knowledge this is the first published report ofmucoepidermoid carcinoma associated with membranousnephropathy it is well known that patients with malignancies may have secondary membranous nephropathy cases have been reported in lung prostate andcolon cancers and even with hematologic malignancies the mechanism of why malignancyrelated secondarymembranous nephropathies can occur remains unknownbut several hypotheses are proposed antibodystimulation from tumor antigens immunologically similarto an endogenous podocyte antigen leading to in situ immune complex formations shed tumor antigens mightform circulating immune complexes that are subsequentlytrapped in the glomerular capillary wall circulating antibodies might also react to the tumor antigens that havealready been planted in the subepithelial location and an extrinsic process including viral infection or underlyingabnormal immune response might be responsible for bothdiseases in this case a tumorassociated immunoreactionmight have had a considerable impact on the developmentof a secondary membranous nephropathy the detailedmechanism of renal interstitial infiltration of eosinophilscontinues to remain uncertainthough tumorsecretedcytokine release might be the causative pathwayabbreviationspla2r phospholipase a2 receptor thsd7a thrombospondin type domaincontaining 7aacknowledgementsnot applicableauthors contributionshf contributed to analyzing and interpretation of data and writing themanuscript tk contributed to interpretation of data and writing themanuscript ti contributed to interpretation of data and writing themanuscript kka contributed to analyzing and interpretation of data andwriting the manuscript hy contributed to managing the patient andassessing the data ha contributed to managing the patient and operationtn contributed to analyzing and interpretation of pathological findings kkicontributed to analyzing and interpretation of data and writing themanuscript the authors read and approved the final manuscriptfundingthere was no fundingavailability of data and materialsall the date supporting our findings is contained within the manuscriptethics approval and consent to participatethe case report was approved by the medical ethics committee of theuniversity of toyama the patient provided written informed consent forparticipation in the study at university of toyamaconsent for publicationthe patient received all information regarding this case report writteninformed consent for publication in bmc nephrology was obtained from thepatientcompeting intereststhe authors declare that they have no competing interestsauthor details1the second department of internal medicine university of toyama sugitani toyama toyama japan 2department ofotorhinolaryngology head neck surgery university of toyama toyamajapan 3department of diagnostic pathology university of toyama toyamajapanreceived december accepted august referencesrow pg cameron js turner dr evans dj white rh ogg cs membranous nephropathy longterm followup and association withneoplasia q j med kimura t on the unusual granulation combined with hyperplastic changesof lymphatic tissue trans soc pathol jpn kuo tt shih ly chan hl kimura's disease involvement of regional lymphnodes and distinction from angiolymphoid hyperplasia with eosinophiliaam j surg pathol guevaracanales jo moralesvadillo r guzm¡narias g cavavergiº ceguerramiller h montesgil je mucoepidermoid carcinoma of the salivaryglands a retrospective study of cases and review of the literature actaodontol latinoam urano m abe m horibe y kuroda m mizoguchi y sakurai k sclerosing mucoepidermoid carcinoma with eosinophilia of the salivaryglands pathol res pract hu g wang s zhong k tumorassociated tissue eosinophilia predictsfavorable clinical outcome in solid tumors a metaanalysis bmc cancergatault s legrand f delbeke m loiseau s capron m involvement ofeosinophils in the antitumor response cancer immunol immunother anagnostopoulos gk sakorafas gh kostopoulos p disseminatedcolon cancer with severe peripheral blood eosinophilia and elevated serumlevels of interleukine2 interleukine3 interleukine5 and gmcsf j surgoncol 0cfujioka bmc nephrology page of natov sn strom ja ucci a relapsing nephrotic syndrome in a patient withkimura's disease and iga glomerulonephritis nephrol dial transplant yamada a mitsuhashi k miyakawa y membranous glomerulonephritisassociated with eosinophilic lymphfolliculosis of the skin report of a caseand review of literature clin nephrol terada n konno a shirotori k fujisawa t atsuta j ichimi r mechanism of eosinophil infiltration in the patient with subcutaneousangioblastic lymphoid hyperplasia with eosinophilia kimura's diseasemechanism of eosinophil chemotaxis mediated by candida antigen and il int arch allergy immunol suppl couser wg primary membranous nephropathy clin j am soc nephrolleeaphorn n kue app thamcharoen n ungprasert p stokes mb knight elprevalence of cancer in membranous nephropathy a systematic review andmetaanalysis of observational studies am j nephrol beck lh jr membranous nephropathy and malignancy semin nephrolpublishers notespringer nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations 0c" | Colon_Cancer |
"adipogenesis is the process through which mesenchymalstem cells mscs commit to the adipose lineage and diï¬erentiate into adipocytes during this process preadipocytescease to proliferate begin to accumulate lipid droplets anddevelop morphologic and biochemical characteristics ofmature adipocytes such as hormoneresponsive lipogenesisand lipolytic programs currently there are mainly twomodels of benign adipocyte diï¬erentiation in vitro one isï¬broid pluripotent stem cells which can diï¬erentiate intonot only adipocytes but also muscle cartilage and othercells there are two kinds of ï¬broid pluripotent stem cellsbone marrow and adipose mesenchymal stem cells anothergroup is ï¬broblastic preadipocytes which have a single direction of diï¬erentiation namely lipid diï¬erentiation including3t3l1 and 3t3f422a cells cancer cells with tumorinitiation ability designated as cancer stem cells cscshave the characteristics of tumorigenesis and the expressionof speciï¬c stem cell markers as well as the longterm selfrenewal proliferation capacity and adipose diï¬erentiationpotential in addition to cscs cancer cells undergoing epithelialmesenchymaltransformation emt havebeen reported to be induced to diï¬erentiate into adipocytes[] lung cancer ncih446 cells can be induced to differentiate into neurons adipocytes and bone cells in vitro the adipogenesis diï¬erentiation treatment is promisingin the p53 gene deletion type of ï¬broblastderived cancer cancer cells with homologous recombination defectssuch as ovarian and breast cancer cells with breast cancersusceptibility genes brca mutations can be inducedto diï¬erentiate by poly adpribose polymerase parp 0cstem cells internationalinhibitors the nuclear receptor peroxisome proliferatoractivated receptor Î pparÎ agonist antidiabetic thiazolidinedione drug can induce growth arrest and adipogenicdiï¬erentiation in human mouse and dog osteosarcoma cells thyroid cancer cells expressing the pparÎ fusion proteinppfp can be induced to diï¬erentiate into adipocytes bypioglitazone adipogenesis can be induced in welldiï¬erentiated liposarcoma wdlps and dediï¬erentiatedliposarcoma ddlps cells by dexamethasone indomethacininsulin and 3isobutyl1methyl xanthine ibmx in this review we highlight some of the crucial transcription factors that induce adipogenesis both in mscs and inincluding the wellstudied pparÎ and ccaatcscsenhancerbinding proteins cebps as well as othercell factors that have been recently shown to have an important role in adipocyte diï¬erentiation we focus on understanding the complex regulatory mechanism of adipocytediï¬erentiation that can contribute to the clinical treatmentof human diseases including those caused by obesity andadipocytes dysfunction especially for the malignant tumorwhich can be transdiï¬erentiated into mature adipocytes adipocyte differentiationcell proliferation and diï¬erentiation are two opposingprocesses and there is a transition between these two processes in the early stages of adipocyte diï¬erentiation theinteraction of cell cycle regulators and diï¬erentiation factors produces a cascade of events which ultimately resultsin the expression of adipocyte phenotype adipogenesishas diï¬erent stages each stage has a speciï¬c gene expression pattern in general adipocyte diï¬erentiation ofpluripotent stem cells is divided into two phases the ï¬rstphase known as determination involves the commitment ofpluripotent stem cells to preadipocytes the preadipocytescannot be distinguished morphologically from their precursor cells but also have lost the potential to diï¬erentiate intoother cell types in the second phase which is known asterminal diï¬erentiation the preadipocytes gradually acquirethe characteristics of mature adipocytes and acquire physiological functions including lipid transport and synthesisinsulin sensitivity and the secretion of adipocytespeciï¬cproteins the diï¬erentiation of precursor adipocytes is also dividedinto four stages proliferation mitotic cloning early diï¬erentiation and terminal diï¬erentiation after the precursors are inoculated into the cell culture plates the cellsgrow exponentially until they converge after reaching contact inhibition the growth rate slows and gradually stagnatesand the proliferation of precursor adipocytes stops which isvery necessary for initiating the diï¬erentiation of precursoradipocytes adipocyte precursors exhibit transient mitosiscalled clonal expansion a process that relies on the actionof induced diï¬erentiation factors some preadipocyte cellsmouse cell lines 3t3l1 3t3f442a undergo one or tworounds of cell division prior to diï¬erentiation whereasother cell lines mouse c3h10t12 diï¬erentiating into adipocyte do not undergo mitosis clonal expansion whether mitotic clonal expansion is required for adiposediï¬erentiation remains controversial however it is certainthat some of the checkpoint proteins for mitosis regulateaspects of adipogenesis [ ] when cells enter the terminaldiï¬erentiation stage the de novo synthesis of fatty acidsincreases significantly the transcription factors and adipocyterelated genes work cooperatively to maintain precursor adipocyte diï¬erentiation into mature adipocytes containing largelipid droplets regulatory pathways inpreadipocytes commitmentadipocyte diï¬erentiation is a complex process in which geneexpression is ï¬nely regulated the most basic regulatory network of adipose diï¬erentiation has not been updated inrecent years but some factors and signaling pathways thatdo aï¬ect adipose diï¬erentiation have been continuouslyreported adipocyte diï¬erentiation is the result of the geneexpression that determines the phenotype of adipocyteswhich is a complex and delicate regulatory process figure wnt signal pathway in adipogenesis wnt signaling isimportant for adipocytes proliferation and diï¬erentiationboth in vitro and in vivo the wnt family of secretedglycoproteins functions through paracrine and autocrinemechanisms to uence cell fate and development wntprotein binding to frizzled receptors initiates signalingthrough catenindependent and independent pathways wnt signaling inhibits adipocyte diï¬erentiation in vitroby blocking the expression of pparÎ and cebpα constitutive wnt10b expression inhibits adipogenesis wnt10b isexpressed in preadipocytes and stromal vascular cells butnot in adipocytes in vivo transgenic expression of wnt10bin adipocytes results in a reduction in white adipose tissuemass and absent brown adipose tissue development wnt10a and wnt6 have also been identiï¬ed as determinantsof brown adipocyte development [ ] wnt5b is transiently induced during adipogenesis and promotes diï¬erentiation indicating that preadipocytes integrate inputs fromseveral competing wnt signals the hedgehog hh signaling pathway mechanismthree vertebrate hh ligands including sonic hedgehogshhindian hedgehog ihh and desert hedgehogdhh have been identiï¬ed and initiated a signaling cascademediated by patched ptch1 and ptch2 receptors [ ]hh signaling had an inhibitory eï¬ect on adipogenesis inmurine cells such as c3h10t12 ks483 calvaria mscslines and mouse adiposederived stromal cells thesecells were visualized by decreased cytoplasmic fat accumulation and the expression of adipocyte marker genes after hhsignaling was inhibited although it is generally agreedthat hh expression has an inhibitory eï¬ect on preadipocytediï¬erentiation the mechanisms linking hh signaling andadipogenesis remain poorly deï¬ned erkmapkppar signal pathway extracellularregulated protein kinase erk is required in the proliferativephase of diï¬erentiation erk activity blockade in 3t3l1 0cstem cells internationaldex insulin demxwnt 10band othersshhpbc smotgfð½p smad3 smad3testosteroneð½catentinarirspi3kaktcrebpkapcrebfoxo1a2tcflef gata23cebpð½mapkg3k3ð½p2cebpð½cebpαppará½»bmpssmad1srebpadipocytegenesfigure regulation pathways in preadipocytes commitment bmp and wnt families are mediators of mscs commitment to producepreadipocytes exposure of growtharrested preadipocytes to diï¬erentiation inducers igf1 glucocorticoid and camp triggers dnareplication leading to adipocyte gene expression due to a transcription factor cascade the dotted line indicates an uncertain molecularregulatory mechanismcells and embryonic stem cells can inhibit adipogenesis inthe terminal diï¬erentiation phase erk1 activity leads topparÎ phosphorylation which inhibits adipocyte diï¬erentiation this implies that erk1 activity must be reduced afteradipocyte proliferation so that diï¬erentiation can proceedthis reduction is mediated in part by mitogenactivatedprotein kinase mapk phosphatase1 mkp1 [ ]these extracellular and intracellular regulation factors causeadipocytespeciï¬c gene expression and eventually lead toadipocyte formation adipocyte differentiationregulatory proteins pparÎ and adipocyte diï¬erentiation pparÎ is a member of the nuclearreceptor superfamily and is both necessaryand suï¬cient for adipogenesis forced expression ofpparÎ is suï¬cient to induce adipocyte diï¬erentiation broblasts indeedthe proadipogenic cebps andkrüppellike factors klfs have all been shown to induceat least one of the two pparÎ promoters in contrast antiadipogenic transcription factor gata functioned in part byrepressing pparÎ expression pparÎ itself has twoisomers the relative roles of pparÎ1 and pparÎ2 in adipogenesis remain an open question pparÎ2 is mainlyexpressed in adipose tissue while pparÎ1 is expressed inmany other tissues although both can promote adipocytediï¬erentiation pparÎ2 could do so eï¬ectively at very lowligand concentration compared with pparÎ1 the twoprotein isoforms are generated by alternative splicing andpromoter usage and both are induced during adipogenesispparÎ1 can also be expressed in cell types other than adipocytes ren et al used engineered zincï¬nger proteins tothe expression ofthe endogenous pparÎ1 andinhibitpparÎ2 promoters in 3t3l1 cells ectopic expression ofpparÎ2 promotes adipogenesis whereas that of pparÎ1does not zhang et al reported that pparÎ2 deï¬ciencyimpairs the development of adipose tissue and insulin sensitivity there are transcriptional cascades between adipocytesgenes including pparÎ and cebpα which are the coreadipocyte diï¬erentiation regulators in the early stage of adipocyte diï¬erentiation the expression of cebp and cebpδincrease which upregulates cebpα expressionfurtheractivate pparÎ pparÎ activating cebpα in turn resultsin a positive feedback pparÎ binding with retinoic acid xreceptor rxr forms diï¬erent heterodimers the variousdimmers can combine with the pparÎ response elementppre and initiate the transcription of downstream genesfor diï¬erentiation into adipocytes cebps participate in adipogenesis and several cebpfamily members are expressed in adipocytesincludingcebpα cebp cebpÎ cebpδ and cebphomologous protein chop the temporal expression of thesefactors during adipocyte diï¬erentiation triggers a cascadewhereby early induction of cebp and cebpδ leads tocebpα expression this notion is further supported by thesequential binding of these transcription factors to severaladipocyte promoters duringadipocyte diï¬erentiationcebp is crucial for adipogenesis in immortalized preadipocyte lines cebp and cebpδ promote adipogenesis atleast in part by inducing cebpα and pparÎ cebpαinduces many adipocyte genes directly and plays an important role in adipose tissue development once cebpα isexpressed its expression is maintained through autoactivation despite the importance of cebps in adipogenesis 0cstem cells internationalthese transcription factors clearly cannot function eï¬cientlyin the absence of pparÎ cebp cannot induce cebpαexpression in the absence of pparÎ which is required torelease histone deacetylase1 hdac1 from the cebpαpromoter furthermore ectopic cebpα expressioncannot induce adipogenesis in pparÎï¬broblasts however cebpα also plays an important role in diï¬erentiated adipocytes overexpression of exogenous pparÎ incebpαdeï¬cient cells showed that although cebpα isnot required for lipid accumulation and the expression ofmany adipocyte genes it is necessary for the acquisition ofinsulin sensitivity [ ] figure human ï¬broblasts withthe ability to diï¬erentiate into adipocytes also do not undergomitotic cloning ampliï¬cation however pparÎ exogenousligands need to be added to promote adipocyte diï¬erentiation therefore it can be inferred that mitotic cloning expansion can produce endogenous ligands of pparÎ bmp and transforming growth factor tgf inadipocyte diï¬erentiation a variety of extracellular factorsaï¬ect the preadipocyte commitment of stem cells includingbone morphogenetic protein bmp transforminggrowth factor tgf insulininsulinlike growthfactor igf1 tumor necrosis factor α and interleukin matrix metalloproteinase ï¬broblast growthfactor fgf and fgf2 bmp and tgf have variedeï¬ects on the diï¬erentiation fate of mesenchymal cells the tgf superfamily members bmps and myostatinregulate the diï¬erentiation of many cell types includingadipocytes tgf inhibitor can promote adipose diï¬erentiation of cancer cells with a mesenchymal phenotypein vitro and transgenic overexpression of tgf impairsadipocyte development inhibition of adipogenesis couldbe obtained through blocking of endogenous tgf with adominantnegative tgf receptor or drosophila mothersagainst decapentaplegic protein smad inhibitionsmad3 binds to cebps and inhibits their transcriptionalactivity including their ability to transactivate the pparÎ2promoter [ ] exposure of multipotent mesenchymalcells to bmp4 commits these cells to the adipocyte lineageallowing them to undergo adipose conversion theeï¬ects of bmp2 are more complex and depend on the presence of other signaling molecules bmp2 alone has little eï¬ecton adipogenesis and it interacts with other factors such astgf and insulin to stimulate adipogenesis of embryonicstem cells bmp2 stimulates adipogenesis of multipotentc3h10t12 cells at low concentrations and can contribute tochondrocyte and osteoblast development at higher concentrations klfs in adipocyte diï¬erentiation during adipocyte differentiation some klf family members are overexpressedsuch as klf4 klf5 klf9 and klf15 while klf16 expression is reduced [ ] klf15 is the ï¬rst klf family members which were identiï¬ed to be involved in adipocytediï¬erentiation its expression increased significantly on thesixth day of 3t3l1 adipocyte diï¬erentiation and peakedon the second day of adipocyte induction in mscs andmouse embryonic ï¬broblasts inhibition of klf15 by sirnaor mutation led to a decrease in pparÎ cebpα fatty acidbinding protein fabp4 and glucose transporter glut4 however overexpression of klf15 in nih3t3cells was found to be associated with lipid accumulation aswell as increases in pparÎ and fabp4 mice with complete absence of klf5 showed embryonal lethality and micewith singlechromosome klf5 knockout showed a significant reduction in white fat in adulthood suggesting thatklf5 plays an important role in adipocyte diï¬erentiationklf5 can be activated by cebp or cebpδ which isinvolved in early adipocyte diï¬erentiation klf5 can beactivated by cebp or cebpδ which is involved in earlyadipocyte diï¬erentiation direct binding of klf5 to thepparÎ2 promoter in combination with cebps inducespparÎ2 expression transfection of klf5 dominantnegative mutants in 3t3l1 cells reduced lipid droplet accumulation and inhibited pparÎ and cebpα expressionwhereas overexpression of wild klf5 significantly promotedadipocyte diï¬erentiation even without exogenous hormonestimulation similar to klf5 klf9 knockdown can inhibitthe expression of a series of adipocyte diï¬erentiation genessuch as pparÎ cebpα and fabp4 hence inhibitingadipocyte diï¬erentiation however klf9 overexpressiondid not upregulate the expression of pparÎ and cebpα in addition klf4 can transactivate cebp by bindingto the region of kb upstream of the cebp promoter and promote lipid diï¬erentiation klf6 can forma complex with histone deacetylase3 hdac3 inhibitingpreadipocyte factor1 pref1 expression and promotinglipid diï¬erentiation klf2 is highly expressed in adiposeprogenitors and its expression decreases during the processof lipid diï¬erentiation overexpressed klf2 can bind to thecaccc region of pparÎ2 proximal promoter and inhibitlipid diï¬erentiation as well as the expression of pparÎcebpα and sterolregulated elementbinding proteinssrebp by inhibiting the promoter activity rnasequence analysisshowed that klfl6 expression wasdecreased on the ï¬rst day of adipocyte diï¬erentiation of3t3l1 cells adipocyte diï¬erentiation was promoted byklf16 knockdown but was inhibited by klf16 overexpression via inhibition of pparÎ promoter activity in addition klf3 and klf7 were also found to play a negativeregulatory role in adipocyte diï¬erentiation [ ] signal transducers and activators of transcriptionstats and adipocyte diï¬erentiation the activated statprotein enters the nucleus as a dimer and binds to the targetgene to regulate gene transcription in the adipocyte diï¬erentiation of mouse 3t3l1 cells the expression of stat1 andstat5 was significantly increased while that of stat3and stat6 was not significantly changed in the adipocyte diï¬erentiation of human subcutaneous adipose precursor cells stat1 expression was significantly decreased while the expression of stat3 and stat5 wasincreased and stat6 expression was unchanged therole of stat1 in adipocyte diï¬erentiation is not clearbecause its expression trend in humans and mice diï¬ersduring the adipocyte diï¬erentiation process early adipocytediï¬erentiation of 3t3l1 cells was inhibited by stat1 0cstem cells internationalklf5srebp1cklf15klf2chopcebpá½»krox20ligandcebpð½cebpð¿gata23ppará½»cebpð¼proadipogenicantiadipogenicgenes of terminaladipocytedifferentiationfigure a cascade of transcription factors that regulate adipogenesis pparÎ is one of the key transcription factors in adipogenesis and thecore of the transcriptional cascade that regulates adipogenesis pparÎ expression is regulated by several proadipogenic blue andantiadipogenic red factors cebpα is regulated through a series of inhibitory proteinprotein interactions some transcription factorfamilies include several members that participate in adipogenesis such as the klfs black lines indicate eï¬ects on gene expression violetlines represent eï¬ects on protein activityagonist interferon Î loss of stat1 in 3t3l1 cells can rescue the inhibition of adipocyte diï¬erentiation caused byprostaglandin factor 2α other studies have found thatstat1 is required for adipose diï¬erentiation and stat1overexpression in c3h10t12 cells can prevent the inhibition of lipid diï¬erentiation caused by bcell lymphoma6knockdown there was no abnormal adipose tissuein stat1 knockout mice stat3 not only aï¬ectsthe proliferation of 3t3l1 cells but also coregulates theiradipocyte diï¬erentiation with high mobility group protein the fabp4 promoter was used to speciï¬callyknock out stat3 in the adipose tissue of mice and theresults showed that mice weight significantly increasedand the adipocyte quantity increased compared with thewildtype mice stat5a and stat5b have diï¬erenteï¬ects on adipocyte diï¬erentiation abnormal adipose tissuewas found in the mice with stat5a or stat5b knockout ordouble knockout and the amount of adipose tissue was onlyoneï¬fth of the original adipose tissue in mice withoutknockdown histone modiï¬cation in adipocyte diï¬erentiation histone deacetylase sirtuin sirt plays an important rolein biological processes such as stress tolerance energymetabolism and cell diï¬erentiation during the adipocyte diï¬erentiation of c3h1012 cells sirt1 expressiondecreased overexpression of sirt1 activated thewnt signal which caused the deacetylation of cateninthe accumulation of catenin in the nucleus could inhibitadipocyte diï¬erentiation sirt1 knockdown resulted inincreased acetylation of the histones h3k9 and h4k16 inthe secreted frizzledrelated protein sfrp and sfrp2 promoters thereby promoting transcription of these genes andpromoting lipid diï¬erentiation forkhead box proteino foxo is a member of the transcription factor foxofamily it can recruit cyclic amp response elementbindingprotein cbphistone acetyltransferase p300 to initiate anacetylation the acetylated foxo1 can be phosphorylatedby phosphorylated protein kinase b pkbakt the phosphorylation of foxo1 by akt inhibits the transcriptionalactivation of foxo1 the acetylation of foxo1 lost the ability of dnabinding aï¬nity and promoted its shuttling fromnuclei to cytoplasm sirt1 and sirt2 can deacetylateand active foxo1 activated foxo1 nonphosphorylatednuclear foxo1 in the nucleus binds to the promoters of target genes encoding p21 p27 and pparÎ and initiates subsequent transcriptions sirt2 inhibits the acetylation andphosphorylation of foxo1 thereby induces the accumulation of activated foxo1 in the nucleus activated foxo1could inhibit adipogenesis via pparÎ [] lysinespeciï¬c histone demethylase lsd1 expression increasedduring the adipocyte diï¬erentiation of 3t3l1 cells lsd1could reduce the dimethylation levels of histone h3k9 andh3k4 in the cebpα promoter region thereby promotingadipocyte diï¬erentiation set domaincontaining setd8 catalyzed the monomethylation of h4k20 andpromoted pparÎ expression the activation of pparÎ transcriptional activity leads to the induction of monomethylatedh4k20 and modiï¬cation of pparÎ and its targets therebypromoting adipogenesis enhancer of zeste homolog ezh2 is a methyltransferase and can bind methyl groupsto histone h3k27 which is also necessary for lipid diï¬erentiation the absence of ezh2 in brown fat precursors results inreduced levels of the wnt promoter histone h3k27me3which is also saved by the ectopic ezh2 expression or theuse of a wntcatenin signal inhibitor in addition histone demethylases such as lysinespeciï¬c histone demethylase lsdkdm kdm6 and histone lysine demethylasephf2 are also involved in adipose diï¬erentiation andkdm2b inhibits transcription factor activator protein 2αpromoter via h3k4me3 and h3k36me2 role of microrna and long noncodingrna in adipogenesismicrorna mir can bind and cut target genes or inhibittarget gene translation endogenous sirna can be producedby the action of dicer enzyme and bind to a speciï¬c proteinto change its cellular location many kinds of mirsare involved in regulating adipocyte diï¬erentiation the 0cstem cells internationalexpression of mir143 increased during the diï¬erentiationof adipose progenitor cells overexpression of mir143promoted gene expression involved in adipose diï¬erentiationand triglyceride accumulation inhibition of mir143 prevented the adipose diï¬erentiation of human fat progenitorcells [ ] additionally mir8 promotes adipocyte diï¬erentiation by inhibiting wnt signaling moreover mir mir103 mir21 mir519d mir210 mir30mir204211 and mir375 also play a certain role in promoting adipocyte diï¬erentiation while mir130 mir448and let7y inhibit lipid diï¬erentiation [ ] in additionto mirs long noncoding rna lncrna is a type of noncoding rna and is important during epigenetic regulationand can form a doublestranded rna complex with mrnacauses protein transcription lncu90926 inhibits adipocytediï¬erentiation by inhibiting the transactivation of pparÎ2 as a novel lncrna hoxaas3 expression increasedduring the adipose diï¬erentiation of mscs and hoxaas3 silencing reduced the marker gene of adipose diï¬erentiation and inhibited the adipose diï¬erentiation zhu et al reported that hoxaas3 interacted with ezh2 toregulate lineage commitment of mscs hoxa as3 canregulate the trimethylation level of h3k27 in the runx2promoter region by binding to ezh2 therefore hoxaas3 is considered to be an epigenetic switch regulating mscslineage speciï¬city adipocyte diï¬erentiationassociatedlncrna can act as a competitive endogenous rna of mir in the process of lipid diï¬erentiation thereby promotingthe expression of sirt1 the target gene of mir204 and thusinhibiting lipid diï¬erentiation the lncrna neat1can also regulate adipocyte diï¬erentiation under the uence of mirna140 other lncrna including lncrnablnc1 and plnc are also involved in regulating adipocytediï¬erentiation [ ] other biochemical response involved inadipocyte differentiation unfolded protein responses in adipocyte diï¬erentiationin the endoplasmic reticulum of eukaryotes unfolded protein response involves three proteinsinositolrequiringenzyme 1α doublestranded rnadependent proteinkinaselike er kinase and activating transcription factoratf 6α knockdown of atf6α aï¬ects the expressionof adipocytes genes and inhibits c3h10t12 adipocyte differentiation the inhibitory eï¬ect of berberine on adipocyte diï¬erentiation of 3t3l1 cells is also due to inducedchop and decorin expressions and this inhibitory eï¬ectis ameliorated by chop knockout in the adipocytediï¬erentiation process of 3t3l1 cells increases in pparÎand cebpα as markers of adipocyte diï¬erentiation wereaccompanied by an increase in the corresponding proteinexpressions of phosphorylated eukaryotic translation initiaeif 2α phosphorylated endoribonucleasetion factorire1α atf4 chop and other unfolded protein responsesendoplasmic reticulum stress inducer or hypoxic endoplasmic reticulum stress can inhibit adipocyte diï¬erentiationadditionally eif2α mutation results in continuous activation or overexpression of chop which also inhibits adipocyte diï¬erentiation after the initiation of adiposediï¬erentiation numerous diï¬erentiationassociated proteinsare synthesized exogenous endoplasmic reticulum stressinducers can lead to excessive endoplasmic reticulumresponse which in turn aï¬ects the synthesis of proteinsrelated to diï¬erentiation and inhibits adipocyte formationfigure role of oxidative stress in adipogenesis during thedirectional diï¬erentiation of mscs mitochondrial complexi and iii and nadph oxidase nox4 are the main sourcesof oxygen species ros production currently it is believedthat ros aï¬ects not only the cell cycle and apoptosis but alsodiï¬erentiation through uencing the signaling pathwaysincluding the wnt hh and foxo signaling cascade duringmscs diï¬erentiation the diï¬erentiation ability ofstem cells is determined by the arrangement of perinuclearmitochondria which speciï¬cally manifests as low atpcellcontents and a high rate of oxygen consumption the lackof these characteristics indicates stem cell diï¬erentiation adipocyte diï¬erentiation is a highly dependent rosactivation factor related to mitosis and cell maturation schroder et al found that exogenous h2o2 could stimulate adipocyte diï¬erentiation of mouse 3t3l1 cells andhuman adipocyte progenitor cells in the absence of insulinh2o2 regulates adipocyte diï¬erentiation of 3t3l1 cells ina dosedependent manner high doses of h2o2 and μm promote adipocyte diï¬erentiation [ ] tormos et al found that ros synthesis increased in humanmscs at the early stage of adipose diï¬erentiation and targeted antioxidants could inhibit lipid diï¬erentiation byknocking down rieske ironsulfur protein and ubiquinonebinding protein ros produced by mitochondrial complexiii was found to be necessary in initiating adipose diï¬erentiation however other studies have shown that theexpression levels of adiponectin and pparÎ were decreasedby using h2o2 mm in 3t3l1 cells free radical nitric oxide no also promotes lipid diï¬erentiationbecause treatment with no inducer hydroxylamine or nosynthase nos substrate arginine can significantly induceadipose diï¬erentiation of rat adipose progenitor cells nosinduced adipose diï¬erentiation mainly via enos rather thaninos ros can induce adipose diï¬erentiation primarily by inhibiting wnt foxo and hh signaling pathwaysthat inhibit lipid diï¬erentiation autophagy in adipocyte diï¬erentiation the increase inautophagosomes during lipid diï¬erentiation indicates thatautophagy may play an important role in lipid diï¬erentiation baerga et al conï¬rmed that the adipocyte diï¬erentiation eï¬ciency was significantly inhibited in mouse embryonic ï¬broblasts lacking autophagyrelated gene atg agene encoding an essential protein required for autophagy knockdown of atg5 in 3t3l1 cells promotesproteasomedependent degradation of pparÎ2therebyinhibiting adipocyte diï¬erentiation zhang reportedthat autophagyrelated gene 7atg7 is also crucial for adipose development atg7deï¬cient mice were slim and onlyhad of white fat compared to wildtype mice and the 0cstem cells internationalcebpð½ geneebf1 geneklf4egr2cebpð½cytosolcebpð¿ genecebpð¿klf5geneppará½» geneklf5nr2f2nfkb11433relasrebf1a2rxrappará½»ppará½»rxra heterodimerppará½»rxracorepressor complexfabp4ligands of ppará½»fam120bthrap3ep300ncoa2ncoa3helz2ncoa1crebbpebf1adipoq geneaidrfcebpð¼ geneznf638znf467cebpð¼ncor1hdac3ncor2 slc2a4 geneglut4 genelep genefabp4 genecdk4ccnd3plin1 genepck1 genefabp4cd36 geneppararxracoactivator complexppará½»fatty acidrxramediatorcoactivator complexangptlgeneppargc1amediator complex consensuslpl genenucleoplasmproteins bind to gene promoterstranscription of genes into proteinsacting on proteins compoundingtgfð½1wnt1wnt10btnf77233adipoqglut4slc2a4 tetramerlepfabp4lipid dropletplin1pck1papa pa4xpalmccd36paangptl4lplfigure regulation of adipocyte diï¬erentiation a regulatory loop exists between pparÎ and cebp activation transcription factor coeebf activates cebpα cebpα activates ebf1 and ebf1 activates pparÎ cebp and cebpδ act directly on the pparÎ gene bybinding its promoter and activating transcription cebpα cebp and cebpδ can activate the ebf1 gene and klf5 the ebf1 and klf5proteins in turn bind the promoter of pparÎ which becomes activated other hormones such as insulin can aï¬ect the expression ofpparÎ and other transcription factors such as srebp1c pparÎ can form a heterodimer with the rxrα in the absence of activatingligands the pparÎrxrα complex recruits transcription repressors such as nuclear receptor corepressor ncor ncor1 andhdac3 upon binding with activating ligands pparÎ causes a rearrangement of adjacent factors corepressors such as ncor2 are lostand coactivators such as transcription intermediary factor tif2 cbp and p300 are recruited which can result in the expression of cyclicampresponsive elementbinding protein creb followed by pparÎ pparÎ expression initiates the expression of downstream genesincluding angiopoietinrelated protein pgar perilipin fabp4 cebpα fatty acid transportrelated proteins carbohydrate metabolismrelated proteins and energy homeostasisrelated proteinslipid metabolism and hormoneinduced lipolysis in the adipocytes were altered autophagy related gene atg4b isactivated by cebp in the process of lipid diï¬erentiationand autophagy activation is necessary for the degradationof klf2 and klf3 two negative regulators of lipid diï¬erentiation these results showed that adipose diï¬erentiation andautophagy are mutually complementary in 3t3l1cells autophagy was inhibited by aspartate ammonia or 0cstem cells internationalmethyladenine at diï¬erent lipid induction periods and days and only autophagy inhibition at days hindered the formation of lipid droplets and the expression of lipid marker genes indicating that autophagy wasvery important in the early stage of lipid diï¬erentiation recent studies showed that lc3 is overexpressed in3t3l1 cells further demonstrating the important role ofautophagy in lipid diï¬erentiation role of alternative splicing in adipogenesis selectivesplicing is uenced by splicing regulators which regulateadipocyte diï¬erentiation by regulating the selective splicingof genes speciï¬c to this process lipin1 is an important regulator in the process of adipocyte diï¬erentiation and includestwo isomers lipin1α and lipin1 which have diï¬erenteï¬ects high expression of lipin1α promotes adipocyte differentiation while that of lipin1 promotes lipid droplet formation in sam68deï¬cient mice the ï¬fth intron ofserinethreonineprotein kinase mtor was retained resulting in unstable and rapid mtor degradation and inhibitionof adipocyte diï¬erentiation furthermore there arefour isomers of pref1 pref1a and pr | Colon_Cancer |
gastroenteropancreatic neuroendocrine neoplasms gep nens as well as neuroendocrine tumors gep nets are heterogeneous tumors that originate from peptidergic neurons and neuroendocrine cells previously described as carcinoid tumors in most nets are indolent tumors compared with other epithelial malignancies however they are reported to have the potential to metastasize even in well differentiated tumors and are resistant to therapies1 data from the surveillance epidemiology and end results seer database indicate that the incidence of nets has increased significantly approximately times reaching casesyear of which gep nets account for approximately to of all nets and the correspondence qiang feng department of colorectal surgery national cancer centernational clinical research center for cancercancer hospital chinese academy of medical sciences and peking union medical college no panjiayuan south road chaoyang district beijing peoples republic of china email fengqiang2008vipsinacomsubmit your manuscript wwwdovepresscomdovepresshttp102147cmars256723 cancer management and research wu this work is published and licensed by dove medical press limited the full terms of this license are available at wwwdovepresscomtermsphp and incorporate the creative commons attribution non commercial unported v30 license httpcreativecommonslicensesbync30 by accessing the work you hereby accept the terms noncommercial uses of the work are permitted without any further permission from dove medical press limited provided the work is properly attributed for permission for commercial use of this work please see paragraphs and of our terms wwwdovepresscomtermsphp 0cwu dovepressincidence and prevalence of colorectal nets are inferior only to those of colorectal adenocarcinoma146 in addition the tumor sites varied markedly by race with the incidence of rectal nets among asian populations increasing from per in to per in which was among white populations147 similarly the incidence of rectal neuroendocrine tumors rnets grew fastest among all nets by approximately times compared with the incidence in accounting for of all nets which makes it the second most common net in china8significantly higher than that for localized colorectal nets endoscopic resection including endoscopic mucosal resection emr and endoscopic submucosal dissection esd and surgery including transanal excisions as well as surgical resections are both effective methods for metastatic tumors somatostatin analogs ssas radiation radiofrequency ablation rfa chemotherapy targeted therapy and peptide receptor radionuclide therapy prrt are all alternatives however the 5year survival rate of nets with lymph node metastasis lnm or distant metastasis is still disappointing with fiveyear overall survival rates of approximately and respectively6911the prognostic factors of colorectal nets have been explored by numerous studies tumor stage location size grade lymphovascular invasion and the status of resection margins are major factors that have been reported to be associated with lnm and poor prognosis12 however these studies were highly heterogeneous which affected the accuracy of the metaanalysis and the effectiveness of these scurrently controversies remain in the treatment of colorectal nets experts from the chinese society of clinical oncology csco agreed that colonic nets greater than cm and less than cm could be completely resected endoscopically when the t stage was less than t2 but the national comprehensive cancer network nccn recommends that these tumors be treated by surgery in accordance with the guidelines for colon adenocarcinoma13the aim of this study was to evaluate the outcomes of colorectal nets explore the risk factors for lymph node metastasis in colorectal nets and identify the prognostic factors for survival outcomesmethodsclinical data collectionbetween and a total of consecutive patients received treatments for colorectal nets in our center we constructed a database of retrospectively collected data from patients medical records including clinical characteristics pathological reports recurrence and survival during the followup periodfor radical resection with lymph node dissection lymph node metastasis was detected by pathological evaluation for local excision such as endoscopic mucosal resection emr endoscopic submucosal dissection esd or transanal excision tae lnm was evaluated through computed tomography ct or magnetic resonance imaging mri before the treatment and during the followup periods the diagnosis of a metastatic lymph node was based on the following criteria size criteria short axis diameter of lymph nodes was greater than mm for round lymph nodes and greater than mm for ovoid lymph nodes morphological abnormalities irregular contour and margin unclear border heterogeneous internal echoes or signal intensity1617 the tumor diameter refers to the longest diameter of the tumor according to pathology reports for patients with distant metastases tumor diameter was determined by endoscopic findings before treatmentpathological diagnosisthe tumor stage was classified according to the american joint committee on cancer ajcc cancer staging manual 7th edition and 8th edition1819 and the tumor grade was classified according to the classification20 for patients before we revised their pathology results and found that they were all neuroendocrine carcinomas necs therefore we classified them as having g3 grade tumors the mitosis count n23 was expressed as the number of mitotic cells in ten highpower fields hpfs from hematoxylin and eosin hestained slides examined with microscopy according to enetswho guidelines g1 grade mitotic image hpfs g2 grade mitotic image hpfs and g3 grade mitotic images2010 hpfs the ki index was calculated as the percentage of cells labeled by immunohistochemistry according to enetswho guidelines g1 grade ki67 positive index g2 grade ki67 positive index to and g3 grade ki67 positive index20 the expression levels of chromogranin a cga n101 and synaptophysin syn n109 were scored according to the percentage of positive cells and the intensity of cell staining the positive cell percentage score was based on the following system points no positive cells point positive cells accounting for to points positive cells accounting for to points submit your manuscript wwwdovepresscom dovepress cancer management and research 0cdovepress wu positive cells accounting for to and points positive cells accounting for to the positive cell staining intensity score was based on the following system points negative point weakly positive points moderate positive and points strong positive the two scores were multiplied together points for negative to points for weak positive to points for moderate positive and to points for strong positive inclusion criteriapatients who were treated in our center for localized and metastatic colorectal nets from to exclusion criteria patients who had colorectal nets combined with other malignancies patients for whom the pathological diagnosis was mixed adenoneuroendocrine carcinoma patients for whom there was insufficient clinical inappropriate pathology reports information or from outside hospitalsrisk factors for lymph node metastasis and prognostic factors related to survival were investigated in all patientsthe primary endpoint was progressionfree survival pfs which was defined as the interval between initial treatment and the first documentation of disease progression or deathstatistical analysisstatistical analysis was performed using spss for mac version spss chicago il usa continuous data are described as means±sds in this study the risk factors for lnm were assessed using pearsons Ï2 test in univariable analysis and logistic regression analyses in multivariable analysis the 5year overall survival os and progression free survival pfs were analyzed with the kaplanmeier method variables were compared with the log rank test and the multivariable analysis for survival outcomes was conducted using the cox proportional hazards model statistical significance was accepted for p values female ratio was the frequencies of grade g1 g2 and g3 nets were and respectively of the patients patients were resected locally by emr by esd and by transanal excision in addition nets were surgically resected including radical resections multivisceral resections and palliative resections due to distant metastasis the remaining patients were treated by systemic treatment due to distant metastasis the most commonly used chemotherapeutic regimen in our center was the ep regimen etoposide and cisplatin as the firstline chemotherapy and the secondline chemotherapy was variable and included the xelox regimen oxaliplatin and capecitabine the folfox regimen oxaliplatin calcium folate and 5fu and everolimus temozolomide and tegafurgimeraciloteracil and combinations between them the tumor diameter was less than cm in patients and the distance from the anal verge was less than cm in patients lnm was found in cases and distant metastasis occurred in patients two patients had radiologically determined lnm after tae in the followup period and one of them went on to undergo radical surgery the other patient was also found to have liver metastasis therefore he was treated with chemotherapy the clinical and histopathological characteristics are summarized in table risk factors for lnmthe risk factors for lnm through univariate analysis were tumor location in the colon p0001 tumor diameter ¥ cm p0001 t stage p0001 tumor grade p0001 and the positive degree of syn p0012 and cga p0049 table in multivariable analyses tumor diameter ¥ cm or ci p0040 and tumor grade g3 or ci p0001 were independent risk factors for lnm in colorectal ntes tumor location in the colon or ci p0083 and tumor grade g2 or ci p0066 might be independent risk factors for lnm even though the p value was greater than table resultsclinical and histopathological characteristics figure a total of patients were included in our study figure the age of the patients was ± years and the male risk factors for survival outcomesthe median followup period was months range months a total of patients died in this cohort in patients with distant metastasis before treatment patients died during chemotherapy cancer management and research submit your manuscript wwwdovepresscom dovepress 0cwu dovepressfigure flowchart of the selection of patientspatients died after multivisceral resections and patients died after palliative resections due to tumor progression in patients without distant metastasis before treatment patients died due to the recurrence of distant metastasis at the liver peritoneum lung pleura and brain and patient died of severe lung infections the 5year progressionfree survival pfs and overall survival os rates of all patients were and respectively the prognostic factors for the 5year pfs and os rate in all patients were age neoadjuvant chemotherapy tumor diameter tumor location tumor grade lnm cga level and treatment method table in the multivariable analysis age ¥ hr ci p00020001 and lnm yes hr ci p00180025 were independent risk factors for 5year pfs and os the cga level [moderate positive hr ci p0010 and strong positive hr ci p0007] were independent risk factors for 5year pfs tumor diameter ¥ cm hr ci p0063 and tumor grade g3 hr ci p0090 were independent risk factors for 5year pfs even though the p value was greater than table comparison of two age groupsin univariable analyses patients were in the 65year group and patients were in the ¥65year group the proportion of tumors with a diameter ¥ cm was significantly higher in the ¥65year group than in the 65year group vs p0016 there were also significantly more patients with lnm in the ¥65year group vs p0041 for t stage the proportion of earlystage t1 patients in the ¥65year group was significantly less than that in the 65year group although p was vs p0086 for treatments there were significantly more patients who were treated with systematic chemotherapy in the ¥65year group vs p0040different operative methods for t1n0m0 colorectal netsthere was no significant difference in tumor grade tumor location surgical margin relapse or 5year os except for tumor diameter p0012 the diameters of tumors resected by emr esd and transanal excision were ± cm ± cm and ± cm respectively table submit your manuscript wwwdovepresscom dovepress cancer management and research 0cdovepress wu table clinical and histopathological characteristics of colorectal nets n135table continued variablesage¥sexmalefemalebmi¥smoking historydrinking historyfamily cancer historyyesnoneoadjuvant chemotherapyyesnoneoadjuvant radiotherapyyesnodistance from the anal verge cm¥tumor locationrectumcolonappendixtreatmentemresdtransanal excisionradical resectionmultivisceral resectionpalliative resectionsystemic treatmenttumor diameter cm¥t stage after chemotherapyxn variableslnmnegativepositivedistant metastasisnegativepositivetumor gradeg1g2g3cga n101negativesyn n109negativen kaplanmeier survival curveskaplanmeier survival curves for os and pfs according to tumor grade diameter location and cga level are shown in figures discussionnets have a relatively good prognosis with a median os time reported to be years months and a 5year os rate of however the survival outcomes varied significantly at different stages of nets the 5year os rate of stage i and ii tumors was reported to be as high as and but dropped to and for stage iii and iv tumors respectively22 according to epidemiological data from the seer database and gkr joint cancer registry the 5year os rates of lymph node metastases stage iiib and distant metastases stage iv are and respectively1610 the 5year os rates of all patients and patients without distant metastasis were and respectively and the 5year pfs rates were and respectively lymph node metastasis is the most important factor that determines the prognosis of nets and the prediction of lymph node continuedcancer management and research submit your manuscript wwwdovepresscom dovepress 0cwu dovepresstable univariable analysis for risk factors for lymph node metastasis n table multivariable analysis for risk factors for lymph node metastasis n135lnm n Ï2 valuep valuen hr cip valueage¥tumor diameter¥ tumor locationrectumcolonappendix tumor gradeg1g2g3t staget0t1t2t3t4cganegativesynnegative notes by logistic regression analyses p values abbreviation hr hazard ratiomost nets are at the g1 phase and g2 or g3 phases account for only to of all nets and have been reported to be risk factors for lnm by numerous studies1223 a multicenter clinical study in china showed that pathological type g3 nec is an independent risk factor affecting the prognosis of patients with rectal nets p similarly sohn et al3 found that the lnm rate of g1 phase rectal nets was only but it increased remarkably to at the g2 phase in our study our results showed that histological tumor grades g2 p and g3 p0001 were independent risk factors for lymph node metastasis lymph node metastasis occurred in of patients with g3 tumors and with g2 tumors but only with g1 tumors the 5year variablessexmalefemaleage¥bmi¥smoking historyyesnodrinking historyyesnotumor locationrectumcolonappendixtumor diameter¥t staget0t1t2t3t4 tumor gradeg1g2g3cga n101negativesyn n109negative notes by pearsons Ï test p values metastasis is necessary for clinicians to choose a suitable treatment the aim of our research was to explore the predictive factors for lymph node metastasis of colorectal nets and assess the current therapeutic algorithmsubmit your manuscript wwwdovepresscom dovepress cancer management and research 0cdovepress wu table prognostic factors for survival outcomestable continued variables5year pfs 5year os Ï2 valuep valueg3lnmyesnocga negativesyn negativetreatment variablessexmalefemalebmi¥age¥smoking historyyesnodrinking historyyesnoneoadjuvant radiotherapyyesnoneoadjuvant chemotherapyyesnotumor locationrectumcolontumor diameter cm¥t stagextumor gradeg1g25year pfs 5year os Ï2 value p value continuedemresdtransanal excisionradical or palliativesurgerysystemic treatmentnotes kaplanmeier method and log rank test p values os rate decreased sharply from to when the tumor grade increased from g3 to g1 however tumor grade was not significant in survival outcomes possibly because tumor grade affects survival outcomes indirectly by directly affecting lymph node statustumor size has been reported to be a strong predictive factor for lymph node metastasis previous studies have shown that a tumor less than mm is usually limited to the submucosa with a low metastasis risk of less than and the 5year survival rate can reach approximately to according to the enets guidelines surgical treatment is recommended if rnets are greater than cm g1g3 or are g3 phase with or without metastasis endoscopic resection is feasible when the tumors are less than cm g12 phase and t1 stage15 the treatment of rnets in western countries and in china is similar but there have been controversies regarding the treatment of 2cmsized cnets chinese experts agree that endoscopic resection can be considered for cnets less than cm however there is cancer management and research submit your manuscript wwwdovepresscom dovepress 0cwu dovepresstable multivariable analysis for survival outcomes5year pfsos hr cip valueage ¥tumor diameter¥tumor locationrectumcolonappendixlnmnoyescganegativesynnegativetumor gradeg1g2g3neoadjuvant chemotherapynoyestreatmentemresdtaesurgerysystemic treatmentnotes by the cox proportional hazards model p values abbreviation hr hazard ratio0019987800003633e1700003066e29200001826e4500343994900007412e29010015970800001686e30no explicit mention of treatment in the enets guidelines and experts from the nccn recommended surgical resection instead of endoscopic resection1315 in our study survival curves were significantly better p0001 among patients with tumors less than cm figure a tumor diameter greater than cm was an independent risk factor for lnm p0040 table and we believe this was due to the small sample size however patients with tumors less than cm had lnm and patients with tumors less than cm also developed lnm the lnm in small nets might be due to the tumor cells extending to the submucosal layer which has abundant lymphatic vessels for them to spread through previous studies have reported that small nets also have malignant potential26 therefore even if tumor size was submit your manuscript wwwdovepresscom dovepress cancer management and research 0cdovepress wu table comparison of different treatment methods for t1n0m0 colorectal netsemresdtransanal excisiong gradeg1g2g3tumor diameter±±±tumor locationrectumcolonappendixsurgical marginpositivenegativerelapse cases5year os rate notes by anova p values Ï2f valuep valuea strong predictive factor for lnm lnm should not be predicted only by tumor size and further examinations such as eus or ct can help us to evaluate the status of lymph nodes more specificallychromogranin a cga and synaptophysin syn are two neuroendocrine differentiation ned immunohistochemistry markers frequently used in nets in univariable analysis there was a significant difference for colorectal nets with different expression levels of cga in terms of both risk factors for lnm and survival outcomes both p005 in multivariable analysis moderate positive p0010 and strong positive cga p0007 were independent risk factors for 5year pfs which has been proven in a wide variety of retrospective analyses27 in addition prospective clinical trials radiant1 and have been performed to assess the prognostic value of cga in advanced nets and the results showed shorter os for patients with elevated cga31 however the increase in the expression level of cga was not proportional to the increase in the lnm rate or 5year os rate and cga was negative in patients with lnm which may affect the accuracy of the prognostic value of cga lindholm et al35 also found that a relevant portion of nets do not show elevated cga levels the major problem is that several confounding factors can including gastrointestinal and affect cga levels figure a pfs curves according to tumor grade b os curves according to tumor gradecancer management and research submit your manuscript wwwdovepresscom dovepress ab050100150200020406080100progressionfree survivalg1g2g3 0cwu dovepressfigure a pfs curves according to tumor diameter b os curves according to tumor diameterfigure a pfs curves according to tumor location b os curves according to tumor locationfigure a pfs curves according to age b os curves according to agesubmit your manuscript wwwdovepresscom dovepress cancer management and research ababab 0cdovepress wu figure a pfs curves according to cga level b os curves according to cga levelcardiovascular disorders or proton pump inhibitor ppi consumption and a variety of other nontumor reasons36 regarding syn there was only a significant difference in the univariable analysis for lnm previous studies have shown that patients with a low level of synaptophysin had a better os rate than those with a high level however the small sample size limited the accuracy of the results37 based on the findings of this research and previous studies38 it can be suggested that ned might affect the survival outcomes of colorectal net patients and markers especially cga might be suitable for clinicians to predict the prognosis of patientsthe location of the tumor is also an important factor affecting the prognosis and treatment of nets tumors in the colon are more common in necs and generally have a worse prognosis with higher metastatic potential than tumors in the rectum the outcomes of neuroendocrine tumors from the right hemicolon of the midgut and from the left hemicolon from the hindgut are not the same the welldifferentiated biological behavior of the left hemicolon is closer to that of the rectum a recent chinese multicenter study found that more than of colonic nets of midgut origin are necs or mixed adenoneuroendocrine carcinomas manecs39 according to statistics from the seer database the 5year survival rate of patients with rnets is which is significantly higher than that of colonic nets in our study the lymph node metastasis rate in the colon was which was significantly higher than that in the rectum the 5year os rate and pfs rate of individuals with lnm in the appendix and in the rectum were significantly better than those of individuals with lnm in the colon p0005 and p0003 respectively which was consistent with previous studies1 based on the findings of this research and previous studies it can be suggested that colonic nets should be completely resectedin our research the survival outcomes of patients years and older in our study were worse than those of patients younger than years p0001 we also found that tumors from elderly patients ¥ years were larger and more advanced than those from younger patients years both p0001 the reason for the poor prognosis in elderly patients may be that elderly patients have lower tolerance to surgical trauma and side effects of chemotherapy because of their weakened an physiological functions which leads to multiple complications4142 therefore when we encounter elderly patients minimally invasive therapies such as laparoscopic surgery could help reduce surgical trauma and chinese herbs can relieve and reduce the adverse events of chemotherapy43for nets in the colon the recommended treatment varies among different guidelines but surgical resection is generally recommended because of the greater likelihood of malignant behavior than with rectal nets endoscopic resection may be considered if the tumor diameter is less than cm and does not reach the muscularis propria for nets in the rectum eus is required before surgery surgical resection is recommended when the tumor diameter is more than cm g3 grade t3 to t4 stage or when there is peripheral lymph node metastasis when the tumor diameter is less than cm g1 or grade and t1 stage endoscopic resection is feasible in other cases the treatment method is determined according to the depth of tumor invasion assessed by eus21cancer management and research submit your manuscript wwwdovepresscom dovepress 050100150200020406080100months progressionfree survivalnegativeweak positivemoderate positivestrong positivep0023050100150200020406080100months overall survivalnegativeweak positivemoderate positivestrong positivep0038ab 0cwu dovepressthis study has some limitations including its retrospective design and the relatively small number of patients included although lnm should be evaluated after radical resection with lymph node dissection we analyzed the risk factors for lnm by ct or mri in those who underwent local excision before the treatment and during the follow up periods and we believe the results are reliable because this study lasted more than years we could investigate the longterm survival outcomes and prognostic factors after different treatments even with the small number of patients finally further studies should be performed to validate our main sthe clinical and pathological characteristics of rectal and colon neuroendocrine tumors are different | Colon_Cancer |
recently extensive evidence has clarified the crucial role of circular rnas circrnas as a protumor or anticancer participant in human malignancies a new circrna derived from oxysterol binding protein like osbpl10 circosbpl10 has not been researched in cervical cancer cc yetmethods the expression of molecules was analyzed by rtqpcr or western blot several functional assays were applied to explore the biological influence of circosbpl10 on cc the interaction between rnas was estimated via luciferase reporter rna immunoprecipitation and rna pulldown assaysresults circosbpl10 characterized with cyclic structure was revealed to possess elevated expression in cc cells circosbpl10 downregulation elicited suppressive impacts on cc cell proliferation and migration interestingly circosbpl10 regulated cc progression by interacting with microrna1179 mir1179 moreover ubiquitin conjugating enzyme e2 q1 ube2q1 targeted by mir1179 was positively regulated by circosbpl10 in cc furthermore enhanced ube2q1 expression or suppressed mir1179 level countervailed the repressive effect of circosbpl10 depletion on the malignant phenotypes of cc cells moreover forkhead box a1 foxa1 was confirmed to induce circosbpl10 expression in cc cellss foxa1induced circosbpl10 facilitates cc progression through mir1179ube2q1 axis highlighting a strong potential for circosbpl10 to serve as a promising therapeutic target in cckeywords circosbpl10 mir1179 ube2q1 foxa1 cc as a frequent type of human gynecological malignancies worldwide cervical cancer cc is depicted as one of the dominating causes contributing to cancerassociated death in women [ ] it is estimated that that nearly correspondence luo0700777337163com lushun1982livecn department of radiotherapy sichuan cancer hospital institute sichuan cancer center school of medicine university of electronic science and technology of china no renmin south road chengdu sichuan china pharmacy department sichuan jinxin women and childrens hospital no jingxiu road jinjiang district chengdu sichuan chinafull list of author information is available at the end of the new cases are diagnosed with cc annually in china cc is also regarded as one of the most prevalent lethal tumors over the past few years in spite of the application of human papillomavirus hpv vaccine in treatment cc is still a major stumbling block for female health [ ] the majority of patients at an early stage of cc are likely to be cured through surgery whereas no or limited efficient therapeutic approaches for those at the advanced stages in order to make advances in the treatment of cc researchers have focused on exploring and developing tumorparticular biomarkers for cc for example melatonin was identified as a new adjuvant agent in treating patients with cc so was curcumin 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 0cyang a0et a0al cancer cell int page of however more efforts should be made in developing new effective therapeutic strategies for cc hence it is imperative to make indepth exploration of the underlying molecular mechanisms in ccnoncoding rnas ncrnas have been commonly considered as the potential key modulators in gene regulation to impact on tumor development [ ] including genetic and epigenetic manners in recent years ncrnas have been indicated as clinical biomarkers in diverse diseases [ ] including cancer [ ] as a member of ncrnas circular rnas circrnas own a covalently closed structure which are tolerant to rnase rmediated degradation [ ] increasing analyses have suggested the abnormal expression of circrnas in various cancers [ ] including cc [ ] emerging researches have testified the implication of circrnas in tumorigenesis and progression via regulation of different biological processes which includes cell proliferation migration and invasion [] emerged as a new circrna circosbpl10 circbase id hsa_circ_0064669 is derived from backsplicing of osbpl10 mrna messenger rna to our knowledge the critical regulatory mechanism of circosbpl10 has not been investigated in cc yetin this study the main purpose was to decipher the potential regulatory role of circosbpl10 in cc data from a series of assays uncovered that foxa1induced upregulation of circosbpl10 contributes to cc progression via mir1179ube2q1 axis revealing that circosbpl10 might be a hopeful biomarker for ccmethodscell culturehuman normal cervical epithelial cells h8 and human cc cells c33a caski hela and siha were bought from chinese academy of sciences shanghai china cells were cultured with dulbeccos modified eagle medium dmem invitrogen carlsbad ca usa adding fetal bovine serum fbs invitrogen penicillinstreptomycin sigmaaldrich milan italy and then incubated in an incubator of co2 at a0°ccell transfectionhela and siha cells were transfected with specific short hairpin rnas shrnas against circosbpl10 shcircosbpl1012 foxa1 shfoxa112 and their corresponding negative control nc shnc as well as pcdna31circosbpl10 pcdna31ube2q1 pcdna31foxa1 and empty pcdna31 ± circrna mini vector empty pcdna31 vector severally the mir mimics mir1179 inhibitor nc mimics and nc inhibitor were synthesized by genepharma shanghai china transfection experiments were executed by lipofectamine invitrogenrealtime quantitative polymerase chain reaction rtqpcrtotal rna of cells was isolated using trizol reagent followed by cdna complementary dna synthesis with reverse transcription kit invitrogen rtqpcr was measured by sybrgreen realtime pcr kit takara tokyo japan operated on biorad cfx96 system takara relative expression level was calculated utilizing δδct method with normalization to glyceraldehyde3phosphate dehydrogenase gapdh or u6 the sequences of primers were presented in additional file a0 table a0s1cell counting kit cckin short cells were plated into a 96well plate after incubation for specific times a0h cells were processed with a0 μl of cck8 reagent for additional a0 h absorbance at a0 nm was measured via a microplate reader olympus tokyo japancolony formation assaytransfected cells were first coated into 6well plates after a0weeks of incubation cells were rinsed with phosphate buffer saline pbs sigmaaldrich san francisco usa fixed in methanol sigmaaldrich and dyed using crystal violet sigmaaldrich the visible colonies were counted manuallyactinomycind actd and a0rnase r treatmentto block transcription a0mgml actinomycin d actd sigmaaldrich or dimethylsulphoxide dmso sigmaaldrich was added into culture medium total rna was cultivated with or without a0uμg of rnase r epicentre technologies madison wi usa for a0min after treatment with actd or rnase r rtqpcr was applied for determining the expression levels of circosbpl10 and osbpl10 mrnanucleic acid electrophoresisconvergent primers and divergent primers were designed to amplify osbpl10 mrna or circosbpl10 the level of circosbpl10 in pcr products from cdna and genomic dna was examined by agarose gels with te trisethylene diamine tetraacetic acid buffer from thermo scientific waltham ma usaterminal deoxynucleotidyl transferasemediated dutp nickend labeling tunel assayapoptosis transfected siha and hela cells were assessed utilizing tunel apoptosis kit invitrogen ²6diamidino2phenylindole dapi sigmaaldrich was employed 0cyang a0et a0al cancer cell int page of to dye above cells the percentage of positive stained cells was observed by fluorescence microscopy olympus and then analyzedflow cytometry analysiscell apoptosis analysis was performed via cell apoptosis analysis kit takara after incubation in 6well plates siha and hela cells were rinsed with pbs and resuspended in binding buffer followed by fixation with icecold ethanol sigmaaldrich cells were doublestained by annexin vfluorescein isothiocyanate and propidium iodide last cell apoptosis rate was detected by flow cytometer bectondickinson ma usamigration assaycell migration abilities were examined using transwell chambers corning ny usa transfected cells with serumfree medium were placed into top compartment while medium with fbs was added into the lower compartment a0h later cells in the lower chamber were immobilized and dyed in methanol and crystal violet separately then migratory cells were then counted in five random chosen fields under a microscope olympuswound healingsiha and hela cells were added in 6well plates for cultivation when cell confluence was scratches were produced in cell layer using sterile pipette tip afterward cells were cleaned using pbs and incubated in a culture medium for a0h images of migrating cells were detected at lastwestern blottotal protein was reaped in radio immunoprecipitation assay ripa lysis buffer beyotime shanghai china bicinchoninic acid bca kit beyotime determined protein concentrations proteins were separated through sodium dodecyl sulfatepolyacrylamide gel electrophoresis sdspage and moved onto polyvinylidene fluoride pvdf membranes the membranes were sealed with skimmed milk and cultivated with primary antibodies against ube2q1 orb77618 biorbyt san francisco california usa and gapdh ab8245 abcam cambridge uk secondary antibody was added for incubating for a0h gapdh was an internal control proteins quantities were evaluated via chemiluminescence detection systemluciferase reporter assaythe wildtype wt and mutant mut binding sites of mir1179 in circosbpl10 or ube2q1 ²utr was subcloned into pmirglo dualluciferase vector to construct circosbpl10wtmut or ube2q1wtmut and plasmids were cotransfected with mir1179 mimics or nc mimics into siha and hela cells respectively the pgl3osbpl10 promoter was cotransfected with pcdna31foxa1 or pcdna31 into cells dualluciferase reporter assay system promega usa detected the luciferase activitysubcellular fractionationusing neper¢ nuclear and cytoplasmic extraction fractions of cytoplasmic and nuclear were separated reagents invitrogen and gathered by rneasy midi kit qiagen hilden germany to determine the cellular localization of circosbpl10 rtqpcr was used to examine the levels of circosbpl10 u6 nuclear control and gapdh cytoplasmic controlrna pulldownbriefly cell lysates were incubated with biotinylated rna including biomir1179wt biomir1179mut and bionc moreover m280 streptavidin magnetic beads sigmaaldrich were added to coculture for a0h the relative enrichment of rnas pulled down in each group were assayed by rtqpcrrna immunoprecipitation riprip assays were progressed with magna riptm rnabinding protein immunoprecipitation kit millipore bedford usa siha and hela cells were lysed with rip lysis buffer followed by incubation with magnetic beads conjugated with antiago2 millipore or antiigg millipore the rtqpcr was performed to evaluate the expression levels of circosbpl10 mir1179 and ube2q1 in the precipitateschromatin immunoprecipitation chipvia magna chip kit millipore chip experiment was achieved dna in cell lysates was interrupted into 300bp chromatin fragments by ultrasound after that lysates were subjected to immunoprecipitation with antifoxa1 or antiigg negative control group the precipitated dna fragments were detected by rtqpcrstatistical analysisexperimental data from at least three independent experiments were shown as mean ± standard deviation sd statistical analysis was performed using graphpad prism software graph pad la jolla ca usa significance in differences between or more groups was analyzed via students t test or oneway analysis of variance anova p had statistical significance in requirements 0cyang a0et a0al cancer cell int page of resultscircosbpl10 is a0highly expressed in a0cc and a0its depletion impedes cc cell proliferation and a0migrationto study the cellular function of circosbpl10 in cc we first applied rtqpcr analysis and unveiled a marked elevation of circosbpl10 expression in cc cell lines compared with a0h8 cells fig a01a then nucleic acid electrophoresis manifested that in siha and hela cells divergent primers could produce circosbpl10 from cdna but not from genomic dna gdna while convergent primers amplified linear osbpl10 from both cdna and gdna fig a0 1b besides osbpl10 mrna was greatly degraded by actd whereas circosbpl10 exhibited as resistant to actd fig a0 1c additionally osbpl10 expression was dramatically reduced whereas circosbpl10 expression demonstrated no evident change after siha and hela cells were treated with rnase r fig a01d then we verified that circosbpl10 expression was lowered in two cc cells after transfection with shcircosbpl1012 while those with shcircosbpl101 showed higher silencing efficiency fig a0 1e subsequently cell proliferation assays depicted a notably weakened proliferation ability of siha and hela cells under circosbpl10 silence fig a0 1f g moreover cell apoptosis capability was proved to be facilitated after silencing circosbpl10 in siha and hela cells fig a01h i furthermore it was uncovered that circosbpl10 deficiency gave rise to attenuated migration ability of siha and hela cells fig a01j k taken together circosbpl10 is expressed at high levels in cc and knockdown of it impairs malignant behaviors in cc cellscircosbpl10 sponges mir in a0ccfor the purpose of investigating the molecular mechanism of circosbpl10 in regulating cc we first detected its cellular sublocalization in siha and hela cells via subcellular fractionation as illustrated in fig a0 2a circosbpl10 was majorly distributed in cytoplasm thus we speculated that circosbpl10 might affect cc via serving as a sponge of specific mirna after searching starbase httpstarb asesysueducn with certain condition clip data strict stringency ¥ degradome data low stringency ¥ three mirnas mir1179 mir27a3p and mir27b3p were revealed to have binding potentials with circosbpl10 fig a02b then we discovered a significant downregulation of mir1179 whereas no apparent changes on the levels of mir27a3p and mir27b3p in cc cell lines compared to normal h8 cells fig a02c therefore mir1179 was chosen for further analysis subsequently circosbpl10 and mir1179 were presented to be conspicuously concentrated in antiago2 group fig a02d afterwards two binding sites between circosbpl10 and mir1179 were predicted via starbase fig a02e moreover we validated that mir1179 bound with circosbpl10 at site fig a02f to further test whether circosbpl10 promoted cc progression via its interaction with mir1179 we mutated the sequence of circosbpl10 recognized by mir1179 as displayed in fig a02g the expression of circosbpl10 was observably elevated in c33a and caski cells after overexpressing circosbpl10 and circosbpl10 mut interestingly it seemed that cell proliferation and migration in c33a and caski cells could be fortified by overexpression of fulllength circosbpl10 but not by upregulation of circosbpl10 with mutated mir1179 binding sites fig a0 2h i indicating that the function of circosbpl10 in cc depended on its binding to mir1179 in circosbpl10 interacts with mir1179 to drive cc progressioncircosbpl10 upregulates ube2q1 level in a0cc by a0sequestering mirto further investigate the downstream mechanism of circosbpl10 in cc starbase was utilized as predicted under certain circumstances clip data strict stringency ¥ degradome data low stringency ¥ four candidates ube2q1 prpf38b cacul1 and luc7l3 were found as the targets of mir1179 fig a03a the following rna pulldown assay demonstrated the distinct enrichment of ube2q1 whereas limited harvest of other three mrnas in biomir1179wt group fig a0 3b more importantly ube2q1 level was cut down in siha and hela cells by circosbpl10 knockdown as well as by augmented mir1179 expression fig a03c and additional file a0 besides rtqpcr obtained a conspicuous elevation on ube2q1 expression in cc cell lines relative to h8 cells at both mrna and protein levels fig a0 3e and see figure on next pagefig circular rna circosbpl10 was highly expressed in cc and knockdown of it suppressed cc progression a circosbpl10 expression was detected by rtqpcr in cc cell lines h8 cells b it was delineated by nucleic acid electrophoresis analysis that divergent primers amplified circosbpl10 from cdna but not from gdna gapdh was a negative control c the resistance of circosbpl10 and osbpl10 mrna to actd in siha and hela cells was analyzed by rtqpcr d rtqpcr assay was conducted to determine the abundance of circosbpl10 and linear osbpl10 mrna in siha and hela cells treated with rnase r normalized to mock treatment e rtqpcr was utilized to analyze the efficacy of circosbpl10 knockdown in siha and hela cells f g the proliferation ability of siha and hela cells transfected with shcircosbpl101 or shnc was evaluated via cck8 and colony formation h i cell apoptosis ability in transfected cells was measured by tunel assay and flow cytometry analysis j k transwell and wound healing assays were conducted to analyze the migration of transfected cells p p 0cyang a0et a0al cancer cell int page of 0cyang a0et a0al cancer cell int page of 0cyang a0et a0al cancer cell int page of see figure on next pagefig circosbpl10 sponged mir1179 in cc a subcellular fractionation was applied to detect the cellular sublocalization of circosbpl10 in siha and hela cells b mir1179 mir27a3p and mir27b3p were predicted via starbase to have the binding capacity with circosbpl10 c mir1179 expression in cc cell lines and h8 cells was detected via rtqpcr d rip assay unveiled the significant enrichment of circosbpl10 and mir1179 in antiago2 group e two binding sites between circosbpl10 and mir1179 were predicted via starbase f luciferase reporter assay validated the interaction between circosbpl10 and mir1179 g the expression of circosbpl10 in siha and hela cells transfected with pcdna31 pcdna31circosbpl10 or pcdna31circosbpl10 mut was analyzed through rtqpcr h i cell proliferation and migration in different groups was analyzed via cck8 and transwell assays respectively p p additional file a0 afterwards the binding sites between ube2q1 and mir1179 were obtained through starbase prediction fig a0 3f importantly we observed declined luciferase activity of ube2q1wt owing to mir1179 upregulation through luciferase reporter assay however no overt changes on that of ube2q1mut were noted between two groups fig a03g more importantly a significant enrichment of circosbpl10 mir1179 and ube2q1 in antiago2 groups was observed via rip analysis fig a03h further ube2q1 expression was visibly upregulated by circosbpl10 overexpression whereas enhanced expression of mutated circosbpl10 had no such influence on ube2q1 expression in these two cc cells fig a03i and additional file a0 in sum circosbpl10 facilitates ube2q1 expression by sponging mir1179 in cccircosbpl10 accelerates cc progression via a0mirube2q1 axisgiven that circosbpl10 sponged mir1179 to upregulate ube2q1 expression in cc we intended to detect whether this mechanism contributed to cc progression herein the efficacy of elevating ube2q1 expression or lowering mir1179 level was analyzed at first by rtqpcr and the outcome appeared to be satisfactory in siha cells fig a04a afterwards we testified that ube2q1 upregulation or mir1179 inhibition could offset circosbpl10 depletionmediated suppressive effect on cell proliferation fig a04b c in addition cell apoptosis facilitated by circosbpl10 knockdown was counteracted by ube2q1 overexpression or mir1179 suppression fig a0 4d e moreover the attenuated cell migration capability in circosbpl10depleted cells was restored by upregulation of ube2q1 or inhibition of mir1179 fig a04f g to conclude circosbpl10 promotes cc progression via mir1179ube2q1 axisfoxa1 activates circosbpl10 expression in a0ccsince the downstream signaling responsible for the regulation of circosbpl10 in cc had been studied we subsequently focused on figuring out its possible upstream mechanism through utilizing ucsc university of california santa cruz httpgenom eucscedu foxa1 seemed to be a probable transcription factor of osbpl10 the host gene of circosbpl10 prior to testify the influence of foxa1 on circosbpl10 expression in cc we first silenced or overexpressed foxa1 with satisfactory efficacies in siha and hela cells fig a0 5a of interest we discovered that the expression of circosbpl10 was distinctly declined by foxa1 depletion whereas overtly augmented by foxa1 upregulation fig a0 5b later on we employed jaspar database httpjaspa rgener egnet and obtained the dna motif of foxa1 fig a05c seen from fig a05d the sequences of osbpl10 promoter were fragmented into five parts p15 interestingly it was verified by chip assay that foxa1 mainly bound to osbpl10 promoter at p4 region fig a0 5e moreover when overexpressing foxa1 in siha and hela cells the luciferase activity of osbpl10 promoterwt was observably increased while there were no evident changes on that of osbpl10 promotermut with mutated foxa1 binding sites predicted in p4 region fig a05f in a word foxa1 activates osbpl10 transcription and thereby facilitates circosbpl10 expression in ccdiscussionmounting evidence has manifested that circrnas serve vital parts in cc initiation and progression for instance circrna smarca5 regulates cc progression by sponging mir620 increased expression of circ_0067934 accelerates cc development by targeting mir545eif3c see figure on next pagefig circosbpl10 upregulated ube2q1 expression by competitively binding with mir1179 in cc a venn diagram showed the overlaps of potential mir1179 targets predicted by pita microt pictar mirmap b the binding capacity between mir1179 and four mrnas was verified through rna pulldown assay c d the expression of ube2q1 in siha and hela cells transfected with different plasmids was detected via rtqpcr and western blot e ube2q1 expression in cc cell lines and h8 cells was detected via rtqpcr f the binding sites between ube2q1 and mir1179 obtained from starbase were displayed g the interaction between ube2q1 and mir1179 was validated by luciferase reporter assay h rip analysis revealed that circosbpl10 mir1179 and ube2q1 coexisted in riscs rnainduced silencing complexes i rtqpcr and western blot were utilized to detect the expression of ube2q1 in indicated cells p 0cyang a0et a0al cancer cell int page of 0cyang a0et a0al cancer cell int page of fig circosbpl10 accelerated cc progression via mir1179ube2q1 axis a the efficacy of ube2q1 overexpression and mir1179 inhibition in siha cells was analyzed by rtqpcr b c the proliferation ability of siha cells under diverse conditions were measured by cck8 and colony formation assays d e the apoptosis ability of transfected cells were measured via tunel assay and flow cytometry analysis f g transwell and wound healing assays were carried out to analyze cell migration under different contexts p 0cyang a0et a0al cancer cell int page of fig foxa1 activated circosbpl10 expression in cc a the efficacy of foxa1 knockdown and overexpression in siha and hela cells was obtained through rtqpcr analysis b the expression of circosbpl10 in different groups was detected by rtqpcr c the dna motif of foxa1 was obtained from jaspar database d fulllength of osbpl10 promoter was fragmented into pieces p15 and jaspar predicted a potential binding site for foxa1 to osbpl10 promoter at p4 region e chip assay proved that the p4 region of osbpl10 promoter was recognized by foxa1 in both siha and hela cells f the interaction between osbpl10 promoter and foxa1 at above predicted sites was testified by luciferase reporter assay p axis circrna hsa_circ_0023404 plays an oncogenic part in cc circosbpl10 derived from backsplicing of osbpl10 mrna is a circular rna that has not been studied in cc but is worth exploring in this research circosbpl10 was evidenced to have a circular structure and possess high levels in cc additionally silenced circosbpl10 exerted suppressive impacts on cc cell proliferation and migrationincreasing researches have elucidated that circrnas might serve as a molecular sponge of specific mirnas which has been suggested to be of significant value in cc [ ] so as to regulate the tumorigenesis and 0cyang a0et a0al cancer cell int page of development of numerous cancers including cc [ ] in current study on the basis of bioinformatics prediction and molecular mechanism experiments mir1179 that was unveiled as an antitumor regulator in some cancers [ ] was screened out and validated to be implicated in circosbpl10 regulated cellular processes in ccpreviously ube2q1 has been depicted as a critical participator in tumor progression [ ] in present study ube2q1 was verified capable of binding with mir1179 and its expression was boosted by circosbpl10 in cc through mir1179 sequestration more importantly the followup rescue experiments indicated that ube2q1 upregulation or mir1179 inhibition could rescue circosbpl10 depletionmediated suppressive effects on malignant phenotypes in ccincreasing researches have indicated that foxa1 expresses at high levels in many cancers such as lung cancer glioma and prostate cancer besides a previous study indicated that foxa1 could directly bind with plod2 promoter and activate plod2 transcription in nsclc similarly we revealed that foxa1 activates osbpl10 transcription and thereby facilitates circosbpl10 expression in cc in this studyin foxa1induced circosbpl10 promotes cc progression by targeting mir1179ube2q1 axis providing novel insights into exploring more effective treatment of cc however the biggest regret is that no clinical data are included in this work and the findings in our study need to be further testified by clinical samples in the futuresupplementary informationsupplementary information accompanies this paper at https doi101186s1293 additional file a0 table a0s1 list of the sequences of primers used in rtqpcr additional file a0 figure s1 the size of marker for western blot gelsabbreviationscircrna circular rna cc cervical cancer ube2q1 ubiquitin conjugating enzyme e2 q1 foxa1 forkhead box a1 ncrnas noncoding rnas fbs fetal bovine serum actd actinomycin d gdna genomic dnaacknowledgementswe are very grateful to all individuals and groups involved in this study authors contributionssy yj xr and df designed this study sy lz fz yj and xr devoted to experiment data curation and interpretation sy yj xr df dh sh and lz were in charge of investigation experiment record figures and writing sl edited the manuscript all authors read and approved the final manuscript fundingthis study was supported by the national natural science foundation of china the national cancer center climbing foundation of chinancc201808b016 department of science and technology of sichuan province20yyjc3815 department of science and technology of sichuan province20gjhz0088 availability of data and materialsresearch data and material are not sharedethics approval and consent to participatenot applicableconsent for publicationnot applicablecompeting intereststhe authors declare that they have no competing interestsauthor details department of gynecological radiotherapy harbin medical university cancer hospital no haping road nangang district harbin heilongjiang china department of radiotherapy sichuan cancer hospital institute sichuan cancer center school of medicine university of electronic science and technology of china no renmin south road chengdu sichuan china pharmacy department sichuan jinxin women and childrens hospital no jingxiu road jinjiang district chengdu sichuan china received september accepted june references jemal a bray f center mm ferlay j ward e forman d global cancer statistics ca torre la bray f siegel rl ferlay j lortettieulent j jemal a global cancer statistics ca tewari ks sill mw long hj 3rd penson rt huang h ramondetta lm landrum lm oaknin a reid tj leitao mm et al improved survival with bevacizumab in advanced cervical cancer new engl j med chen w zheng r zeng h zhang s he j annual report on status of cancer in china chinese j cancer res testing for cervical cancer new recommendations from the american cancer society american society for colposcopy and cervical pathology and american society for clinical pathology ca burki tk cervical cancer screening and risk with age lancet oncol 2014153e107 noordhuis mg fehrmann rs wisman gb nijhuis er van zanden jj moerland pd ver loren van themaat e volders hh kok m ten hoor ka et al involvement of the tgfbeta and betacatenin pathways in pelvic lymph node metastasis in earlystage cervical cancer clin cancer res shafabakhsh r reiter rj mirzaei h teymoordash sn asemi z melatonin a new inhibitor agent for cervical cancer treatment j cell physiol ghasemi f shafiee m banikazemi z pourhanifeh mh khanbabaei h shamshirian a amiri moghadam s arefnezhad r sahebkar a avan a et al curcumin inhibits nfkb and wntβcatenin pathways in cervical cancer cells pathol res pract perez ds hoage tr pritchett jr ducharmesmith al halling ml ganapathiraju sc streng ps smith di long abundantly expressed noncoding transcripts are altered in cancer hum mol genet guttman m donaghey j carey bw garber m grenier jk munson g young g lucas ab ach r bruhn l et al lincrnas act in the circuitry controlling pluripotency and differentiation nature 0cyang a0et a0al cancer cell int page of khani p nasri f khani chamani f saeidi f sadri nahand j tabibkhooei a mirzaei h genetic and epigenetic contribution to astrocytic gliomas pathogenesis j neurochem mirzaei h stroke in women risk factors and clinical biomarkers j cell biochem saeedi borujeni mj esfandiary e baradaran a valiani a ghanadian m codo±erfranch p basirat r alonsoiglesias e mirzaei h yazdani a molecular aspects of pancreatic βcell dysfunction oxidative stress microrna and long noncoding rna j cell physiol asma v zahra s ahmad m soheila m sima f hamid rm afshin n amir s hamed m long noncoding rnas as epigenetic regulators in cancer curr pharm des mirzaei h yazdi f salehi r mirzaei h sirna and epigenetic aberrations in ovarian cancer j cancer res ther qu s zhong y shang r zhang x song w kjems j li h the emerging landscape of circular rna in life processes rna biol qu s yang x li x wang j gao y shang r sun w dou k li h circular rna a new star of noncoding rnas cancer lett shabaninejad z vafadar a movahedpour a ghasemi y namdar a fathizadeh h pourhanifeh mh savardashtaki a mirzaei h circular rnas in cancer new insights into functions and implications in ovarian cancer j ovarian res naeli p pourhanifeh mh karimzadeh | Colon_Cancer |
" prognostic markers play an essential role in the proper management of communityacquiredpneumonia this research work aimed to evaluate the association of rdw and or mpv with mortality andmorbidity in patients with cap to improve the yield of already used prognostic scoresresults the current study enrolled patients with communityacquired pneumonia cap out of them patients improved while died it was noticed that each of delta mpv and rdw p hadpositive significant correlation with psi and curb65 delta mpv and rdw was significantly higher in patients whodied ± vs ± p for delta mpv and ± vs ± p for rdw initial rdw and rising mpv during hospitalization for cap is associated with more severe clinicalcharacteristics and high mortality moreover the use of rdw and delta mpv in patients admitted with cap canimprove the performance of prognostic scaleskeywords communityacquired pneumonia red cell distribution width mean platelet volume communityacquired pneumonia cap is the fourthleading cause of death all over the world and plays animportant role of morbidity and mortality [] scoringsystems have an essential role in the management of patients with cap and currently there are several severityscores in use such as pneumonia severity index psicurb65 however these severity scores have some limitations and variations for example the curb65 andcrb65 are crude scores for rapid assessment of thehighrisk patients while psi is believed to be useful foridentifying lowrisk patients therefore there is effort toimprove the prognostic value of these severity scores correspondence randaezzeldin98gmailcom32561department of chest diseases faculty of medicine assiut university assiutegyptfull list of author information is available at the end of the several biomarkers have been checked and verified foruse in cap which could improve the prognostic performance of severity scores [ ] however some ofthese biomarkers are nonspecific and not sensitiveothers are expensive and are not always available immediately mean platelet volume mpv is done as a routine laboratory test that is measured in complete bloodcount and it is considered a marker of platelet functionand activation [ ] a single elevated mpv measurement has been found to be associated with increasedmorbidity and mortality in various patient populations[ ] patients hospitalized with communityacquiredpneumonia cap are found to be at an increased risk ofdeath in the hospital and following discharge []the prognostic significance of mpv has been studied inonly two small studies on cap patients which werebased on single mpv determinations [ ] the clinical characteristics and prognosis oftimedependent 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 0cfarghly the egyptian of bronchology page of mpv changes have not been investigated in the cappopulation we hypothesized that the mpv may reflectplatelet activity and may be associated with an impairedhost response according to this hypothesis an increasing mpv may be associated with poor outcomes andmay predict inhospital mortality in icu patients withsevere pneumonia to test our hypothesis we examinedmpv alterations in patients with severe pneumonia whohad been admitted or transferred to the icured cell distribution width rdw is defined as a coefficient of variation of circulating red cells it is affectedby changes of red cell volume and is measured in theroutine complete blood count cbc few years earlyrdw used in the clinical practice to diagnose differenttypes of anemia moreover elevated rdw had a prognostic role in the outcome of some diseases like cardiovascular disease rheumatoid arthritis colon cancer andmetabolic syndrome [ ] furthermorefew researches have reported rdw as a prognostic predictorof mortality in different populations the exactmechanism of variation in rdw is still unknown but itis mostly associated with the process of oxidative stressand inflammation which reflects the prognostic role ofrdw to our knowledge rdw use does not implyany additional cost because it is routinely provided aspart of the whole blood count by hemocytometryseveral studies support the hypothesis that rdw maybe a useful parameter for gathering either diagnostic orprognostic information on a variety of cardiovascularand thrombotic disorders [ ] although the link between rdw and cardiovascular disease is unclear in recent years rdw has been associated with capoutcomes especially with 30day and 90day mortalityand complicated hospitalization [ ] this researchwork aimed to validate the role of rdw and mpv aspromising markers of mortality and morbidity in patients with cap to improve the yield of already used severity scoresmethodsthis prospective study included adult years ofage patients admitted to chest department and ricuof assiut university hospital with a diagnosis of capbetween october and october patients wereprospectively recruited within h of their arrival capwas defined as an acute disease with a radiological infiltrate that was not previously present and not due to another known cause and was associated with symptomsof lower respiratory tract infection exclusion criteriawere severe immunodepression hiv infection or severe hematological diseasesimmunosuppressivetherapy prednisone or equivalent dose of mg dailyfor weeks or any immunosuppressive regime therapy azathioprine cyclosporine cyclophosphamide andor other immunosuppressant drugs leukopenia leukocyte per mm3 or neutropenia neutrophils per mm3 andor chemotherapy in the previousyear pulmonary abscess radiological cavitation aspiration pneumonia and obstructive pneumoniapossible nosocomial origin days from hospital discharge and known active neoplasia all patientswere followed up during their hospital stay and thosewith a definitive diagnosis other than cap were excluded all of the patients were followed up until beingdischarged our study primary outcome variable was inhospital mortality of patients with cap the secondaryfig outcome of patients with cap 0cfarghly the egyptian of bronchology page of table baseline characteristics of both groupsimproved n died n ± ± p valuetable correlation between curb65 and psi in correlation todelta mpv and rdwdelta mpvrppsicurb65 rdwrp date expressed as r strength of correlation p significance of correlation pvalue was significant if mpv mean platelet volume rdw red celldistribution width psi pneumonia severity indexage yearsexmalefemalesmokingcurrent smokingnonestopped smokerexsmokerscomorbid diseasescurb65psiclassiiiiiivvlaboratory datardw delta mpvwbcs 109lplatelets 109lpao2 fio2hospital stay dayneed to mvtransfer to icuradiological findings ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ph pao2 urea nainhaled or oral corticoids and antibiotics were recordedon admission the following data were also recordeddays of duration of disease cap signs and symptomsfever cough sputum dyspnea pleural pain vital constants at the ed temperature respiratory and heartrates arterial pressure and oxygen saturation analyticdataglucose hematocrithemoglobin red cell distribution width rdw andmean platelet volume mpv number of lobes involvedand type of radiological condensation alveolar interstitial or mixed and complications respiratory cardiologic renal neurologic digestive and others noninvasive mechanical ventilation nimv need of icuand invasive mechanical ventilation imv psi andcurb65 scores were calculated for all patients all patients were admitted to the hospital for ¥ hsample collection and processingblood was collected from anticubital fossa by experienced phlebotomists using a standardized atraumaticprotocol using clean venipuncture and minimum tourniquet pressure needles used were gauge specimens were maintained at room temperature °cnot placed on ice refrigerator or water bath tubes keptcapped upright at room temperature not exposed to vibration excessive mixing or agitation specimens containing any evidence of clotting were discarded twosamples of ml of venous blood in standard tubes contain ethylenediamine tetraacetic acid edta anticoagulant for complete blood count cbc the first one atthe time of admission and the second one after daysof admission the cbc sample was examined within has recommended by bcsh guideline to avoidbias due to excessive platelet swelling and to minimizevariation due to sample aging mpv and other bloodcount parameters were measured by an automatedanalyzer advia 2120i jermany with lh controlsystem in our laboratory the range of normal mpvvalues is fl for analysis of timedependentmpv changes patients were categorized according toδmpv mpv on discharge minus mpv on admissioninto patients with no rising mpv δmpv fl andpatients with rising mpv δmpv ¥ fl rdw wasunilobar pneumonia multilobar pneumonia effusion positive blood culture positive sputum culture data expressed as mean sd frequency percentage p value was significantif mv mechanical ventilation icu intensive care unitoutcome variables were length of hospital stay intensivecare unit icu admission and mechanical ventilator requirement this study protocol was approved by thelocal ethics committee and informed consent was obtained from all patients or next of kindata collectionage sex tobacco and alcohol consumption comorbidconditions diabetes liver chronic kidney hearth andcerebrovascular diseases nonactive neoplasia bronchiectasis and previous cap and previous therapies 0cfarghly the egyptian of bronchology page of fig correlation between delta mpv and psireported as a part of the cbc results rdw is the standard deviation of mcv and was measured in as percentage a single rdw had been recorded from cbc ofpatients on admission in our laboratory the range ofnormal rdw values is statistical analysisdata was collected and analyzed those using spss statistical package for the social science version ibmand armonk ny continuous data was expressed inform of mean ± sd or median range while nominaldata was expressed in form of frequency percentageindependent risk factors of mortality had been determined by multivariate regression analysisreceiver operator curve was used to determine cutoffof rdw and delta mpv for prediction of inhospitalmortality in patient with communityacquired pneumonia pearson correlation was used to determine correlation between psi and curb65 with rdw and deltampv level of confidence was kept at hence pvalue was significant if resultsthe currentstudy included with communityacquired pneumonia cap out of them patients improved while died as shown in fig the characteristic data of the enrolled patients are summarized in table mean age of improved patients was ± years majority of them were malesand of them were smokers mean age of patients who died was ± years and ofthem were females it was noticed that patientswho died were smokers it was noticed that patients who improved and patients who diedhad coexisting comorbidities with significant differencesbetween both groups it was noticed that class of psi wasii iii iv and v in and respectively of improved patients and in and of patients died respectively mean psi was significantly higher in patientswho died ± vs12339 ± p moreover curb65 was significantly higher in patientswho died in comparison to improved patients ± vs ± p fig correlation between delta mpv and curb65 0cfarghly the egyptian of bronchology page of fig correlation between rdw and psidelta mpv and rdw was significantly higher in patients who died ± vs ± p fordelta mpv and ± vs ± p for rdw our research also detected that comorbiddiseases transfer to icu and need for mechanical ventilation were highly frequent in patients who died moreover patients who died had longer duration of hospitalstay on radiological findings pleural effusion and unilobar pneumonia were presented in and ofpatients who died vs and of improved patients respectively while multilobar pneumonia wasmore frequent in patients who died blood culture waspositive only in of patients who improved vs of patients who diedthe current study also discovered that each of δ mpvand rdw had positive significant correlation with psiand curb65 p as shown in table and figs and based on the current study table the following variables were predictors of inhospital mortality in patients withcap with adjusted r2 was curb65 or ci p psi or ci p rdw or ci p delta mpv or ci p it was worthwhile to notice that rdw at cutoff point had sensitivity and specificity for predictionof mortality in patient with cap with area under curve while delta mpv at cutoff point had sensitivityand specificity for prediction of death in patient withcap with area under curve as shown in table andfig discussionin the recent few years cap had been considered one ofthe leading causes of death worldwide thereforefig correlation between rdw and curb65 0cfarghly the egyptian of bronchology page of table predictors of inhospital mortality in patients with capp valueodds ratioage confidence intervalsexcomorbiditiescurb65psirdwdelta mpvp value was significant if mpv mean platelet volume rdw red celldistribution width psi pneumonia severity index capcommunityacquired pneumoniaaugmentation of the conventional severity scoreslikethe psi and curb65 became a must to identify patients with high risk for a complex course as the predictive performance of these scores alone may be limitedseveral researches have detected that discovering newbiomarkers could augment the validity of these severityscores [ ]in this prospective study we planned to assess the validity of the mean platelet volume change and rdw asbiomarkers for assessing the severity of cap it wasworthwhile to know that no previous study has beendone at assiut university hospital to assess those twobiomarkers in patients with cap the main potentialmechanism for rising mpv in patient population is severe inflammation caused by pneumonia in severe infection increased release of thrombopoietin and variousinflammatory cytokines such as interleukin1 and and tumor necrosis factorα result in increased thrombopoiesis and enhanced expression of younger largeplatelets into the blood circulation [ ] on theother hand rising mpv may be attributed to increasedthrombocyte consumption in the peripheral tissue andspleeninduced by severe inflammatory status [ ] communityacquired pneumonia is an infectiousdisease that results in inflammatory and oxidative stresstable performance of rdw and delta mpv in prediction ofmortality in capsensitivityspecificitypositive predictive valuenegative predictive valuecutoff pointrdw delta mpv area under curvep valuep value was significant if rdw red cell distribution width mpv meanplatelet volume cap communityacquired pneumonia to the host if these stresses are severe mortality will beincreased the underlying pathophysiological mechanisms for a relationship of rising mpv with poor prognosis and mortality are not fully understood the mainpotential explanation can be increased platelet activation[ ] larger thrombocytes are known to be functionally metabolically and enzymatically more active thansmaller ones the greater activation of enlarged plateletsresults in increased release of procoagulant substancessuch as thromboxane a2 βthromboglobulin and surface proteins [ ] consequent hyperaggregability ofplatelets extended vasoconstriction and endothelial dysfunction may contribute to an increased shortterm riskof cardiovascular thrombosis and death in patients withrising mpv [ ]our research detected that a high rdw and risingmpv were significantly related with increased risk ofdeath in patients with cap as delta mpv and rdw wassignificantly higher in patients who died ± vs ± p for delta mpv and ± vs ± p for rdw our results are inagreement with braun who detected thatrdw was associated with high risk of death and disability in young patients admitted with cap in this retrospective study brawn in a cohort of patients of years or older hospitalized due to cap demonstratedthat elevated rdw was independently associated with complicated hospitalization length of stay days and admission to icu and 90day mortality irrespective of hemoglobin levels in line with the results of our study lee also identified a high rdwis a prognostic factor for 30day cap mortality ageis significant prognostic factor in various diseasesincluding cap in our study the mean age of improvedpatients with cap was ± years while meanage of patients who died was ± years thusour findings support rdw as a significant prognosticfactor in patients with cap across all ages these resultsare in agreement with braun their finding is similar to our results however they only includedpatients who were younger than years which theycited as a limitation of their study this study revealedthat each of psi and curb65 had positive significantcorrelation with delta mpv and rdw p ourresults are also in line with gorelik whostated that rising mpv during hospitalization for cap isassociated with a more severe clinical profile and mortality than no rise in mpv they found that mpv valuesabove the cutoff at discharge were associated with anincreased risk of mechanical ventilation and death during hospitalization and shortened survival following discharge based on the current study the following variableswere predictors of inhospital mortality in patients with 0cfarghly the egyptian of bronchology page of fig diagnostic performance of delta mpv and rdw in prediction of inhospital mortalitycap with adjusted r2 was curb65 or ci p psi or ci p rdw or ci p delta mpv or ci p indeed in our patient population a risein mpv was associated with higher pneumonia severityscoresin our study the mortality prediction of both the psiand curb65 was improved by the addition of rdwand delta mpv as severity criteria the exact mechanisms of an association of rdw with the mortality ofcap need to be determined it has been suggested thatinflammation and oxidative stress affect red cell homeostasis a previous study revealed that rdw displayed astrong graded association with inflammatory biomarkersin general outpatient populations and anotherstudy indicated that serum antioxidant levels includingselenium and carotenoids were associated with rdw inolder women in our data patients with a higherrdw had a tendency toward higher severity indexscores and the overall mortality was also higher in patients with a higher rdwthere are some limitations of our research work firstthe study included one group of cap patients who wereadmitted in our assiut chest department and ricuthus it cannot be generalized to all patients with capsecond larger studies of large numbers of patients needto be done to investigate the value of mean plateletvolume change and rdw as prognostic markers incommunityacquired pneumoniasrdw and rising mpv during hospitalization for cap isassociated with more severe clinical characteristics andhigh mortality we suggestthat repeated mpv andrdw determination throughout hospitalization may improve risk stratification for cap patientsabbreviationscap communityacquired pneumonia cbc complete blood count crb confusion respiratory rate blood pressure age ¥ curb65 confusionurea respiratory rate blood pressure age ¥ δmpv mean platelet volumechange ed emergency department icu intensive care unit rdw blood celldistribution widthacknowledgementsto all work team who do their best to do this research in a perfect way andto all patients involved in this study and their parentsauthors contributionssf and ra carried out historytaking and physical examination of all participants in addition to obtaining blood samples participated in the sequencealignment and drafted the manuscript they also analyzed and interpretedthe patients data re and da carried out laboratory investigations and participated in the revision of the manuscript all authors read and approved thefinal manuscriptfundingno fund was taken from any institute or companyavailability of data and materialsdata and materials are available 0cfarghly the egyptian of bronchology page of ethics approval and consent to participatethe study obtained approval from the ethical committee at the faculty ofmedicine assiut university and a written consent was taken from theparticipants no reference number is usually given for approved studies inour universityconsent for publicationconsent for publication was taken from all authorscompeting intereststhe authors declare that they have no competing interestsauthor details1department of chest diseases faculty of medicine assiut university assiutegypt 2department of clinical pathology faculty of medicine assiutuniversity assiut egyptreceived april accepted august references mandell la wunderink rg anzueto a infectious diseasessociety of americaamerican thoracic society consensus guidelines on themanagement of communityacquired pneumonia in adults clin infect dis44suppl 2s27s72almirall j bolibar i vidal j epidemiology of community acquiredpneumonia in adults a populationbased study eur respir j armstrong gl conn la pinner rw trends in infectious diseasemortality in the united states during the 20th century jama menendez r martinez r reyes s biomarkers improve mortalityprediction by prognostic scales in communityacquired pneumonia thoraxlee jh kim j kim k albumin and creactive protein haveprognostic significance in patients with communityacquired pneumonia jcrit care chu sg becker rc berger pb mean platelet volume as apredictor of cardiovascular risk a systematic review and metaanalysis jthromb haemost noris p melazzini f balduini cl new roles for mean platelet volumemeasurement in the clinical practice platelets ar©valolorido jc carreterog³mez j ¡lvarezoliva a guti©rrezmonta±o cfern¡ndezrecio jm najarrodiez f mean platelet volume in acutephase of ischemic stroke as predictor of mortality and functional outcomeafter 1year j stroke cerebrovasc dis wasilewski j desperak p hawranek m prognostic implicationsof mean platelet volume on short and longterm outcomes among patientswith nonstsegment elevation myocardial infarction treated withpercutaneous coronary intervention a singlecenter large observationalstudy platelets dabbah s hammerman h markiewicz w relation between redcell distribution width and clinical outcomes after acute myocardialinfarction am j cardiol 105312e331sangoi mb da silva sh da silva je relation between red bloodcell distribution width and mortality alter acute myocardial infarction int jcardiol 146278e280 montagnana m cervellin g meschi t the role of red blood celldistribution width in cardiovascular and thrombotic disorders clin chemlabmed 504635e641lee jh chung hj kim k red cell distribution width as aprognostic marker in patients with communityacquired pneumonia am jemerg med 31172e79 braun e domany e kenig y elevated red cell distribution widthpredicts poor outcome in young patients with community acquiredpneumonia crit care 154r194 harrison p mackie i mumford a briggs c liesner r winter m machin s guidelines for the laboratory investigation of heritable disorders ofplatelet function br j haematol bello s fandos s lasierra ab minchole e panadero c simon al de pabloog menendez r torres a red blood cell distribution width [rdw]and longterm mortality after communityacquired pneumonia acomparison with proadrenomedullin respir med 1091193e1206 ware j corken a khetpal r platelet function beyond hemostasis andthrombosis curr opin hematol becchi c al malyan m fabbri lp marsili m boddi v boncinelli s mean platelet volume trend in sepsis is it a useful parameter minervaanestesiol kitazawa t yoshino y tatsuno k ota y yotsuyanagi h changes inthe mean platelet volume levels after bloodstream infection haveprognostic value intern med kamath s blann ad lip gy platelet activation assessment andquantification eur heart j gorelik o tzur i barchel d almozninosarafian d swarka m feldman iblcohen n izhakian s a rise in mean platelet volume duringhospitalization for communityacquired pneumonia predicts poorprognosis a retrospective observational cohort study bmc pulmonarymedicine lippi g targher g montagnana m relation between red bloodcell distribution width and inflammatory biomarkers in a large cohort ofunselected outpatients arch pathol lab med semba rd patel kv ferrucci l serum antioxidants andinflammation predict red cell distribution width in older women thewomen's health and aging study i clin nutr publishers notespringer nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations musher dm thorner ar communityacquired pneumonia n engl jmed jain s self wh wunderink rg communityacquired pneumoniarequiring hospitalization among us adults n engl j med eurich dt marrie tj minhassandhu jk tenyear mortality aftercommunityacquired pneumonia a prospective cohort am j respir critcare med karadagoncel e ozsurekci y kara a karahan s cengiz ab ceyhan m the value of mean platelet volume in the determination ofcommunity acquired pneumonia in children ital j pediatr golcuk y golcuk b bilge a irik m dikmen o combination of meanplatelet volume and the curb65 score better predicts 28day mortality inpatients with communityacquired pneumonia am j emerg med felker gm allen la pocock sj red cell distribution width as anovel prognostic marker in heart failure data from the charm programand the duke databank j am coll cardiol ani c ovbiagele b elevated red blood cell distribution width predictsmortality in persons with known stroke j neurol sci patel kv semba rd ferrucci l red cell distribution width andmortality in older adults a metaanalysis j gerontol a biol sci med sci 0c" | 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 |
microbiota involves communities ofhepatitis is generally known as an ammation of the liver that can be caused by hepaticand nonhepatic viruses can be caused by alcohol can be drug induced and can be caused byautoimmunity gut microbiota composition is known to be associated with disease pathogenesishowever dynamic alteration of the gut microbiota in disease pathogenesis is not wellunderstoodsymbiotic as well as pathogenicmicroanisms found in anisms ie plants and animals microbiota of a healthy individualshows more of commensalism or symbiosis without causing any disease these microbes mainlycolonize humans during birth or shortly thereafter and remain throughout the course of life thesecan be found in many areas like skin respiratory tract urinary tract and digestive tract whilebrain lungs and the circulatory system are free of microbes approximately microbes arepresent in a healthy individual gut minemura and shimizu therefore gut microbiota hasan important role to modulate the immune system in disease progression or recoverycommensaltranslocation of microbes or their metabolic products cause intestinal ammation leadingto impairment of the primary barrier hill there is limited available informationregarding the role of gut microbiota in hepatitis which makes it important to majorly focus onclinical data of gut microbiota linked with hepatitis b and c virusgut microbiotagut or gastrointestinal tract starts from the mouth and ends at the back passage anus gut helpsin the digestion of food by absorbing energy and nutrients majority of gut microbiota to contains good bacteria and only to are harmful bacteria in diï¬erent parts of the intestineedited bymilan surjittranslational health science andtechnology institute thsti indiareviewed byjawed iqbaljamia millia islamia indiabinod kumarloyola university chicagounited statescorrespondencenirupma trehanpatitrehanpatigmailcomspecialty sectionthis was submitted tovirus and hosta section of the frontiers in cellular and infectionmicrobiologyreceived march accepted june published august citationsehgal r bedi o and trehanpati n role of microbiota inpathogenesis and management ofviral hepatitisfront cell infect microbiol 103389fcimb202000341frontiers in cellular and infection microbiology wwwfrontiersinaugust volume 0csehgal microbiota and liver diseasesbajaj in mouth and upper respiratory tract normalï¬ora is more of the commensal bacteria like streptococcusmoraxella neisseria and haemophilus very few species ofbacteria are present in the stomach and small intestine whilethe large intestine and colon contain dense population ofmicrobes ie up to cellsg along with bacteria many othermicroanisms like fungi protists archaea and viruses alsosymbiotically harbor in the gutthere are four dominant phyla of bacteria present in thegut and they are firmicutes bacteroidetes actinobacteriaand proteobacteria khanna and tosh most importantgenera in which bacteria belong are bacteroides clostridiumfaecalibacterium eubacterium ruminococcus peptococcuspeptostreptococcus and biï¬dobacterium fern¡ndez some of the fungal species that also coexist in the gut arecandida saccharomyces aspergillus penicillium rhodotorulatrametes pleospora sclerotinia bullera and galactomycesamong others raimondi functions of gut microbiotagut microbiota plays an important but diverse role such asbarrier eï¬ect vitamin synthesis and fermentation residentbacteria of the gut acts as a barrier and protect the intestinalmucosa from invasion of the other potential pathogens hooper many factors including diet age medicationillness stress and lifestyle uence the gut microbiota whichhave a great impact on disease pathogenesis in fact manybacteria ie bacteroides eubacterium propionibacterium andfusobacterium are instrumental in the synthesis of vitamins kand b ie folate b12 and biotin canny and mccormick they are also involved in the fermentation of nondigestible carbohydrates for the production of shortchain fattyacids scfas which are helpfulin maintaining metabolichomeostasis in addition to the production of scfa glycolysisand pentose phosphate pathway also produce butyrate whichpromotes the growth of lactobacilli and biï¬dobacteria bacteriain the colon venegas various studies supported thefact that nutrients derived from microbiota play a pivotal rolein the normal functioning of the hepatic system li zheng moratalla jiminez cremer wang 2017agut microbiota in liver diseasescommensal bacteria play a decisive role in maintaining immunehomeostasis figure and also guard immune reactions atmucosal surfaces ichinohe intestinal microï¬ora isa dynamic and complex ecosystem which helps in proliferationgrowth and diï¬erentiation of epithelial cells to ï¬ght infectionsand improve immunity despite its crucial role in the synthesisfolate scfa and peroxides gut microbiotaof vitamin kacts as a chief environmental as well as etiologicalfactorfor the progression of many liver diseasesohara andshanahan particularly gut microbiota has a largeruence on alcoholic liver disease nonalcoholic fatty liverdisease viral hepatitishepatitis b and c autoimmunehepatitis aih primary sclerosing cholangitis psc andprimary biliary cholangitismohamadkhani pbclactobacillus biï¬dobacterium saccharomyces boulardii andlactobacillus plantarum play a bigger role in the managementof various metabolic disorders and hepatitis mohamadkhaniincludingvirusesandseveralpathogensintestinalmicroanisms use mucous membranes as a doorwaykarst hepatic viruses breach the intestinal permeabilityleading to gut dysbiosis and release proammatory cytokinesinstrumental in developing liver cirrhosis and hcc it is alsoobserved that the use of probiotics reduces the tolerogenicresponse and enhances the mucosal defense against viralpathogens rigoadrover m del lactobacillusalone can uence the production of interferon by modulatingthe antiviral eï¬ects of vitamin a lee and ko themixture of various probiotics and biï¬dobacterium with galactooligosaccharides and fructooligosaccharides has a defensiveeï¬ect against rotavirus infection by aggregating the productionof tnfα il4 ifnÎ and tlr2 expression rigoadrover mdel in most of the liver disease especially cirrhosisdysbiosis of the gut increases proteobacteria enterobacteriaceaeand veillonellaceae while it decreases bacteroidetes andlachnospiraceae sanduzzi zamparelli recentlythe cirrhosis dysbiosis ratio cdr is coined for deï¬ningthe changes in gut microbiome in cirrhosis patients withbeneï¬cial lachnospiraceae and ruminococcaceae and harmfulenterobacteriaceae bacteria bajaj other groupshave also associated patients with severe cirrhosis and hepaticencephalopathy with overgrowth of enterobacteriaceae bacteriachen role of gut microbiota in hepaticviral infectionsacute viral hepatitis due to hepatitis a and e viral infectionsis a major community health problem especially in developingcountries hepatitis a and e cause acute infection whichcould be shortlived and selfclearing unless the subjects areimmunocompromised or in transplant settings acute hepatitise infection also becomes detrimental and lifethreatening duringpregnancy aï¬ecting both the mother and the childboth hepatitis a and e are rna viruses thattransmitthrough oral fecal routes lemon and may havedevastating eï¬ects on intestinal microï¬ora it was observedthat administration of the healthy probiotic bacterium likeenterococcus faecium ncimb aï¬ects the reduction as wellas the removal of enteric hev viruses in pigs kreuzer however there is lack of relevant data in humansas per the world health anization who hepatitisb virus hbv infection caused deaths in and million diagnosed with chronic infection in similarly hepatitis c virus hcv caused deaths with anestimated million diagnosed with chronic infection in both these viruses cause chronic infections at in hbv andmore than in hcv leading to cirrhosis and hepatocellularcarcinoma hepatic viruses have evolved mechanisms to avoidtheir detection from the host innate and adaptive immunityfrontiers in cellular and infection microbiology wwwfrontiersinaugust volume 0csehgal microbiota and liver diseasesfigure protective role of fecal microbiota transplantation and use of probiotics in immune restorationand characterized as viral escape visvanathan itis observed that chronic hepatitis patients have largertranslocation of the intestinal microbiota lu li bacterialtranslocation cause intestinalammation viadysregulation of immune cell overgrowth of pathogenic bacteriaas well as dysfunction of the primary barrier hill xu also supported the fact that intestinal ï¬oraloses homeostasis during dysbiosis which in fact helps theadvancement of hepatitis viral infection xu itthereforeis now understood that during chronicitycommensal microbiota have greater impact not only on viral hostcell interaction but also on viral replicationin viral hepatitis few harmful bacteria like escherichia colienterobacteriaceae enterococcus faecalis and faecalibacteriumprausnitzii directly alter the proï¬le of good intestinal microbiotawith a lower number of intestinallactic acid species suchas lactobacillus pediococcus weissella and leuconostoc bajaj chen some of the bacterial species ieneisseria e coli enterobacteriaceae e faecalis f prausnitziiand gemella are also found responsible for the progression ofhepatitis b and c virusrelated cirrhosis and primary biliarycirrhosis chen mohamadkhani candidais also frequently found in patients with hepatitis brelatedcirrhosis cui role of gut microbiota in hepatitis b viralinfectiondysbiosis of gut microbiota in chronic hepatitis b infectionaï¬ects disease pathogenesis and causes liver failure in alarge proportion lps lipopolysaccharides from the outermembrane of gramnegative bacteria help in the activationof innate immune response by recognizing tlrs especiallytlr2 and hbv infection leads to progressive declinein butyrateproducing bacteria however lpsproducinggenera is enriched in hbv infection in hbv infection abeneï¬cial bacterium lachnospiraceae plays a role in themanagement of hbv infection via reduction in lps sectionand bacterialtranslocation chen ren studies have shown the role of faecalibacteriumpseudobutyrivibrioruminoclostridiumprevotella alloprevotella and phascolarctobacterium in potentialantiammatory scfa activity which increases the abundanceof butyrate compared to normal subjects liu lu have demonstrated that copy numbers of fprausnitzii e faecalis enterobacteriaceae biï¬dobacteria andlactic acid bacteria lactobacillus pediococcus leuconostocand weissella have marked variation in the intestine of hbvcirrhotic patients during hbv infection dysbiosis in theoral microbiota was observed and yellow tongue coating issuggestive of a reduction in bacteroidetes but an increaselachnoclostridiumfrontiers in cellular and infection microbiology wwwfrontiersinaugust volume 0csehgal microbiota and liver diseasesin proteobacteria zhao also suggested positivecorrelation of neisseriaceae with the serum hbvdnacirrhotic patients with hbv infection showed a signiï¬cantdecrease in the biï¬dobacteriaceaeenterobacteriaceae be ratiolu while yun observed no diï¬erence in thebe ratio in hbsag with normal or high alt and in noncirrhotic hbv carriers yun it means the be ratiois disturbed only in cirrhosis however other study observedthat the megasphaera genus from the firmicutes phylum wasabundant in the hbsag high alt group than the normal altin patients with normal alt butyrateproducing bacteria likeanaerostipes are more in feces compared to hbsagve yun it is interesting to note that both megasphaeraand anaerostipes produce scfa as a byproduct of lactatefermentation and butyrate however butyrate is known asanticarcinogenic and antiammatory and plays a role inoxidative stress hamer another study suggests thatchronic hepatitis b infected cirrhotic patients exhibit a decreasein biï¬dobacteria and lactobacillus levels while signiï¬cantlyincreasing enterococcus and enterobacteriaceae levels comparedto healthy individualsbacterial translocation is also observed in the developmentof hepatocellular carcinoma hcc recently wang havedeï¬ned the serum zonulin as an intestinal permeability markerand showed its association with afp levels in hbvassociatedliver cirrhosis and hcc they are helpful in correlating it withadvanced stages of the diseases fasano the use of probiotic in hbvinfected patients showed beneï¬tand suggested that probiotic vsl3 plays an important role in themanagement of hbv viral infection dhiman role of gut microbiota in hepatitis c viralinfectionchronic hepatitis c infection is another leading cause ofcirrhosis hcc and in some casesliver failure and deathin majority enterobacteriaceae and bacterioidetes increased inchronic hcv patients but firmicutes found to be decreasedhcv infection cause marked elevation in lps which issuggestive of microbial translocation and ammation duringdisease progression dolganiuc inoue on the other hand it was observed that antiviral treatment ofhcv with ribavirin rbv and immune modulator pegylatedinterferon pegifn has no direct impact on gut dysbiosisin factit increases the production of bile acids which isimportant for gut microbiota ponziani somepathogenic bacteria such as enterobacteriaceae staphylococcusand enterococcus decreased the bile acid in hcvinfectedcirrhotic patients which normalized after a directacting antiviraltreatment oral directacting antivirals daas were also foundto be helpful in improving gut especially lachnospira and doreagenera and restored tnfα levels p©rezmatute but after daa treatment expression of calprotectin zo1and lps was found more in hcv patients with cirrhosis itwas also suggested that during hcv infection l acidophilusand biï¬dobacterium spp can act as a supportive supplementwith antiviral and antibacterial activities dore immune response in hcv patients can be stimulated by usefulmicrobiota via activation of cd3 cells and cd56 nk cellcounts which were explained by doskali and furthersuggested that good ï¬ora increases the cytotoxic eï¬ects of nkcells against viral infected cells inhibiting the replication of hcvuse of probiotics in hcvinfected patients with cirrhosis wassigniï¬cantly beneï¬cial preveden another hepatic virus hepatitis d virus is a new playerand not much is known about it yet it was also suggestedthat endotoxemia in hcv and hdv patientstobe multifactoriallikely depending on impaired phagocyticfunctions and reduced tcellmediated antibacterial activitykefalakes and rehermann seemsmicrobiota modulates molecularsignaling in hepatitisactivereceptorthe keycomponent of gramnegative bacterialps isie enterobacteriaceae thefor lps iscd14tlr4md2 receptor complex on induction whichsecretes many proammatory cytokines including tumornecrosis factorα il1 il6 and chemokines through the nfκbsignaling fooladi seki and schnabl bryant to cause liver injury in the intestinal tract lpsdownregulates the expression of various tight junction proteinszo1 and closed protein by increasing the permeability ofthe intestinal mucosa and enters the blood ï¬ow through theportal venous system park in liver kupï¬er cells asspecialized macrophages are induced by the lpstlr4 pathwayfor the release of immunosuppressive mediators such as il10which in turn suppress the release of ammatory mediatorsby kupï¬er cells dixon in this way during viralhepatitis virus speciï¬c immune responses are suppressed andultimately inhibit eï¬cient clearing of bacteria as well as virusesin addition to lps unmethylated cpg dna bacterialdnarna bacterial cell wall also contains teichoic acidpeptidoglycan and specialized proteins ï¬agellin bacterialdnarna is recognized by tlrs as well as all components ofcellwalllike teichoic acid and peptidoglycan also recognized bytlr2 while tlr5 got activated by ï¬agellin dsrna bacteriaare recognized by tlr3 ssrna activates receptors of bothtlr7 and tlr8 all these tlrs ultimately stimulate the jakstat pathway hepatitis viruses are also recognized by tlrs inthe liver or in the intestine and activate downstream signalingpathways mencin unmethylated cpg dnas are found abundantly in thelactobacillus family ie l casei l plantarum l rhamnosus andothers like biï¬dobacteria proteobacteria and bacteroidetes inthe intestinal ï¬ora of animals unmethylated cpg dna is sensedby tlr9 expressed on various mononuclear cells and stimulatesboth innate immune response as well as adaptive immuneresponse krieg kauppila activation the ofcpgtlr9 pathway stimulates downstream molecules of myd88such as irak4 traf6 and irak1 ultimately triggering nfκb and mapk signaling pathways these downstream pathwayshelp in the activation of dcs for the secretion of cytokines andfrontiers in cellular and infection microbiology wwwfrontiersinaugust volume 0cfrontiersincellluarandifnectionmcroboogyiilwwwfrontiersniaugustlvoumearticeltable randomized fmt clinical trials for the treatment of chronic hepatitis b infectionsnostudy titlestudy typeno ofsubjectsinterventiontreatmentstatusphaseprimary outcomemeasuresrandomized controlled trialcomparing the efï¬cacy andsafety of fmt in hepatitis breactivation leads to acuteon chronic liver failurelocationinstitute of liver and biliarysciencesnew delhi delhi indiastudy on effect of intestinalmicrobiota transplantationin chronic hepatitis blocation zhongshanhospital afï¬liated to xiamenuniversityxiamen fujian chinainterventionalclinical trialdrug tenofovirdrug fecal microbiotatransplantation fmtcompletedcompletedtransplant free survival[time frame months]interventionalclinical trialother intestinalmicrobiota transplantdrug antiviral agentsrecruitingnachange of serum hepatitis bvirus e antigenhbeag level[time frame months]serum hepatitis b virus eantigenhbeag levels ismeasured in scosecondary outcome measuresclinicaltrialsgovsehgaletalidentiï¬ernct02689245nct03429439reduction in hepatitis b virus dnalevel ¥ log [time frame weeks]improvement in meld model forend stage liver disease score[time frame weeks]change of serum hepatitis b virussurface antigenhbsag level [timeframe months] serumhepatitis b virus surfaceantigenhbsag levels is measuredin iumlchange of serum antihepatitis bvirus e antigenantihbe [timeframe months] appearanceof serum antihepatitis b virus eantigenantihbe suggest the abilityof body to resistant hbvchange of serum antihepatitis bvirus surface antigenantihbs[time frame months]appearance of serum antihepatitisb virus surface antigenantihbssuggest the ability of body toresistant hbvchanges of gut microbiota [timeframe months] alpha andbeta diversity of gi microbiota byhighthroughput sequencing 16srrna on baseline line and months after treatmentrelief of constipation [time frame months] relief of diarrhea[time frame months] reliefof abdominal pain [time frame months] the onset andduration of constipation will beassessed by evaluation scoretable of gastrointestinalsymptomsimcrobotaiandlveriidseases 0csehgal microbiota and liver diseaseschemokines krieg kauppila chronic hbvpatients have reduced lactobacillus and biï¬dobacteria both arerich in unmethylated cpg dna levels ultimately aï¬ecting thecpg dnatlr9 pathway and immune response on hbv linand zhang role of fecal microbialtransplantation fmt in viralhepatitisfmt mainly involves the insertion of healthy microbiota in thediseased gut in brief fecal matter derived from a healthy familymember of the patient receiving the same diet as the patient isprocessed and introduced in the intestinal tract of the patientthese have minimal side eï¬ects and proved helpful in reinstatinghealthy gut ï¬ora in the patient fmt administration can bedone using several routes such as oral nasogastric nasoduodenalnasojejunal endoscopic rectal and colonoscopic or midguttransendoscopic enteral tubing cui tang for cirrhotic patients with dysbiosis small bowel route ismost aï¬ected while mostly used route is oral delivery in severealcoholic hepatitis sah in comparison to steroids fmt isassociated with decreased disease severity and improved survivalearlier wang 2017b have observed that fmt restored thecognitive function liver function indexes and tlr response incarbon tetrachloride ccl4induced acute hepatitis in ratswoodhouse have observed in a profit clinical trialthe beneï¬ts of fecal microbiota transplantation in the smallbowel of cirrhotic patients woodhouse meiglani also observed that cirrhotic patients with antibioticresistant clostridioides diï¬cile infection cdi responded wellafter fmt treatment in factfecal microbiota of alcoholresistant mice when given to alcoholsensitive mice has reducedbacteroidetes and increased actinobacteria as well as firmicutesand protected steatosis development ferrere limited studies are published yet on fmt administration inalcoholrelated liver disease however all these studies showedimmense beneï¬t of fmt bajaj observed the recoveryof cognitive function and hepatic encephalopathy in patientsunder clinical trial after administration of fmt studies recentlypublished from our center have found better eï¬ciency of fmtreferencesbajajj s heuman d m hylemon p b sanyal a the cirrhosis dysbiosisb monteith p changescomplicationsin the gut microbiomej hepatolassociated with cirrhosisj white mratio deï¬nesand its101016jjhep2013bajaj j s kassam z fagan a gavis e a liu e cox i j fecal microbiota transplant from a rational stool donor improveshepatic encephalopathy a randomized clinical trial hepatology 101002hep29306bryant c e symmons m and gay n j tolllike receptor signallingthrough macromolecular protein complexes mol immunol 101016jmolimm201406033in severe alcoholic patients than standard medical treatmentsarin there are only a couple of randomizedfmt clinical trials for chronic hepatitis b infected patientstable recently groups have addressed how fmt is modulatingimmunity in gut and liver mucosaassociated invariant tmait cells are found abundant in liver to ofintrahepatic t cells gut peripheral blood as well as lungs gao have observed that functional mait cells were altered insah resulting in more bacterial infection in patients alterationin circulating mait cells is observed with defective antibacterialcytokinecytotoxic response against the infection gao they believe that fmt administration has a profoundeï¬ect on the expression of mait cells in alcoholrelated diseasessummary and conclusionlikeruminoclostridiumgut microbiota has an important role in viral alcoholicand metabolic liver diseases gut microbiota plays a crucialrole in modulating the tolllike receptors nfκb signalingjanus kinasesignal transducer and transcription jakstatpathway and cd4t cell activation numerous usefulmicrobiotasfaecalibacteriumlachnoclostridium prevotella alloprevotella pseudobutyrivibrioand phascolarctobacterium play an important role in potentiatingantiammatory short chain fatty acid scfa activity andincreased the butyrate abundance which play a crucial role inthe management of various hepatitisrelated viral infectionsfecal microbiota transplantation became an attractive andsafest mode of treatment for the management of various liverdiseases especially in severe alcoholic hepatitis despite recentpublications there are still gaps in understanding the role ofmicrobiota in viral hepatitis especially in acute hav and hevviral infections therefore there is a need to explore more inthese infectionsauthor contributionsrs and ob written the review nt provide valuablesuggestions corrected and revised all authors contributed to the and approved the submitted versioncanny g o and mccormick b a bacteria in the intestine helpfulresidents or enemies from within infection and immunity am soc microbiol 101128iai0018708chen y ji f guo j shi d fang d and li l dysbiosis of smallintestinal microbiota in liver cirrhosis and its association with etiology sci rep 101038srep34055chen y yang f lu h wang b chen y lei d characterization of fecal microbial communities in patients with liver cirrhosishepatology 101002hep24423cremer j arnoldini m and hwa t eï¬ect of water ï¬ow and chemicalenvironment on microbiota growth and composition in the human colon procnatl acad sci usa 101073pnas1619598114cui b feng q wang h wang m peng z li p fecalmicrobiota transplantation through midgut for refractory c rohns diseasefrontiers in cellular and infection microbiology wwwfrontiersinaugust volume 0csehgal microbiota and liver diseasessafety feasibility and eï¬cacy trial results j gastroenterol hepatol 101111jgh12727cui l morris a and ghedin e the human mycobiome in health anddisease genome med 101186gm467dhiman r k rana b agrawal s garg a chopra m thumburu k k probiotic vsl\\ reduces liver disease severity and hospitalization inpatients with cirrhosis a randomized controlled trial gastroenterology 101053jgastro201408031dixon l j barnes m tang h pritchard m t and nagy l e kupï¬ercells in the liver compr physiol 101002cphyc120026dolganiuc a norkina o kodys k catalano d bakis g marshall c viral and host factors induce macrophage activation and loss of tolllikereceptor tolerance in chronic hcv infection gastroenterology 101053jgastro200708003dore g ward j and thursz m hepatitis c disease burden andstrategies to manage the burden guest editors mark thursz gregory doreand john ward j viral hepat 21suppl 101111jvh12253doskali m tanaka y ohira m ishiyama k tashiro h chayama k possibility of adoptive immunotherapy with peripheral bloodderived cd3cd56 and cd3cd56 cells for inducing antihepatocellularcarcinoma and antihepatitis c virus activity j immunother 101097cji0b013e3182048c4efasano a intestinal permeability and its regulation by zonulin diagnosticand therapeutic implications clin gastroenterol hepatol 101016jcgh201208012fern¡ndez m f reinap©rez i astaj m rodriguezcarrillo aplazadiaz j and fontana l breast cancer and its relationshipwith the microbiotaj environ res public health int 103390ijerph15081747ferrere g wrzosek l cailleux f turpin w puchois v spatz m fecal microbiota manipulation prevents dysbiosis and alcoholinduced liver injury in mice j hepatol 101016jjhep2016fooladi a i tavakoli h and naderi a detection of enterotoxigenicjin domestic dairy productsisolatesiranstaphylococcus aureusmicrobiol gao b ma j and xiang x mait cells a novel therapeutic target foralcoholic liver disease gut 101136gutjnl2017315284hamer h m jonkers d venema k vanhoutvin s troost f and brummerr j the role of butyrate on colonic function aliment pharmacol ther 101111j13652036200703562xhill d a hoï¬mann c abt m c du y kobuley d kirn t j metagenomic analyses reveal antibioticinduced temporal and spatial changesin intestinal microbiota with associated alterations in immune cell homeostasismucosal immunol 101038mi2009132hooper l v xu j falk p g midtvedt t and gordon j i amolecular sensor that allows a gut commensal to control its nutrient foundationin a competitive ecosystem proc natl acad sci usa 101073pnas96179833ichinohe t pang i k kumamoto y peaper d r ho j h murray ts microbiota regulates immune defense against respiratorytract uenza a virus infection proc natl acad sci usa 101073pnas1019378108inoue t nakayama j moriya k kawaratani h momoda r ito k gut dysbiosis associated with hepatitis c virus infection clin infectdis 101093cidciy205jiminez j a uwiera t c abbott d w uwiera r r and inglis gd impacts of resistant starch and wheat bran consumption onenteric ammation in relation to colonic bacterial community structuresand shortchain fatty acid concentrations in mice gut pathog 101186s1309901601496karst s m the uence of commensal bacteria on infection with entericviruses nat rev microbiol 101038nrmicro201525kauppila j h karttunen t j saarnio j nyberg p salo t gravesd e short dna sequences and bacterial dna induceesophageal gastric and colorectal cancer cell invasion apmis 101111apm12016kefalakes h and rehermann b ammation drives an alteredphenotype of mucosalassociated invariant t cells in chronic hepatitis d virusinfection j hepatol 101016jjhep201905024khanna s and tosh p k a clinicians primer on the role of themicrobiome in human health and disease mayo clin proc 101016jmayocp201310011kreuzer s machnowska p amus j sieber m pieper r schmidt m f feeding of the probiotic bacterium enterococcus faecium ncimb diï¬erentially aï¬ects shedding of enteric viruses in pigs vet res krieg a m therapeutic potential of tolllike receptor activation natrev drug discov 101038nrd2059lee h and ko g antiviral eï¬ect of vitamin a on norovirus infection viamodulation of the gut microbiome sci rep 101038srep25835lemon s m ott j j van damme p and shouval d typea viral hepatitis a summary and update on the molecular virologyepidemiology pathogenesis and preventionj hepatol 101016jjhep201708034li d yan p abousamra a b chung r and butt a proton pumpinhibitors are associated with accelerated development of cirrhosis hepaticdecompensation and hepatocellular carcinoma in noncirrhotic patients withchronic hepatitis c infection results from erchives aliment pharmacolther 101111apt14391li h gao z zhang j ye x xu a ye j sodium butyratestimulates expression of ï¬broblast growth factor in liver by inhibition ofhistone deacetylase diabetes 102337db110846lin l and zhang j role of intestinal microbiota and metabolitesand human diseases bmc immunolon gut homeostasis 101186s1286501601873liu q li f zhuang y xu j wang j mao x alterationin gut microbiota associated with hepatitis b and nonhepatitis virus relatedhepatocellular carcinoma gut pathog 101186s1309901802816lu h wu z xu w yang j chen y and li l intestinal microbiotawas assessed in cirrhotic patients with hepatitis b virus infection microb ecol 101007s0024801098018meiglani a alimirah m ramesh m and salgia r fecal microbiotatransplantation for clostriodioides diï¬cile infection in patients with chronicliver disease int j hepatol mencin a kluwe j and schwabe r f tolllike receptors as targets inchronic liver diseases gut 101136gut2008156307minemura m and shimizu y gut microbiota and liver diseases worldj gastroenterol 103748wjgv21i61691mohamadkhani aintestinal microbialcommunity in hepatocarcinogenesis in chronic hepatitis b cancer med 101002cam41550 on the potential role ofmoratalla a gmezhurtado i santacruz a moya ¡ peir g zapater p protective eï¬ect of biï¬dobacterium pseudocatenulatum cect against induced bacterial antigen translocation in experimental cirrhosisliver int 101111liv12380ohara a mand shanahan ftherapeutic potential clin gastroenterol hepatolfor 101016jcgh200612009 gut microbiota miningpark e j thomson a b and clandinin m t protection of intestinaloccludin tight junction protein by dietary gangliosides in lipopolysaccharideinduced acute ammation j pediatr gastroenterol nutr 101097mpg0b013e3181ae2ba0p©rezmatute p ±iguez m villanuevamill¡n m j reciofern¡ndez e andv¡zquez a m s¡nchez s c shortterm eï¬ects of directactingantiviral agents on ammation and gut microbiota in hepatitis cinfectedpatients eur j inter med 101016jejim201906005ponziani f r putignani l paroni sterbini f petito v picca a del chiericof uence of hepatitis c virus eradication with directactingantivirals on the gut microbiota in patients with cirrhosis aliment pharmacolther 101111apt15004preveden t scarpellini e mili´c n luzza f and abenavoli l gut microbiota changes and chronic hepatitis c virus infection expert revgastroenterol hepatol frontiers in cellular and infection microbiology wwwfrontiersinaugust volume 0csehgal microbiota and liver diseasesraimondi s amaretti a gozzoli c simone m righini l candelieref in the human gutits proï¬ling phenotyping and colonization front microbiol 103389fmicb201901575 longitudinalsurvey offungiren z li a jiang j zhou l yu z lu h gut microbiomeanalysis as a tooltowards targeted noninvasive bioma | Colon_Cancer |
"the kinetics and localization of the reactions of metabolism are coordinated by the enzymes that catalyze themthese enzymes are controlled via a myriad of mechanisms including inhibitionactivation by metabolitescompartmentalization thermodynamics and nutrient sensingbased transcriptional or posttranslational regulationall of which are influenced as a network by the activities of metabolic enzymes and have downstream potential toexert direct or indirect control over protein abundances considering many of these enzymes are active only whenone or more vitamin cofactors are present the availability of vitamin cofactors likely yields a systemsinfluence overtissue proteomes furthermore vitamins may influence protein abundances as nuclear receptor agonistsantioxidants substrates for posttranslational modifications molecular signal transducers and regulators ofelectrolyte homeostasis herein studies of vitamin intake are explored for their contribution to unraveling vitamininfluence over protein expression as a body of work these studies establish vitamin intake as a regulator of proteinabundance with the most powerful demonstrations reporting regulation of proteins directly related to the vitaminof interest however as a whole the field has not kept pace with advances in proteomic platforms and analyticalmethodologies and has not moved to validate mechanisms of regulation or potential for clinical applicationkeywords proteomics big data vitamin metabolism precision nutrition molecular nutritionintroductionregulatory mechanismscellular metabolism is a system of chemical reactions inwhich cells harness the energy stored in the chemicalbonds of substrate molecules to perform their biologicalfunctions maintain homeostasis or to synthesize buildingblocks for structural maintenance or cellular division thekinetics of these reactions are dependent on the activity ofthe proteins which catalyze them thus proteins are keymodulators of metabolismmetabolic activity also exerts network control over itselfby a diverse array of mechanisms which finely tune proteinexpression responses via nutrient sensing machineries products or intermediates of a metabolic pathway caninhibit or activate metabolic enzymes eg malate inhibitsthe succinate dehydrogenase complex and fructose correspondence nv83cornelledudivision of nutritional sciences cornell university ithaca ny usa26bisphosphate activates phosphofructokinase theoxidative status of a cell can drive the directionality ofredox reactions and impact abundances of redox reactioncatalyzing proteins eg the keap1nrf2 network responds to oxidative stress by upregulating expression ofantioxidantfunctioning proteins splicevariant or isozyme expression can impact relative pathway utilization atmetabolic network nodes eg splice variants and isozymesof pyruvate and lactate dehydrogenase respectively impactthe bridge between glycolysis and the tricarboxylic acidtca cycle [ ] additionally local metabolite concentrations and thermodynamics can dictate the directionalityof reactions catalyzed by compartmentspecific isozymeseg reductive activity of isocitrate dehydrogenase can beconfined to the cytosolspecific isozyme the impactsof the abovementioned regulations are closely monitoredby nutrient sensing proteins which initiate molecularevents altering protein activation and expression eg 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 0cjeong and vacanti nutrition metabolism page of serinethreonine kinase ampactivated protein kinasemammalian target of rapamycin and sterol regulatoryelementbinding protein are part of overlapping proteinnetworks that orchestrate proteinexpression and posttranslational modification responses to nutrient availability[ ] considering that many metabolic enzymes do notfunction in isolation and as detailed in the sections thatfollow require vitamin cofactors to stabilize intermediatesdonateaccept electrons shuttle substrates and hold reactants in close proximity vitamin status is a critical consideration when examining proteinmediated regulation ofmetabolism and the impacts of metabolism on proteinexpressionin addition to their potential regulatory roles ascofactors vitamins orchestrate other direct or indirectmechanisms influencing protein abundance retinoicacid vitamin a interacts with nuclear receptors impacting gene transcription ascorbic acid vitamin cimpacts oxidative status and associated protein networks and is reported to exhibit epigenetic regulation overprotein expression vitamin d regulates calcium signaling machinery activates nuclear receptors and exertshormonal regulation over protein expression [ ]and niacin vitamin b3 and biotin vitamin b7 can beincorporated as posttranslational modifications impacting protein function [ ]herein studies on systemic intake dietary injectionoral gavage of vitamins and their impacts on tissueproteomes are examined and their contributions tounraveling vitaminbased regulation of protein expression and tissue function are explored the currentwork is intended to provide informationto understand each vitamins figs and molecularfunctions and highlight its role as a cofactor or substrate in the reactions of central metabolism fig tables s1 s2 s3 s4 s5 s6 s7 s8 s9 s10 s11 s12and s13 finally this work is intended as a resourcefor identifying regulation of proteins related to vitaminmetabolism in published works the public domain ofproteomic data sets is ever expanding but is rarelysearched for effects related to vitamin metabolism tothat end all proteins are specified by their hugogene nomenclature committee hgnc gene symbolor the hgnc gene symbol of the human orthologwhen identified in another species and proteins requiring a vitamin as a cofactor or substrate are tabulated tables s1 s2 s3 s4 s5 s6 s7 s8 s9 s10s11 s12 and s13proteomics platformsproteomics platforms ofthe discussed studies areprovided to place them on a technological timelineplatforms are described with the terms orbitrap qtofquadrupole timeofflight tripletof triple time offlight qqq triplequadrupole 2dgems twodimensional gel electrophoresis mass spectrometry and2dge in brief orbitrap platforms are the workhorses ofmodern proteomics because their high achievable massresolutions combined with high sensitivity are bestsuited for maximizing the number of proteins identifiedin a complex sample [ ] though qtof and tripletof instruments capable of maintaining mass resolutionfig fat soluble vitamin structures 0cjeong and vacanti nutrition metabolism page of fig water soluble vitamin structuresat higher scan speeds hold a substantial influence inthis arena within the categories of orbitrap qtof andtripletof there are major technological advances notdiscussed here qqq platforms are best suited for quantifying a predetermined list of proteins lower scan speedsand mass resolution render them less capable thanorbitrap qtof or tripletof systems for nontargetedapplications advances in nanoflow liquid chromatography coupled directly to mass spectrometry haveimproved proteomic depth by orders of magnitude overthat achievable by 2dgems where the upstream selection of protein spots predates the modern definition ofnontargeted proteomics similarlyidentifying differentially intense protein spots using 2dge alone is considered an important milestone in the development ofproteomics but is rarely discussed outside the topic of thefields historyvitamin regulation of tissue proteomesvitamin avitamin a exists in alcohol aldehyde acid and esterforms known as retinol retinal retinoic acid and retinylesters respectively fig several carotenoids areprecursors to vitamin a including α and βcarotene βcarotene is converted to two molecules of retinalby beta carotene oxygenases bco1 or bco2 retinal is an important component of rhodopsin rho aprotein in rod cells responsible for detecting low levelsof light thus night blindness is telltale characteristic of vitamin a deficiency retinoic acid serves as asignaling molecule acting through nuclear retinoic acidrara rarb rarg and retinoid x rxra rxrbrxrg receptors which regulate growth and differentiation [ ] cellular and anismal trafficking ofvitamin a is dependent on retinolretinoic acid bindingproteins rbp family crabp1 crabp2 and retinolesterification via lecithin retinol acyltransferase lrat retinal is oxidized to retinol via aldehyde dehydrogenases aldh family and retinol is oxidized to retinoicacid by retinol dehydrogenases rdh and dhrs families in addition to inducing night blindness vitamin adeficiency adversely impacts cellular growth bone development and antibodybased immune responses in an orbitrapbased study of mouse embryo headstoxic levels of prenatal retinoic acid exposure intendedto model an established risk factor for craniofacial birthdefects are reported to induce abundance alterations inproteins associated with craniofacial development and 0cjeong and vacanti nutrition metabolism page of fig schematic of vitamin involvement in reactions of central carbon metabolism the depicted lipid bilayer represents the inner mitochondiralmembrane abbreviations defined in the abbreviations section vitamins specified by alphanumeric designations 0cjeong and vacanti nutrition metabolism page of neural crest processes in a parallel tripletofbased study of gerbil plasma and 2dgemsbased studyof gerbil liver and white adipose tissue a few dozen protein abundances linked to a handful of biological processesare reported to respond to dietary retinol βcarotene lutein or lycopene though process or pathway enrichmentanalyses are not reported as the authors discuss plasmawas not depleted of common highly abundant proteinsupstream of analysis by mass spectrometry which areknown to adversely impact data quality in anorbitrapbased study of plasma from nepalese childrendozens of proteins are associated with circulating carotenoid abundances potentiating development oflowcostantibodybased tests for carotenoid deficiencies apair of 2dgemsbased studies link tissue function toprotein abundance responses to vitamin a status in micebrains and bovine muscle vitamin b1thiamine vitamin b1 is composed of linked pyrimidineand thiazole rings decorated with methyl amine andalkylhydroxyl functional groups fig thiamineistransported through the plasma membrane viathiamine transporters slc19a2 and slc19a3 andthen twice phosphorylated on the alkylhydroxyl functional group by thiamine pyrophosphokinase tpk1rendering it active as thiamine diphosphate tdp tdp is a cofactor for enzymes catalyzing the oxidativedecarboxylation of ketoacids including the pyruvatedehydrogenase complex pdha pdhb pdhx dlatdld the oxoglutarate dehydrogenase complex ogdhdlst dld and the branched chain keto acid dehydrogenase complex bckdha dbt dld it is also acofactor for transketolase tkt in the nonoxidativebranch of the pentose phosphate pathway independent from its role as a cofactor thiamine is believed toregulate ion transport activity in the nervous system vitamin b1 deficiency is marked by a broad range ofneurological respiratory and cardiovascular pathophysiologies and is termed beriberi symptoms of beriberi aredifficult to directly link to the molecular functions ofvitamin b1 in a 2dgemsbased study of type diabetic andhealthy control subjects authors report treatment withthiamine reduces albumin alb abundance in urine indicating the vitamin serves a protective role of kidneyfunction in a qtofbased study of rat thalamiunder thiamine deficiency glyceraldehyde3phosphatedehydrogenase gapdh is the most upregulated protein fold while regulated proteins are most enrichedin the synaptic vesicle cycle pathway according to thekegg database proteomic changes are accompaniedby diminished performances on cognitive tests vitamin b2riboflavin vitamin b2 is composed of an isoalloxazinering and a bound ribitol fig it is activated byriboflavin kinase rfk forming flavin mononucleotidefmn and by flavin adenine dinucleotide synthase flad1 forming flavin adenine dinucleotide fad bound fmn or fad serves as an electron carrierfor redoxreactioncatalyzing proteins flavoproteinsincludingcomplexsdha sdhb sdhc sdhd the pyruvate dehydrogenase complex pdha pdhb pdhx dlat dldacylcoa dehydrogenases acads and methylenetetrahydrofolate reductase mthfr dehydrogenasethesuccinateriboflavin deficiency in humans predominantly occursin combination with that of other nutrients howeveranimal studies link it to impaired fetal and intestinaldevelopment [ ]iron absorption and lipidmetabolism [ ]in a qtofbased study of duck livers riboflavin deficiency is accompanied by a reduced abundance of smallchainspecific acylcoenzyme a dehydrogenases acadsfor which riboflavin serves as a cofactor and concordantelevation of hepatic small chain fattyacid lipid contentdramatic decreases in protein abundance are reported forinpp1 involved in inositol signaling thrsp purportedregulator of lipid metabolism bdh2 a regulator of lipidmetabolism fxn involved in mitochondrial ironsulfurcomplex assembly and ndufs1 a subunit of electrontransport chain complex i in a qtofbased study ofmaternal riboflavin deficiency reductions in fetal duck hepatic tca cycle betaoxidation and electron transport chainproteins are reported with idh3a being the lone memberof these pathways whose abundance increases vitamin b3niacin vitamin b3 is inclusive of nicotinic acid and nicotinamide fig which are converted to their mononucleotide forms by nicotinate phosphoribosyltransferase naprt and nicotinamide phosphoribosyltransferase namptrespectively both forms of the mononucleotide aresubsequently converted to their adenosine dinucleotideforms by nicotinamidenicotinic acid mononucleotidenmnat1 nmnat2 nmnat3adenylyltransferasesnicotinamide adenine dinucleotide nad is a cofactorform of the vitamin whereas nicotinic acid dinucleotide issubsequently converted to nad by nad synthase nadsyn1 nad is reduced to nadh by oxidative reactions of glycolysis the tca cycle and βoxidation andsubsequently serves as a redox equivalent carrier to theelectron transport chain and to regenerate reducedascorbic acid vitamin c glutathione and thioredoxin nad can also be phosphorylated by nadkinases nadk nadk2 to form a distinct redox shuttlingcofactor nadp nadp is reduced by reactions in the 0cjeong and vacanti nutrition metabolism page of oxidative pentose phosphate pathway g6pd pgd andother enzymes eg me1 me3 idh1 idh2 to nadphnadph provides reducing equivalents for biosynthetic reactions in fatty acid cholesterol and deoxyribonucleotidesynthesis outside its role as a reducing equivalentshuttle nad provides adenine dinucleotide phosphateadp ribose for synthesis of the second messenger cyclicadenosine monophosphate camp via the activity of adenylate cyclases adcy family nad also providesadpribose and polyadpribose for post translationalmodifications of proteins via activity of adpribosyl transferases art family and adp ribose polymerases parpfamily [ ] camp and protein polyadpribosylationare important mediators of cell signaling and proteinexpression niacin is synthesized from tryptophan butin small quantities relative to a healthydietary intake deficiency known as pellagraismarked by dermatitis and severe gastrointestinalneurological pathophysiologies which are fatalif untreated no proteomic studies on systemic intakeof vitamin b3 were found at the time of writing thisreviewvitamin b5pantothenic acid vitamin b5 is composed of a moleculeof pantoic acid bound to βalanine fig itsprimary metabolic function is as an acylcarrier pantothenic acid is a substrate in the first reaction of coenzyme a coa biosynthesis catalyzed by pantothenatekinases pank1 pank2 pank3 pank4 coa isa substrate for enzymes catalyzing the oxidative decarboxylation of ketoacids including the pyruvate dehydrogenase complex pdha pdhb pdhx dlat dldthe oxoglutarate dehydrogenase complex ogdh dlstdld and the branched chain keto acid dehydrogenasecomplex bckdha dbt dld [] acyl speciesare activated by conjugation with coa and are substratesin or products of glycolysis the tca cycle fattyacidsynthesisβoxidation cholesterol synthesis ketogenesisbranchedchain amino acid catabolism and proteinacetylationoglcnacylation finally phosphopanthetheine product of pank proteins activities is acofactor of the acyl carrier protein domain of fatty acidsynthase fasn vitamin b5 deficiency is rare andusually accompanied by that of other nutrients burning of the feet and numbness in the toes is a characteristic manifestation along with variety of other symptoms no proteomic studies on systemic intake ofvitamin b5 were found at the time of writing this reviewvitamin b6vitamin b6 has aldehyde alcohol and amine forms fig of which the phosphorylated aldehyde form pyridoxalphosphate acts as a cofactor to over enzymes allthree forms of vitamin b6 are phosphorylated by pyridoxalkinase pdxk both the phosphorylated alcohol andamine forms pyridoxine phosphate and pyridoxaminephosphate are converted to pyridoxal phosphate bypyridoxine phosphate oxidase pnpo pyridoxalphosphate is a cofactor for enzymes catalyzing decarboxylase reactions in gammaaminobutyric acid gad1 gad2 and serotonindopamine biosynthesis ddc aswell as for enzymes catalyzing transamination reactionseg got1 got2 gpt gpt2 cysteine synthesiscth heme synthesis alas1 alas2 carnitinesynthesis3hydroxy6ntrimethyllysine aldolase geneunidentified andsphingolipid synthesis sptlc1 sptlc2 pyridoxalphosphate is also an important cofactor for enzymes ofonecarbon metabolism shmt1 and shmt2 andglycogen catabolism pygl and pygm vitamin b6deficiency is rare because of its availability in many foodsand pathophysiologies can be diverse niacin synthesis kynuin a tripletofbased study of streptozotocininduceddiabetic rat hippocampi pyridoxamine treatment prevented longterm recognition memory impairment andregulated protein abundances in a number of diversepathways notably upregulating half of the proteins involved in ubiquinol biosynthesis in a 2dgemsbased study of mice hippocampi the abundances ofphosphoglycerate mutase pgam1 and cannabinoidreceptorinteracting protein cnrip1 are reported tobe elevatedreduced respectively upon administration ofpyridoxine proteomic changes are accompanied by improved novel object recognition the plasma membrane byvitamin b7biotin vitamin b7 is composed of a fusedring structurebound to a valeric acid side chain fig it istransported acrossthesodiumdependent solute carriers slc5a6 and slc19a3[ ] as a cofactorposttranslational modificationbiotin covalently binds lysine residues it is a cofactor for pyruvate carboxylase pc acetylcoa carboxylase acaca propionylcoa carboxylase pcca andthe methylcrotonylcoa carboxylase complex mccc1mccc2 histones are also biotinylated regulatinggene expression the posttranslational modification occurs via the activity of holocarboxylase synthetasehlcs biotin deficiency is rare and has wide ranging pathophysiologies eating raw egg whites can preventitsabsorption leading to deficiency because of its affinityfor avidin a chemical in egg whites that is denaturedupon cooking this observation led to the vitaminseventual discovery no proteomic studies onsystemic intake of vitamin b7 were found at the time ofwriting this review 0cjeong and vacanti nutrition metabolism page of vitamin b9the term folate vitamin b9 is inclusive of a group ofcompounds composed of a pteridine ring linked to paraaminobenzoic acid with a mono or polyglutamate tailfig in its reduced form tetrahydrofolate aonecarbon unit crosslinks as ch or ch2 aminegroups on the ring structure and aminobenzoic acid orbinds the secondary amine as a formyl group on theaminobenzoic acid group [ ] this onecarbonunit is utilized in the synthesis of purines and thymidineconversion of homocysteine to methionine interconversion of serine and glycine and catabolism of histidinereactions collectively termed onecarbon metabolism[ ] at the cellular level onecarbon metabolismis tightly regulated by compartmentalization [ ] while wholebody folate homeostasis is predominantly maintained by the liver through the enterohepaticcycle folate deficiency induces megaloblastic macrocyticanemia and fetal neural tube defects purportedly via itsadverse impact on nucleotide synthesis [ ] lowintake of folate is also linked to cardiovascular disease[ ] neurodegenerative disease [ ] alzheimers disease [ ] and cancer [] in an orbitrapbased study of follicle fluid of womenundergoing in vitro fertilization the folate supplementedgroup is reported to have elevated abundances of apolipoproteins from high density lipoproteins and reducedreactive protein c crp the study is performed onwomen who did not become pregnantin aqtofbased study of a folatedeficiencyinduced intestinal neoplasia mouse model the combinatorial impactsof folate deficiency and methylene tetrahydrofolate reductase heterozygous deletion mthfr are reported toimpact protein abundances spanning diverse cellularfunctions however of samples are discarded as outliers and the simultaneous examination of mthfr anddietary folate deficiency does not allow proteomic adaptations to be attributed to either in isolation in a2dgemsbased study of adult rats aortic calmodulincalm1 calcium signaling protein abundances arepositively correlated with folate dose while abundancesof triose phosphate isomerase tpi1 glycolysis transgelin tagln cytoskeleton and glutathione stransferasealpha gsta3 reductive detoxification respond inversely in an 2dgemsbased study of rat liversprdx6 and gpx1 are reported to be elevated whilecofilin cfl1 is reported to be depleted under folatedeficiency other studies report protein abundancedifferences due to folate intake in rat urinary exosomesqqqbased human plasma 2dgems fetal brain tissue from pregnant mice fed ethanol2dgems pregnant rat livers 2dgems fetal rat livers 2dgems adult rat livers andbrains 2dgems and livers of piglets born tofolate deficient mothers 2dgems vitamin b12cobalamin vitamin b12 encompasses a group of molecules with four linked pyrrole ring derivatives forming a corrin ring and a cobalt atom bound at thecenter of the corrin ring the cobalt atom also binds a56dimethylbenzimidazole nucleotide and a functionalgroup fig the identity of the functionalgroup distinguishes the vitamin b12 compounds ascyanocobalamin hydroxycobalamin hydrocobalaminnitrocobalamin deoxyadenosylcobalamin also called adenosylcobalamin and methyl cobalamin [ ] methylcobalamin serves as a coenzyme in the conversion ofhomocysteine to methionine by methionine synthase mtrin the cytosol and adenosylcobalamin is required forconversion of lmethylmalonylcoa to succinylcoa bymethylmalonylcoa mutase mut in mitochondria vitamin b12 deficiency is closely related to folatedeficiency and can lead to megaloblastic anemia by impairment in the activity of methionine synthase mtr 5methyl tetrahydrofolate cannot be converted toonecarbon donors required for purine and thymidinesynthesis without vitamin b12 as a cofactor thus interfering with dna synthesis and erythrocyte production vitamin b12 deficiency is also linked to neurological disorders independent of anemia ruoppolo and colleagues performed a 2dgemsbased study of lymphocytes isolated from methylmalonicacidemia with homocystinuria cobalamin deficiency typec mmachc patients an inborn error in metabolismmarked by inactivity of the mmachc gene productreceiving a standard treatment of hydroxycobalaminbetaine folate and carnitine protein products of me2glud1 and gpd2 genes involved in anaplerosis andredox equivalent shuttling are upregulated while variant of protein pyruvate kinase muscle isozyme pkmand lactate dehydrogenase b ldhb are downregulatedrelative to lymphocytes isolated from healthy control donors in a 2dgebased study of adult rat cerebralspinal fluid protein abundance shifts are reported topeak after several months on a cobalamin deficient dietmodest shifts or after a total gastrectomy more severeshifts and return to near control values at later timepoints in a 2dgemsbased study glutathione stransferase p gstp1 abundances are diminished andglutathione peroxidase gpx1 abundances are elevated in rat pup kidneys under maternal vitamin b12deficient and maternal folate deficient conditions suggesting maternal dietary intake of these vitamins impacts offspring kidney redox homeostasis mechanisms 0cjeong and vacanti nutrition metabolism page of in a similar 2dgemsbased study of maternal vitaminb12 deficiencythe same group reports that severaldozen rat kidney pup proteins revert to control levelsupon administration of vitamin b12 at birth additionally diminished abundance of betaoxidation proteinsin kidneys of pups born to vitamin b12 deficientmothers is accompanied by elevated ppara apositive regulator offatty acid oxidation suggestingattempted compensation at the cellular levelvitamin cvitamin c ascorbic acid is absorbed at the brushborder and distributed to cells throughout the body bythe sodiumdependent plasma membrane solute carriersslc23a1 and slc23a2 the oxidized form of vitamin c dehydroascorbate is also transported via plasmamembrane glucose transporters slc2a1 slc2a3 andslc2a4 also known as glut1 glut3 and glut4 and reduced intracellularly to ascorbic acid byglutathione and the activity of thioredoxin reductases txnrd1 txnrd2 or txnrd3 vitamin c is a cofactor in the function of prolyl andlysyl hydroxylases which consume oxygen and alphaketoglutarate to form the hydroxylated amino acid residueand succinate the fe2 of these enzymes is restoredfrom fe3 by oxidation of vitamin c in the presenceof oxygen prolyl hydroxylases egln1 egln2 egln3also known as phd2 phd1 phd3 respectively hydroxylate the hif1a protein providing a necessary signal for itsdegradation and preventing a hypoxic response at the cellular level prolyl and lysyl hydroxylase activities arealso necessary for posttranslational modifications to formfunctional collagen lysyl hydroxylases includeplod1 plod2 and plod3 vitamin c serves anearly identical function in reducing fe3 as a cofactor fortrimethyllysine dioxygenase tmlh which catalyzes thefirst reaction in carnitine biosynthesis carnitine isessential for fatty acid catabolism in the mitochondria asonly fatty acyl carnitines formed via the activity of carnitine palmitoyl transferases cpt1a cpt1b and cpt1ccross the inner mitochondrial membrane through thesolute carrier slc25a20 vitamin c similarly servesas a cofactor for tyrosine hydroxylase th which catalyzes the first reaction in catecholamine eg dopamineepinephrine and norepinephrine synthesis additionally vitamin c serves and as a general antioxidant vitamin c deficiency leads to the condition knownas scurvy with symptoms largely attributed to malformedconnective tissue due to improperly folded collagen in a orbitrapbased study on a pig model of hemorrhagicshock vitamin c administration is reported to impactplasma protein abundances in the complement pathwayand those in polytrauma related processes including thestabilization of adamts13 abundance an importantregulator of clot formation an orbitrapbased studyof endoplasmic reticulum enriched fractions of livers inwerner syndrome mouse models identifies around adozen proteins whose abundances are impacted by administration of vitamin c a qtofbased study ofzebrafish reports upregulation of glutamate dehydrogenase glud1 and downregulation of pyruvate kinasemuscle isozyme pkm upon administration of vitamin cin a vitamin e deficient in a qqqbased study of human plasma ascorbic acid concentrationis reported to be inversely related to vitamin d bindingprotein gc abundance 2dgemsbased studiesidentify protein abundance regulations in mouse modelsof sarcoma metastases in the liver and tumor nodules of adenocarcinoma due to administration of vitaminc another 2dgemsbased study reports polypeptide abundance shifts in hemodialysis patient plasma uponvitamin c supplementation vitamin dvitamins d2 and d3 are respectively distinguished by theirergosterol and cholesterol backbones though onlyvitamin d3 is synthesized in animals both can be converted to active forms exposure of 7dehydrocholesterolan intermediate in cholesterol synthesis to ultravioletradiation in the skin and subsequent isomerization produces cholecalciferol vitamin d3 fig whether dehydrocholesterol is derived from cholesterol via activityof 7dehydrocholesterol reductase dhcr7 or synthesizedde novo in the skin is disputed 7dehydrocholesterolis successively hydroxylated by activity of cytochrome p450enzymes eg cyp2r1 and cyp27b1 in the liver and kidney to its active 125oh2 cholecalciferol [125oh2d3]form transport of vitamin d and its metabolitesoccurs bound to vitamin d binding protein gc ergocalciferol is the vitamin d2 equivalent of cholecalciferol and is activated analogously 125oh2d3 influences cellular function via nuclearreceptordependent and nuclear receptorindependentmechanisms the former involves 125oh2d3boundvitamin d receptor vdr forming a heterodimer complex with a retinoid x receptor rxra rxrb rxrgand subsequently binding vitamin d response elementsregulating transcription of genes largely involved modulating calcium and phosphorous transport andmaintaining homeostasis by regulating their absorptionin the kidneysintestines and bones [ ] therapidonsetreceptorindependent of 125oh2d3 are mediated by a membraneassociated rapid response steroid binding protein identifiedextracellularimpactsnuclear 0cjeong and vacanti nutrition metabolism page of as pdia3 and diversely impact cell growth survivaland immune response deficiency in vitamin d impairs bone mineralizationcausing rickets in infantschildren and osteomalacia inadults vitamin d deficiency is also linked tocardiovascular diseases [ ] cancer [ ]neurologicalimpairments [ ] and autoimmunediseases [ ] though underlying mechanisms arenot completely understoodin an orbitrapbased study of mouse fetal and postnatal lung tissue maternal vitamin d deficiency is reflectedin total proteome adaptations which are unexpectedlystrongest at postnatal day opposed to fetal time pointsimpacted proteins include several associated with lungdevelopment an orbitrapbased study of a mousebrain tissue model of remyelination in multiple sclerosisreports calcium binding protein abundances to be upregulated upon treatment with 125oh2d3 consistentwith the vitamin's regulatory role over calcium absorption in an orbitrapbased study of serum fromoverweight adults vitamin d deficiency is reported todifferentially affect abundances of proteins related toblood coagulation in males and females howeverabundances of these proteins are likely impacted by theproduction of serum from whole blood the authors report quantifying proteins table an impressiveanalytical depth for serum in a 2dgebased studyvitamin d deficient children are reported to have diminished serum abundances of adiponectin adipoq in a separate 2dgebased study the same groupreports fetuinb fetub to be elevated in the plasma ofobese vitamin d deficient children compared with theirvitamin d sufficient counterparts however theauthors do not directly identify fetub and rely on comparison of their findings to those of another study two 2dgemsbased studies of rat left ventricular andaortic tissueidentify proteins whose abundances respo | Colon_Cancer |
" microwave ablation mwa is widely used to treat unresectable primary and secondary malignanciesof the liver and a limited number of studies indicate that ablation can cause not only necrosis at the in situ site butalso an immunoreaction of the whole body this study aimed to investigate the effects of mwa on cytokines inpatients who underwent mwa for a hepatic malignancymethods patients admitted to the oncology department in the first affiliated hospital of soochow universitybetween june and february were selected peripheral blood was collected from patients with a hepaticmalignancy treated with mwa the levels of cytokines il2 ifnÎ tnfα il12 p40 il12 p70 il4 il6 il8 il10and vascular endothelial growth factor vegf were detected with a milliplex® map kit the comparison times wereas follows before ablation h after ablation days after ablation and days after ablation data were analyzedusing a paired sample ttests and spearmans correlation analysisresults a total of patients with hepatic malignancies were assessed there were significant differences in il2il12 p40 il12 p70 il1 il8 and tnfα at h after mwa significant increases 2fold vs before ablation wereobserved in il2 il1 il6 il8 il10 and tnfα after mwa elevated il2 and il6 levels after ablation werepositively correlated with energy output during the mwa procedures wa treatment for hepatic malignancies can alter the serum levels of several cytokines such as il2 and il6keywords microwave ablation hepatic malignancy cytokines il2 il6 immunoregulation primary and secondary malignancies of the liver have asubstantial impact on morbidity and mortality worldwidein china hepatocellular carcinoma hcc has the secondhighest mortality rate of malignancies the treatmentof primary and secondary hepatic malignancies via correspondence lengbengsudaeducn jing zhao qiang li and merlin muktiali contributed equally to this work2department of oncology the first affiliated hospital of soochow universitysuzhou china5division of neurosurgery city of hope beckman research institute duartecalifornia usafull list of author information is available at the end of the interventional imaging therapy is undertaken by investigators in the field of interventional radiology and possibly bya smaller group of practitioners known as interventionaloncologists whose major focus is cancer care via minimally invasive approaches [ ] recently percutaneous ablation therapy has been widely accepted as a radicaltreatment method for hcc and its fiveyear survival rateis similar to that of resection microwave ablationmwa is widely used to treat unresectable hcc and recurrent hcc and has the advantages of minimal invasiona good curative effect and no side effects due to radiationor chemotherapy immune checkpoint inhibitors icis the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0czhao bmc cancer page of such as pd1pdl1 and ctla4 antibodies have beenwidely applied in several cancers and studies have indicated that ici treatment could enhance the effect of ablation evidence hasindicated that hyperthermicdestruction causes the release of a large population of heterogeneous tumor antigens and inflammatory cytokinesmay play crucial roles in this process cytokines aremediators that regulate a broad range of processes involved in the pathogenesis of cancer several cytokineswhich can arise from either tumor cells or immunocytes such as tumor necrosis factor tnfα interleukinil1 il6 il8 il10 and vascular endothelial growthfactor vegf have been linked with cancers and can either promote or inhibit tumor development the serumlevels of cytokines differ during cancer development although cytokines have been found to be altered after anticancer treatment such as chemotherapy and radiotherapy[ ] few investigations have focused on cytokines beforeand after mwa it is still unknown whether the above cytokines changed before andor after mwa in patientswith hepatic malignancies in this study we investigatedthe effects of mwa on the serum levels of cytokines inpatients with hepatic malignanciesmethodspatients and samplesthe patient population examined in this study was derivedfrom the first affiliated hospital of soochow universitypatients were admitted to the oncology department between june and february the total number ofpatients was with liver metastases and primaryliver cancers the inclusion criterion was a tumor locatedat a hepatic site either primary or metastases all patients with metastatic hepatic malignances should be givensystematic treatments chemotherapy or target therapyand get at least stable disease sd or partial responsepr for more than days informed consent for blooddraw and the relevant therapy was obtained from all patients the protocol was approved by the human ethicscommittee of the first affiliated hospital of soochowuniversity and was conducted in accordance with thedeclaration of helsinki all written informed consent wasobtained from all participants and clearly stated wholeblood ml was drawn into edta anticoagulant tubeson days to before and h days and days afterablation mostly on the last day of the course for cytometry and cytokine analysesablation procedurethe ablation procedure used in this research was mwathe puncture site and pathway were determined underthe guidance of a computed tomography ct scanlocal infiltration anesthesia was achieved by using lidocaine the placement of microwave ablation probeswas guided by a ct scan or ultrasonic device and allprobes were placed at the maximum diameter layerdouble probes were employed when the maximumdiameter of the tumor was up to cm the power andtime of ablation were designed for each patient in therange of w and min respectively basedon the size number and position of the tumor theboundaries of ablation zones were designed as extended cm upon the tumor sitecytokine detectiona milliplex map kit with human cytokinechemokinepanels that measured ifnÎ il2 il6 il8 il10 il12p40 il12 p70 il1 tnfα and vegf was utilized according to the manufacturers instructions briefly chemically dyed antibodybound beads were mixed withstandard or sample incubated overnight at °c washedand then incubated with a biotinylated detection antibodyafter the beads were washed they were incubated with astreptavidin phycoerythrin complex and the mean fluorescent intensities were quantified on a luminex analyzer luminex corporation all samples were measured in duplicate standard curves of known concentrations of recombinant human cytokineschemokines wereused to convert fluorescence units to cytokine concentration units pgml the minimum detectable concentrations were as follows ifnÎ pgml il2 pgmlil12 p40 pgml il12 p70 pgml il1 pgml il6 pgml il8 pgml il10 pgml tnfα pgml and vegf pgml all resultsbelow the minimum concentrations were processed as theminimum concentrationsstatistical analysisibm spss statistics software was used for the statistical analysis along with graphpad prism for figurecreations normally distributed numerical data areexpressed as the mean ± standard deviation and nonnormally distributed numerical data are expressed as themedian and confidence interval ci cytokinesat different times were compared using a onetailedpaired ttest spearmans correlation analysis was executed to determine the correlation between clinical indexes and cytokine levels p indicates a significantdifferenceresultsclinical characteristics of the enrolled patientsas shown in table a total of patients with tumorslocated on the liver liver metastases primary livercancers were analyzed the patients cytokine levelswere compared according to time before treatment h after treatment days after treatment and daysafter treatment 0czhao bmc cancer page of table clinical characteristics of the patients enrolled n characteristicsexmalefemaleagepathogenesisprimarysecondaryprimary site for metastatic hepatic malignancescolon rectalpancreasstomachebreastothersmaximum tumor length mmablation probe usedablation time minaverage power per probe w ± ± ± ± average energy time à power time à power¼¼ time and power indicate the time and power respectively ofdifferent probes used during the operation ± ifnÎ il12 p40 and il12 p70 were slightly increasedafter mwa treatmentas shown in table and fig the median level ofifnÎ before the mwa treatment was pgml ci pgml at days and days after themwa treatment there was a slight increase comparedto that premwa with median levels of pgml ci pgml and pgml ci pgml respectively the median level of il p40 before the mwa treatment was pgml ci pgml there was a slight increase to pgml ci pgml days postmwathe median il12 p70 level before the mwa treatmentwas pgml ci pgml and increasedto pgml ci pgml days afterthe mwa treatment and to pgml ci pgml days postmwa no significant alteration in the vegf median level was detected after themwa treatmentil2 il1 il6 il8 and il10 were elevated over 2foldafter the mwa treatmentas shown in table fig and fig the median levelof il2 before the mwa treatment was pgml ci pgml there was a significant increase at h postmwa with a median level of pgml ci pgml the median level ofil1 before the mwa treatment was pgml ci pgml and a significantincrease wasnoted days after the mwa treatment pgml ci pgml the median level of il6before the mwa treatment was pgml ci pgml and significantly increased daysafter the mwa treatment pgml ci pgml the median level ofil8 before themwa treatment was pgml ci pgml and increased significantly to pgml ci pgml days after the mwa treatmentthe median level of il10 before the mwa treatmentwas pgml ci pgml and increasedsignificantly days after the mwa treatment pgml ci pgml the median level oftnfα before the mwa treatment was pgml ci pgml and increased significantlyto pgml ci pgml days afterthe mwa treatmentlevelselevated il2 and il6 levels after ablation were positivelycorrelated with energy output during mwato further evaluate the relationship between the increased cytokineand mwa treatment weemployed the concept of energy time à power time à power time and power indicated thetime and power of different probes used in the operation to reflect total hyperthermic damage to hepatictissues during the mwa procedure as shown in table and fig the il2 levels at h postmwa and the il levels at days postmwa illustrated significant correlations with energy the relative indexes were and respectivelydiscussionas technology continues to develop other types of localtherapy such as radiotherapy chemical ablation andhyperthermal ablation for primary and metastatic livercancer are increasingly being used mwa for liver malignances is reserved for patients who cannot undergosurgical removal or for whom other treatments havefailed a consensus guideline was recently developed to address indications for mwa in these patientsthermal ablation is a process that heats the target tissueto a temperature that causes immediate coagulative necrosis usually over °c terminal treatment requiresthat a necrotic area surrounds the target site with anadditional 10mm margins however in the liverhigh tissue perfusion and large blood vessels can cause aheat sink effect around the ablation zone making itdifficult to achieve terminal ablation the heat sink 0czhao bmc cancer page of table median levels of cytokines before and after mwacytokineifnÎil2premwa pgml ci ci ci ci ci ci ci ci ci ci h postmwa pgml ci ci ¼ ci ci ci ci ci ci ci ci il12 p40il12 p70il1il6il8il10tnfαvegf p vs premwa ¼ 2fold vs premwa days postmwa pgml ci ci ci ci ci ¼ ci ¼ ci ¼ ci ¼ ci ¼ ci days postmwa pgml ci ci ci ci ci ci ci ci ci ci effect can lead to sublethal temperatures and the retention of malignant cells thereby increasing the likelihoodof local tumor progression ltp however an incompletely ablated zone containing immune cells andcancer cells as well as functional vessels could establisha serious inflammatory site that may provide tumorspecific antigens cytokines and activated immune cellsin our study significant increases in the secretion ofchemokines il8 proinflammatory cytokines il1il12 ifnÎ and tnfα and antiinflammatory cytokines il10 were observed after mwa il8 is mainlyproduced by macrophages the classical biological activity of il8 is to attract and activate neutrophils whichcan lead to a local inflammatory response however recent studies have indicated that il8 both macrophageand cancer cellderived can recruit myeloidderivedsuppressor cells mdscs into the tumor microenvironment eventually inhibiting antitumor immunity andpromoting cancer progression [ ] il1 is mainlyproduced by macrophages b cells and nk cells couldproduce il1 under certain circumstances generallycells can only synthesize and secrete il1 after beingstimulated by foreign antigens or mitogens il1 couldpromote the th1 response promoting the activation ofdendritic cells dcs and cytotoxic t lymphocytesctls il12 is mainly produced by b cells and macrophages human il12 is a heterodimer with two subunits p40 kd and p35 kd which areinactivated in isolated form in general il12 functionsas a combination of two subunits il12 p70 while p40alone possesses partial functions of il12 p70 its mentionable that il12 p40 and p35 are not expressed inequal proportions so the amounts of il12 p40 and il p70 are different in one cell il12 can stimulate theproliferation of activated t cells and promote the differentiation of th0 cells into th1 cells moreover il12could induce the cytotoxic activity of ctls and nk cellsand promote the secretion of several cytokines such asifnÎ and tnfα previous research indicatedthat tnfα may play a crucial role in mwa in combination with immunotherapy notably our data illustrated that the il12 results were consistent with thoseof ifnÎ after the ablation operation but not with thoseof tnfα this result indicated that upregulation ofifnÎ may be a major effect of the il12 increase aftermwa on the other handan antiinflammatory and immunosuppressive cytokine wasevaluated after mwa il10 is a multicellularderivedmultifunctional cytokine that regulates cell growth anddifferentiation and could participate in inflammatoryand immune responses il10 was reported to increaseafter thermal ablation in the literature [ ] strategiesto inhibit il10induced immunosuppression after thermal ablation treatment would be of interestil10asablation therapy can mediate antitumor immunity astumor tissue necrosis caused by ablation may release various antigens that eventually form a kind of in situ vaccination moreover ablative therapy can not onlydirectly kill cancer cells in situ but also regulate immunecells and promote the immune function of patients withliver cancer [ ] many immunoregulatory cytokineswere released or expressed after thermal ablation notablythe cytokines released after thermal ablation can regulatethe positive and negative aspects of the cancer immunecycle previously researchers demonstrated that proinflammatory cytokines such as il1 il6 il8 il18 andtnfα were increased several hours or days after thermalablation [ ] to our knowledge terminal tumorthermal ablation may not only cause local heat injury intissues surrounding the tumor site but also induce a systemic reaction this systemic reaction would becaused by different mechanisms first interventional operation may result in trauma to the liver although this procedure is very minimally invasive the healing process maycause alteration of some cytokines second heat injurycould cause acute thermal necrosis in liver and tumor 0czhao bmc cancer page of fig levels of cytokines before and after mwa treatment slightly increased ifnÎ il12 p40 and il12 p70 levels after mwa treatment over fold enhancement of il2 h postmwa and of il1 il6 il8 il10 and tnfα d postmwa p 0czhao bmc cancer page of fig trends in cytokines significantly altered after mwa treatment the levels of il2 at h postmwa il1 at d postmwa il6 at dpostmwa il8 at d postmwa and il10 at d postmwa were elevated over 2fold compared to the levels premwatable correlation between the ablation energy and significantly elevated cytokinesenergyvsil2 h postmwaenergyvsil1 d postmwaenergyvsil6 d postmwaenergyvsil8 d postmwaenergyvsil10 d postmwaenergyvstnfα d postmwaspearmans rp value onetailed p 0czhao bmc cancer page of fig correlation between the ablation energy and the serum levels of il2 and il6 the serum levels of il2 at h postmwa and il6 at dpostmwa were positively correlated with energy output during the mwa procedureand nonspecifictissues and release of necrotic tissue fragments into bloodcould cause immunological reactions including nonspecific and specific reactions generally cytokines affectedby wound healingimmunologicalreactions do not last longer than those affected by specificimmunologicalreactions ablation treatmentinducedspecific immunological reactions are more complicatedand could affect more immunocytes [ ] which wouldmake this process last longer than other reactions theseexplanations may be the reason why the cytokine changeslasted different durations moreover cytokines affected bythe second manner would be positively correlated withthe ablation scale which is why we employed the energyindex in our ablation operation design to receive a terminal ablation larger tumor would cost higher energy including higher power and longer duration time terminaltumorthermal ablation would release tumorrelatedneoantigen to blood circulation eventually induce a systemic reaction this reaction is dependent on the scale ofthermal injury and the local immunological microenvironment of the tumor our findings indicated that il2 andil6 were significantly altered after the ablation procedureand positively correlated with mwa energy il2 is commonly derived from activated t cells primarily th1 cellsil2 can stimulate t cells to proliferate and differentiateactivate natural killer nk cells and macrophages and enhance the functions of cytotoxic t lymphocytes ctls our data illustrated that il2 is significantly increased at h after mwa indicating that il2 may induce a nonspecific immune response after mwa but il decreased after h postmwa in our study suggesting that the il2induced immune response may not belong lasting mentionable many cytokines detected il8il1 il12 were mainly derived from macrophagewhich was a widely distributed antigen presenting cellthis result support the theory that mwa could releasefragment of cancer cells into blood as neoantigen macrophages could response to this proceed and cause a systemic immunoreaction additional cytokines alterationsuch as il6 after ablation may be no anspecific inliver evidences indicate that increase of il6 was not onlyoccurred in liver ablation researches focus on lung cancerincluding primary lung cancer and pulmonary metastasesdemonstrated that serum il8 il1 il6 il10 il12and tnfα were significantly raised after radiofrequencythermal ablation moreover joseph found that imageguided thermal ablation of tumors located in lung liver orsoft tissues increases plasma levels of il6 and il10 another question remain unveiled was if our result wascancerspecific we checked literature about cytokinemodulation after thermal ablation in benign diseases andonly got limit evidences based on benign thyroid nodules and adenomyosis according to these literatureil6 levels did not show any significant difference aftertreatment compared with pretreatment values indicatingthat elevation of il6 may be caused by tumour antigenreleased by ablation treatment however the ablationenergy used in thyroid nodules was much lower thanliver and lung which would lead to a false negativein cytokine detection to the research about adenomyosis on the other hand experiment design was determined to followup the il6 at months afterhifu ablation as our data demonstrated mostly cytokines were return to premwa level after monthdetection after months may miss the modulation ofil6 overall few evidences support that some of thecytokines were altered in a cancerspecific mannerwhile no solid results could confirm that further animal experiments were required to make a clarifieddata and answer this question 0czhao bmc cancer page of thetumorassociated immunein recent years ablationinduced systemic effects suchasresponse haveattracted increased attention de baere t first reported two cases of spontaneous regression of multiplepulmonary metastases occurring after radiofrequencyablation of a single lung metastasis although growing evidence suggests that thermal ablation can inducespontaneous regression of the socalled abscopal effecton distant tumors the incidence rate of such an effect israre probably due to uncontested immunological activation caused by one ablation treatment and the lack ofimmuneamplification management in it was described that in situ tumor destruction can provide a useful antigen source forthe induction of antitumorimmunity however clinical studies could not sufficiently utilize such an effect until the development ofimmune checkpoint inhibitors icis [ ] icis suchas pd1pdl1 and ctla4 antibodies are widely applied in several cancers and studies have indicated thatici treatment could enhance the effect of ablation evidence indicates that hyperthermic destruction causesthe release of a large population of heterogeneous tumorantigens and inflammatory cytokines may play crucialroles in this process however opposite evidence indicated that incomplete radiofrequency ablation couldinduce inflammation which may accelerates tumor progression and hinders pd1 immunotherapy suggesting that ablation treatment may promote tumorprogression our data demonstrated that il6 was significantly increased after mwa treatment il6 is derived from monocytes macrophages dcs th2 cells andsometimes cancer cells and it plays a key role in t cellproliferation and survival the role of il6 appearsto be rather complex korn classified il6 as differentiation factor which could involve in differentiation ofth17 cells however il6 does not direct the commitment to the th1 or th2 cell lineage but controls theproliferation and survival of immunocytes cooperatingwith other cytokines such as tgf tnf or il21 for instance il6 activated stat3 pathway in naivecd4 t cells in the presence of the morphogen tgfbpromotes the population expansion of th17 cells recent evidence indicates that il6 plays an indispensable role in t cellinfiltration to the tumor sitewhich could benefit immunomodulatory therapy incontrast il6 can increase mdscs inhibit the development and maturation of dendritic cells dcs and inhibit the polarization of th1 cells eventuallyresulting in negative immunomodulatory effects according to muneeb ahmeds work the adjuvant uses ofa nanop smallinterfering rna sirna can besuccessfully used to target the il6mediated locoregional and systemic effects of thermal ablation il6 knockout via a nanop antiil6 sirna in mice coulddecrease the local vegf level at the ablation site therefore how to utilize the positive effect of il6 whileavoiding the negative effect after mwa needs further investigation preclinical research indicated that il6 andpdl1 blockade combination therapy reduced tumorprogression in animal models [ ] thus an antiil strategy after ablation should be considered whencombined with ici therapy previous studies and ourshave demonstrated that most cytokine levels returned topretreatment levels days after ablation this resultsuggests that h to days after ablation may be optimal timing for additional immunomodulatory therapysour results reported here support the evidence for terminal tumor thermal ablation could cause heat injury totissues surrounding the tumor site and release neoantigento blood circulation eventually induce a systemic reactionthis reaction could lead to a detectable alteration of cytokine levels further investigation is required to revealwhether the cytokines altered by mwa treatment couldaffect cancer progression whether positive or negativeabbreviationsmwa microwave ablation hcc hepatocellular carcinoma icis immunecheckpoint inhibitors tnf tumor necrosis factor il interleukinvegf vascular endothelial growth factor sd stable disease pr partialresponse ct computed tomography ci confidence interval ltp likelihoodof local tumor progression mdscs myeloidderived suppressor cellsctls cytotoxic t lymphocytes nk natural killer sirna small interfering rnaacknowledgementsnot applicableauthors contributionsjz conceptualization data curation writingoriginal draft and writingreview and editing ql conceptualization and writingreview and editingmm conceptualization and writingreview and editing brconceptualization and writingreview and editing and collect samples yhexecute milliplex assay and collect data dpl patient enrollment executemwa ablation and collect samples zl execute mwa ablation and collectsamples dml patient enrollment execute mwa ablation and collectsamples yx execute milliplex assay and collect data mt conceptualizationand writingreview and editing rl conceptualization data curation formalanalysis visualization writingoriginal draft and writingreview and editingall authors have read and approved the manuscriptfundingthis work was supported by the national natural science foundation ofchina the natural science foundation ofjiangsu province of china bk20140295 the jiangsu governmentscholarship for oversea studies js2018179 and the six one projects forhighlevel health personnel in jiangsu province lgy2018077availability of data and materialsthe datasets used andor analysed during the current study are availablefrom the corresponding author on reasonable requestethics approval and consent to participatethe protocol was approved by the human ethics committee of the firstaffiliated hospital of soochow university and was conducted in accordancewith the declaration of helsinki patients were informed that the bloodsamples were stored by the hospital and potentially used for scientific 0czhao bmc cancer page of research and that their privacy would be maintained all written informedconsent was obtained from all participants and clearly statedconsent for publicationnot applicablecompeting intereststhere is no financial or personal relationship with other people oranizations that could inappropriately influence bias this workauthor details1department of radiation oncology the first affiliated hospital of soochowuniversity suzhou china 2department of oncology the first affiliatedhospital of soochow university suzhou china 3department of lymphatichematologic oncology jiangxi cancer hospital nanchang china4department of interventional radiology the first affiliated hospital ofsoochow university suzhou china 5division of neurosurgery city of hopebeckman research institute duarte california usareceived january accepted august referencesfu j wang h precision diagnosis and treatment of liver cancer in chinacancer lett bruix j han kh gores g llovet jm mazzaferro v liver cancer approachinga personalized care j hepatol suppls144rognoni c ciani o sommariva s bargellini i bhoori s cioni r facciorussoa golfieri r gramenzi a mazzaferro v transarterial radioembolizationfor intermediateadvanced hepatocellular carcinoma a budget impactanalysis bmc cancer nault jc sutter o nahon p gannecarrie n seror o percutaneoustreatment of hepatocellular carcinoma state of the art and innovations jhepatol yin j dong j gao w wang y a case report of remarkable response toassociation of radiofrequency ablation with subsequent atezolizumab instage iv nonsmall cell lung cancer medicine baltimore 20189744e13112shi l chen l wu c zhu y xu b zheng x sun m wen w dai x yang m pd1 blockade boosts radiofrequency ablationelicited adaptiveimmune responses against tumor clin cancer res lippitz be cytokine patterns in patients with cancer a systematic reviewlancet oncol 2013146e218jin yb zhang gy lin kr chen xp cui jh wang yj luo w changes ofplasma cytokines and chemokines expression level in nasopharyngealcarcinoma patients after treatment with definitive intensitymodulatedradiotherapy imrt plos one 2017122e0172264kim mj jang jw oh bs kwon jh chung kw jung hs jekarl dw lee schange in inflammatory cytokine profiles after transarterial chemotherapy inpatients with hepatocellular carcinoma cytokine gillams a goldberg n ahmed m bale r breen d callstrom m chen mhchoi bi de baere t dupuy d thermal ablation of colorectal livermetastases a position paper by an international panel of ablation expertsthe interventional oncology sans frontieres meeting eur radiol ahmed m solbiati l brace cl breen dj callstrom mr charboneau jwchen mh choi bi de baere t dodd gd 3rd imageguided tumorablation standardization of terminology and reporting criteriaa 10yearupdate radiology chiang j hynes k brace cl flowdependent vascular heat transfer duringmicrowave thermal ablation conf proc ieee eng med biol soc huang hw influence of blood vessel on the thermal lesion formationduring radiofrequency ablation for liver tumors med phys najjar yg rayman p jia x pavicic pg jr rini bi tannenbaum c ko jhaywood s cohen p hamilton t myeloidderived suppressor cellsubset accumulation in renal cell carcinoma parenchyma is associated withintratumoral expression of il1beta il8 cxcl5 and mip1alpha clin cancerres alfaro c teijeira a onate c perez g sanmamed mf andueza mp alignanid labiano s azpilikueta a rodriguezpaulete a tumorproducedinterleukin8 attracts human myeloidderived suppressor cells and elicitsextrusion of neutrophil extracellular traps nets clin cancer res kundu m roy a pahan k selective neutralization of il12 p40 monomerinduces death in prostate cancer cells via il12ifngamma proc natl acadsci u s a onishi h kuroki h matsumoto k baba e sasaki n kuga h tanaka mkatano m morisaki t monocytederived dendritic cells that capture deadtumor cells secrete il12 and tnfalpha through il12tnfalphanfkappabautocrine loop cancer immunol immunother yu z geng j zhang m zhou y fan q chen j treatment of osteosarcomawith microwave thermal ablation to induce immunogenic cell deathoncotarget yang w wang w liu b zhu b li j xu d ni y bai l liu gimmunomodulation characteristics by thermal ablation therapy in cancerpatients asia pac j clin oncol 2018145e490erinjeri jp thomas ct samoilia a fleisher m gonen m sofocleous ctthornton rh siegelbaum rh covey am brody la imageguidedthermal ablation of tumors increases the plasma level of interleukin6 andinterleukin10 j vasc interv radiol den brok mh sutmuller rp van der voort r bennink ej figdor cg ruerstj adema gj in situ tumor ablation creates an antigen source for thegeneration of antitumor immunity cancer res zerbini a pilli m laccabue d pelosi g molinari a negri e cerioni sfagnoni f soliani p ferrari c radiofrequency thermal ablation forhepatocellular carcinoma stimulates autologous nkcell responsegastroenterology zhang h hou x cai h zhuang x effects of microwave ablation on tcellsubsets and cytokines of patients with hepatocellular carcinoma minim | Colon_Cancer |
"gut microbiota composition influences the balance between human health and disease increasing evidencesuggests the involvement of microbial factors in regulating cancer development progression and therapeuticresponse distinct microbial species have been implicated in modulating gut environment and architecture thataffects cancer therapy outcomes while some microbial species offer enhanced cancer therapy response othersdiminish cancer treatment efficacy in addition use of antibiotics often to minimize infection risks in cancer causesintestinal dysbiosis and proves detrimental in this review we discuss the role of gut microbiota in cancerdevelopment and therapy we also provide insights into future strategies to manipulate the microbiome and gutepithelial barrier to augment therapeutic responses while minimizing toxicity or infection riskskeywords intestinal dysbiosis cancer development cancer therapy microbial therapy human intestinal microbiota is essentialfor microbialhomeostasis regulation of metabolism and immune tolerance intestinal dysbiosis occurs when there are alteredratios of healthy microbial flora along with changes in theirdiversity and density such changes may lower mucus layerthickness reduce antimicrobial defense and disrupt theepithelial tightjunction barriers to allow increased translocation of intestinal bacteria and bacterial products intothe systemic circulation and trigger inflammation andimmune responses circulating bacterial products such asendotoxin genotoxin and trimethylamine oxide have beenimplicated in many human disorders including metabolicsyndrome cardiovascular complications atherosclerosisand thrombosis and various neoplastic conditions intestinal dysbiosis may also affect adaptive immunity by correspondence seahhlimyahoocom1division of hematology and oncology suny downstate health sciencesuniversity clarkson avenue room b5495 brooklyn new york usa2division of hematology and oncology department of medicine new yorkmedical college valhalla new york usamodulating the functions of t lymphocytes and promotingtumor immune escapewhile increased translocation of intestinal luminal content is associated with carcinogenesis and poor therapeuticresponse the causeeffect relationship is often bidirectionalin this review we will discuss the role of gut microbes inmodulating tumor immunity intestinal permeability andcancer development next we will highlight the effects ofintestinal dysbiosis and increased permeability in cancertherapy finally we will explore the options to improve guthealth to enhance the efficacy of cancer therapyintestinal immunity and permeabilitythe intestinal architecture and microbiota regulateinnate and adaptive immunity disruption of the architecture andor microbiota affects these functions therelationships between the different players in the intestinal microenvironment is summarized in fig the composition of microbes in the gut dictatesmucus layer thickness and production of antimicrobialsignals in germfree mice mucus layer and effector t the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cdutta and lim biomarker research page of fig interplay between different factors involved in gut immunity and permeability a the intestinal epithelial cells containing paneth cellsgoblet cells enterocytes and enteroendocrine cells coordinate with intraepithelial lymphocytes to generate a functional immune responsepaneth cells secrete antimicrobial peptides and goblet cells produce mucus to cover the epithelial layer this mucus layer prevents adhesion ofmicrobes to the epithelial cells lamina propria situated under the mucus layer contains peyers patches and immune cells including antigenpresenting cells apcs like dendritic cells dcs t cells and b cells pattern recognition receptors prrs such as tolllike receptors tlrs onepithelial cells interact with microbederived pathogenassociatedmolecular patterns pamps such as lipopolysaccharide lps to activatemyd88dependent signaling dcs travel to mesenteric lymph nodes mln and promote the differentiation of na¯ve t cells to regulatory t tregcells that migrate to other sites treg cells secrete il10 to elicit an antiinflammatory response b dysbiosis decreases mucus layer thickness andshortchain fatty acid scfas production this affects the secretion of antimicrobial peptides and allows microbes to come in close proximity tothe epithelial cells reduction in scfas influences gut barrier dysfunction as a result the gut luminal content also translocated and spreadedthrough the systemic circulation to trigger local and systemic immune responses in addition to pamps damps released from damaged intestinalepithelium interact with prrs to facilitate expression of macrophages and maturation of dcs mature dcs promote the differentiation of na¯ve tcells to effector t cells such as t helper cells th1 th2 th17 th1 release tnfα and ifnÎ and th17 secrete il17 to recruit polymorphonuclearneutrophils pmns these cytokines create a proinflammatory condition 0cdutta and lim biomarker research page of cells are absent [ ] microbes secrete shortchain fattyacids scfas such as propionate and butyrate thatprevent microbial binding to the epithelial cells and helpmaintain barrier function and immune homeostasisbutyrate promotes tightjunction formation [ ] andactivates peroxisome proliferatoractivated receptor gammapparÎto enhance epithelial oxygen consumptionresulting in reduced emanation of oxygen from the mucosalsurface it helps in maintaining an anaerobic condition inthe gut lumen needed for colonization of obligate anaerobes this intestinal microenvironment determines thecomposition of resident bacterial species for example onlyclostridium lactobacillus and enterococcus are enrichedon the epithelial surface and in the mucus layer whereasbacteroides bifidobacterium streptococcus enterobacteriaceae enterococcus clostridium and lactobacillus are allpredominant in the intestinal lumen dysbiosis increases inflammatory signals that shiftthe metabolism of enterocytes epithelial hypoxia iseliminated and increased oxygenation results in therelease of more oxygen from the mucosal surfacesince only facultative anaerobes can respire oxygendysbiosisinduced shift in epithelial oxygenation altersgut microbial community from obligate to facultativeanaerobes intestinal pathogens such as proteobacteria produce genotoxins like colibactin and cytolethaldistending toxin cdt to induce inflammation andhost deoxyribonucleic acid dna damage that initiates tumor formation dysbiosis also decreasesmucus layer thickness reduces scfa production anddamages mucosal barrier allowing pathogenassociatedmolecular patterns pamps to interact with pattern recognition receptors prrs and activate tolllike receptortlr 24myeloid differentiation primary response protein myd88 signaling pathways in addition changes inmicrobial composition and density triggers epithelial releaseof damageassociated molecular patterns damps such asextracellular adenosine triphosphate atp cytoplasmiccalreticulin high mobility group box hmgb1 proteinsendogenous nucleic acids and intracellular proteins tointeract with prrs prr engagementtriggers a proinflammatory condition that causes tissue damage and localinflammation microbiotadriven tlr immune signalinghas been implicated in cancer formation and modificationof treatment efficacy [] for example cpg oligodeoxynucleotides that mimic bacterial dna acts as a pamp totrigger a tlr9dependent tlr4 activation and tumor necrosis factor tnfα production by tumorinfiltratingmyeloidderived cells mice bearing el4 lymphomamc38 colon carcinoma and b16 melanoma when treatedwith cpg oligodeoxynucleotides show reduced tumrowth and enhanced survival rate the beneficial effects ofcpg oligodeoxynucleotides were positively associated withthe abundance of alistipes shaii in the gut effects of intestinal microbiota on cancerdevelopmentintestinal microbes can influence local and distantcarcinogenesis through infection and microbial productsor by modulating tumor immunosurveillance this isaccomplished via altering the balance between the rateof cell proliferation and apoptosis triggering chronicinflammation andor immunosuppression or changingthe metabolism of the products produced by host andmicrobes in this section we will discuss how intestinaldysbiosisrelated permeability may contribute to tumorigenesis in different anscolorectal cancerfusobacterium nucleatum a gramnegative mucosaadherent anaerobic bacteria has been implicated in theinitiation and progression of colorectal cancer crc[ ] fada an adhesion molecule on f nucleatumbinds to host ecadherin to enter epithelial cells this activates the wntβcatenin pathway leading toan increased secretion of inflammatory cytokines including il6 il8 and tnfα and upregulation of nuclearfactor kappa light chain enhancer of activated b cellsnfκb that facilitates crc development in addition itattracts myeloidderived suppressor cells and the autotransporter protein fap2 interacts with the human inhibitoryreceptor t cellimmunoreceptor with ig and itimdomains tigit to create a tumor immunosuppressivemicroenvironment f nucleatum may also induce chemoresistance by modulating the tlr4myd88 signalingpathway following 5fluoruracil treatment in crc patients an increased abundance of f nucleatum along with clostridium difficile and species ofstreptococcus campylobacter and leptotrichia has beendemonstrated in tumor tissue and fecal materials []f nucleatummediated colorectal carcinogenicity occursdownstream of apc introduction of f nucleatum resultedin rapid onset of colonic tumors in mice deficient in onecopy of adenoma polyposis coli apc apcmin gene both intestinal dysbiosis and loss of apc disruptepithelial tightjunctions and mucus layer [ ] andallow increased infiltration of f nucleatum and other nonresidential microbes to drive crc development the roleof defective gut barrier in crc has been confirmed inmucin 2knockout muc2 mice in which the lack ofgastrointestinal mucin resulted in spontaneous crc development therefore dysbiosisinduced gut permeabilitymay play an important role in tissue enrichment of fnucleatum and increased risks for crchepatobiliary cancerthe liver is chronically exposed to intestinal microbiotaand its products via the portal vein intestinal dysbiosisand increased permeability enhance translocation of gut 0cdutta and lim biomarker research page of microbiota to trigger inflammation and chronic liver disease that predisposes patients to the development of hepatocellular cancer alteration in bile acid metabolism due tochanges in clostridium spp suppress anticancer immunity in mice eradication of grampositive bacteria by oralvancomycin inhibits secondary bile acid conversion resulting in the upregulation of chemokine cxc motif ligandcxcl16 in liver sinusoidal endothelial cells cxcr16recruits natural killer t nkt cells in the tumor microenvironment and kill tumor cells in a cd1ddependentmanner in addition gut microbiotaderived lipopolysaccharides lps promote tumor progression in liver cancerby activating the tlr4 signaling in a study involving cholangiocarcinoma patients bile ducttissues haddistinct dominance of dietziaceae pseudomonadaceae andoxalobacteraceae members pancreatic cancergut microbiota influences the development of pancreaticcancer through activating tlr4 signaling the stromain pancreatic tumor harbors an abundance of microbiotaespecially bifidobacterium pseudolongum compared tonormal pancreas this helps in creating an immunosuppressive environment by differentially activating distincttlrs in monocytes pancreatic adenocarcinoma has anenrichment of proteobacteria synergistetes and euryarchaeota longer survival is observed in patients with amore diverse intratumor microbial composition primarilyof sachharopolyspora pseudoxanthomonas streptomycesand bacillus clausii tumoral colonization with mycoplasma hyorhinis and gammaproteobacteria is associatedwith gemcitabine resistance antibiotics diminishmyeloidderived suppressor cells and increase antitumorm1 macrophages to promote th1 differentiation of cd4t cells and cd8 t cell activation in the tumor cotreatment of gemcitabine with ciprofloxacin abrogatedgammaproteobacteriainduced chemotherapy resistance the efficacy of immune checkpoint inhibitors icistherapy is also enhanced by antibiotics lung cancerwhile local microbiota is important there are reportsthat gut microbiome may also contribute to lung cancerdevelopment lung cancer patients demonstrated an abundance in intestinal enterococcus and depletion in bifidobacterium and actinobacteria they are also enrichedwith veillonella bacteroides and fusobacterium depletedof dialister enterobacter escherichiashigella fecalibacterium and kluyvera in nonsmall celllung cancernsclc patients butyrate producers such as faecalibacterium prausnitzii clostridium leptum clostridial cluster iruminococcus spp clostridial cluster xiva and roseburiaspp were significantly reduced since butyrate isessential for preserving mucosal homeostasis reduction ofintestinal butyrate producers may imply a compromised intestinal barrier in these patientshematologic malignanciesdysbiosisinduced intestinal permeability affects mucosaassociated lymphoid tissue malt and plays a significantrole in hematologic malignancies composition of intestinalmicrobiota is responsible for maintaining the pool of bonemarrow myeloid cells preleukemic myeloproliferationis driven by microbial signals in teneleven translocation2tet2deficient mice [ ] these mice show increasedinfiltration of inflammatory cells disrupted mucosal barrierand increased translocation of bacteria [ ] it wassuggested that dysfunction of small intestinal barrier andleakage of microbes can occur due to tet2 mutation in occurrence of tet2hematopoietic compartmentmutation intestinal dysbiosis and leaky gut is common inleukemia and lymphomaacute myeloid leukemia aml and acute lymphoblasticleukemia all patients have a compromised intestinalbarrier [] fecal microbiota in all patients showedlower microbial diversity they were enriched in enterococcaceae porphyromonadaceae and bacteroidetes mainlyb fragilis and depleted in blautia erysipelotrichiales lachnospiraceae and clostridiales members [ ] abundanceof staphylococcaceae and streptococcaceae have also beenreported in pediatric all and adult aml [ ]helicobacter pylori is associated to malt lymphoma and chamydophila psittacito ocular maltlymphoma while borrelia burgdorferi was linked tocutaneous bcell nonhodgkin lymphoma two studiesdid not find significant risk of borrelia burgdorferi in thedevelopment of nonhodgkin lymphoma [ ] abundance of proteobacteria is a predictor for neutropenicfever and enrichments of enterococcaceae and streptococcaceae are strong predictors of infectious complications inall similarly higher gut microbiota diversity inmultiple myeloma is associated with reduced risk fordisease relapse all patients with infectious complications have an abundance of brevundimonas diminuta andagrobacterium tumefaciens whereas faecalibacteriumprausnitzii producer of scfas is completely absent similar findings have been reported in nonhodgkinlymphoma with infectious complications effects of intestinal microbiota on cancer therapythe efficacy of cancer treatment is in parts dependenton normal immune function since gut microbiota playsa crucial role in modulating immune response it is notsurprising that dysbiosis affects treatment outcomesprophylactic antibiotics are commonly used for cancerpatients undergoingallogeneichematopoietic stem cell transplantation allohsct toreduce the risk of neutropeniaassociated infectionchemotherapyand 0cdutta and lim biomarker research page of however antibiotic use causes intestinal dysbiosis thatresults in negative outcomes including poor treatmentresponse and toxicity and the development of clostridium difficile infection cdi in addition to antibioticsopioid analgesics for cancer pain management may alsotrigger dysbiosis opioid analgesics impair intestinal motility and promote bacterial overgrowth resulting in dysbiosisand gut permeability intestinal dysbiosis induces mucosal injury and triggers the release of damps damps have a dual andbidirectional effect on cancer although damps exertimmunosurveillance and immunemediated cell death toeliminate tumor cells and protect against cancer development chronic inflammation induced by damps maypromote tumor initiation damps released by apoptoticcells from cancer therapy may also induce chemoresistance and promote metastasis for example tlr78expressed on tumor cells may bind damps loxoribinefor tlr7 and poly u for tlr8 and promote chemoresistance through the activation of nfκb and the upregulation of bcl2 damps may also activate tlr9on human breast prostate and lung cancer cells to trigger tumor invasion and metastasis [ ] given theclinical significance of dysbiosismediated mucosal injuryand permeability in cancer we will in this section discusshow the treatment outcome by various cancer therapymay be affected by intestinal microflora and permeabilitychemotherapy and radiation therapyintestinal microbial composition and mucosal barrier function influence chemotherapeutic outcome and the effect isbidirectional while dysbiosis can exacerbate chemotherapy drug toxicity and reduce its efficacy chemotherapy canitself cause dysbiosis although prevalence of certain intestinal microbes in the gastrointestinal tract offer beneficialeffects others contribute to chemoresistance and drug toxicity this multiplepathway effect is best covered by timer mechanisms translocation of microbes immunomodulation metabolism and enzymatic effects on drugsand reduced microbial diversity these mechanistic effectsalter chemotherapy efficacy and toxicity and risks forinfections for example translocation of microbes due tochemotherapy induceddysbiosis and disruption of mucosal barrier can increase the risk of infection howevercertain chemotherapy drugs such as cyclophosphamideand doxorubicin damage intestinal barrier for the translocation of commensal bacteria into secondary lymphnodes to elicit antitumor immune response vancomycin prophylaxis inhibits antitumor effects of cyclophosphamide in fibrosarcoma inoculated mice irinotecanused for crc treatment is transformed into its active formsn38 by tissue carboxylesterase it is detoxified in theliver by host udpglucuronosyltransferases into inactiveglucuronide sn38g and excreted into the gut via bileducts in the gut bacterial βglucuronidases reconvertssn38g into active sn38 which causes severe intestinaltoxicity and diarrhea streptomycin inhibits irinotecanabsorption and reduces epithelial carboxylesterase activityand diarrhea ciprofloxacin inhibit βglucuronidases and low dose amoxapine βglucuronidases inhibitorsuppress irinotecanassociated diarrhea in rats table provides a selection of chemotherapeutic agents affectingand affected by intestinal microbial composition andpermeabilitylocal pelvic irradiation damages intestinal epitheliumand barrier integrity and produce reactive oxygen speciesirradiation increase alistipes and decrease prevotella inmice in gynecologic cancer patients receiving pelvicradiotherapy firmicutes and fusobacterium were significantly decreased in addition to reduced diversitysignificant enrichment of clostridium iv roseburia andphascolarctobacterium was associated with radiationenteropathy in pelvic cancer patients the effects oftotal body irradiation which is a preparative regimen forallohsct that causes dysbiosis and gastrointestinaltoxicity is discussed in more details in the allohsctsection belowimmunotherapycancer cells often create an immunosuppressive microenvironment to mediate tumor immune escape this immune escape mechanism may be reversed by icis directedat cytotoxic t lymphocyteassociated antigen ctla4programmed death receptor pd1 or pd1 ligandspdl1 since intestinal microbes influence local and systemic antitumor immune reaction by modulating prrspamps and dampsintestinal dysbiosis may impacttreatment outcome figure illustrates how the potentialmechanisms ofthe antitumor immune responses aredownregulated by intestinal dysbiosis the effects of intestinal microbiome on responses to icis have been discussed previously [ ] broadspectrum antibioticsbefore during or after icis therapy alter intestinal microbiome and resulted in lower tumor response rate inferiorprogressionfree survival and reduced overall survival responses to inhibition of ctla4 by ipilimumab inmouse models of mca205 sarcoma ret melanomaand mc38 colon carcinoma were inferior in germfreeor in broadspectrum antibiotic treated mice poorresponses were associated with decrease in intestinalbacteroides thetaiotaomicron bacteroides uniformis andburkholderia cepacia and increase in clostridiales suchdysbiosis was also associated with mucosal damage andcolitis oral feeding with either bacteroides thetaiotaomicron or bacteroides fragilis individually or with acombination of bacteroides fragilis and burkholderiacepacia restored the antitumor effects of ctla4 blockade through augmentation of th1 responses in tumor 0cdutta and lim biomarker research page of table selection of chemotherapeutic agents and the bidirectional effects between the chemotherapy and intestinal microbiotachemotherapy drugcisplatintoxicity infectioncdi ototoxicity effects on gut changes in microbiotadamages mucosal barrier by impairing dnareplication of rapidly proliferating epithelialcells facilitates translocation of gut bacteriacommensal gut bacteria influencesgenotoxicity by inducing reactive oxygenspecies ros production and recruitment ofpathogenic th17 cells in the tumormicroenvironment independently ofimmunity elicited by immunogenic celldeath microbial interventionantibiotics against grampositive bacteriaabrogate antitumor chemotoxicityincrease tumor size and decrease survivalratecisplatin alone show better responsecompared to a combined treatment ofcisplatin and antibiotics in mice with lungcancer the combination treatmentincreased tumor size and decreasedsurvival ratelactobacillus acidophilus restores antitumorefficacy following antibiotic treatment[ ]restoration of gut microbiota andepithelial integrity by fmt andtreatment with dmethionine [ ]prevent infections and ototoxicity withoutaffecting tumor chemotoxicityfmt increases a muciniphilaabundance and reduces cipn oral butyrate supplementation improvesgut barrier by reducing inflammation andmucositis antibiotics reduce mucositis and cytokineproduction but also diminish antitumorefficacy and promote chemotherapyresistance antibiotics against grampositive bacteriareduce th17 responses and subsequentdevelopment of cyclophosphamideresistancereestablishment of e hirae alone restoresantitumor activity e hirae decreases tumorinfiltrating tregsbarnesiella intestinihominis accumulates inthe colon and increases the number ofintratumoral ifnÎproducing Îδt cellse hirae and b intestinihominissynergistically stimulate local and systemicimmunity to improve anticancer effects nod1nod2 mice having abundant bintestinihominis demonstrate increased Îδtcells in tumor beds and enhancedcyclophosphamide efficacy paclitaxel5fluoruracilincreases gut permeability as indicated by5fold elevation in circulating lpsbindingprotein and systemic inflammation reduces abundance of roseburiaporphyromonadaceae and akkermanisamuciniphila [ ]reduces clostridium spp and increasesmembers of proteobacteria mainlyenterobacteriaceae damages mucosal barrierchemotherapyinducedperipheral neuropathicpain cipn cdi [ ]mucositis along the entiregastrointestinal tract cdi [ ]cyclophosphamidecdi triggers disruption of gut barrier by alteringbacterial compositiongrampositive bacteria such asenterococcus hirae lactobacillus johnsoniiand l murinus translocate from gut intomesenteric lymph nodes and spleen this enhances immune responses by theproduction of interferon gamma ifnÎ andactivation of th17 cellsdraining lymph nodes and promotion of maturation oftheintratumoral dendritic cells dcscombination treatment of bacteroidesandburkholderia cepacia prevented intestinal damage andrefractory colitisin additionfragilisfecal microbiota analysis of melanoma patients beforeand after ipilimumab treatment showed a change in therelative proportions ofenterotypeclusters cluster a was dominated by prevotella spwhereas clusters b and c by different bacteroides sppfecal microbiota transplantation fmt from patientsinto tumorbearing germfree mice showed that onlyfecal material from cluster c resulted in colonizationthree dominantwith bacteroides thetaiotaomicron or bacteroides fragilisand enhanced ipilimumab response in another study ofipilimumab in mice vancomycin treatment resulted in amore severe manifestation of colitis whereas oraladministration of bifidobacterium ameliorated the sideeffects similarly melanoma patients with increasedabundance of bacteroidaceae rikenellaceae and barnesiellaceae members responded better to ctla4 antibodies however a different study in ipilimumabtreated melanoma patients found that bacteroides spp were associatedwith decreased response whereas faecalibacterium andother firmicutes members improved clinical outcome 0cdutta and lim biomarker research page of fig potential antitumor immune mechanisms induced by intestinal dysbiosis a in the presence of intact mucosal barrier and signals fromcommensal microbiota effector t cell activation is modulated by t cell receptor tcr ligation with major histocompatibility complex mhc classi and costimulation of cd80cd86 and cd28 binding of cytotoxic t lymphocyteassociated antigen ctla4 receptor to antictla4 antibodyon treg impairs its effector tcell inhibitory function it also downregulates ctla4 expression on apc ligation of repressive receptorprogrammed death receptor pd1 and its ligand pdl1 to antipd1 and antipdl1 antibodies respectively activate effector tcell proliferationand function activated effector t cells interact with tumor cells and release cytokines to induce tumor cell death b signals from unfavorablemicrobes due to dysbiosis upregulates ctla4 pd1 and pdl1 expression to inhibit tcell activation ctla4 on treg binds to cd80cd86 onantigen presenting cell apc cd80cd86 on apc also disengages from cd28 and binds to ctla4 on effector t cells pdl1 the ligand of pd1is expressed on antigen presenting cell apc and tumor cells pd1 on effector t cells ligates to pdl1 on apc and tumor cells these activitiesinhibit effector tcell activation reduces immune checkpoint inhibitor ici efficacy and causes tumor escape 0cdutta and lim biomarker research page of patients with higher abundance of faecalibacteriumand improved response to ctla4 antibodies showedhigher incidence of enterocolitis and lower level of treg inperipheral blood thus the beneficial effects of specificand isolated gut microbes may depend on the commensalassociation with other microbial species and may differ between humans and micepd1 blockade may also be modulated by intestinalmicrobiota melanoma patients who responded to pd1blockade had increased abundance of enterococcus faeciumcollinsella aerofaciens bifidobacterium adolescentis klebsiella pneumoniae veillonella parvula parabacteroidesmerdae lactobacillus sp and bifidobacterium longumwhereas in nonresponders the intestinal microbiome wasenriched in ruminococcus obeum and roseburia intestinalis another study found higher abundance of faecalibacterium species in responders and enrichment with bacteroides thetaiotaomicron escherichia coli and anaerotruncuscolihominis in nonresponders clinically nonsmallcell lung cancer nsclc and renal cell carcinoma rccpatients experienced increased resistance to pd1 blockadeafter antibiotic treatment these patients had shorterprogressionfree survival as well as overall survival in thisstudy response to pd1 blockade correlated with higherfecal abundance of akkermansia muciniphila fmt fromresponders to germfree or antibiotictreated mice improved the outcome of pd1 blockade administration ofa muciniphila after fmt from nonresponders restoredresponsesimilarly intestinal microbiota may influence the outcome of chimeric antigen receptor t cell car t therapypatients with complete response to cd19 car ttherapyexhibited enrichment of oscillospiraceae ruminococcacaeae and lachnospiraceae in their intestinal microbiomewhereas patients who did not attain a complete responseshowed increased abundance of peptostreptococcaceae effectiveallogenic hematopoietic stem cell transplantationalthough allohsct issomein treatinghematological malignancies the immunosuppressive agentsbroadspectrum antibiotics and chemoradiation used withthe transplant often induce intestinal dysbiosis gut permeability and impaired systemic immune response highermicrobiota diversity is associated with longterm survivaland lower diversity in gut microflora is associated with reduced overall survival and higher transplantrelated mortalityfollowing allohsct [ ] severe infections that occurdue to intestinal dysbiosis such as cdi and vancomycinresistant enterococci vre infections are also associatedwith higher treatmentrelated mortality [] allohsctdisrupts the equilibrium of bacterial composition in feceswith a dominance of enterococcus streptococcus and proteobacteria members [ ] and reduces beneficial bacteriasuch as faecalibacterium and ruminococcus higherabundance of blautia was found to be associated with improved overall survival moreover allohsct patientswith reduced risk of relapse had an enrichment of eubacterium limosum ofgraftversushost diseaseone of the major complications of allohsct is thedevelopmentgvhdoccurrence of cdi during allohsct increases the riskof gvhd besides the loss of overall microbial diversityreduction in beneficial faecalibacterium blautia lactobacillus and ruminococcus and increased abundance ofenterococcus and clostridiales was observed in gvhd[ ] patients without gvhd had increasedabundance of parabacteroides and bacteroides in theirpretransplant feces in a preclinical study reducedgvhd and improved overall survival was observed afterthe administration of the probiotics lactobacillus rhamnosus gg alone or in combination with ciprofloxacindue to the preservation of gut mucosal integrity in therecipient mice restoration of normalintestinalmicrobiome by fmt has been found to benefit patientswith steroidrefractory gvhd [ ] multipleclinical trials are currently ongoing to investigate howmanipulation of gut microbiota using dietary intervention and fmt might reduce the risk of gvhdmanipulation of intestinal microbiome and barrierto improve outcome of cancer therapeuticsifintestinal dysbiosis and its associated increased gutpermeability are associated with cancer development andtherapyrelated complications and treatment outcomes itfollows that intervention of the intestinal microbiomeandor gut barrier may alter cancer outcome in thissection we will explore three broad approaches fig that might be investigated nonselective modificationof intestinal microbiome using fmt semiselectivemodification of intestinal microbiome using antibioticsand biologic modification of intestinal barrier we willdiscuss the challenges and obstacles each of the approaches may encounternonselective modification of intestinal microbiome usingfmtmodification of the intestinal microbiome is theoreticallybest accomplished by fmt unmanipulated fmt will notonly replete the dysbiotic intesti | Colon_Cancer |
gon§ales junior w effect of treatment with injectable progesterone at the onset of super precocious tairessincronization protocol on tai pregnancy rates of nellore heifers in animal reproduction for the annual meeting of the brazilian embryo technology society sbte august florianopolis brazil animal reproduction suppl pugliesi g a novel strategy for resynchronization of ovulation in nelore cows using injectable progesterone p4 and p4 releasing devices to perform two timed inseminations within days reprod domest anim 101111rda13475 pugliesi g use of doppler ultrasonography in embryo transfer programs feasibility and field results anim reprod 1021451 19843143ar201 siqueira l g b color doppler flow imaging for the early detection of nonpregnant cattle at days after timed artificial insemination j dairy sci 103168jds20136814 pugliesi g efficacy of doppler ultrasonography to detect nonpregnant nelore cows and heifers submitted to three timedai in days in reproduction ed international 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applications to sage data biostatistics 101093biost atist icskxm03 benjamini y hochberg y controlling the false discovery rate a practical and powerful approach to multiple testing j r stat soc ser b methodol 101111j251761611995tb020 31x huang d w sherman b t lempicki r a systematic and integrative analysis of large gene lists using david bioinformatics resources nat prot 101038nprot andersen c l jensen j l orntoft t f normalization of realtime quantitative reverse transcriptionpcr data a modelbased variance estimation approach to identify genes suited for normalization applied to bladder and colon cancer data sets cancer res 10115800085472can040496 pfaffl m w a new mathematical model for relative quantification in realtime rtpcr nucl acids res 101093nar299e45 acknowledgementsthis study was funded by the s£o paulo research foundation fapesp the authors would like to thank all students from the molecular endocrinology physiology laboratory lfemfmvzusp project 03gdcr17 we thank the administration of pirassununga campus of the university of s£o paulo the laboratory of animal biotechnology of the esalqusp and ourofino animal health and alta genetics brazil for donating the hormones and semen respectivelyauthors contributionsccr conceived the study performed pcr analyses and wrote the manuscript scdsa performed the bioinformatics analysis gddm assisted with sample processing igm performed the ultrasonography evaluations llc contributed with the rnaseq analysis amgd assisted with data analyses and provide expertise in pcr data analysis mb contributed with critical review of the manuscript gp is the pi of the project provided the financial support expertise in experimental design statistical analysis and correct the manuscript all authors reviewed and approved the manuscriptcompeting interests the authors declare no competing interestsadditional informationsupplementary information is available for this paper at 101038s4159 correspondence and requests for materials 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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 |
the introduction of combined conventional cytostatics and pathwayspecific inhibitors has opened new treatment options for several cancer types including hematologic neoplasia such as leukaemias as the detailed understanding of the combinationinduced molecular effects is often lacking the identification of combinationinduced molecular mechanisms bears significant value for the further development of interventional approachesmethods combined application of conventional cytostatic agents cytarabine and dexamethasone with the pi3kinhibitor idelalisib was analysed on cellbiologic parameters in two acute prob lymphoblastic leukaemia ball cell lines in particular for comparative characterisation of the molecular signatures induced by the combined and mono application whole transcriptome sequencing was performed emphasis was placed on pathways and genes exclusively regulated by drug combinationsresults idelalisib cytostatics combinations changed pathway activation for eg retinoblastoma in cancer tgfb signalling cell cycle and dnadamage response to a greater extent than the two cytostatics alone analyses of the top regulated genes revealed that both combinations induce characteristic gene expression changes a specific set of genes was exclusively deregulated by the drug combinations matching the combinationspecific antiproliferative cellbiologic effects the addition of idelalisib suggests minor synergistic effects which are rather to be classified as additivekeywords pik3inhibition acute lymphoblastic leukaemia idelalisib cytostatics drug combinations cytarabine dexamethasonecorrespondence hugomuruaescobarmedunirostockde department of medicine clinic iii hematologyoncologypalliative care rostock university medical center rostock germanyfull list of author information is available at the end of the acute lymphoblastic leukaemia all is a malignant disease which is characterized by a clonal proliferation of lymphoid progenitor cells most commonly of bcells all affects children as well as elderly individuals 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 0csklarz a0et a0al cancer cell int page of with a significantly different outcome while children are reported to have a longterm survival probability of approximately [ ] in adults relapsefree survival is lower than patients showing mixedlineage leukaemia mll rearrangements display even lower survival rates []all therapy is currently dominated by the application of cytostatic agents according to the current clinical practice guidelines however severe side effects development of drug resistance and relapse limit the therapeutic success the introduction of pathway specific tyrosine kinase inhibitors tki such as imatinib and immunotherapeutics such as the anticd20 antibody rituximab have advanced curative treatment in chronic leukaemia the 3kinase pi3k represents a key molecule within the b cell receptor bcr pathway and different tkis are currently evaluated to target this kinasephosphatidylinositol45bisphosphate idelalisib idel is a selective pi3k pathway inhibitor targeting the δ subunit [ ] mono and combined administration of idel were approved for the treatment of indolent nonhodgkin lymphoma and chronic lymphocytic leukaemia [] however postmarketing surveillance suggested increased mortality caused by infections as a side effect of idel the molecular mechanisms leading to this observed side effect are not yet fully understood [ ]idel preferentially targets the deltasubunit pi3kδp110δ of the pi3k kinase which plays a key role in signal transduction cell proliferation and survival energy metabolism cellular motility and cell cycle progression the kinase is highly activated in several tumour types of different origins [ ] consequently pi3k is targeted in several novel therapeutic approaches [ ] table a0the introduction of tkis such as idel enabled the evaluation of new drug combinations potentially featuring lower doses of the individual drugs and thereby reducing side effects and drug resistance in turn understanding the respective combination modes of action is critical for a rational selection of the best candidate drugs introduction of nextgeneration sequencing such as whole genome and exome sequencing and rnasequencing provided profound knowledge of the disease acting molecular mechanisms especially rnasequencing has been of considerable value as mrna allowed to characterise drug combination action as well as drug combination induced effects therefore in the present study the cellbiological and molecular effects of the pi3kinhibitor idel in mono and combined drug application with the conventionally used cytostatics cytarabine arac and dexamethasone dex on proball cells were investigated cellbiological assays analysing cell proliferation metabolism and apoptosis induction revealed combinationspecific table pathway analysis of a0 rs411 and a0 sem exposed to a0 arac dex and a0 idel and a0 two drugs combined arac idel dex idelpathway regulation arac idel vs arac vs idelcell lineretinoblastoma in cancer uptgfbeta signaling pathway downtgfbeta signaling pathway upsids susceptibility pathway uptnfalpha signaling pathway uppathway regulation dex idel vs dex vs idelproteasome degradation downcytoplasmic ribosomal proteins updex idel micrornas in cardiomyocyte hypertrophy upectoderm differentiation upretinoblastoma in cancer downcell cycle downdna replication downg1 to s cell cycle downdna damage response down ranking positions of the pathways and amount of corresponding genes in parentheses are representedaracpathway ranking position corresponding genesarac idel dex idelidel rs411semcell liners411sem 0csklarz a0et a0al cancer cell int page of enhanced antiproliferative effects of the combined drug applications comparative whole transcriptome sequencing analyses identified pathways and gene signatures specifically regulated by the respective drug combinations within the top20 modulated pathways the retinoblastoma in cancer tgfb signalling cell cycle and dnadamage response were predominantly affected by the combination in order to identify key player genes using these pathways the top20 modulated were analysed revealing a gene set exclusively regulated by the drug combination in both cell lines this gene set featured cyp3a4 steap1 slitrk1 ackr3 and ccl25 some of these genes are reported to be deregulated in leukaemic cells thus exclusively regulation by drug combination may explain the rather additive effectsresultsidel enhances the a0antiproliferative and a0antimetabolic effect of a0arac and a0dexin rs411 cells enhanced effects on proliferation inhibition were observed for combinations of arac idel ± and dex idel ± compared to the respective mono drug applications arac ± dex ± idel ± control fig a01a comparison of cell count and metabolic activity wst1assay fig a0 1b revealed a reduction in cell numbers while barely decreasing metabolic activity for arac and arac idel ± and ± in sem cells all combinations arac dex ± arac idel ± dex idel ± resulted in an enhanced antiproliferative effect compared to the respective mono applications arac ± dex ± or idel ± fig a0 1d akin to rs411 the incubation with arac ± and arac idel ± resulted in a decreased metabolic activity fig a01e the observed reduction of metabolism did not match the observed reduction in cell numbersidel boosts the a0apoptotic effect of a0dex in a0rs411incubation with dex idel resulted in a significantly higher apoptosis rate ± compared to the respective mono substance application dex ± idel ± control ± in rs411 cells fig a0 1c in sem only the combination arac dex resulted in an increased amount of early and late apoptotic cells ± compared to the respective mono substances arac ± dex ± control ± fig a01f in fig a01g are exemplarily the plots of the flow cytometry analysis additionally all plots are shown in the supplementary file additional file a0 facs plotsin summary biological assays revealed enhanced antiproliferative effects triggered by combined application of idel with arac and dex respectively therefore we further investigated drug exposure induced effects on gene and pathway regulation by rnasequencing for all drug combinations and mono applicationscombined drug application of a0idel with a0arac and a0dex induces enhanced changes in a0gene expressiondrug combination induces an a0enhanced amount of a0regulated genesin rs411 genes were differentially expressed by arac idel exposure compared to the respective control cells while mono application of arac modulated and idel genes see additional file a0 supplement tables thereby an overlap of genes in all three conditions was identified fig a02aalso dex idel exposure modulated more genes compared to dex or idel here an overlap of genes was detected by both mono and as well as combined drug application fig a02bin sem the arac idel combination led akin to rs411 to a stronger gene modulation compared to arac and idel thereby genes were modulated by both the mono and the combined drug application fig a0 2c moreover dex idel exposure resulted in a higher amount of regulated genes in comparison to their respective mono applications dex idel all three conditions showed an overlap of genes in totalin summary for both cell lines and both drug combinations the total number of genes modulated by these combinations exceeds the absolute number of the respective mono application especially the conditions arac vs arac idel and dex vs dex idel showed a high overlap of modulated genes heatmaps of the top100 regulated genes are shown in the additional file a0 supplement figureshowever the comparison of the up and downregulated genes revealed a higher amount of upregulated genes by arac idel exposure in both cell lines moreover dex idel led nearly to a similar amount of up and downregulated genes in both cell linesdrug combinations induced stronger changes of a0gene expression levelsin rs411 we observed a higher range of gene expression level changes by arac idel to in comparison to arac to and idel to an enhanced range was also observed by exposure with dex idel to in contrast to the respective mono application dex to idel to 0csklarz a0et a0al cancer cell int page of srmesa] sll ec dedees[ tnuoc llecd] sllec dedees [ tnuoc llecproliferationb] lortnoc[ ytivitca cilobateme] lortnoc[ ytivitca cilobatemmetabolismapoptosisc]evitisop ipvnixenna[ sllec degamadf]evitisop ipvnixenna[ sllec degamadgieddoiimudporpiaracdexidelaracideldexidelsrmesannexin v fitc fig prob all cell lines rs411 ac and sem df exposed with arac dex and idel and two drugs combined aracdex aracidel dexidel for h influence of mono and combined application on a d proliferation cell count b e proliferation and metabolism wst proliferation assay and c f apoptosis annexin vpi staining a pairwise students ttest compared to control cells and single compounds reveals significance p ¤ p ¤ p ¤ [n ¥ ] g plots of apoptotic annexin v fitc and propidium iodide and necrotic cells annexin v fitc and propidium iodide detected by flow cytometry analysis at h data are representative of three independent experiments further plots are represented in the additional file facs plots 0csklarz a0et a0al cancer cell int page of arac vs idel vs aracideldex vs idel vs dexidelasrcmesbdfig prob all cell lines rs411 a b and sem c d exposed with arac vs idel vs aracidel and dex vs idel vs dexidel venndiagrams represent the differential expressed genes deg of each sample and there overlap among these samplesas with rs411 a higher range was observed in sem cells by combined drug incubation with arac idel to compared with mono application arac to idel to similar effects were detected for dex idel to exposure dex to idel to arac idel and a0dex idel modulated combination specific gene setsin rs411 arac idel exposure led to a set of genes which were exclusively regulated by this combination but not by dex idel in contrast dex idel led to selective modulation of genes which were only regulated by this combined drug application see additional file a0 supplement tablesin sem arac idel led to an exclusively regulation of genes which were not modulated by the other drug combination exposure to dex idel resulted led to a set of exclusively effected genes which were not regulated by arac idel see additional file a0 supplement tablesin summary the combined application of idel with arac or dex resulted in regulation of an exclusively gene set and also in higher gene expression levels further the specific combinations induce characteristic expression changes 0csklarz a0et a0al cancer cell int page of combined drug application of a0idel with a0arac or a0dex leads to a0combination specific pathway modulationa combination of arac idel or dex idel led to specifically enhanced pathway modulation compared to the respective mono applications absolute numbers of genes included in the respective pathways were increased in the following we focussed on pathways that did not rank within the top30 deregulated pathways within the mono applications but ranked in the top20 pathways during combined setup overview in table a0 a complete listing table in the additional file a0 xlsfile section additive pathways genesarac idel modulates tgfbeta signalling in a0rs411 and a0sem and a0further three cell line specific pathwaysin rs411 cells the combination of arac idel led to upregulated genes corresponding to the retinoblastoma in cancer pathway while the respective mono applications modulated only four arac respectively two idel genes respectively accordingly the pathway ranked on position of the top modulated pathways arac position idel position further tgfbeta signalling showed more downregulated genes after arac idel exposure compared to the mono applications arac genes idel genes combined pathway ranking on position arac position idel position in sem with respect to the modulation of the tgfbeta signalling pathway a higher number of downregulated genes was found by arac idel in comparison to the respective mono applications arac idel this pathway ranked on position for the combined application arac position idel nafurther tnfalpha signalling was found on position of the top upregulated pathways arac position idel na modulating combination specific genes compared to arac genes and idel genes accordingly tgfalpha signalling was found to be a major target of idel combination induced pathway modulationfurther upregulation of genes corresponding to the sids susceptibility pathway was observed by arac idel exposure compared to the respective mono applications arac genes idel genes this pathway was found at position of the list of the top20 regulated pathways arac idel nadex idel exposure induces extensive cytoplasmic ribosomal proteins retinoblastoma in a0cancer and a0cell cycle pathway deregulationwhile arac idel modulated four different pathways in total in both cell lines the induced observed modulation by the addition of idel to dex resulted in a high number of combination specific pathway deregulations in total nine different pathways were modulated by dex idel in both cell lines interestingly eight of nine pathways were found to be significantly deregulated in sem upregulated downregulatedthereby the total number of modulated genes exceeded the number of genes modulated by the respective mono application in pathways such as cytoplasmic ribosomal proteins upregulated retinoblastoma in cancer and cell cycle both downregulated while genes belonging to the cytoplasmic ribosomal proteins pathway were found upregulated by dex idel only five genes were found upregulated by dex and by idel a similar pattern was observed for the retinoblastoma in cancer pathway dex idel downregulation of genes vs dex genes vs idel gene as well as for the cell cycle pathway dex idel genes vs dex genes vs idel genesfurther pathways showing a similar combination specific enhanced modulation were pathways such as micrornas in cardiomyocyte hypertrophy ectoderm differentiation dna replication g1 to s cell cycle and dna damage response in sem cells and the proteasome degradation pathways in rs411 detailed listing in additional file a0 xlsfile section additive pathways genesin summary the additional application of idel enhances the observed effects of arac and dexidel combination modulated pathways showed characteristic gene deregulationsdex idel as well as arac idel combination induced specific gene modulation not found in either of both mono applications in total arac idel led to an exclusive modulation of genes annotated with the four drug combination specific pathways log foldchange to the dex idel combination induced exclusive modulation of in total of genes within the drug combination specific modulated pathways log foldchange to exemplarily dex idel exposure led to exclusive modulation of the cell division cycle a cdc25a log foldchange cell division cycle cdc6 log foldchange and myosin lightchain kinase gene mylk log foldchange genesa detailed listing of all affected genes of both cell lines can be found in the additional file a0 xlsfile section additive pathways genesidel combination specific pathways show enhanced gene expression regulationas described above the addition of idel to arac or dex led to exclusive gene regulations as well as increased 0csklarz a0et a0al cancer cell int page of log gene numbers belonging to to the top deregulated pathways additionally the respective combinations led to enhanced log foldchanges for a set of specific genes summary is given in the additional file a0 supplement tables thereby the respective range in the combinations exceeded the respective mono application detailed listing in the additional file a0 xlsfile section additive pathways genes as mentioned before arac idel led to a drug combination specific pathway modulation of four pathways further investigation revealed a deregulation of genes by arac idel log foldchange range to while arac deregulates genes log foldchange range to and idel genes log foldchange range to incubation with dex idel led to a deregulation of nine drug combination specific modulated pathways from these pathways dex idel deregulated genes log foldchange range to while dex deregulated genes log foldchange range to and idel none exemplarily genes such as aristaless related homeobox arx and zinc finger and btb domain containing zbtb16 were upregulated by dex application arx log foldchange zbtb16 log foldchange and stronger deregulated by the drug combination dex idel arx log foldchange zbtb16 log foldchange for a detailed comparison of the combined and mono application induced expression changes see additional file a0 xlsfile section additive pathways genes arac exposure led to an upregulation of distalless homeobox a0 dlx2 log foldchange while the addition of idel induced a log foldchange for the combined applicationtop drug combination modulated genes revealed combination specific modes of a0actionto further explore combination specific acting mechanisms the top20 deregulated genes pathway independent log foldchanges combined drug exposure were compared to the corresponding expression values of the respective mono applications additional file a0 xlsfile section rs411top20 genes aidi and semtop20 genes aidi akin to the observed exclusive gene modulations within the top ranking pathways comparable effects were found when analysing the general pathway independent top20 deregulated genesarac idel modulates histone genes predominantlyarac idel exposure led in both cell lines to a downregulation of different histones thereby hist1h2bo was found the only histone which ranked within the top downregulated genes for both cell linesin rs411 twelve histones belong to the general top downregulated genes thereby the observed log log foldchange of the histones hist1h1e hist1h2ah hist1h1d hist1h2bm and hist1h3b were found stronger deregulated given the observed log foldchanges compared to both respective mono applications range log foldchange arac idel to vs arac to vs idel to further the histones hist1h3i and hist1h3f were exclusively found to be downregulated by arac and arac idel while not being affected by idel interestingly both histones were more affected by arac idel log foldchange and compared to arac log foldchange and further the histones foldchange hist1h2aj hist1h2bf log foldchange hist12ad log foldchange foldchange and hist1h3g hist1h2bo log foldchange were found to be exclusively downregulated by the arac idelin sem histones as hist1h2bb log foldchange and also hist1h2bo log foldchange were only affected by arac idel while histones as hist1h4b hist1h2be hist1h4j hist1h2bg and hist1h3a were downregulated by arac and stronger affected by arac idel log foldchanges are detailed listed in the additional file a0 xlsfile section semtop20 genes aidiin addition to the mentioned histones both cell lines showed a specific pattern of the remaining combination specific top20 deregulated genes in rs411 downregulation of the genes small nucleolar rna snora12 log foldchange nucleoside diphosphate kinase nme1nme2 log foldchange and eukaryotic translation initiation factor 5alike eif5al1 log foldchange and an upregulation of camp responsive element binding protein like creb3l3 log foldchange and transmembrane and immunoglobulin domain containing tmigd2 log foldchange were foundin sem cytochrome p450 family subfamily a member cyp3a4 log foldchange six transmembrane epithelial antigen of the prostate steap1 log foldchange and potassium voltagegated channel subfamily j member kcnj1 log foldchange represents genes which were found only downregulated by the combination arac ideldex idel leads to a0regulation of a0similar top modulated genes in a0both a0cell linesin contrast the exposure with dex idel induced a higher number of genes commonly deregulated in both cell lines in both cell lines two genes were found upregulated as well as eight downregulated ranking within the top20 deregulated genesto arac idel 0csklarz a0et a0al cancer cell int page of as ring finger protein rnf175 was downregulated in both cell lines by dex and dex idel exposure in rs411 rnf175 was significantly more affected by the drug combination dex log foldchange dex idel log foldchange in sem rnf175 showed a similar expression level with dex idel exposure log foldchange as with dex log foldchange also zbtb16 described above olfactory receptor family subfamily a member orc7a5 and olfactory receptor family subfamily c member or7c1 were upregulated and more affected by the drug combination in both cell linesadditionally some genes were exclusively deregulated by drug combination in rs411 leucinerich repeatcontaining protein slitrk1 log foldchange and matrilin matn4 log foldchange were only downregulated by the drug combination dex idel in sem atypical chemokine receptor ackr3 log foldchange and cc motif chemokine ligand ccl25 log foldchange were also only downregulated by dex idel in both cell lines the top20 upregulated genes did not contain any gene which was exclusively regulated by the drug combination dex idel the here reported genes comprise a short overview and more genes are listed in the additional file a0 xlsfile section rs411top20 genes aidi semtop20 genes aididiscussionthe combined application of idel and arac or dex resulted in enhanced antiproliferative effects depending on the addressed cell line the combination led to enhanced antiproliferative effects on the cellular level characteristic gene regulation and expression thereby the specific exclusively regulated genes and pathways were identified in both mllpositive proball cell lines the focus here is on mono and combined therapy of maximum two agents in cell lines and adds insights into the previously gained knowledge of expression profiling as well as fusion gene detection in patients with ball using standard treatment regimen containing arac and dex [ ]addition of a0idel to a0arac results in a0pronounced antiproliferative effects independent from a0aracsensitivitya different aracsensitivity characterises both cell lines while a0µm arac exposure reduces rs411 proliferation to approx half sem proliferation is inhibited to nearly by the 100fold lower concentration the addition of idel induced in both cell lines independently from their characteristic aracsensitivity an enhanced decrease of proliferation interestingly arac exposure led to an increase of metabolic activity in both cell lines while the addition of idel leads to a proportional ratio of remaining cells and corresponding metabolic activity further the addition of idel initiates an increase of the number of regulated genes and stronger modulated gene expression levels further the addition of idel led to a modulation of genes belonging to the tgfbeta signalling pathway in both cell lines this pathway is an essential regulator of proliferation differentiation migration and cell survival additionally several genes regulating cellular key processes as proliferation and cell cycle were found regulated explicitly by the addition of idel exemplarily various histones with direct effect on dnapackaging were found downregulated and thus influence dnareplication further the elongation factor eif5al1 was found exclusively downregulated by arac idel in rs411 both mechanisms show a specific enhancement of cell division critical checkpoints which could be mediators of the observed cellular responsegenes snora12 nme1nme2 cyp3a4 and steap1 were exclusively downregulated by arac idel these genes are described to be found overexpressed in cancer of different origins while snora12 is found upregulated in lung cancer nme1nme2 upregulation is described to promote the survival of aml cells steap1 overexpression is detected in different cancer types and was associated with a poorer prognosis for aml multiple myeloma diffuse large b cell lymphoma and colorectal cancer accordingly the observed exclusively downregulation potentially represents a molecular mechanism resulting in the enhanced antiproliferative effects of arac idel further we detected a selective downregulation of cyp3a4 cyp3a4a290g polymorphism that resulted in overexpression was found in many acute myeloid leukaemia aml samples additionally an overexpression in breast cancer had been detected cyp3a4 is responsible for the detoxification of more than of the drugs on the other hand we discovered an exclusive downregulation of kcnj1 this gene is reported to inhibit proliferation and metastasis in renal cell carcinoma currently data of kcnj1 for leukaemia is missing the examined downregulation of these genes by arac idel may contribute to the more potent effect of the drug combination in comparison to the respective mono applicationwhile the previous represents the loss of prooncologic cellular features also gain of function modulations were observed leading to the enhanced antiproliferative molecular mechanism exemplarily creb3l3 was found exclusively upregulated while dlx2 expression was found stronger upregulated by the combination creb3l3 overexpression suppresses proliferation in hepatoma 0csklarz a0et a0al cancer cell int page of cells and has been described to be downregulated in hepatocellular carcinoma dlx2 is reported to be downregulated in paediatric precursor balls carrying mllrearrangement and may be induced during metabolic stressinduced necrosis these functional gene modulations represent candidate mechanisms mediating the observed enhanced antiproliferative effects in the all cell linesinterestingly in all similar cell biological effects were observed showing that the addition of pi3k or mtor inhibitors to arac was able to induce enhanced antiproliferative effects in a0vitro lately comparative observations were described for the combination of the pi3kδ inhibitor puquitinib with arac in an aml xenograft model addition of a0idel to a0dex leads to a0enhanced antiproliferative effects in a0glucocorticoidsensitive and a0resistant cellsthe addition of idel to dex resulted in an enhanced antiproliferative effect and higher apoptosis rates in glucocorticoid gcresistant and sensitive proball cell lines both cell lines are characterized by a translocation between chromosome hsa4 and hsa11 in general the presence of this translocation is associated with gcresistance and often observed in cases of relapses however only sem cells established from a 5yearsold girl at relapse show reduced sensitivity to dex while rs411 established from a 32yearold woman at relapse are considered as highly sensitive while in sem a0µm dex exposure inhibited the rate of proliferation to approx a half rs411 proliferation was found decreased to nearly by the 1000fold diluted concentration the addition of idel to dex led to a strongly dexsensitizing effect on both cell lines sem proliferation was further reduced to approx while rs411 proliferation was reduced to nearly additionally the observed apoptosis inducing effect of dex was found increased by the addition of idel in both cell linesakin to the effects observed for arac idel the addition of idel to dex induced specific pathways modulations and also induced exclusive gene expression influencing key regulators such as cell cycle and dnareplication thereby key mediato | Colon_Cancer |
" the empirical dietary index for hyperinsulinemia edih score is a validated foodbased dietary scorethat assesses the ability of wholefood diets to predict plasma cpeptide concentrations although the edih hasbeen extensively applied and found to be predictive of risk of developing major chronic diseases its influence oncancer survival has not been evaluated we applied the edih score in a large cohort of colorectal cancer patients toassess the insulinemic potential of their dietary patterns after diagnosis and determine its influence on survivaloutcomesmethods we calculated edih scores to assess the insulinemic potential of postdiagnosis dietary patterns andexamined survival outcomes in a sample of stage iiii colorectal cancer patients in the nurses health studyand health professionals followup study cohorts multivariableadjusted cox regression was applied to computehazard ratios hr and confidence intervals ci for colorectal cancerspecific mortality and allcause mortalitywe also examined the influence of change in diet from pre to postdiagnosis period on mortalityresults during a median followup of years there were deaths which included colorectal cancerspecific deaths in the multivariableadjusted analyses colorectal cancer patients in the highest compared tolowest edih quintile had a greater risk of dying from colorectal cancer hr ci and a greater risk of allcause death hr 95ci compared to patients who consumed low insulinemicdiets from pre to postdiagnosis period patients who persistently consumed hyperinsulinemic diets were at higherrisk of colorectal cancer death hr151 95ci and allcause death hr 95ci our findings suggest that a hyperinsulinemic dietary pattern after diagnosis of colorectal cancer isassociated with poorer survival interventions with dietary patterns to reduce insulinemic activity and impactsurvivorship are warrantedkeywords colorectal cancer survival insulinemic dietary patterns insulin cpeptide correspondence fredtabungosumcedu1division of medical oncology department of internal medicine the ohiostate university college of medicine west 12th avenue 302b wisemanhallccc columbus oh usa2the ohio state university comprehensive cancer center arthur g jamescancer hospital and richard j solove research institute columbus oh usafull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0ctabung bmc cancer page of canceristhefourth most colorectalcommonlydiagnosed cancer in the united states while there ishigh potential for dietary patterns as a modifiable riskfactor for colorectal cancer development [ ] verylimited evidence exists among colorectal cancer survivors for example in a recent review we identified s published up to that reported on theassociation between dietary patterns and colorectalcancer development but only about five s onthe association between dietary patterns and outcomesamong colorectal cancer survivors [] the evidenceshowed that the western dietary pattern often characterized by high intakes ofred andprocessed meats desserts and potatoesis associatedwith higher risk of allcause mortality but generally notwith colorectal cancerspecific mortality in patients withcolorectal cancer the prudent dietary pattern oftencharacterized by high intakes of fruits vegetables wholegrains and poultry showed similar results with inverseassociations for allcause mortality but no consistent association with colorectal cancerspecific mortality []higher adherence to other dietary patterns including themediterranean diet score dietary approaches to stophypertension meal plan american cancer society cancerprevention guidelines score healthy eating index scorewere generally associated with lower risk of allcausemortality but the associations were inconsistent acrossstudies [ ]refined grainschronic diseasesfurther research is therefore needed to clarify ifdietary patterns are importantfor colorectal cancerprognosis and if dietary changes can maximally impactoverall and cancerspecific survival biomarkerbaseddietary patterns may be helpfulin this regard forexample hyperinsulinemia and insulin resistance areconsidered important underlying mechanisms linkingpoor dietary patterns and lifestyle behaviors to the development of multipleincludingcolorectal cancer [] studies have shown positiveassociations between circulating cpeptide concentrations a marker of beta cell secretory activity and colorectal cancer risk and prognosis [] therefore adietary pattern associated with hyperinsulinemia may bemore predictive of outcomes following colorectal cancerdiagnosis than a dietary pattern not associated with thispathway we previously derived the empirical dietaryindex for hyperinsulinemia edih score to assess thepotential of dietary patterns to influence insulinemia which has been extensively applied in large cohortstudies and found to be predictive of nonfasting cpeptide concentrations [ ] longterm weight gain risk of developing colorectal cancer other digestive system cancers and other cancers however the influence of dietary insulinemic potentialon cancer survival outcomes has not yet been evaluatedthe objective of the current study is to apply the edihscore in a large cohort of colorectal cancer patients toassess the insulinemic potential of their dietary patternsafter diagnosis and determine its influence on survivaloutcomesmethodsstudy populationwe used data from the nurses health study nhs andthe health professionals followup study hpfs twoongoing cohorts in the united states hpfs was initiatedin and enrolled male health professionalsbetween the ages of and years nhs initiatedin enrolled registered female nurses aged to years fig data on medical lifestyleand other healthrelated factors was collected at baselineand have been updated every years thereafter ethicalapproval for our study was provided by the harvardth chan school of public health and those of participating registries as required and the institutional reviewboards of the brigham and womens hospital studyparticipants provided consent by completing and submitting study questionnaires participants were free toterminate participation in the study at any timeassessment of diet and the empirical dietary index forhyperinsulinemia edih scorein both cohorts diet was assessed using a validated selfadministered food frequency questionnaire ffq thatassessed how often on average participants consumed astandard portion size of various foods in the past yearin the nhs diet was assessed in andevery years thereafter whereas in the hpfs diet wasassessed in and every years thereafter theedih score developed to empirically measure the insulinemic potential of whole diets using food groups hasbeen described in detail briefly thirtynine foodgroups were entered into stepwise linear regressionmodels to identify a dietary pattern most predictive ofplasma cpeptide levels the edih score represents aweighted sum of food groups with higher scores indicating hyperinsulinemic diets hyperinsulinemia andlower scores indicating low insulinemic diets the foodgroups contributing to lower edih scores are winecoffee fullfat dairy products whole fruit and green leafyvegetables whereas the food groups contributing to highedih scores arelowfat dairy products french frieslowenergy beverages cream soups processed meat redmeat margarine poultry nondark fish high tomatoesenergy beverage and eggs in the current study we calculated edih scores foreach participant based on the selfadministered ffqspostdiagnosis edih score was calculated based on the 0ctabung bmc cancer page of fig flow chart describing the flow of participants from the full cohorts to the final analytic sample in the nurses health study nhs andhealth professionals followup study hpfsfirst ffq returned at least months but not more than years after colorectal cancer diagnosis thus avoidingdiet assessment during active cancertherapy themedian time from diagnosisto postdiagnosis dietassessment was years prediagnosis edih score wascalculated based on the cumulative average of edihscores up to the last diet assessment before colorectalcancer diagnosis the median time from prediagnosisdiet assessment to diagnosis was yearspatients with colorectal cancer and mortality assessmentwhen a colorectal cancer diagnosis was reported duringthe previous years on the followup biennial questionnaires we requested permission to obtain hospital records and pathology reports blinded study physiciansthen reviewed these records and recorded data on tumorcharacteristics for nonrespondents the national deathindex was used to identify deaths and ascertain anydiagnosis of colorectal cancer that contributed to deathafter years of followup for disease diagnoses in nhs and to in hpfs we identified patients with pathologically confirmed colorectalcancer we excluded participants who died before in nhs or in hpfs had reported any cancerexcept nonmelanoma skin cancer before colorectalcancer diagnosis who died at diagnosis who did nothave prediagnosis diet or postdiagnosis diet patientswho did not complete a diet assessment between months and years after diagnosis or had diet assessedoutside of this period who had diabetes at colorectalcancer diagnosis and patients with stage iv or unknownstage at diagnosis therefore the current analysis included patients with stage i ii or iii colorectal cancerincluding participants from hpfs and from nhs fig deaths were ascertained throughreporting by family for persistent nonresponders wequeried the national death index with their names up 0ctabung bmc cancer page of to june for nhs and january for hpfs cause of death was assigned by blinded physicianscovariate assessmentboth cohorts assessed covariate data eg medical historylifestyle and health factorsthrough selfadministeredquestionnaires every years these factors included physical activity smoking habits alcohol intake multivitaminuse endoscopy status regular use of aspirin and othernonsteroidal antiinflammatory drugs nsaids familyhistory of colorectal cancer weight height menopausalstatus and postmenopausal hormone use only forwomenin both cohorts as previously described dietassessment was conducted every years [ ]statistical analysiswe categorized the edih score into quintiles withcohortspecific cutoffs then pooled the data for analysispersontime of followup was calculated from the dateof postdiagnosis diet assessment to death or to lastfollowup date january in hpfs or june innhs whichever was first we used the kaplanmeiermethod to generate survival curves by quintiles of edihscore and tested group differences highest vs lowestquintile using the logrank test for this test the edihscore was adjusted for total energy intake and bmi usingthe residual methodcox proportional hazards regression was used to calculate hazard ratios hrs of colorectal cancerspecificdeath or allcause death in edih quintiles quintile cutpoints were created separately by sex and applied in thepooled sample given that participants must survivefrom diagnosis until postdiagnosis diet assessment weused time since diagnosis as the underlying time scale toaccount for left truncation due to staggered entry thecox models were tested for the assumption of proportionality using timecovariate interaction terms andstratified by age sex and stage we fitted two models tothe data as follows model minimally adjusted modelincluded bmi demographic factors sex age at diagnosis and tumor characteristics stage subsite within thecolon grade of tumor differentiation model fullyadjusted model included all the covariates in model and postdiagnosisfactors packyears ofsmoking physical activity regular aspirin use pre topostdiagnosis weight change total alcohol intake andprediagnosis dietary pattern edih score testforlinear trend of risk across edih quintiles was performedusing the median postdiagnosis edih score in eachedih quintile as a continuous variable in the cox regression models and interpreting the pvalue of thisvariable as the pvalue for linear trendlifestyleto determine how changes in the insulinemic potential of diet before and after diagnosis influence survivalwe dichotomized pre and postdiagnosis edih scores atthe median and used to create a change variable withlow indicating a score below the median and high ascore above the median lowlow consistently lowdietary insulinemic potential before and after diagnosisie both scores below the median lowhigh patientsconsuming low insulinemic diets before diagnosis andmore hyperinsulinemic diets after diagnosis highlowpatients consuming hyperinsulinemic diets before diagnosis and then changed towards low insulinemic dietsafter diagnosis and highhigh patients who consistentlyconsumed hyperinsulinemic dietary patterns before andafter diagnosis we then applied these dietary patternchanges in multivariableadjusted cox models to examine risk of death from colorectal cancer and from othercauseschange preand postdiagnosisfrom postdiagnosis weightwe conducted exploratory subgroup analysesincategories of the following potential effect modifiers sexweightand prediagnosis edih score we categorized prediagnosisedih at the median median and ¥ median weightchange was calculated by subtracting prediagnosisweightthe continuousweight change variable was categorized as follows thosewho gained more than kg had a stable weight kg to kg or lost more than kg we also conducted subgroup analyses by time since diagnosis years ¥ years and age at diagnosis years ¥years tests of interaction between postdiagnosis edihscore and the potential effect modifiers were assessed byentering in the modelthe cross product of postdiagnosis edih score and the stratification variable andevaluated by the wald test all analyses were performedusing sas for unix all pvalues were two sidedresultscharacteristics of patients women with colorectal cancer after diagnosis are shown in table meanage at diagnosis was years and mean postdiagnosisbmi was kgm2 with of patients classified asoverweight or obese regarding disease stage hadstage i or ii and had stage iii disease during amedian followup of years there were allcausedeaths which included colorectal cancerspecificdeaths median overall survival by cancer stage was years for those with stage i disease years for thosewith stage ii and years for those with stage iiidisease fortyone percent of patients maintained astable weight between kg and kg between theprediagnosis and postdiagnosis period while lostmore than kg body weight and gained kgbody weight in the same period colorectal cancer patients with the most hyperinsulinemic dietary patterns 0ctabung bmc cancer page of table postdiagnosis characteristics of colorectal cancer patients by quintiles of postdiagnosis empirical dietary index forhyperinsulinemia edih score n characteristictotal population n quintiles of the empirical dietary index for hyperinsulinemia edih scoreabquintile quintile to to n n quintile to n quintile to n quintile to n female age at diagnosisdage at diagnosis by sexdfemalemalecurrent smoker packyears of smokingbody mass index kgm2overweight bmi ¥ obese bmi ¥ physical activity methweekcphysical activity methweekcd by sex femalemale nonalcohol drinkers regular aspirin use location of cancer in the colon proximal colondistal colonrectumunspecifiedstage at diagnosis stage istage iistage iiimedian survival time yearsmedian survival time by staged years stage istage iistage iiiweight change categoriesd stable weight to kggained more than kglost more than kg avalues are means sd for continuous variables and percentages for categorical variables and are standardized to the age distribution of the study populationbedih scores were adjusted for total energy intakecmetabolic equivalents from recreational and leisuretime activitiesdvalue is not age adjustedafter diagnosis quintile tended to have higher bodyweight and lower physical activity for example theaverage bmi among those classified in quintile was kgm2 and the average physical activity was methourweek compared with kgm2 and methourweek among those in quintile alsopatients consuming the most hyperinsulinemic dietarypatterns were less likely to have stage i disease and theyexperienced shorter survival times table patients consuming low insulinemic dietary patternshad higher intakes of wholegrains nuts vegetables wholefruits and coffee and lower intakes of refined grainscream soup eggs french fries butter margarine sugarsweetened beverages red meat and processed meat in 0ctabung bmc cancer page of table median 5th 95th percentile food and nutrient intake profiles of colorectal cancer patients by quintiles of postdiagnosisempirical dietary index for hyperinsulinemia edih scoretotal population n quintiles of the empirical dietary index for hyperinsulinemia edih scoreabquintile n quintile n quintile n quintile n quintile n foods servingsweekprocessed meatred meat highenergy sugary beverages lowenergy sugary beverages margarinebutterfrench friesnondark fisheggslowfat dairycream souprefined grainstomatopoultrydark fishfullfat dairycoffeeteawhole fruitfruit juicepotatoesgreenleafy vegetablesdarkyellow vegetablesother vegetablesnuts total alcohol intake drinksweek whole grainsnutrient profile total carbohydrates gdtotal protein gd branchedchain amino acids gd total fat gdtotal fiber gd avalues are means sd for continuous variables and percentages for categorical variables and are standardized to the age distribution of the study populationbedih scores were adjusted for total energy intaketerms of the nutrient profile resulting from this postdiagnosis dietary pattern patients consuming a lowinsulinemic dietary pattern had higher intakes of totalcarbohydrates and total fiber and lower intakes of total fattotal protein and branchedchain amino acids table kaplanmeier curves by quintiles of edih score areshown in fig with patients consuming a lowinsulinemic diet quintile experiencing better survivalfor colorectal cancerspecific and overall mortalitycompared to those consuming hyperinsulinemic dietsquintile in the multivariableadjusted analyses wefound that a hyperinsulinemic postdiagnosis dietarypattern was associated with higher risk of colorectalcancerspecific mortality and allcause mortality table 0ctabung bmc cancer page of fig kaplanmeier curves of a colorectal cancerspecific and b overall survival among patients with colorectal cancer by quintile of postdiagnosis empirical dietary index for hyperinsulinemia edih score logrank pvalues were calculated to test group differences quintile vs and adjusted for postdiagnosis total energy intake and postdiagnosis body mass indexcomparing colorectal cancer patients classified in the highestedih quintile to those in the lowest quintile there was a higher risk of colorectal cancerspecific death hr 95ci ptrend and a higher risk of allcause death hr 95ci ptrend afteraccounting for prediagnosis dietary insulinemic potentialamong other confounding variables table in relation to changes in the insulinemic potential ofthe diet before and after diagnosis patients whoconsumed a more hyperinsulinemic dietary patternconsistently before and after diagnosis were at higherrisk of dying from colorectal cancer hr ci and from other causes hr ci compared to patients who consistentlytable hazard ratios ci for colorectal cancerspecific and allcause mortality among patients with colorectal cancer byquintile of postdiagnosis edih scorestatistical modelcolorectal cancerspecific mortalityquintiles of the empirical dietary index for hyperinsulinemia edih scorequintile quintile quintile quintile ptrendquintile deathspatients aliveminimallyadjusted model reference fully adjusted model reference allcause mortalitydeathspatients aliveminimallyadjusted model reference fully adjusted model reference the minimallyadjusted models was adjusted for age at diagnosis postdiagnosis body mass index total energy intake sex race year of diagnosis cancer stagegrade of tumor differentiation and location of primary tumor within the colon the fullyadjusted model was additionally adjusted for postdiagnosis physicalactivity postdiagnosis pack years of smoking postdiagnosis regular aspirin use weight change pre to postdiagnosis postdiagnosis total alcohol intake andprediagnosis edih score 0ctabung bmc cancer page of fig hazard ratios for the association of change in dietary insulinemic potential between prediagnosis diet and postdiagnosis diet and risk ofdying form colorectal cancer crcsurvival and from all causes combined overall survival edih scores were dichotomized at the median lowlow the reference category represents participants who persistently consumed low insulinemic diets below the median edih from pre to postdiagnosis period lowhigh are those who changed from low insulinemic diets towards more hyperinsulinemic diets highlow represents thosewho changed from consuming hyperinsulinemic diets prior to diagnosis towards consuming low insulinemic diets after diagnosis whereas highhigh represents those who persistently consumed hyperinsulinemic diets prior to diagnosis and after diagnosis the number of deaths patientsalive in the four categories were as follows crcsurvival lowlow lowhigh highlow highhigh overallsurvival lowlow lowhigh highlow highhigh models were adjusted for age at diagnosis postdiagnosisbody mass index total energy intake sex race year of diagnosis cancer stage grade of tumor differentiation location of primary tumor withinthe colon postdiagnosis physical activity postdiagnosis pack years of smoking postdiagnosis regular aspirin use weight change pre to postdiagnosis postdiagnosis total alcohol intake and prediagnosis edih scoreconsumed a low insulinemic dietary pattern before andafter diagnosis fig in subgroups of potential effect modifiers risk of colorectal cancerspecific mortality was significantly elevatedamong women and among those who lost body weightthose who were consuming a hyperinsulinemic dietarypattern before diagnosis and those younger than years for these subgroup analysesinteractions werestatistically significant only for sex in allcause mortalitytable discussionin the current study we showed that habitual consumption of hyperinsulinemic dietary patterns after colorectalcancer diagnosis or consumption of a hyperinsulinemicdietary pattern consistently before and after diagnosiswas associated with higher risk of dying from colorectalcancer and from all causes combinedthe insulinemic potential of diet was first estimatedby the insulin index which is based on a conceptsimilar to the more widely used glycemic index thatcharacterizes carbohydratecontaining foods accordingto their ability to raise blood glucose concentrationspostprandially compared with a reference food glucoseor white bread though carbohydrate content isone important factor influencing insulin response foodscan also stimulate insulin secretion in a carbohydrateindependent manner the insulin index directly quantifies the postprandial insulinemic potential of a food andtakes into account foods with a low or no carbohydratecontent it is important to understand that theinsulin index which was used in most previous studiesof dietary insulinemic potential and colorectal cancersurvival is conceptually and technically different fromthe edih and essentially uncorrelated spearman r the principle ofthe insulin index is how aparticularfood item stimulated insulin secretionindependent of underlying insulin resistance whereasthe edih is primarily driven by insulin resistance forcolorectal cancer the only other paper using the edihwas on cancer incidence both the insulin index and glycemic index assess thepostprandial shortterm effects of the diet unlike theedih score which predicts integrated insulin exposureie both fasting and nonfasting based on habituallongterm dietary intake postdiagnosis insulinindex and insulin load have been linked to higher risk ofdying from colorectal cancer [ ] higher dietary insulin load and insulin index after diagnosis of colorectalcancer were associated with increased risk of colorectalcancerspecific and overall mortality the association of postdiagnosis glycemic indices with colorectal 0ctabung bmc cancer page of table subgroup analyses of the association between dietary insulinemic potential and colorectal cancerspecific and allcausemortalitysubgroupptrendpinteractionquartile quartile quartile deathspatientsaliveedih quintilesquartile colorectal cancerspecific mortalitysexmenwomenweight change post minus prediagnosis weightstable weight to kgweight gain kgweight loss kgprediagnosis edih score median¥ medianage group at diagnosis years¥ yearstime since diagnosis years¥ yearsallcause mortalitysexmenwomenweight change post minus prediagnosis weightstable weight to kgweight gain kgweight loss kgprediagnosis edih score median¥ medianage group at diagnosis years¥ yearstime since diagnosis years¥ years ref ref ref ref ref ref ref ref ref ref ref ref ref ref ref ref ref ref ref ref ref ref models were adjusted for age at diagnosis postdiagnosis body mass index total energy intake sex race year of diagnosis cancer stage grade of tumordifferentiation and location of primary tumor within the colon postdiagnosis physical activity postdiagnosis pack years of smoking postdiagnosis regular aspirinuse weight change pre to postdiagnosis and postdiagnosis total alcohol intake and prediagnosis edih scorecancer prognosis has been inconsistent whereas onestudy found higher risk of colorectal cancer recurrenceand death associated with higher glycemic load but nothigher glycemic index another found no associationbetween glycemic load or glycemic index and colorectalcancer survival glycemic scores are primarilyreflective ofthe postprandial glucose responses ofcarbohydratecontaining foods whereas the edih scoredirectly reflects insulin increases induced by componentsof the dietary pattern that may or may not be contributing to calories eg coffee current study findingstherefore suggest that the direct effect of the diet on 0ctabung bmc cancer page of insulin may be more important than the effect of diet onglucose for colorectal cancer prognosis though theglycemic index is a measure of the shortterm postprandial effect of the diet on glucose concentrations it ispossible that such a habitual dietary pattern could overtime lead to sustained hyperinsulinemia and insulin resistance which could then mediate colorectal cancerprognosis however a previous study in these cohortsdid not observe an association between an overall lowcarbohydrate diet score and colorectal cancer or overallmortality although those who consumed a plantrichlowcarbohydrate diet which emphasized plant sourcesof fat and protein with moderate consumption of animalproducts had lower risk of colorectal cancerspecificmortality insulin is a growth factor and major regulator of cellmetabolism and its effects in target cells are mediatedby the insulin receptor a transmembrane protein withenzymatic activity evidence suggest that insulinstimulates growth mainly via its own receptor and notthe igf1 receptor and that in many cancer cells theinsulin receptor is overexpressed and the a isoformwhich has a predominant mitogenic effectis morerepresented than the b isoform the metabolicpathway stimulated by the activated insulin receptor toregulate glucose protein and lipid metabolism involvesthe pi3kakt pathwaycharacteristicsprovide a selective growth advantage to cancer cellswhen exposed to insulin therefore all conditions ofhyperinsulinemia both endogenousdiabetes metabolic syndrome obesity and exogenouseg hyperinsulinemic diets which also influence someof the endogenous conditions [ ] willincreasecancer risk and mortality theseegtypefor most ofalthough interactionsthe subgroupanalyses were not statistically significant some of thefindings merit some discussion the associations werestronger among women than among men which may berelated to severalfactors the larger sample size andstatistical power in our evaluation of women potentialconfounding with age as women were younger on average than men and a true biological interaction basedupon endocrine and associated metabolic factors wealso observed that there were worse outcomes amongpatients who lost weight than among those who maintained a stable weightto postdiagnosisperiod which may be consistent with complications ofprogressing disease leading to poor diet intakefrom premajor strengths of our study include the use of afoodbased edih score that is correlated with circulating cpeptide concentrations [ ] we had access tocomprehensive pre and postdiagnosis data on diet andimportant covariates which reduces the potential for residual confounding and recall bias our findings alsoaccounted for potential bias from staggered entry due todifferences between participants in the time betweendiagnosis and postdiagnosis diet assessment limitations to be considered in interpreting our findings include potential measurement error in the selfreporteddietary and lifestyle data though prior studies in thehpfs and nhs that evaluated the relative validity offfq data have shown reasonably good correlations between ffq and diet records [ ] though we adjusted for several potential confounding variables ahyperinsulinemic dietary pattern may be associated withother factors not included in the current study therefore we cannot completely rule out confounding byunmeasured variables given that we did not have information on cancer treatment which could influence dietary choices of cancer patients or modify the diet andsurvival association we adjusted all analyses by cancerstage at diagnosis which is the principal determinant ofcolorectal cancer treatmentin this large prospective study a higher edih scorereflecting higher insulinemic potential of the diet wasassociated with higher risk of death from colorectalcancer and from all causes taken together our resultssuggest that this association may be mediated partlythrough mechanisms involving hyperinsulinemia interventions with dietary patterns to reduce insulinemia mayenhance survivorship among colorectal cancer patientsabbreviationsbmi body mass index ci confidence interval edih empirical dietary indexfor hyperinsulinemia score ffq food frequency questionnairehpfs health professionals followup study hr hazard ratio methourweek metabolic equivalent hours per week nhs nurses health study nsaids nonsteroidal antiinflammatory drugs pi3kakt phosphatidylinositol kinaseprotein kinase b sas® statistical analysis software®acknowledgementswe would like to thank the participants and staff of the nurses health studyand health professionals followup study for their valuable contributions asw | Colon_Cancer |
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 gouttefangeas c actively personalized vaccination trial for newly diagnosed glioblastomanature 101038s415860180810y ott pa hu zt keskin db shukla sa sun j bozym dj animmunogenic personal neoantigen vaccine for patients with melanomanature 101038nature22991 sahin u derhovanessian e miller m kloke bp simon p lower m personalized rna mutanome vaccines mobilize polyspeciï¬c therapeuticimmunity against cancer nature 101038nature23003 carreno bm magrini v beckerhapak m kaabinejadian s hundal jpetti aa a dendritic cell vaccine increases the breadth anddiversity of melanoma neoantigenspeciï¬c t cells science 101126scienceaaa3828 tanyial personalized cancerjl bobisse s ophir e tuyaerts s roberti a genolet rantitumorett cell10eaao5931 101126scitranslmedaao5931eï¬ectively mobilizessci transl medimmunityovarianvaccineincancer van rooij n van buuren mm philips d velds a toebes m heemskerkb tumor exome analysis reveals neoantigenspeciï¬c tcell reactivity rizvi na hellmann md snyder a kvistb p makarov v havel jjlandscape determines sensitivity to pd1 blockade in mutationalnonsmall cell lung cancer science 101126scienceaaa1348 van allen em miao d schilling b shukla sa blank c zimmer l genomic correlates of response to ctla4 blockade in metastatic melanomascience 101126scienceaad0095 dudley me wunderlich jr robbins pf yang jc hwu p schwartzentruberafterregression and autoimmunity in patientsal cancerrepopulation withdjetclonal 101126science1076514lymphocytesantitumorscience dudley me yang jc sherry r hughes ms royal r kammula u adoptive cell therapy for patients with metastatic melanoma evaluation ofintensive myeloablative chemoradiation preparative regimens j clin oncol 101200jco2008165449 rosenberg sa yang jc sherry rm kammula us hughes ms phan gq durable complete responses in heavily pretreated patients with metastaticmelanoma using tcell transfer immunotherapy clin cancer res 10115810780432ccr110116 besser mj shapirafrommer rlevy d adoptive transfer ofpatients with metastatic melanomaitzhaki o treves aj zippel dbtumorltrating lymphocytes inintenttotreat analysis and eï¬cacyfrontiers in oncology wwwfrontiersinaugust volume 0cli and dingisolating neoantigenspeciï¬c t cellsafter failure to prior immunotherapies clin cancer res 10115810780432ccr130380 andersen r donia m ellebaek e borch th kongsted piversentz longlasting complete responses in patients with metastaticmelanoma after adoptive cell therapy with tumorltrating lymphocytesand an attenuated il2 regimen clin cancer res 10115810780432ccr151879 stevanovic s draper lm langhan mm campbell te kwong mlwunderlich jr complete regression of metastatic cervical cancer aftertreatment with human papillomavirustargeted tumorltrating t cells jclin oncol 101200jco2014589093 huang j elgamil m dudley me li yf rosenberg sa robbins pf tcells associated with tumor regression recognize frameshifted products ofthe cdkn2a tumor suppressor gene locus and a mutated hla class i geneproduct j immunol 104049jimmunol172106057 zhou jh dudley me rosenberg sa robbins pf persistence of multipletumorspeciï¬c tcell clones is associated with complete tumor regression ina melanoma patient receiving adoptive cell transfer therapy j immunother lu yc yao x li yf elgamil m dudley me yang jc mutatedppp1r3b is recognized by t cells used to treat a melanoma patientwho experienced a durable complete tumor regression j immunol 104049jimmunol1202830 robbins pf lu yc elgamil m li yf gross c gartner j mining exomic sequencing data to identify mutated antigens recognizedby adoptively transferred tumorreactive t cells nat med 101038nm3161 lu yc yao x crystaljs li yf elgamil m gross c eï¬cient identiï¬cation of mutated cancer antigens recognized by t cellsassociated with durable tumor regressions clin cancer res 10115810780432ccr140433 prickett td crystal js cohen cj pasetto a parkhurst mr gartner jj durable complete response from metastatic melanoma after transfer ofautologous t cells recognizing mutated tumor antigens cancer immunolres 10115823266066cir150215 stevanovic s pasetto a helman sr gartner jj prickett td howieb landscape ofin successfulimmunotherapy of virally induced epithelial cancer science 101126scienceaak9510immunogenic tumor antigens tran e turcotte s gros a robbins pf lu yc dudley me cancerimmunotherapy based on mutationspeciï¬c cd4t cells in a patient withepithelial cancer science 101126science1251102 yossef r tran e deniger dc gros a pasetto a parkhurst mr enhanced detection of neoantigenreactive t cells targeting unique andshared oncogenes for personalized cancer immunotherapy jci insight 101172jciinsight122467 vinay ds kwon bs role of 41bb in immune responses semin immunol 101006smim19980157 wattstthcelltnftnfrresponsesfamilyannuof 101146annurevimmunol23021704115839revimmunolmembersincostimulation cannons jl lau p ghumman b de benedette ma yagita h okumurak 41bb ligand induces cell division sustains survival and enhanceseï¬ector function of cd4 and cd8 t cells with similar eï¬cacy j immunol 104049jimmunol16731313 halstead es mueller ym altman jd katsikis pd in vivo stimulation ofcd137 broadens primary antiviral cd8 t cell responses nat immunol 101038ni798 wolï¬ m kuball j ho wy nguyen h manley tj bleakley m activationinduced expression of cd137 permits detection isolation andexpansion of the full repertoire of cd8 t cells responding to antigenwithout requiring knowledge of epitope speciï¬cities blood 101182blood200611056168 ye qral cd137song dg poussin m yamamoto t best a li csnaturallyetoccurring tumorreactive t cells in tumor clin cancer res 10115810780432ccr130945accuratelyidentiï¬esenrichesandfor parkhurst m gros a pasetto a prickett t crystaletalisolation of tcelltumorassociatedpmutatedlymphocytes based on cd137 expression clin cancer res 10115810780432ccr162680speciï¬callyfromreceptorsantigensjs robbinsreactive withtumorltrating seliktaroï¬r s merhavishoham eitzhaki o yunger s markel gschachter j selection of shared and neoantigenreactive t cells foradoptive cell therapy based on cd137 separation front immunol 103389ï¬mmu201701211 makkoukacancerchesterimmunotherapyccd137 101016jejca201509026kohrt herationaleforeurj canceranti chen l coinhibitory molecules of the b7cd28 family in the control oftcell immunity nat rev immunol 101038nri1349 greenwaldrjfreemangjsharpeahfamilyimmunol 101146annurevimmunol23021704115611revisitedannurevtheb7 tran e robbins pf lu yc prickett td gartner jj jia l tcelltransfer therapy targeting mutant kras in cancer new engl j med 101056nejmoa1609279 sharpe ah wherry ej ahmed r freeman gj the function of programmedcell death and its ligands in regulating autoimmunity and infection natimmunol 101038ni1443 zacharakis n chinnasamy h black m xu h lu yc zheng zl immune recognition of somatic mutations leading to completedurable regression in metastatic breast cancer nat med 101038s4159101800408 gros a robbins pf yao x li yf turcotte s tran e pd1identiï¬es the patientspeciï¬c cd8 tumorreactive repertoire ltratinghuman tumors 101172jcij clin invest stronen e toebes m kelderman s van buuren mm yang ww van rooijn targeting of cancer neoantigens with donorderived t cell receptorrepertoires science 101126scienceaaf2288 yarchoan m johnson ba lutz er laheru da jaï¬ee em targetingneoantigens to augment antitumour immunity nat rev cancer 101038nrc2016154 tran e robbins pf rosenberg sa final common pathway of human cancerimmunotherapy targeting random somatic mutations nat immunol 101038ni3682 schumacher tn schreiber rd neoantigens in cancer immunotherapyscience 101126scienceaaa4971 inozume t hanada ki wang qj ahmadzadeh m wunderlich jr rosenbergsa selection of cd8pd1 lymphocytes in fresh humanmelanomas enriches for tumorreactive t cells j immunother 101097cji0b013e3181fad2b0 ahmadzadeh m johnson la heemskerk b w | Colon_Cancer |
microbiota involves communities ofhepatitis is generally known as an ammation of the liver that can be caused by hepaticand nonhepatic viruses can be caused by alcohol can be drug induced and can be caused byautoimmunity gut microbiota composition is known to be associated with disease pathogenesishowever dynamic alteration of the gut microbiota in disease pathogenesis is not wellunderstoodsymbiotic as well as pathogenicmicroanisms found in anisms ie plants and animals microbiota of a healthy individualshows more of commensalism or symbiosis without causing any disease these microbes mainlycolonize humans during birth or shortly thereafter and remain throughout the course of life thesecan be found in many areas like skin respiratory tract urinary tract and digestive tract whilebrain lungs and the circulatory system are free of microbes approximately microbes arepresent in a healthy individual gut minemura and shimizu therefore gut microbiota hasan important role to modulate the immune system in disease progression or recoverycommensaltranslocation of microbes or their metabolic products cause intestinal ammation leadingto impairment of the primary barrier hill there is limited available informationregarding the role of gut microbiota in hepatitis which makes it important to majorly focus onclinical data of gut microbiota linked with hepatitis b and c virusgut microbiotagut or gastrointestinal tract starts from the mouth and ends at the back passage anus gut helpsin the digestion of food by absorbing energy and nutrients majority of gut microbiota to contains good bacteria and only to are harmful bacteria in diï¬erent parts of the intestineedited bymilan surjittranslational health science andtechnology institute thsti indiareviewed byjawed iqbaljamia millia islamia indiabinod kumarloyola university chicagounited statescorrespondencenirupma trehanpatitrehanpatigmailcomspecialty sectionthis was submitted tovirus and hosta section of the frontiers in cellular and infectionmicrobiologyreceived march accepted june published august citationsehgal r bedi o and trehanpati n role of microbiota inpathogenesis and management ofviral hepatitisfront cell infect microbiol 103389fcimb202000341frontiers in cellular and infection microbiology wwwfrontiersinaugust volume 0csehgal microbiota and liver diseasesbajaj in mouth and upper respiratory tract normalï¬ora is more of the commensal bacteria like streptococcusmoraxella neisseria and haemophilus very few species ofbacteria are present in the stomach and small intestine whilethe large intestine and colon contain dense population ofmicrobes ie up to cellsg along with bacteria many othermicroanisms like fungi protists archaea and viruses alsosymbiotically harbor in the gutthere are four dominant phyla of bacteria present in thegut and they are firmicutes bacteroidetes actinobacteriaand proteobacteria khanna and tosh most importantgenera in which bacteria belong are bacteroides clostridiumfaecalibacterium eubacterium ruminococcus peptococcuspeptostreptococcus and biï¬dobacterium fern¡ndez some of the fungal species that also coexist in the gut arecandida saccharomyces aspergillus penicillium rhodotorulatrametes pleospora sclerotinia bullera and galactomycesamong others raimondi functions of gut microbiotagut microbiota plays an important but diverse role such asbarrier eï¬ect vitamin synthesis and fermentation residentbacteria of the gut acts as a barrier and protect the intestinalmucosa from invasion of the other potential pathogens hooper many factors including diet age medicationillness stress and lifestyle uence the gut microbiota whichhave a great impact on disease pathogenesis in fact manybacteria ie bacteroides eubacterium propionibacterium andfusobacterium are instrumental in the synthesis of vitamins kand b ie folate b12 and biotin canny and mccormick they are also involved in the fermentation of nondigestible carbohydrates for the production of shortchain fattyacids scfas which are helpfulin maintaining metabolichomeostasis in addition to the production of scfa glycolysisand pentose phosphate pathway also produce butyrate whichpromotes the growth of lactobacilli and biï¬dobacteria bacteriain the colon venegas various studies supported thefact that nutrients derived from microbiota play a pivotal rolein the normal functioning of the hepatic system li zheng moratalla jiminez cremer wang 2017agut microbiota in liver diseasescommensal bacteria play a decisive role in maintaining immunehomeostasis figure and also guard immune reactions atmucosal surfaces ichinohe intestinal microï¬ora isa dynamic and complex ecosystem which helps in proliferationgrowth and diï¬erentiation of epithelial cells to ï¬ght infectionsand improve immunity despite its crucial role in the synthesisfolate scfa and peroxides gut microbiotaof vitamin kacts as a chief environmental as well as etiologicalfactorfor the progression of many liver diseasesohara andshanahan particularly gut microbiota has a largeruence on alcoholic liver disease nonalcoholic fatty liverdisease viral hepatitishepatitis b and c autoimmunehepatitis aih primary sclerosing cholangitis psc andprimary biliary cholangitismohamadkhani pbclactobacillus biï¬dobacterium saccharomyces boulardii andlactobacillus plantarum play a bigger role in the managementof various metabolic disorders and hepatitis mohamadkhaniincludingvirusesandseveralpathogensintestinalmicroanisms use mucous membranes as a doorwaykarst hepatic viruses breach the intestinal permeabilityleading to gut dysbiosis and release proammatory cytokinesinstrumental in developing liver cirrhosis and hcc it is alsoobserved that the use of probiotics reduces the tolerogenicresponse and enhances the mucosal defense against viralpathogens rigoadrover m del lactobacillusalone can uence the production of interferon by modulatingthe antiviral eï¬ects of vitamin a lee and ko themixture of various probiotics and biï¬dobacterium with galactooligosaccharides and fructooligosaccharides has a defensiveeï¬ect against rotavirus infection by aggregating the productionof tnfα il4 ifnÎ and tlr2 expression rigoadrover mdel in most of the liver disease especially cirrhosisdysbiosis of the gut increases proteobacteria enterobacteriaceaeand veillonellaceae while it decreases bacteroidetes andlachnospiraceae sanduzzi zamparelli recentlythe cirrhosis dysbiosis ratio cdr is coined for deï¬ningthe changes in gut microbiome in cirrhosis patients withbeneï¬cial lachnospiraceae and ruminococcaceae and harmfulenterobacteriaceae bacteria bajaj other groupshave also associated patients with severe cirrhosis and hepaticencephalopathy with overgrowth of enterobacteriaceae bacteriachen role of gut microbiota in hepaticviral infectionsacute viral hepatitis due to hepatitis a and e viral infectionsis a major community health problem especially in developingcountries hepatitis a and e cause acute infection whichcould be shortlived and selfclearing unless the subjects areimmunocompromised or in transplant settings acute hepatitise infection also becomes detrimental and lifethreatening duringpregnancy aï¬ecting both the mother and the childboth hepatitis a and e are rna viruses thattransmitthrough oral fecal routes lemon and may havedevastating eï¬ects on intestinal microï¬ora it was observedthat administration of the healthy probiotic bacterium likeenterococcus faecium ncimb aï¬ects the reduction as wellas the removal of enteric hev viruses in pigs kreuzer however there is lack of relevant data in humansas per the world health anization who hepatitisb virus hbv infection caused deaths in and million diagnosed with chronic infection in similarly hepatitis c virus hcv caused deaths with anestimated million diagnosed with chronic infection in both these viruses cause chronic infections at in hbv andmore than in hcv leading to cirrhosis and hepatocellularcarcinoma hepatic viruses have evolved mechanisms to avoidtheir detection from the host innate and adaptive immunityfrontiers in cellular and infection microbiology wwwfrontiersinaugust volume 0csehgal microbiota and liver diseasesfigure protective role of fecal microbiota transplantation and use of probiotics in immune restorationand characterized as viral escape visvanathan itis observed that chronic hepatitis patients have largertranslocation of the intestinal microbiota lu li bacterialtranslocation cause intestinalammation viadysregulation of immune cell overgrowth of pathogenic bacteriaas well as dysfunction of the primary barrier hill xu also supported the fact that intestinal ï¬oraloses homeostasis during dysbiosis which in fact helps theadvancement of hepatitis viral infection xu itthereforeis now understood that during chronicitycommensal microbiota have greater impact not only on viral hostcell interaction but also on viral replicationin viral hepatitis few harmful bacteria like escherichia colienterobacteriaceae enterococcus faecalis and faecalibacteriumprausnitzii directly alter the proï¬le of good intestinal microbiotawith a lower number of intestinallactic acid species suchas lactobacillus pediococcus weissella and leuconostoc bajaj chen some of the bacterial species ieneisseria e coli enterobacteriaceae e faecalis f prausnitziiand gemella are also found responsible for the progression ofhepatitis b and c virusrelated cirrhosis and primary biliarycirrhosis chen mohamadkhani candidais also frequently found in patients with hepatitis brelatedcirrhosis cui role of gut microbiota in hepatitis b viralinfectiondysbiosis of gut microbiota in chronic hepatitis b infectionaï¬ects disease pathogenesis and causes liver failure in alarge proportion lps lipopolysaccharides from the outermembrane of gramnegative bacteria help in the activationof innate immune response by recognizing tlrs especiallytlr2 and hbv infection leads to progressive declinein butyrateproducing bacteria however lpsproducinggenera is enriched in hbv infection in hbv infection abeneï¬cial bacterium lachnospiraceae plays a role in themanagement of hbv infection via reduction in lps sectionand bacterialtranslocation chen ren studies have shown the role of faecalibacteriumpseudobutyrivibrioruminoclostridiumprevotella alloprevotella and phascolarctobacterium in potentialantiammatory scfa activity which increases the abundanceof butyrate compared to normal subjects liu lu have demonstrated that copy numbers of fprausnitzii e faecalis enterobacteriaceae biï¬dobacteria andlactic acid bacteria lactobacillus pediococcus leuconostocand weissella have marked variation in the intestine of hbvcirrhotic patients during hbv infection dysbiosis in theoral microbiota was observed and yellow tongue coating issuggestive of a reduction in bacteroidetes but an increaselachnoclostridiumfrontiers in cellular and infection microbiology wwwfrontiersinaugust volume 0csehgal microbiota and liver diseasesin proteobacteria zhao also suggested positivecorrelation of neisseriaceae with the serum hbvdnacirrhotic patients with hbv infection showed a signiï¬cantdecrease in the biï¬dobacteriaceaeenterobacteriaceae be ratiolu while yun observed no diï¬erence in thebe ratio in hbsag with normal or high alt and in noncirrhotic hbv carriers yun it means the be ratiois disturbed only in cirrhosis however other study observedthat the megasphaera genus from the firmicutes phylum wasabundant in the hbsag high alt group than the normal altin patients with normal alt butyrateproducing bacteria likeanaerostipes are more in feces compared to hbsagve yun it is interesting to note that both megasphaeraand anaerostipes produce scfa as a byproduct of lactatefermentation and butyrate however butyrate is known asanticarcinogenic and antiammatory and plays a role inoxidative stress hamer another study suggests thatchronic hepatitis b infected cirrhotic patients exhibit a decreasein biï¬dobacteria and lactobacillus levels while signiï¬cantlyincreasing enterococcus and enterobacteriaceae levels comparedto healthy individualsbacterial translocation is also observed in the developmentof hepatocellular carcinoma hcc recently wang havedeï¬ned the serum zonulin as an intestinal permeability markerand showed its association with afp levels in hbvassociatedliver cirrhosis and hcc they are helpful in correlating it withadvanced stages of the diseases fasano the use of probiotic in hbvinfected patients showed beneï¬tand suggested that probiotic vsl3 plays an important role in themanagement of hbv viral infection dhiman role of gut microbiota in hepatitis c viralinfectionchronic hepatitis c infection is another leading cause ofcirrhosis hcc and in some casesliver failure and deathin majority enterobacteriaceae and bacterioidetes increased inchronic hcv patients but firmicutes found to be decreasedhcv infection cause marked elevation in lps which issuggestive of microbial translocation and ammation duringdisease progression dolganiuc inoue on the other hand it was observed that antiviral treatment ofhcv with ribavirin rbv and immune modulator pegylatedinterferon pegifn has no direct impact on gut dysbiosisin factit increases the production of bile acids which isimportant for gut microbiota ponziani somepathogenic bacteria such as enterobacteriaceae staphylococcusand enterococcus decreased the bile acid in hcvinfectedcirrhotic patients which normalized after a directacting antiviraltreatment oral directacting antivirals daas were also foundto be helpful in improving gut especially lachnospira and doreagenera and restored tnfα levels p©rezmatute but after daa treatment expression of calprotectin zo1and lps was found more in hcv patients with cirrhosis itwas also suggested that during hcv infection l acidophilusand biï¬dobacterium spp can act as a supportive supplementwith antiviral and antibacterial activities dore immune response in hcv patients can be stimulated by usefulmicrobiota via activation of cd3 cells and cd56 nk cellcounts which were explained by doskali and furthersuggested that good ï¬ora increases the cytotoxic eï¬ects of nkcells against viral infected cells inhibiting the replication of hcvuse of probiotics in hcvinfected patients with cirrhosis wassigniï¬cantly beneï¬cial preveden another hepatic virus hepatitis d virus is a new playerand not much is known about it yet it was also suggestedthat endotoxemia in hcv and hdv patientstobe multifactoriallikely depending on impaired phagocyticfunctions and reduced tcellmediated antibacterial activitykefalakes and rehermann seemsmicrobiota modulates molecularsignaling in hepatitisactivereceptorthe keycomponent of gramnegative bacterialps isie enterobacteriaceae thefor lps iscd14tlr4md2 receptor complex on induction whichsecretes many proammatory cytokines including tumornecrosis factorα il1 il6 and chemokines through the nfκbsignaling fooladi seki and schnabl bryant to cause liver injury in the intestinal tract lpsdownregulates the expression of various tight junction proteinszo1 and closed protein by increasing the permeability ofthe intestinal mucosa and enters the blood ï¬ow through theportal venous system park in liver kupï¬er cells asspecialized macrophages are induced by the lpstlr4 pathwayfor the release of immunosuppressive mediators such as il10which in turn suppress the release of ammatory mediatorsby kupï¬er cells dixon in this way during viralhepatitis virus speciï¬c immune responses are suppressed andultimately inhibit eï¬cient clearing of bacteria as well as virusesin addition to lps unmethylated cpg dna bacterialdnarna bacterial cell wall also contains teichoic acidpeptidoglycan and specialized proteins ï¬agellin bacterialdnarna is recognized by tlrs as well as all components ofcellwalllike teichoic acid and peptidoglycan also recognized bytlr2 while tlr5 got activated by ï¬agellin dsrna bacteriaare recognized by tlr3 ssrna activates receptors of bothtlr7 and tlr8 all these tlrs ultimately stimulate the jakstat pathway hepatitis viruses are also recognized by tlrs inthe liver or in the intestine and activate downstream signalingpathways mencin unmethylated cpg dnas are found abundantly in thelactobacillus family ie l casei l plantarum l rhamnosus andothers like biï¬dobacteria proteobacteria and bacteroidetes inthe intestinal ï¬ora of animals unmethylated cpg dna is sensedby tlr9 expressed on various mononuclear cells and stimulatesboth innate immune response as well as adaptive immuneresponse krieg kauppila activation the ofcpgtlr9 pathway stimulates downstream molecules of myd88such as irak4 traf6 and irak1 ultimately triggering nfκb and mapk signaling pathways these downstream pathwayshelp in the activation of dcs for the secretion of cytokines andfrontiers in cellular and infection microbiology wwwfrontiersinaugust volume 0cfrontiersincellluarandifnectionmcroboogyiilwwwfrontiersniaugustlvoumearticeltable randomized fmt clinical trials for the treatment of chronic hepatitis b infectionsnostudy titlestudy typeno ofsubjectsinterventiontreatmentstatusphaseprimary outcomemeasuresrandomized controlled trialcomparing the efï¬cacy andsafety of fmt in hepatitis breactivation leads to acuteon chronic liver failurelocationinstitute of liver and biliarysciencesnew delhi delhi indiastudy on effect of intestinalmicrobiota transplantationin chronic hepatitis blocation zhongshanhospital afï¬liated to xiamenuniversityxiamen fujian chinainterventionalclinical trialdrug tenofovirdrug fecal microbiotatransplantation fmtcompletedcompletedtransplant free survival[time frame months]interventionalclinical trialother intestinalmicrobiota transplantdrug antiviral agentsrecruitingnachange of serum hepatitis bvirus e antigenhbeag level[time frame months]serum hepatitis b virus eantigenhbeag levels ismeasured in scosecondary outcome measuresclinicaltrialsgovsehgaletalidentiï¬ernct02689245nct03429439reduction in hepatitis b virus dnalevel ¥ log [time frame weeks]improvement in meld model forend stage liver disease score[time frame weeks]change of serum hepatitis b virussurface antigenhbsag level [timeframe months] serumhepatitis b virus surfaceantigenhbsag levels is measuredin iumlchange of serum antihepatitis bvirus e antigenantihbe [timeframe months] appearanceof serum antihepatitis b virus eantigenantihbe suggest the abilityof body to resistant hbvchange of serum antihepatitis bvirus surface antigenantihbs[time frame months]appearance of serum antihepatitisb virus surface antigenantihbssuggest the ability of body toresistant hbvchanges of gut microbiota [timeframe months] alpha andbeta diversity of gi microbiota byhighthroughput sequencing 16srrna on baseline line and months after treatmentrelief of constipation [time frame months] relief of diarrhea[time frame months] reliefof abdominal pain [time frame months] the onset andduration of constipation will beassessed by evaluation scoretable of gastrointestinalsymptomsimcrobotaiandlveriidseases 0csehgal microbiota and liver diseaseschemokines krieg kauppila chronic hbvpatients have reduced lactobacillus and biï¬dobacteria both arerich in unmethylated cpg dna levels ultimately aï¬ecting thecpg dnatlr9 pathway and immune response on hbv linand zhang role of fecal microbialtransplantation fmt in viralhepatitisfmt mainly involves the insertion of healthy microbiota in thediseased gut in brief fecal matter derived from a healthy familymember of the patient receiving the same diet as the patient isprocessed and introduced in the intestinal tract of the patientthese have minimal side eï¬ects and proved helpful in reinstatinghealthy gut ï¬ora in the patient fmt administration can bedone using several routes such as oral nasogastric nasoduodenalnasojejunal endoscopic rectal and colonoscopic or midguttransendoscopic enteral tubing cui tang for cirrhotic patients with dysbiosis small bowel route ismost aï¬ected while mostly used route is oral delivery in severealcoholic hepatitis sah in comparison to steroids fmt isassociated with decreased disease severity and improved survivalearlier wang 2017b have observed that fmt restored thecognitive function liver function indexes and tlr response incarbon tetrachloride ccl4induced acute hepatitis in ratswoodhouse have observed in a profit clinical trialthe beneï¬ts of fecal microbiota transplantation in the smallbowel of cirrhotic patients woodhouse meiglani also observed that cirrhotic patients with antibioticresistant clostridioides diï¬cile infection cdi responded wellafter fmt treatment in factfecal microbiota of alcoholresistant mice when given to alcoholsensitive mice has reducedbacteroidetes and increased actinobacteria as well as firmicutesand protected steatosis development ferrere limited studies are published yet on fmt administration inalcoholrelated liver disease however all these studies showedimmense beneï¬t of fmt bajaj observed the recoveryof cognitive function and hepatic encephalopathy in patientsunder clinical trial after administration of fmt studies recentlypublished from our center have found better eï¬ciency of fmtreferencesbajajj s heuman d m hylemon p b sanyal a the cirrhosis dysbiosisb monteith p changescomplicationsin the gut microbiomej hepatolassociated with cirrhosisj white mratio deï¬nesand its101016jjhep2013bajaj j s kassam z fagan a gavis e a liu e cox i j fecal microbiota transplant from a rational stool donor improveshepatic encephalopathy a randomized clinical trial hepatology 101002hep29306bryant c e symmons m and gay n j tolllike receptor signallingthrough macromolecular protein complexes mol immunol 101016jmolimm201406033in severe alcoholic patients than standard medical treatmentsarin there are only a couple of randomizedfmt clinical trials for chronic hepatitis b infected patientstable recently groups have addressed how fmt is modulatingimmunity in gut and liver mucosaassociated invariant tmait cells are found abundant in liver to ofintrahepatic t cells gut peripheral blood as well as lungs gao have observed that functional mait cells were altered insah resulting in more bacterial infection in patients alterationin circulating mait cells is observed with defective antibacterialcytokinecytotoxic response against the infection gao they believe that fmt administration has a profoundeï¬ect on the expression of mait cells in alcoholrelated diseasessummary and conclusionlikeruminoclostridiumgut microbiota has an important role in viral alcoholicand metabolic liver diseases gut microbiota plays a crucialrole in modulating the tolllike receptors nfκb signalingjanus kinasesignal transducer and transcription jakstatpathway and cd4t cell activation numerous usefulmicrobiotasfaecalibacteriumlachnoclostridium prevotella alloprevotella pseudobutyrivibrioand phascolarctobacterium play an important role in potentiatingantiammatory short chain fatty acid scfa activity andincreased the butyrate abundance which play a crucial role inthe management of various hepatitisrelated viral infectionsfecal microbiota transplantation became an attractive andsafest mode of treatment for the management of various liverdiseases especially in severe alcoholic hepatitis despite recentpublications there are still gaps in understanding the role ofmicrobiota in viral hepatitis especially in acute hav and hevviral infections therefore there is a need to explore more inthese infectionsauthor contributionsrs and ob written the review nt provide valuablesuggestions corrected and revised all authors contributed to the and approved the submitted versioncanny g o and mccormick b a bacteria in the intestine helpfulresidents or enemies from within infection and immunity am soc microbiol 101128iai0018708chen y ji f guo j shi d fang d and li l dysbiosis of smallintestinal microbiota in liver cirrhosis and its association with etiology sci rep 101038srep34055chen y yang f lu h wang b chen y lei d characterization of fecal microbial communities in patients with liver cirrhosishepatology 101002hep24423cremer j arnoldini m and hwa t eï¬ect of water ï¬ow and chemicalenvironment on microbiota growth and composition in the human colon procnatl acad sci usa 101073pnas1619598114cui b feng q wang h wang m peng z li p fecalmicrobiota transplantation through midgut for refractory c rohns diseasefrontiers in cellular and infection microbiology wwwfrontiersinaugust volume 0csehgal microbiota and liver diseasessafety feasibility and eï¬cacy trial results j gastroenterol hepatol 101111jgh12727cui l morris a and ghedin e the human mycobiome in health anddisease genome med 101186gm467dhiman r k rana b agrawal s garg a chopra m thumburu k k probiotic vsl\\ reduces liver disease severity and hospitalization inpatients with cirrhosis a randomized controlled trial gastroenterology 101053jgastro201408031dixon l j barnes m tang h pritchard m t and nagy l e kupï¬ercells in the liver compr physiol 101002cphyc120026dolganiuc a norkina o kodys k catalano d bakis g marshall c viral and host factors induce macrophage activation and loss of tolllikereceptor tolerance in chronic hcv infection gastroenterology 101053jgastro200708003dore g ward j and thursz m hepatitis c disease burden andstrategies to manage the burden guest editors mark thursz gregory doreand john ward j viral hepat 21suppl 101111jvh12253doskali m tanaka y ohira m ishiyama k tashiro h chayama k possibility of adoptive immunotherapy with peripheral bloodderived cd3cd56 and cd3cd56 cells for inducing antihepatocellularcarcinoma and antihepatitis c virus activity j immunother 101097cji0b013e3182048c4efasano a intestinal permeability and its regulation by zonulin diagnosticand therapeutic implications clin gastroenterol hepatol 101016jcgh201208012fern¡ndez m f reinap©rez i astaj m rodriguezcarrillo aplazadiaz j and fontana l breast cancer and its relationshipwith the microbiotaj environ res public health int 103390ijerph15081747ferrere g wrzosek l cailleux f turpin w puchois v spatz m fecal microbiota manipulation prevents dysbiosis and alcoholinduced liver injury in mice j hepatol 101016jjhep2016fooladi a i tavakoli h and naderi a detection of enterotoxigenicjin domestic dairy productsisolatesiranstaphylococcus aureusmicrobiol gao b ma j and xiang x mait cells a novel therapeutic target foralcoholic liver disease gut 101136gutjnl2017315284hamer h m jonkers d venema k vanhoutvin s troost f and brummerr j the role of butyrate on colonic function aliment pharmacol ther 101111j13652036200703562xhill d a hoï¬mann c abt m c du y kobuley d kirn t j metagenomic analyses reveal antibioticinduced temporal and spatial changesin intestinal microbiota with associated alterations in immune cell homeostasismucosal immunol 101038mi2009132hooper l v xu j falk p g midtvedt t and gordon j i amolecular sensor that allows a gut commensal to control its nutrient foundationin a competitive ecosystem proc natl acad sci usa 101073pnas96179833ichinohe t pang i k kumamoto y peaper d r ho j h murray ts microbiota regulates immune defense against respiratorytract uenza a virus infection proc natl acad sci usa 101073pnas1019378108inoue t nakayama j moriya k kawaratani h momoda r ito k gut dysbiosis associated with hepatitis c virus infection clin infectdis 101093cidciy205jiminez j a uwiera t c abbott d w uwiera r r and inglis gd impacts of resistant starch and wheat bran consumption onenteric ammation in relation to colonic bacterial community structuresand shortchain fatty acid concentrations in mice gut pathog 101186s1309901601496karst s m the uence of commensal bacteria on infection with entericviruses nat rev microbiol 101038nrmicro201525kauppila j h karttunen t j saarnio j nyberg p salo t gravesd e short dna sequences and bacterial dna induceesophageal gastric and colorectal cancer cell invasion apmis 101111apm12016kefalakes h and rehermann b ammation drives an alteredphenotype of mucosalassociated invariant t cells in chronic hepatitis d virusinfection j hepatol 101016jjhep201905024khanna s and tosh p k a clinicians primer on the role of themicrobiome in human health and disease mayo clin proc 101016jmayocp201310011kreuzer s machnowska p amus j sieber m pieper r schmidt m f feeding of the probiotic bacterium enterococcus faecium ncimb diï¬erentially aï¬ects shedding of enteric viruses in pigs vet res krieg a m therapeutic potential of tolllike receptor activation natrev drug discov 101038nrd2059lee h and ko g antiviral eï¬ect of vitamin a on norovirus infection viamodulation of the gut microbiome sci rep 101038srep25835lemon s m ott j j van damme p and shouval d typea viral hepatitis a summary and update on the molecular virologyepidemiology pathogenesis and preventionj hepatol 101016jjhep201708034li d yan p abousamra a b chung r and butt a proton pumpinhibitors are associated with accelerated development of cirrhosis hepaticdecompensation and hepatocellular carcinoma in noncirrhotic patients withchronic hepatitis c infection results from erchives aliment pharmacolther 101111apt14391li h gao z zhang j ye x xu a ye j sodium butyratestimulates expression of ï¬broblast growth factor in liver by inhibition ofhistone deacetylase diabetes 102337db110846lin l and zhang j role of intestinal microbiota and metabolitesand human diseases bmc immunolon gut homeostasis 101186s1286501601873liu q li f zhuang y xu j wang j mao x alterationin gut microbiota associated with hepatitis b and nonhepatitis virus relatedhepatocellular carcinoma gut pathog 101186s1309901802816lu h wu z xu w yang j chen y and li l intestinal microbiotawas assessed in cirrhotic patients with hepatitis b virus infection microb ecol 101007s0024801098018meiglani a alimirah m ramesh m and salgia r fecal microbiotatransplantation for clostriodioides diï¬cile infection in patients with chronicliver disease int j hepatol mencin a kluwe j and schwabe r f tolllike receptors as targets inchronic liver diseases gut 101136gut2008156307minemura m and shimizu y gut microbiota and liver diseases worldj gastroenterol 103748wjgv21i61691mohamadkhani aintestinal microbialcommunity in hepatocarcinogenesis in chronic hepatitis b cancer med 101002cam41550 on the potential role ofmoratalla a gmezhurtado i santacruz a moya ¡ peir g zapater p protective eï¬ect of biï¬dobacterium pseudocatenulatum cect against induced bacterial antigen translocation in experimental cirrhosisliver int 101111liv12380ohara a mand shanahan ftherapeutic potential clin gastroenterol hepatolfor 101016jcgh200612009 gut microbiota miningpark e j thomson a b and clandinin m t protection of intestinaloccludin tight junction protein by dietary gangliosides in lipopolysaccharideinduced acute ammation j pediatr gastroenterol nutr 101097mpg0b013e3181ae2ba0p©rezmatute p ±iguez m villanuevamill¡n m j reciofern¡ndez e andv¡zquez a m s¡nchez s c shortterm eï¬ects of directactingantiviral agents on ammation and gut microbiota in hepatitis cinfectedpatients eur j inter med 101016jejim201906005ponziani f r putignani l paroni sterbini f petito v picca a del chiericof uence of hepatitis c virus eradication with directactingantivirals on the gut microbiota in patients with cirrhosis aliment pharmacolther 101111apt15004preveden t scarpellini e mili´c n luzza f and abenavoli l gut microbiota changes and chronic hepatitis c virus infection expert revgastroenterol hepatol frontiers in cellular and infection microbiology wwwfrontiersinaugust volume 0csehgal microbiota and liver diseasesraimondi s amaretti a gozzoli c simone m righini l candelieref in the human gutits proï¬ling phenotyping and colonization front microbiol 103389fmicb201901575 longitudinalsurvey offungiren z li a jiang j zhou l yu z lu h gut microbiomeanalysis as a tooltowards targeted noninvasive bioma | Colon_Cancer |
" microwave ablation mwa is widely used to treat unresectable primary and secondary malignanciesof the liver and a limited number of studies indicate that ablation can cause not only necrosis at the in situ site butalso an immunoreaction of the whole body this study aimed to investigate the effects of mwa on cytokines inpatients who underwent mwa for a hepatic malignancymethods patients admitted to the oncology department in the first affiliated hospital of soochow universitybetween june and february were selected peripheral blood was collected from patients with a hepaticmalignancy treated with mwa the levels of cytokines il2 ifnÎ tnfα il12 p40 il12 p70 il4 il6 il8 il10and vascular endothelial growth factor vegf were detected with a milliplex® map kit the comparison times wereas follows before ablation h after ablation days after ablation and days after ablation data were analyzedusing a paired sample ttests and spearmans correlation analysisresults a total of patients with hepatic malignancies were assessed there were significant differences in il2il12 p40 il12 p70 il1 il8 and tnfα at h after mwa significant increases 2fold vs before ablation wereobserved in il2 il1 il6 il8 il10 and tnfα after mwa elevated il2 and il6 levels after ablation werepositively correlated with energy output during the mwa procedures wa treatment for hepatic malignancies can alter the serum levels of several cytokines such as il2 and il6keywords microwave ablation hepatic malignancy cytokines il2 il6 immunoregulation primary and secondary malignancies of the liver have asubstantial impact on morbidity and mortality worldwidein china hepatocellular carcinoma hcc has the secondhighest mortality rate of malignancies the treatmentof primary and secondary hepatic malignancies via correspondence lengbengsudaeducn jing zhao qiang li and merlin muktiali contributed equally to this work2department of oncology the first affiliated hospital of soochow universitysuzhou china5division of neurosurgery city of hope beckman research institute duartecalifornia usafull list of author information is available at the end of the interventional imaging therapy is undertaken by investigators in the field of interventional radiology and possibly bya smaller group of practitioners known as interventionaloncologists whose major focus is cancer care via minimally invasive approaches [ ] recently percutaneous ablation therapy has been widely accepted as a radicaltreatment method for hcc and its fiveyear survival rateis similar to that of resection microwave ablationmwa is widely used to treat unresectable hcc and recurrent hcc and has the advantages of minimal invasiona good curative effect and no side effects due to radiationor chemotherapy immune checkpoint inhibitors icis the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0czhao bmc cancer page of such as pd1pdl1 and ctla4 antibodies have beenwidely applied in several cancers and studies have indicated that ici treatment could enhance the effect of ablation evidence hasindicated that hyperthermicdestruction causes the release of a large population of heterogeneous tumor antigens and inflammatory cytokinesmay play crucial roles in this process cytokines aremediators that regulate a broad range of processes involved in the pathogenesis of cancer several cytokineswhich can arise from either tumor cells or immunocytes such as tumor necrosis factor tnfα interleukinil1 il6 il8 il10 and vascular endothelial growthfactor vegf have been linked with cancers and can either promote or inhibit tumor development the serumlevels of cytokines differ during cancer development although cytokines have been found to be altered after anticancer treatment such as chemotherapy and radiotherapy[ ] few investigations have focused on cytokines beforeand after mwa it is still unknown whether the above cytokines changed before andor after mwa in patientswith hepatic malignancies in this study we investigatedthe effects of mwa on the serum levels of cytokines inpatients with hepatic malignanciesmethodspatients and samplesthe patient population examined in this study was derivedfrom the first affiliated hospital of soochow universitypatients were admitted to the oncology department between june and february the total number ofpatients was with liver metastases and primaryliver cancers the inclusion criterion was a tumor locatedat a hepatic site either primary or metastases all patients with metastatic hepatic malignances should be givensystematic treatments chemotherapy or target therapyand get at least stable disease sd or partial responsepr for more than days informed consent for blooddraw and the relevant therapy was obtained from all patients the protocol was approved by the human ethicscommittee of the first affiliated hospital of soochowuniversity and was conducted in accordance with thedeclaration of helsinki all written informed consent wasobtained from all participants and clearly stated wholeblood ml was drawn into edta anticoagulant tubeson days to before and h days and days afterablation mostly on the last day of the course for cytometry and cytokine analysesablation procedurethe ablation procedure used in this research was mwathe puncture site and pathway were determined underthe guidance of a computed tomography ct scanlocal infiltration anesthesia was achieved by using lidocaine the placement of microwave ablation probeswas guided by a ct scan or ultrasonic device and allprobes were placed at the maximum diameter layerdouble probes were employed when the maximumdiameter of the tumor was up to cm the power andtime of ablation were designed for each patient in therange of w and min respectively basedon the size number and position of the tumor theboundaries of ablation zones were designed as extended cm upon the tumor sitecytokine detectiona milliplex map kit with human cytokinechemokinepanels that measured ifnÎ il2 il6 il8 il10 il12p40 il12 p70 il1 tnfα and vegf was utilized according to the manufacturers instructions briefly chemically dyed antibodybound beads were mixed withstandard or sample incubated overnight at °c washedand then incubated with a biotinylated detection antibodyafter the beads were washed they were incubated with astreptavidin phycoerythrin complex and the mean fluorescent intensities were quantified on a luminex analyzer luminex corporation all samples were measured in duplicate standard curves of known concentrations of recombinant human cytokineschemokines wereused to convert fluorescence units to cytokine concentration units pgml the minimum detectable concentrations were as follows ifnÎ pgml il2 pgmlil12 p40 pgml il12 p70 pgml il1 pgml il6 pgml il8 pgml il10 pgml tnfα pgml and vegf pgml all resultsbelow the minimum concentrations were processed as theminimum concentrationsstatistical analysisibm spss statistics software was used for the statistical analysis along with graphpad prism for figurecreations normally distributed numerical data areexpressed as the mean ± standard deviation and nonnormally distributed numerical data are expressed as themedian and confidence interval ci cytokinesat different times were compared using a onetailedpaired ttest spearmans correlation analysis was executed to determine the correlation between clinical indexes and cytokine levels p indicates a significantdifferenceresultsclinical characteristics of the enrolled patientsas shown in table a total of patients with tumorslocated on the liver liver metastases primary livercancers were analyzed the patients cytokine levelswere compared according to time before treatment h after treatment days after treatment and daysafter treatment 0czhao bmc cancer page of table clinical characteristics of the patients enrolled n characteristicsexmalefemaleagepathogenesisprimarysecondaryprimary site for metastatic hepatic malignancescolon rectalpancreasstomachebreastothersmaximum tumor length mmablation probe usedablation time minaverage power per probe w ± ± ± ± average energy time à power time à power¼¼ time and power indicate the time and power respectively ofdifferent probes used during the operation ± ifnÎ il12 p40 and il12 p70 were slightly increasedafter mwa treatmentas shown in table and fig the median level ofifnÎ before the mwa treatment was pgml ci pgml at days and days after themwa treatment there was a slight increase comparedto that premwa with median levels of pgml ci pgml and pgml ci pgml respectively the median level of il p40 before the mwa treatment was pgml ci pgml there was a slight increase to pgml ci pgml days postmwathe median il12 p70 level before the mwa treatmentwas pgml ci pgml and increasedto pgml ci pgml days afterthe mwa treatment and to pgml ci pgml days postmwa no significant alteration in the vegf median level was detected after themwa treatmentil2 il1 il6 il8 and il10 were elevated over 2foldafter the mwa treatmentas shown in table fig and fig the median levelof il2 before the mwa treatment was pgml ci pgml there was a significant increase at h postmwa with a median level of pgml ci pgml the median level ofil1 before the mwa treatment was pgml ci pgml and a significantincrease wasnoted days after the mwa treatment pgml ci pgml the median level of il6before the mwa treatment was pgml ci pgml and significantly increased daysafter the mwa treatment pgml ci pgml the median level ofil8 before themwa treatment was pgml ci pgml and increased significantly to pgml ci pgml days after the mwa treatmentthe median level of il10 before the mwa treatmentwas pgml ci pgml and increasedsignificantly days after the mwa treatment pgml ci pgml the median level oftnfα before the mwa treatment was pgml ci pgml and increased significantlyto pgml ci pgml days afterthe mwa treatmentlevelselevated il2 and il6 levels after ablation were positivelycorrelated with energy output during mwato further evaluate the relationship between the increased cytokineand mwa treatment weemployed the concept of energy time à power time à power time and power indicated thetime and power of different probes used in the operation to reflect total hyperthermic damage to hepatictissues during the mwa procedure as shown in table and fig the il2 levels at h postmwa and the il levels at days postmwa illustrated significant correlations with energy the relative indexes were and respectivelydiscussionas technology continues to develop other types of localtherapy such as radiotherapy chemical ablation andhyperthermal ablation for primary and metastatic livercancer are increasingly being used mwa for liver malignances is reserved for patients who cannot undergosurgical removal or for whom other treatments havefailed a consensus guideline was recently developed to address indications for mwa in these patientsthermal ablation is a process that heats the target tissueto a temperature that causes immediate coagulative necrosis usually over °c terminal treatment requiresthat a necrotic area surrounds the target site with anadditional 10mm margins however in the liverhigh tissue perfusion and large blood vessels can cause aheat sink effect around the ablation zone making itdifficult to achieve terminal ablation the heat sink 0czhao bmc cancer page of table median levels of cytokines before and after mwacytokineifnÎil2premwa pgml ci ci ci ci ci ci ci ci ci ci h postmwa pgml ci ci ¼ ci ci ci ci ci ci ci ci il12 p40il12 p70il1il6il8il10tnfαvegf p vs premwa ¼ 2fold vs premwa days postmwa pgml ci ci ci ci ci ¼ ci ¼ ci ¼ ci ¼ ci ¼ ci days postmwa pgml ci ci ci ci ci ci ci ci ci ci effect can lead to sublethal temperatures and the retention of malignant cells thereby increasing the likelihoodof local tumor progression ltp however an incompletely ablated zone containing immune cells andcancer cells as well as functional vessels could establisha serious inflammatory site that may provide tumorspecific antigens cytokines and activated immune cellsin our study significant increases in the secretion ofchemokines il8 proinflammatory cytokines il1il12 ifnÎ and tnfα and antiinflammatory cytokines il10 were observed after mwa il8 is mainlyproduced by macrophages the classical biological activity of il8 is to attract and activate neutrophils whichcan lead to a local inflammatory response however recent studies have indicated that il8 both macrophageand cancer cellderived can recruit myeloidderivedsuppressor cells mdscs into the tumor microenvironment eventually inhibiting antitumor immunity andpromoting cancer progression [ ] il1 is mainlyproduced by macrophages b cells and nk cells couldproduce il1 under certain circumstances generallycells can only synthesize and secrete il1 after beingstimulated by foreign antigens or mitogens il1 couldpromote the th1 response promoting the activation ofdendritic cells dcs and cytotoxic t lymphocytesctls il12 is mainly produced by b cells and macrophages human il12 is a heterodimer with two subunits p40 kd and p35 kd which areinactivated in isolated form in general il12 functionsas a combination of two subunits il12 p70 while p40alone possesses partial functions of il12 p70 its mentionable that il12 p40 and p35 are not expressed inequal proportions so the amounts of il12 p40 and il p70 are different in one cell il12 can stimulate theproliferation of activated t cells and promote the differentiation of th0 cells into th1 cells moreover il12could induce the cytotoxic activity of ctls and nk cellsand promote the secretion of several cytokines such asifnÎ and tnfα previous research indicatedthat tnfα may play a crucial role in mwa in combination with immunotherapy notably our data illustrated that the il12 results were consistent with thoseof ifnÎ after the ablation operation but not with thoseof tnfα this result indicated that upregulation ofifnÎ may be a major effect of the il12 increase aftermwa on the other handan antiinflammatory and immunosuppressive cytokine wasevaluated after mwa il10 is a multicellularderivedmultifunctional cytokine that regulates cell growth anddifferentiation and could participate in inflammatoryand immune responses il10 was reported to increaseafter thermal ablation in the literature [ ] strategiesto inhibit il10induced immunosuppression after thermal ablation treatment would be of interestil10asablation therapy can mediate antitumor immunity astumor tissue necrosis caused by ablation may release various antigens that eventually form a kind of in situ vaccination moreover ablative therapy can not onlydirectly kill cancer cells in situ but also regulate immunecells and promote the immune function of patients withliver cancer [ ] many immunoregulatory cytokineswere released or expressed after thermal ablation notablythe cytokines released after thermal ablation can regulatethe positive and negative aspects of the cancer immunecycle previously researchers demonstrated that proinflammatory cytokines such as il1 il6 il8 il18 andtnfα were increased several hours or days after thermalablation [ ] to our knowledge terminal tumorthermal ablation may not only cause local heat injury intissues surrounding the tumor site but also induce a systemic reaction this systemic reaction would becaused by different mechanisms first interventional operation may result in trauma to the liver although this procedure is very minimally invasive the healing process maycause alteration of some cytokines second heat injurycould cause acute thermal necrosis in liver and tumor 0czhao bmc cancer page of fig levels of cytokines before and after mwa treatment slightly increased ifnÎ il12 p40 and il12 p70 levels after mwa treatment over fold enhancement of il2 h postmwa and of il1 il6 il8 il10 and tnfα d postmwa p 0czhao bmc cancer page of fig trends in cytokines significantly altered after mwa treatment the levels of il2 at h postmwa il1 at d postmwa il6 at dpostmwa il8 at d postmwa and il10 at d postmwa were elevated over 2fold compared to the levels premwatable correlation between the ablation energy and significantly elevated cytokinesenergyvsil2 h postmwaenergyvsil1 d postmwaenergyvsil6 d postmwaenergyvsil8 d postmwaenergyvsil10 d postmwaenergyvstnfα d postmwaspearmans rp value onetailed p 0czhao bmc cancer page of fig correlation between the ablation energy and the serum levels of il2 and il6 the serum levels of il2 at h postmwa and il6 at dpostmwa were positively correlated with energy output during the mwa procedureand nonspecifictissues and release of necrotic tissue fragments into bloodcould cause immunological reactions including nonspecific and specific reactions generally cytokines affectedby wound healingimmunologicalreactions do not last longer than those affected by specificimmunologicalreactions ablation treatmentinducedspecific immunological reactions are more complicatedand could affect more immunocytes [ ] which wouldmake this process last longer than other reactions theseexplanations may be the reason why the cytokine changeslasted different durations moreover cytokines affected bythe second manner would be positively correlated withthe ablation scale which is why we employed the energyindex in our ablation operation design to receive a terminal ablation larger tumor would cost higher energy including higher power and longer duration time terminaltumorthermal ablation would release tumorrelatedneoantigen to blood circulation eventually induce a systemic reaction this reaction is dependent on the scale ofthermal injury and the local immunological microenvironment of the tumor our findings indicated that il2 andil6 were significantly altered after the ablation procedureand positively correlated with mwa energy il2 is commonly derived from activated t cells primarily th1 cellsil2 can stimulate t cells to proliferate and differentiateactivate natural killer nk cells and macrophages and enhance the functions of cytotoxic t lymphocytes ctls our data illustrated that il2 is significantly increased at h after mwa indicating that il2 may induce a nonspecific immune response after mwa but il decreased after h postmwa in our study suggesting that the il2induced immune response may not belong lasting mentionable many cytokines detected il8il1 il12 were mainly derived from macrophagewhich was a widely distributed antigen presenting cellthis result support the theory that mwa could releasefragment of cancer cells into blood as neoantigen macrophages could response to this proceed and cause a systemic immunoreaction additional cytokines alterationsuch as il6 after ablation may be no anspecific inliver evidences indicate that increase of il6 was not onlyoccurred in liver ablation researches focus on lung cancerincluding primary lung cancer and pulmonary metastasesdemonstrated that serum il8 il1 il6 il10 il12and tnfα were significantly raised after radiofrequencythermal ablation moreover joseph found that imageguided thermal ablation of tumors located in lung liver orsoft tissues increases plasma levels of il6 and il10 another question remain unveiled was if our result wascancerspecific we checked literature about cytokinemodulation after thermal ablation in benign diseases andonly got limit evidences based on benign thyroid nodules and adenomyosis according to these literatureil6 levels did not show any significant difference aftertreatment compared with pretreatment values indicatingthat elevation of il6 may be caused by tumour antigenreleased by ablation treatment however the ablationenergy used in thyroid nodules was much lower thanliver and lung which would lead to a false negativein cytokine detection to the research about adenomyosis on the other hand experiment design was determined to followup the il6 at months afterhifu ablation as our data demonstrated mostly cytokines were return to premwa level after monthdetection after months may miss the modulation ofil6 overall few evidences support that some of thecytokines were altered in a cancerspecific mannerwhile no solid results could confirm that further animal experiments were required to make a clarifieddata and answer this question 0czhao bmc cancer page of thetumorassociated immunein recent years ablationinduced systemic effects suchasresponse haveattracted increased attention de baere t first reported two cases of spontaneous regression of multiplepulmonary metastases occurring after radiofrequencyablation of a single lung metastasis although growing evidence suggests that thermal ablation can inducespontaneous regression of the socalled abscopal effecton distant tumors the incidence rate of such an effect israre probably due to uncontested immunological activation caused by one ablation treatment and the lack ofimmuneamplification management in it was described that in situ tumor destruction can provide a useful antigen source forthe induction of antitumorimmunity however clinical studies could not sufficiently utilize such an effect until the development ofimmune checkpoint inhibitors icis [ ] icis suchas pd1pdl1 and ctla4 antibodies are widely applied in several cancers and studies have indicated thatici treatment could enhance the effect of ablation evidence indicates that hyperthermic destruction causesthe release of a large population of heterogeneous tumorantigens and inflammatory cytokines may play crucialroles in this process however opposite evidence indicated that incomplete radiofrequency ablation couldinduce inflammation which may accelerates tumor progression and hinders pd1 immunotherapy suggesting that ablation treatment may promote tumorprogression our data demonstrated that il6 was significantly increased after mwa treatment il6 is derived from monocytes macrophages dcs th2 cells andsometimes cancer cells and it plays a key role in t cellproliferation and survival the role of il6 appearsto be rather complex korn classified il6 as differentiation factor which could involve in differentiation ofth17 cells however il6 does not direct the commitment to the th1 or th2 cell lineage but controls theproliferation and survival of immunocytes cooperatingwith other cytokines such as tgf tnf or il21 for instance il6 activated stat3 pathway in naivecd4 t cells in the presence of the morphogen tgfbpromotes the population expansion of th17 cells recent evidence indicates that il6 plays an indispensable role in t cellinfiltration to the tumor sitewhich could benefit immunomodulatory therapy incontrast il6 can increase mdscs inhibit the development and maturation of dendritic cells dcs and inhibit the polarization of th1 cells eventuallyresulting in negative immunomodulatory effects according to muneeb ahmeds work the adjuvant uses ofa nanop smallinterfering rna sirna can besuccessfully used to target the il6mediated locoregional and systemic effects of thermal ablation il6 knockout via a nanop antiil6 sirna in mice coulddecrease the local vegf level at the ablation site therefore how to utilize the positive effect of il6 whileavoiding the negative effect after mwa needs further investigation preclinical research indicated that il6 andpdl1 blockade combination therapy reduced tumorprogression in animal models [ ] thus an antiil strategy after ablation should be considered whencombined with ici therapy previous studies and ourshave demonstrated that most cytokine levels returned topretreatment levels days after ablation this resultsuggests that h to days after ablation may be optimal timing for additional immunomodulatory therapysour results reported here support the evidence for terminal tumor thermal ablation could cause heat injury totissues surrounding the tumor site and release neoantigento blood circulation eventually induce a systemic reactionthis reaction could lead to a detectable alteration of cytokine levels further investigation is required to revealwhether the cytokines altered by mwa treatment couldaffect cancer progression whether positive or negativeabbreviationsmwa microwave ablation hcc hepatocellular carcinoma icis immunecheckpoint inhibitors tnf tumor necrosis factor il interleukinvegf vascular endothelial growth factor sd stable disease pr partialresponse ct computed tomography ci confidence interval ltp likelihoodof local tumor progression mdscs myeloidderived suppressor cellsctls cytotoxic t lymphocytes nk natural killer sirna small interfering rnaacknowledgementsnot applicableauthors contributionsjz conceptualization data curation writingoriginal draft and writingreview and editing ql conceptualization and writingreview and editingmm conceptualization and writingreview and editing brconceptualization and writingreview and editing and collect samples yhexecute milliplex assay and collect data dpl patient enrollment executemwa ablation and collect samples zl execute mwa ablation and collectsamples dml patient enrollment execute mwa ablation and collectsamples yx execute milliplex assay and collect data mt conceptualizationand writingreview and editing rl conceptualization data curation formalanalysis visualization writingoriginal draft and writingreview and editingall authors have read and approved the manuscriptfundingthis work was supported by the national natural science foundation ofchina the natural science foundation ofjiangsu province of china bk20140295 the jiangsu governmentscholarship for oversea studies js2018179 and the six one projects forhighlevel health personnel in jiangsu province lgy2018077availability of data and materialsthe datasets used andor analysed during the current study are availablefrom the corresponding author on reasonable requestethics approval and consent to participatethe protocol was approved by the human ethics committee of the firstaffiliated hospital of soochow university and was conducted in accordancewith the declaration of helsinki patients were informed that the bloodsamples were stored by the hospital and potentially used for scientific 0czhao bmc cancer page of research and that their privacy would be maintained all written informedconsent was obtained from all participants and clearly statedconsent for publicationnot applicablecompeting intereststhere is no financial or personal relationship with other people oranizations that could inappropriately influence bias this workauthor details1department of radiation oncology the first affiliated hospital of soochowuniversity suzhou china 2department of oncology the first affiliatedhospital of soochow university suzhou china 3department of lymphatichematologic oncology jiangxi cancer hospital nanchang china4department of interventional radiology the first affiliated hospital ofsoochow university suzhou china 5division of neurosurgery city of hopebeckman research institute duarte california usareceived january accepted august referencesfu j wang h precision diagnosis and treatment of liver cancer in chinacancer lett bruix j han kh gores g llovet jm mazzaferro v liver cancer approachinga personalized care j hepatol suppls144rognoni c ciani o sommariva s bargellini i bhoori s cioni r facciorussoa golfieri r gramenzi a mazzaferro v transarterial radioembolizationfor intermediateadvanced hepatocellular carcinoma a budget impactanalysis bmc cancer nault jc sutter o nahon p gannecarrie n seror o percutaneoustreatment of hepatocellular carcinoma state of the art and innovations jhepatol yin j dong j gao w wang y a case report of remarkable response toassociation of radiofrequency ablation with subsequent atezolizumab instage iv nonsmall cell lung cancer medicine baltimore 20189744e13112shi l chen l wu c zhu y xu b zheng x sun m wen w dai x yang m pd1 blockade boosts radiofrequency ablationelicited adaptiveimmune responses against tumor clin cancer res lippitz be cytokine patterns in patients with cancer a systematic reviewlancet oncol 2013146e218jin yb zhang gy lin kr chen xp cui jh wang yj luo w changes ofplasma cytokines and chemokines expression level in nasopharyngealcarcinoma patients after treatment with definitive intensitymodulatedradiotherapy imrt plos one 2017122e0172264kim mj jang jw oh bs kwon jh chung kw jung hs jekarl dw lee schange in inflammatory cytokine profiles after transarterial chemotherapy inpatients with hepatocellular carcinoma cytokine gillams a goldberg n ahmed m bale r breen d callstrom m chen mhchoi bi de baere t dupuy d thermal ablation of colorectal livermetastases a position paper by an international panel of ablation expertsthe interventional oncology sans frontieres meeting eur radiol ahmed m solbiati l brace cl breen dj callstrom mr charboneau jwchen mh choi bi de baere t dodd gd 3rd imageguided tumorablation standardization of terminology and reporting criteriaa 10yearupdate radiology chiang j hynes k brace cl flowdependent vascular heat transfer duringmicrowave thermal ablation conf proc ieee eng med biol soc huang hw influence of blood vessel on the thermal lesion formationduring radiofrequency ablation for liver tumors med phys najjar yg rayman p jia x pavicic pg jr rini bi tannenbaum c ko jhaywood s cohen p hamilton t myeloidderived suppressor cellsubset accumulation in renal cell carcinoma parenchyma is associated withintratumoral expression of il1beta il8 cxcl5 and mip1alpha clin cancerres alfaro c teijeira a onate c perez g sanmamed mf andueza mp alignanid labiano s azpilikueta a rodriguezpaulete a tumorproducedinterleukin8 attracts human myeloidderived suppressor cells and elicitsextrusion of neutrophil extracellular traps nets clin cancer res kundu m roy a pahan k selective neutralization of il12 p40 monomerinduces death in prostate cancer cells via il12ifngamma proc natl acadsci u s a onishi h kuroki h matsumoto k baba e sasaki n kuga h tanaka mkatano m morisaki t monocytederived dendritic cells that capture deadtumor cells secrete il12 and tnfalpha through il12tnfalphanfkappabautocrine loop cancer immunol immunother yu z geng j zhang m zhou y fan q chen j treatment of osteosarcomawith microwave thermal ablation to induce immunogenic cell deathoncotarget yang w wang w liu b zhu b li j xu d ni y bai l liu gimmunomodulation characteristics by thermal ablation therapy in cancerpatients asia pac j clin oncol 2018145e490erinjeri jp thomas ct samoilia a fleisher m gonen m sofocleous ctthornton rh siegelbaum rh covey am brody la imageguidedthermal ablation of tumors increases the plasma level of interleukin6 andinterleukin10 j vasc interv radiol den brok mh sutmuller rp van der voort r bennink ej figdor cg ruerstj adema gj in situ tumor ablation creates an antigen source for thegeneration of antitumor immunity cancer res zerbini a pilli m laccabue d pelosi g molinari a negri e cerioni sfagnoni f soliani p ferrari c radiofrequency thermal ablation forhepatocellular carcinoma stimulates autologous nkcell responsegastroenterology zhang h hou x cai h zhuang x effects of microwave ablation on tcellsubsets and cytokines of patients with hepatocellular carcinoma minim | Colon_Cancer |
the world health anization who indicated thatliver cancer was the sixth mostcommon cancer and the fourth leading cause of cancer deaths worldwide in witha global death toll of the risk factors for liver cancer include hepatitisb hepatitis c alcoholic liver disease nonalcoholic fatty liver disease and cirrhosis at present surgery remains the ï¬rstfor liver cancer howevertreatmentline ofedited by¢ngela sousauniversity of beira interior portugalreviewed bychen lingfudan university chinasamuel silvestreuniversity of beira interior portugalcorrespondencetonghong wangcellwwadmcgmhtw these authors have contributedequally to this workspecialty sectionthis was submitted topharmacology of anticancer drugsa section of the frontiers in oncologyreceived april accepted june published august citationchen cy fang jy chen ccchuang wy leu yl ueng shwei ls cheng sf hsueh c andwang th 2omethylmagnolol a magnololderivative suppresses hepatocellularcarcinoma progression via inhibitingclass i histone deacetylaseexpression front oncol 103389fonc202001319frontiers in oncology wwwfrontiersinaugust volume 0cchen 2omethylmagnolol suppresses hdac expressionchemotherapy or radiation therapy is the preferred choice forpatients with advanced liver cancer who cannot undergo surgicalresection most chemotherapeutic drugs however often havelarge side eï¬ects and significantly impact patients quality of life therefore the development of eï¬ective therapeutic drugswith minimal side eï¬ects has been at the forefront of livercancer researchdue to its advantages such as high speciï¬city and low sideeï¬ects targeted therapy has become the main modality of cancertreatment however carcinogenic factors are multifactorialand often complicated this complexity is further aggravatedby tumor heterogeneity therefore drugs against a singletarget often demonstrate limited eï¬cacy even sorafenib which isrecognized as the most eï¬ective targeted drug against liver canceronly prolongs patient survival by ¼ months thus inclinical practice targeted therapy is often used in conjunctionwith other treatment modalities such as chemotherapy andradiation therapy to improve therapeutic outcomes recent studies have shown that the occurrence oflivercancer is closely associated with genetic and epigenetic variations common epigenetic regulatory mechanisms includedna methylation histone modiï¬cation and noncoding rnaregulation previous studies have reported that histonedeacetylase hdac overexpression is common in hepatitis bvirus hbvinfected liver cancer patients and couldlead to carcinogenesis as hdacs regulate the deacetylation ofhistone and nonhistone proteins thereby coordinating geneexpression or protein activation histone protein deacetylationleads to its tighter binding of the surrounding dna consequentlyinhibiting gene expression in the bound region alternativelynonhistone protein acetylation not only is closely associatedwith its protein activity but also aï¬ects its ability to bind otherproteins or dna thereby indirectly regulating the expressionof other genes and their encoded proteins the known hdac types found in humans can be categorized intofour classes class i hdac1 hdac2 hdac3 and hdac8class iia hdac4 hdac5 hdac7 and hdac9 class iibhdac6 and hdac10 class iii sir2like enzymes comprisingseven sirtuins and class iv hdac11 among these classi hdac overexpression is observed in most cancer typesincluding liver cancer class i hdac overexpressioncan inhibit the expression of multiple tumorsuppressor genessuch as p21 and p53 thereby promoting carcinogenesis moreover these hdacs are therapeutic targets for multipleanticancer treatments hdac inhibitors including trichostatina vorinostat suberoylanilide hydroxamic acid saha trapoxina and valproic acid are eï¬ective in the treatment of lung breastand esophageal cancers whereas saha has been approved bythe food and drug administration fda for the treatment of tcell lymphoma furthermore recent studies have foundthat the combined use of an hdac inhibitor with sorafenib cansubstantially improve the treatment eï¬cacy of sorafenib in livercancer however most hdac inhibitors have significantside eï¬ects which are the reason for the primary bottleneck totheir clinical usethe application of traditional chinese herbal medicine indisease treatment has become increasingly popular in recentyears compared to western medicine chinese herbal medicineis an alternative treatment option that can be eï¬ective andintroduces fewer side eï¬ects owing to the developmentof component separation technologies the active ingredientsof traditional chinese medicines have been extracted and theirfunctions identiï¬ed these compounds can act at lower eï¬ectivedoses and produce more speciï¬c therapeutic eï¬ects amongthem artemisinin and curcumin are used and have shown goodoutcomes in cancer treatment other extracts such asresveratrol and chrysin exert an anticancer stem cell csceï¬ect and may provide an alternative approach to managecancers magnolia oï¬cinalis is a traditional chinese medicinal plantits extractcommonly used in southeast asian countriesmagnolol a phenolic compound has proven antibacterialantioxidant and antiammatory activities and its anticancerand antiangiogenic activities have also been recently veriï¬ed however the mechanism of its anticancer eï¬ects is yetto be elucidated in the present study we modiï¬ed magnololand synthesized a methoxylated derivative 2omethylmagnololmm1 in addition to testing the antihepatocellular carcinomahcc activities of magnolol and its derivative mm1 we alsoused cell and animal models to clarify their modes of actionthereby elucidating the feasibility of their clinical applicationsmaterials and methodscell culturehuman hcc cell lines huh7 and hepg2 were purchased fromthe american type culture collection manassas va usaand donated by dr chauting yeh of chang gung memorialhospital respectively human skin ï¬broblasts hfbs werekindly provided by dr panchyr yang of taiwan universitythe cells were maintained in dulbeccos modiï¬ed eagle mediumgibco gaithersburg md usa containing fetal bovineserum fbs and cultured at ¦c with carbon dioxide ina humidiï¬ed incubator culture medium chemical compoundsand fbs were purchased from life technologies grand islandny usacompounds and antibodiesmagnolol was purchased from shanghai bs biotech co ltdshanghai china mm1 was prepared as described by lin the purity of magnolol and mm1 was asdetermined by highprecision liquid chromatography hplcanalysis magnolol and mm1 were each dissolved in dimethylsulfoxide dmso to obtain a stock concentration of mmwhich was then stored at ¦c before use dmso vvwas used as the vehicle control sorafenib was purchased fromsigmaaldrich st louis mo usa antibodies against humanclass i hdacs hdac1 hdac2 hdac3 and hdac8 acetylhistone h3 acetylp53 p53 p21 ki67 ecadherin ncadherinvimentin snail slug and actin were purchased from genetexirvine ca usa and cell signaling technology beverlyma usa the antibody to cyclin d1 was purchased fromabclonal technology woburn ma usa and the antibodiesagainst cdk4 were purchased from proteintech rosemont ilfrontiers in oncology wwwfrontiersinaugust volume 0cchen 2omethylmagnolol suppresses hdac expressionusa secondary antibodies were purchased from santa cruzbiotechnology santa cruz ca usarealtime reversetranscriptionpolymerase chain reactionanalysistotal rna from huh7 and hepg2 cells were extracted usingtoolsmart rna extractor biotools co ltd taiwan andrneasy mini kit qiagen gaithersburg md usa accordingto the manufacturers instructions complementary dna wassynthesized using a toolscript mmlv rt kit biotoolsco ltd taiwan quantitative realtime polymerase chainreaction pcr assays using the taqman gene expressionkit applied biosystems foster city ca usa tools sybr qpcr mix biotools co ltd taiwan and anabi steponeplustm system applied biosystems were usedto detect p21 expression using glyceraldehyde 3phosphatedehydrogenase as an internal controlwestern blot analysishuh7 and hepg2 cells were treated with magnolol mm1or dimethyl sulfoxide dmso for h followed by lysis inripa lysis buï¬er biotools co ltd taiwan containingprotease inhibitors cell lysates 30µg protein were subjectedto western blotting as described previously using actin asa loading control the relative intensities of the protein bandswere quantiï¬ed using imagequant software ge healthcarepiscataway nj usain vitro cell proliferation assaythe proliferation capacity of magnololmm1treated cells wasexamined using an xcelligence realtime cell analyzerroche life science indianapolis in usa according to themanufacturers standard protocoltranswell migration and invasion assaythe migration and invasion capacities of magnololmm1treated cells were analyzed using a transwell migration assay asdescribed previously cell cycle analysiscells were trypsinized washed twice and ï¬xed with ethanolat ¦c for h the ï¬xed cells were subsequently incubatedin phosphatebuï¬ered saline containing triton x100 mmoll ethylenediaminetetraacetic acid and mgmlribonuclease a at ¦c for h cells were stained with propidiumiodide µgml at ¦c for min and cell cycle distributionwas measured using a bd facs calibercell apoptosis assaythe apoptosis status of huh7 cells was determined using adeadendtm fluorometric terminal deoxynucleotidyl transferasedutp nick end labeling tunel assay kit promega madisonwi usa according to the manufacturers protocol in summaryhuh7 cells were grown on chamber slides and treated withdiï¬erent concentrations of magnolol or mm1 for h thecells were ï¬xed with paraformaldehyde for min at roomtemperature and subsequently subjected to the tunel assayapoptotic cells were examined using a ï¬uorescence microscopemagniï¬cation images of ï¬ve random ï¬elds per dish wereexamined for each experimenttumor formation assay in nude micesixweekold male balbc nude mice were purchased fromthe national laboratory animal center taipei taiwan andmaintained under speciï¬c pathogenfree conditions animalexperiments were performed under an approved protocol inaccordance with the guidelines for the animal care andethics commission of chang gung memorial hospital iacucapproval no approval date the micewere injected subcutaneously with huh7 cells in µlof saline with matrigel [bd biosciences] into both ï¬anksall tumors were allowed to grow for week before the initiationof drug treatment at the start of the second week mice withtumors were intraperitoneally injected three times a week with µl of magnolol or mm1 µmol in µl of dmso oran equal volume of dmso which served as a control twentyeight days after drug administration the mice were euthanizedand the tumors were subjected to immunohistochemical stainingimmunohistochemistrythe tumors from the mice were ï¬xed in paraformaldehydeovernight dehydrated and embedded in paraï¬n paraï¬n blockswere sliced into 2mmthick sections and ï¬oated onto glass slidesthe tissue sections were deparaï¬nized and the expression ofhdac1 hdac2 p21 cyclin d1 cdk4 ki67 ecadherin ncadherin vimentin and snail in the tissues were detected asdescribed previously statistical analysescomparisons between groups were analyzed using studentsttests the results are expressed as the mean ± standarddeviation the halfmaximalinhibitory concentration ic50values were determined by nonlinear regression analysisusing graphpad prism version graphpad software incla jolla ca usa all statistical analyses were performedusing the statistical package for the social sciences version and microsoft excel all pvalues were twosided with p considered to indicate a statisticallysignificant diï¬erenceresults2omethylmagnolol mm1 has superiorinhibitory effects on hepatocellularcarcinoma hcc cell growth metastasisand invasionto determine whether magnolol and mm1 exhibited anticanceractivities againstlineshepg2 and huh7 were treated with diï¬erent concentrationsof magnolol and mm1 to analyze their eï¬ects on cell growththe results suggested that both magnolol and mm1 significantlyinhibited hcc cell growth compared to the control grouptreated with dmso magnolol inhibited the growth of the twoliver cancer figure 1a hcc cellfrontiers in oncology wwwfrontiersinaugust volume 0cchen 2omethylmagnolol suppresses hdac expressionfigure compared to magnolol 2omethylmagnolol mm1 demonstrates a greater ability to inhibit hepatocellular carcinoma hcc cell growth a chemicalstructures of mm1 and magnolol bf human skin ï¬broblasts huh and hepg2 cells were treated with different concentrations and µm ofmagnolol or mm1 and the cell proliferation status was analyzed using an xcelligence realtime cell analyzer the results are shown as the mean ± standarddeviation of three independent experiments significant differences compared with the vehicle groups p p g effect of magnolol and mm1 oncell cycle progression in huh7 cells cells were treated with the indicated concentrations of magnolol or mm1 for h cell cycle distribution was measured bypropidium iodide staining and quantiï¬ed by ï¬ow cytometry the quantitative results are shown in h i effects of magnolol and mm1 on apoptosis in huh7 cellsterminal deoxynucleotidyl transferase dutp nick end labeling tunel staining was used to observe the apoptotic cells under a ï¬uorescence microscopemagniï¬cation green punctate staining represents tunelpositive cells white arrowfrontiers in oncology wwwfrontiersinaugust volume 0cchen 2omethylmagnolol suppresses hdac expressionfigure 2omethylmagnolol mm1 and magnolol inhibit hepatocellular carcinoma cell migration and invasion by suppressing epithelialmesenchymal transitionemt ab comparisons of migration capacities of huh7 and hepg2 cells treated with magnolol or mm1 in transwell assays cd invasion assays usingmatrigelcoated polyethylene terephthalate membrane inserts eg western blotting showing the expression of emtrelated proteins in huh7 and hepg2 cells aftertreatment with magnolol and mm1 quantitative results are shown in fh the results are shown as the mean of three independent experiments significantdifferences compared with the vehicle control groups p p p cell lines from µm onward with increasing eï¬ects in a dosedependent manner however mm1 displayed a significantlystronger inhibitory eï¬ect on cell growth than magnolol atsimilar concentrations indicating a greater tumorsuppressiveactivity than that of magnolol figures 1cf the halfmaximalinhibitory concentration ic50 of magnolol toward huh7 andhepg2 cells was ¼ and µm respectively which is similarto results from other studies while the ic50 of mm1in huh7 and hepg2 cells was and µm respectivelymoreover only a slight inhibitory eï¬ect was observed on thegrowth of the hfb cell line at the highest concentrations ofmagnolol and mm1 figure 1b this ï¬nding indicated thatmagnolol and mm1 selectively inhibited hcc cell growth withlow toxicity to normal cellsflow cytometry analysis to further understand the potentialuences of magnolol and mm1 on the cell cycle showed thatfrontiers in oncology wwwfrontiersinaugust volume 0cchen 2omethylmagnolol suppresses hdac expressionfigure 2omethylmagnolol mm1 and magnolol inhibit class i histone deacetylase hdac expression in hepatocellular carcinoma cell lines ac huh7 andhepg2 cells were treated with magnolol mm1 or vehicle for h the expression levels of hdacs and were determined by western blotting quantitativeresults are shown bd e the levels of acetylated histone h3 in hepg2 cells were examined by western blotting the quantitative results are shown in f themeasurement data are expressed as the mean ± standard deviation of three independent experiments p p p treatment with magnolol and mm1 caused cells to stagnateat the g1 phase figures 1gh additionally even at highconcentrations magnolol and mm1 treatment did not causeapoptosis figure 1i these ï¬ndings suggest that magnolol andmm1 inhibited cell growth by causing cell cycle arrestone of the primary reasons that liver cancer is diï¬cult tocure is the strong invasion and metastasis ability of tumorcells to investigate the eï¬ects of magnolol and mm1 on themetastasis and invasion ability of hcc cells we performeda transwell migration assay the results indicated that bothmagnolol and mm1 had inhibitory eï¬ects on cell migrationability figures 2ab similar inhibitory eï¬ects were alsoobserved on the invasion abilities of hcc cells at similarconcentrations figures 2cd consistent with the results of thecell growth analysis mm1 displayed higher inhibitory eï¬ects onthe migration and invasion capacities of hcc cells compared tothose of magnolol at similar concentrationsconsidering thattheepithelialmesenchymaltransitionemt is an important process for tumor metastasis we alsomeasured the expression levels of emtrelated proteins suchas ncadherin ecadherin and slug to determine whethermagnolol and mm1 inhibited hcc migration and invasionby regulating emt the results showed significantly lowerexpression of emtpromoting proteins ncadherin and slugin magnolol and mm1treated cells compared to that in thecontrol group figures 2eh these ï¬ndings suggested thatmagnolol and mm1 inhibited hcc migration and invasion bysuppressing emtfrontiers in oncology wwwfrontiersinaugust volume 0cchen 2omethylmagnolol suppresses hdac expressionfigure 2omethylmagnolol mm1 and magnolol induce the expression of the tumorsuppressor gene p21 and the acetylation of p53 a huh7 and hepg2cells were treated with the indicated concentrations of magnolol or mm1 for h the p21 rna levels were examined by quantitative realtime reversetranscriptionpolymerase chain reaction b expression levels of p21 and downstream proteins in huh7 cells were analyzed by western blot using actin as aninternal control quantitative results are shown in c p p p d hepg2 cells were treated with magnolol or mm1 for h and the levelsof acetylated p53 were examined by western blot quantitative results are shown in e error bars represent mean ± standard deviation from three independentexperiments p p p magnolol and mm1 inhibit class i histonedeacetylase expression in hcc cellsprevious studies suggestthat magnolol could inhibit nonsmall cell lung cancer progression by inhibiting class i hdacexpression additionally the overexpression of class ihdacs commonly observed in liver cancer patients is associatedwith liver cancer progression to determine whether theantihcc eï¬ects of magnolol and mm1 were exerted byinhibiting class i hdacs western blot analysis was performedto examine the expression of class i hdacs in hcc cells treatedwith magnolol and mm1 the results indicated that treatmentwith magnolol and mm1 considerably inhibited the expressionof hdac and proteins additionally the inhibitoryeï¬ect of mm1 on class i hdacs was significantly higher thanthat of magnolol at similar concentrations figures 3adanother western blot analysis performed to investigate theassociation between magnolol or mm1 treatment and thedegree of acetylation of histone h3 in hcc cell lines showedsubstantially higher histone h3 acetylation in cells treated withmagnolol and mm1 compared to that in the control groupfigures 3ef these ï¬ndings indicated that magnolol and mm1promoted histone acetylation by inhibiting hdac expressionmagnolol and mm1 induce p21 expressionand p53 acetylationhdacs can regulate the degree of deacetylation of histoneand nonhistone proteins thereby suppressing gene expressionprevious studies have reported that class i hdacs inducecarcinogenesis by inhibiting the expression ofthe tumorsuppressor gene p21 and activating the tumorsuppressor proteinp53 thus realtime rtpcr and western blot analyseswere performed to identify the eï¬ects of magnolol and mm1 onthe expression and activation of p21 and p53 the results showedsubstantially higher expression of p21 mrna and protein inhuh7 and hepg2 cells treated with magnolol and mm1 andlower expression of cell cycle regulatory proteins such as cdk4and cyclin d1 than in the control group figures 4acthese ï¬ndings suggested that magnolol and mm1 could inducep21 gene expression thereby impeding cell cycle progressionfurthermore the fact that the degree of p53 protein acetylationincreased with increasing magnolol and mm1 concentrationsfigures 4de suggested that magnolol and mm1 couldpromote the activation of p53 tumorsuppressor proteinsmagnolol and mm1 enhance the antihcceffect of sorafenibprevious studies suggestthat the combined use of hdacinhibitors and sorafenib could enhance the antitumor eï¬ect ofsorafenib to understand whether the combined useof magnololmm1 and sorafenib showed compounded eï¬ectsmagnololmm1 and sorafenib were administered individuallyand concurrently to hcc celllines the cell proliferationassay and ï¬ow cytometry were performed to analyze cellproliferation and cell cycle progression the results indicatedthat individual treatment with magnololmm1 or sorafenib ledto cell stagnation at the g1 phase and induced cell apoptosis incontrast the concurrent administration of magnololmm1 andfrontiers in oncology wwwfrontiersinaugust volume 0cchen 2omethylmagnolol suppresses hdac expressionfigure 2omethylmagnolol mm1magnolol and sorafenib show a synergistic antihepatocellular carcinoma effect ab huh7 and hepg2 cells were treatedwith µm magnololmm1 and µm sorafenib individually or in combination the cell proliferation status was analyzed using an xcelligence realtime cellanalyzer the data are expressed as the mean ± standard deviation from three independent experiments c the cell cycle status in huh7 cells treated withmagnololmm1sorafenib was examined by ï¬ow cytometry d effects of magnololmm1 and sorafenib alone or in combination on cell proliferation in humanï¬broblastssorafenib substantially improved the toxic eï¬ect on hcc celllines figures 5ac these ï¬ndings veriï¬ed that the combineduse of magnolol and sorafenib could enhance the eï¬cacy ofantiliver cancer treatmentsafety ofthethe eï¬ects ofto understand thecombined use ofmagnololmm1 and sorafenibthe abovecompounds on human ï¬broblasts hfb alone or in combinationwere tested figure 5d we found that magnolol and mm1 didnot significantly aï¬ect the growth of hfb whereas sorafenibslightly inhibited the growth of hfb however when mm1 ormagnolol are used in combination with sorafenib it can reducethe toxicity of sorafenib to hfbmagnolol and mm1 inhibit tumor growth inmiceto conï¬rm that magnolol and mm1 demonstrated the sameinhibitory eï¬ects on hcc cells in vivo and veriï¬ed theabovementioned regulatory mechanism a mouse xenograftmodel was established by injecting huh7 cellsinto thebacks of mice and subsequently administering magnolol ormm1 periodically via intraperitonealinjection the resultssuggested that compared to the control group that onlyreceived dmso the administration of either magnolol or mm1significantly inhibited tumor growth in mice in addition theinhibitory eï¬ect of mm1 was superior to that of magnololfigures 6ac furthermorethe weights and liver tissuemorphology of mice treated with magnolol or mm1 did notchange considerably nor were there any significant abnormalitiesin the serological test results for the two groups of micefigures 6df indicating that neither treatment was toxic tothe micemouse tumor tissues were sectioned and subjected toimmunohistochemical staining to analyze the expression of classi hdacs and p21 cdk4 cyclin d1 ki67 and emtrelatedgenes our results were consistent with those from in vivoexperiments that is dramatic decreases in class i hdacs cdk4cyclin d1 ki67 and emtpromoted protein expression andincreased p21 and ecadherin expression in tumor tissues of micetreated with magnolol or mm1 figures 6gh these ï¬ndingsconï¬rm that magnolol and mm1 induce the expression of theabove tumorsuppressor genes by inhibiting class i hdacsthereby inhibiting hcc growth and metastasis figure frontiers in oncology wwwfrontiersinaugust volume 0cchen 2omethylmagnolol suppresses hdac expressionfigure 2omethylmagnolol mm1 and magnolol inhibit tumor growth in mice a a total of huh7 cells were injected into the dorsal ï¬anks of nude micen per group subsequently the mice were intraperitoneally injected three times per week with µl of magnolol or mm1 [ µmol in µl of dimethylsulfoxide dmso] or an equal volume of dmso representative images show the tumor xenografts at weeks postimplantation b tumor tissues were collected atthe end point c tumor weights at end point d body weights measured during the experiment p e serological test results of the three groups ofmice f hematoxylin and eosin he staining of mouse liver tissue sections magniï¬cation gh immunohistochemical staining showing the effect ofmagnolol or mm1 on class i histone deacetylases p21 cdk4 cyclin d1 ki67 and emtrelated protein expression in mouse xenograft tumors magniï¬cation discussionin the present study we tested the antihcc eï¬ects of magnololand its methoxylated derivative mm1 and elucidated their modesof action both the cell and the animal models showed thatmagnolol and mm1 inhibited hcc cell and tumor growthalthough the inhibitory eï¬ect of mm1 was superior to that ofmagnolol at similar concentrations additionally we found thatmagnolol and mm1 inhibited cell cycle progression and tumrowth by inhibiting class i hdac expression and promotingp21 expression and p53 acetylation to the best of our knowledgethis is the ï¬rst study to report the antihcc activity of mm1 andits superior potential for liver cancer treatment compared to thatof magnololdue to their extensive range of gene regulation hdacsaï¬ect multiple physiological processes including cell growthdiï¬erentiation and apoptosis previous studies have suggestedthat abnormal hdac expression is closely associated with theoccurrence of various diseases including cancer and thereforeidentiï¬ed hdacs as key therapeutic targets among hdac types substantial expression of class i hdacsis commonly observed in most cancer types including hdacs and in breast cancer hdac in lung cancer hdacs and in colorectal cancer and hdacs andfrontiers in oncology wwwfrontiersinaugust volume 0cchen 2omethylmagnolol suppresses hdac expressionfigure schematic representation summarizing the antihepatocellular carcinoma mechanisms of magnolol or 2omethylmagnolol mm1 mm1 and magnololinhibited cell cycle progression and tumor growth by inhibiting class i histone deacetylase expression and promoting p21 expression and p53 acetylation in liver cancer considering this we focused on theinhibitory eï¬ects of magnolol and mm1 on class i hdacs toinvestigate the feasibility of their clinical applications howeverdue to the overexpression of other types of hdacs in othercancer types it is necessary to analyze the inhibitory eï¬ectsof these compounds on other types of hdacs to determinewhether they can be used for the treatment of other cancersfurthermore although we discussed the eï¬ects of magnolol andmm1 on the tumorsuppressor genes p21 and p53 these resultsmay only partially explain the anticancer mechanism of magnololand mm1 future studies will continue to investigate the eï¬ectsof these two compounds on the regulation of other tumorsuppressor pathways to better understand the mechanisms bywhich they act to suppress tumorswe observed that magnolol and mm1 enhanced p21expression by inducing histone acetylation thereby inhibitingcyclin d1 and cdk4 activities as well as cell cycle progressionadditionally magnolol and mm1 also induce p53 proteinacetylation which not only enhances its stability but alsoimproves its ability to bind to the target gene promoter therebyupregulating the expression of downstream tumorsuppressenes such as p21 and bax these results indicated thatmagnolol and mm1 could regulate p21 expression via both directand indirect pathways and consequently inhibit tumor growthour previous studies conï¬rmed that replacing the hydroxylfunctional group of magnolol with a methoxy group couldincrease the lipophilicity of the methoxylated derivative andimprove its skin delivery ability and antiammatory activity in another study we also conï¬rmed thatthe sameconcentration of mm1 could induce increased expression of thetumor suppressor long noncoding rna gas5 compared tomagnolol and exert a greater inhibitory eï¬ect on skin cancer cells consistent with previous research we found that the sameconcentration of mm1 could yield better antiliver cancer activitycompared to that of magnolol this may be due to the betterlipophilicity and cell uptake eï¬ciency of mm1 compared to thoseof magnolol as it has higher eï¬cacy at the same concentrationthis methoxylation could also increase the mucosal absorptionrate of the compound enhancing the ï¬exibility of the route ofadministration and its clinical applicabilitymultiple clinicalfor hdac inhibitorstrials have shown excellent outcomesincluding chidamide panobinostatfrontiers in oncology wwwfrontiersinaugust volume 0cchen 2omethylmagnolol suppresses hdac expressionvorinostat and saha in the treatment of many cancers among these saha was the ï¬rst hdac inhibitor approved bythe fda for the treatment of tcell lymphoma it can speciï¬callybind to the zinccontaining catalytic domains of class i ii and vihdacs thus inhibiting their enzymatic activities in addition totcell lymphoma saha has shown promise in treating cancersof the breast lungs and prostate additionally the combined useof hdac inhibitors with other clinical anticancer medicationsshows compounded eï¬ects for example the combineduse of saha and bortezomib promotes nasopharyngeal cancercell apoptosis and the combined use of romidepsin withcisplatin and gemcitabine enhances their therapeutic eï¬ectsagainst triplenegative breast cancer in the present study wefound that magnolol and mm1 inhibit the growth of hcc cellsby suppressing the expression of class i hdac which is diï¬erentfrom the mechanism of action of saha however we alsoobserved that the combined use of magnololmm1 and sorafenibsubstantially enhanced their antiproliferative eï¬ects on hcccells the ï¬ndings indicate the potential of using magnololmm1as an adjuvant in combination with sorafenib in liver cancertreatment future studies will continue to investigate the optimalcombination and dosage of magnololmm1 and existing clinicaldrugs including sorafenib and sahasorafenib is an fdaapproved kinase inhibitor that inhibitsthe activation of tyrosine kinases such as vegfr pdgfr andraf family kinases it has also been reported to inducethe expression of p21 and p53 which is the maintumor suppressor regulatory pathway of magnolol and mm1before fully elucidating the interaction between these drugs andmolecules we cannot assume that the additive antihcc eï¬ectof magnololmm1 and sorafenib is entirely due to the activationof the p21 and p53 tumor suppression pathways howeverwe believe thatthese molecules should play an importantregulatory role in addition we observed that the combined useof magnololmm1 and sorafenib not only substantially enhancedtheir antiproliferative eï¬ects on hcc cells but also reducedthe toxicity of sorafenib monotherapy in normal cells furtherstudies are also required to determine the mechanisms by whichmagnololmm1 reduces the physiological toxicity of sorafenibin conclusion although hdac inhibitors have been usedextensively for the treatment of various cancers their sideeï¬ects remain a bottleneck to their clinical application in thisstudy we synthesized a methoxylated derivative of magnololmm1 and veriï¬ed its superior antihcc activity over magnololadditionally it can enhance the therapeutic eï¬ect of sorafenibwhen used in conjunction and does not present physiologicaltoxicity thus mm1 is a suitable combination therapeuticadjuvant to improve the therapeutic eï¬cacy of anticancer drugsdata availability statementthe raw data supporting the conclusions of this will bemade available by the authors without undue reservationethics statementthis animal study was reviewed and approved by the animalcare ethics commission of chang gung memorial hospitaliacuc approval no approval date author contributionsthw jyf and cyc conceptualization cyc and thwdata curation ccc wyc and yll formal analysis thwlsw and sfc investigation ch and jyf method | Colon_Cancer |
environmental exposure to arsenite as3 has a strong association with the development ofhuman urothelial cancer uc and is the 5th most common cancer in men and the 12th mostcommon cancer in women muscle invasive urothelial cancer miuc are grouped into basalor luminal molecular subtypes based on their gene expression profile the basal subtype ismore aggressive and can be associated with squamous differentiation characterized byhigh expression of keratins krt1 and and epidermal growth factor receptoregfr within the tumors the luminal subtype is less aggressive and is predominatelycharacterized by elevated gene expression of peroxisome proliferatoractivated receptamma pparÎ and forkhead box protein a1 foxa1 we have previously shown thatas3transformed urothelial cells ast exhibit a basal subtype of uc expressing genesassociated with squamous differentiation we hypothesized that the molecular subtype ofthe ast cells could be altered by inducing the expression of pparÎ andor inhibiting theproliferation of the cells nontransformed and ast cells were treated with troglitazonetg pparg agonist μm pd153035 pd an egfr inhibitor μm or a combination oftg and pd for days the results obtained demonstrate that treatment of the ast cellswith tg upregulated the expression of pparÎ and foxa1 whereas treatment with pddecreased the expression of some of the basal keratins however a combined treatment oftg and pd resulted in a consistent decrease of several proteins associated with the basalsubtype of bladder cancers krt1 krt14 krt16 p63 and tfap2a our data suggeststhat activation of pparÎ while inhibiting cell proliferation facilitates the regulation of genesinvolved in maintaining the luminal subtype of uc in vivo animal studies are needed toaddress the efficacy of using pparÎ agonists andor proliferation inhibitors to reduce tumradestage of miuca1111111111a1111111111a1111111111a1111111111a1111111111open accesscitation mehus aa bergum n knutson pshrestha s zhou xd garrett sh activation of pparÎ and inhibition of cellproliferation reduces key proteins associated withthe basal subtype of bladder cancer in as3transformed urotsa cells one e0237976 101371 pone0237976editor karl x chai university of central floridaunited statesreceived may accepted july published august copyright mehus 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 paper and its supporting informationfilesfunding seema somji und school of medicineand health sciences pilot grant das allundergraduate research and core facilities ndinbre idea program p20 gm103442 from thenational institute of general medical sciences nih one 101371 pone0237976 august one 0ccompeting interests the authors have declaredthat no competing interests existactivation of luminal pathway in basal bladder cancerintroductionbladder cancer bc is the ninth most common cancer diagnosed worldwide and in theamerican cancer society estimated that about new cases of bc would be identified inthe us and about deaths would occur from bladder cancer among bcs urothelialcell carcinomas uc are the most common being the second most diagnosed cancer of thegenitourinary tract behind prostate cancer [ ] it is the 5th most common cancer in menand the 12th most common cancer in women urothelial cancers are classified as muscle invasive miuc or nonmuscleinvasivenmiuc nonmuscleinvasive urothelial cancers have a lower tendency to progress whereasmiucs have a high rate of metastasis and a year survival rate of approximately bothmiuc and nmiuc have been subtyped into various groups with the basal and luminal subtype being the most prominent the luminal subtype of human uc includes the majority ofthe early stage noninvasive ucs and a significant number of miucs this subtype isenriched for papillary histology is less aggressive and has a more favorable patient outcome [ ] basal classified tumors have a poorer overall survival compared to luminal tumors they often exhibit squamous differentiation are aggressive and found exclusively inmiuc that metastasize and spread to distal ans about of miucs arise independent of the papillary pathway have poor outcomes and an overall year survival rate of environmental exposure to arsenite as3 has a strong association with the developmentof human uc the increased risk of uc correlates to the same endemic areas of the worldwhere populations have been identified with arsenicinduced skin cancer [] we havedeveloped a cell culture model of arsenicinduced urothelial cancer by exposing the immortalized nontumorigenic urothelial cell line urotsa to arsenite these transformed cell lines produce tumors in athymic mice that express genes for keratin krt and asignature pattern highly similar to the basal subtype of human miuc [ ] the tumorshave a histology similar to urothelialtransitional cell carcinomas with focal areas of squamousdifferentiation [ ] which is associated with poor prognosis [ ]the molecular mechanism driving a tumor towards a basalsquamous subtype is currentlyunknown in a recent study yamashita show that transcription factor activatingprotein alpha tfap2a is expressed at high levels in basalsquamous bladder cancerenriched in areas of squamous differentiation and is associated with increase lymph nodemetastasis and distant recurrence of the disease the study also shows that increased expression of tfap2a can facilitate the expression of other transcription factors such as tumor protein p63 tp63p63 also known as p63 which is known to be associated with the basalsubtype of uc palmbos demonstrated that p63 binds to the transcriptional regulatory regions of the gene ataxiatelangiectasia group d complementing gene atdc alsoknown as trim29 and krt14 thus increasing their expression the study further showedthat both krt14 and trim29 promote the invasion of the basal subtype of uc in vitro and invivo the luminal subtype of uc is associated with the expression of the transcriptional factorsforkhead box protein a1 foxa1 gata binding protein gata3 and peroxisome proliferatoractivated receptor gamma pparÎ [ ] the activation of pparÎ with an agonistcan represses the expression of tfap2a and inhibit squamous differentiation in vitro the exact role of pparÎ signaling in carcinogenesis is somewhat unclear however theexpression of pparÎ in bladder cancers is a favorable prognostic marker both in vivoand in vitro studies indicate that pparÎ ligands such as troglitizone can promote differentiation inhibit cellular proliferation induce autophagy and enhance apoptosis in bladder cancer one 101371 pone0237976 august one 0cactivation of luminal pathway in basal bladder cancer[] likewise suppressing cellular proliferation with epidermal growth factor receptoregfr inhibitors has been used preclinically to reduce basallike muscle invasive bladdertumor growth although the egfr inhibitors did not have the same efficacy in nonbasalliketumors the goal of this study was to determine if the activation of pparÎ and inhibition of cellproliferation in the urotsa parent and the as3transformed urotsa isolates would repressthe expression of genes involved in maintaining the basalsquamous type of bladder cancerand induce genes that were associated with the luminaldifferentiated state of bladder cancermaterials and methodsanimalsathymic nude ncrnunu mice purchased from envigo were used in these studies themice were housed four to a cage at Ëc under a 12hour lightdark cycle food and water wasavailable ad libitum mouse heterotransplants of the urotsa transformed cell lines as3 andas4 and the rt4 cell line were produced by subcutaneous injection at a dose of x cellsin the dorsal thoracic midline of athymic nude ncrnunu mice this study adhered to allrecommendation dictated in the guide for the care and use of laboratory animals of thenih tumor sizes were assessed weekly and the animals were sacrificed when the size of thetumor was approximately cm or when dictated by clinical conditions euthanizationwas done by co2 asphyxiation and conformed to the american veterinary medical association guideline on euthanasia death was confirmed by ascertaining cardiac and respiratoryarrest following which the ans and tumor were harvested care of taken to ensure thatthere was no distress to the animals during the procedure the protocol was approved by theuniversity of north dakota animal care committee iacuc16122ccell culturethe urotsa parent cells and two of the as3transformed isolates as3 and as4 were cultured in in dulbecos modified eagles medium dmem supplemented with vv fetalbovine serum as described previously the cells were subcultured at a ratio usingtrypsinedta and the cultures were fed fresh growth medium every three days the urotsaparent cell line has been authenticated using short tandem repeat str analysis the as3transformed isolates used in the current study have been previously characterized for its ability to form colonies in soft agar form spheroids when grown in ultralow attachment flasksand form tumors when injected subcutaneously in immunecompromised mice [ ]the as3 can also form tumors upon intraperitoneal injection for drug treatmentsurotsa parent and the as3transformed isolates as3 and as4 were grown to confluence inserum containing medium following which the cells were incubated with a serum freemedium consisting of a mixture of dmem and hamss f12 growth medium for h thecells were then exposed to either dimethyl sulfoxide dmso the drug vehicle troglitizonetg μm a pparÎ agonist pd153035 pd μm an epidermal growth factor receptoregfr inhibitor or a combination of tg and pd tgpd for and hours the concentrations of the drugs were chosen based on preliminary studiesvisualization of dapistained cellstoxic effects of tg and pd on the urotsa cells was determined by visualization of 406diamidino2phenylindole dapistained nuclei as described previously by this laboratory atthe indicated time points the cell monolayers were washed twice with phosphate buffered one 101371 pone0237976 august one 0cactivation of luminal pathway in basal bladder cancersaline pbs following which the cells were fixed for min with ethanol and rehydratedwith 1ml pbs the rehydrated cells were stained with 10μl dapi 10μgml in distilled waterrna isolation and realtime pcr analysistotal rna was isolated using tri reagent molecular research center as described previously the expression of various genes was assessed with realtime reverse transcriptionpolymerase chain reaction rtpcr using primers that were purchased commercially frombiorad laboratories the genes along with the catalog number of the primers are listed insupplemental material s1 table total rna μg was transcribed to cdna using theiscript cdna synthesis kit biorad laboratories the amplification of the cdna was performed using the itaq universal sybr green supermix biorad laboratories with μlcdna and μm primers in a total volume of μl in a cfx96 realtime detection systembiorad laboratories amplification was monitored by sybr green fluorescence cyclingparameters consisted of a s hotstart followed by cycles of denaturation at Ëc for sannealing at Ëc for s and extension at Ëc for s which gave optimal amplificationefficiency the resulting levels were normalized to βactin expression assessed by the sameassay the threshold cycles cts for βactin and the resulting delta cts for the target genes arereported in s2 tablewestern blot analysiswestern blot analysis was performed as described previously the cell pellets were dissolved in 1x radioimmunoassay precipitation assay ripa lysis buffer supplemented withpmsf protease inhibitor cocktail and sodium orthovandate santa cruz biotechnology thecell suspension was sonicated and the lysate was centrifuged to remove cellular debris proteinlysates were quantified using the pierce bca protein assay kit thermoscientific pierceprior to loading samples were reduced and denatured the protein extracts were separated ontgx anykd sdspolyacrylamide gels purchased from biorad laboratories and transferred toa μm hybondp polyvinylidene difluoride membrane using semidry transfer the blotswere blocked in trisbuffered saline tbs containing tween20 tbst and wvbovine serum albumin bsa for min at room temperature the membranes were probedovernight at Ëc with the primary antibody diluted in wv bovine serum albumin allantibodies were purchased from commercial suppliers and were validated against known positive and negative expressing cell lines by western analysis prior to use in experimental protocols the source of the antibody along with their catalog numbers are reported in s3 tableafter washing times for minutes each wash in tbst the membranes were incubated withthe antimouse or antirabbit secondary antibody for min at room temperaturethe blots were visualized using the clarity¢ western ecl blotting substrate bioradlaboratoriesimmunohistochemical stainingserial sections were cut at μm and immersed in preheated target retrieval solutiondako in a steamer for min the sections were allowed to cool to room temperature andimmersed into tbst for min the primary antibodies used in this study along with theirdilutions and catalogue numbers are listed in s3 table the primary antibodies were localizedusing dako peroxidase conjugated envision plus for rabbit or mouse primary antibodies atroom temperature for min liquid diaminobenzidine dako was used for visualizationcounter staining was performed for sec at room temperature using readytousehematoxylin dako slides were rinsed in distilled water dehydrated in graded ethanol one 101371 pone0237976 august one 0cactivation of luminal pathway in basal bladder cancercleared in xylene and coverslipped two pathologists judged the presence and degree ofimmunereactivity in the specimensstatistical analysisall experiments were performed in triplicate and the results are expressed as the mean ± semstatistical analyses were performed using graphpad prism1 software version using oneway anova with tukeys or dunnetts posthoc testing for gene expression statistics wererun on the delta cycle threshold δct values that were generated from normalization to βactin levels unless otherwise stated the level of significance was p005resultseffect of troglitizone and pd153035 on the viability and morphology ofurotsa parent and ast cellsthe urotsa parent and ast cells as3 and as4 were treated with either dmso controltroglitizone tg μm pd153035 pd μm or tg and pd tgpd for hr as seenin fig 1a1d there was no change in the morphology of the urotsa parent cells with varioustreatments there was a change in the morphology of the as3 and as4 cells when treatedwith tg fig 1f and 1j and tgpd fig 1h and 1l the cells looked more differentiatedformed mounds and resembled the intermediate cells of the bladder there was a decrease inthe number of urotsa parent cells treated with pd and tgpd when compared to the cellstreated with tg alone or with dmso fig 1m there was also a decrease in the number ofas4 cells when treated with tg and tgpd when compared to the dmso treated cells fig1o there was no significant decrease in the number of as3 cells in any of the treatmentgroups fig 1n an examination demonstrated the lack of dead cells in the treated groups andthe decrease in cell number compared to the dmso group could be due to lack of proliferationandor increased differentiation of cellseffect of pparÎ activation and egfr inhibition on the expression ofluminal genesthe transcription factors pparÎ foxa1 and gata3 play a role in the establishment of theluminal subtype of bladder cancer [ ] studies done by varley have shown thatagonistdependent activation of pparÎ with simultaneous inhibition of egfr phosphorylation in normal human urothelial cells increases the effectiveness of the pparÎ agonist in thepresent study we investigated the effect of the pparÎ agonist tg and an egfr inhibitor pdon the expression of luminal transcriptional factors in the urotsa parent cells and the astcells expression levels for the target genes in this study are reported for a hr hr and hr timecourse for the parent as3 and as4 cells s1 s2 and s3 figs respectively for simplicity the hr gene and protein levels are reported in the main body of the manuscripttreatment with tg increased the expression of pparÎ in the urotsa parent fig 2a i iv andv cell line a similar effect was seen in as3 fig 2b i iv and v and as4 fig 2c i iv and vcell lines treatment with pd did not induce the expression of pparÎ in the urotsa parentfig 2a iv and v or as3 fig 2b iv and v cells but there was a small increase in pparÎ protein in the as4 cells fig 2c iv and v treatment with both tg and pd tgpd increasedthe expression levels of pparÎ mrna in the urotsa parent cells but there was no increase inthe protein levels there was no increase in mrna expression in the as4 cells but there was aslight increase in the protein levels fig 2c i iv and v one 101371 pone0237976 august one 0cactivation of luminal pathway in basal bladder cancerfig morphology and viability of urotsa parent and ast cells the urotsa parent ad and ast cells as3 eh and as4 il were treated witheither dmso control troglitizone tg μm pd153035 pd μm or tg and pd tgpd for hr the measurements were performed in triplicatesand the values reported are mean percentage of control ± sem ordinary oneway anova was performed followed by tukeys posthoc test bars withdiffering letters indicate significant differences p 101371 pone0237976g001foxa1 gene and protein expression in the urotsa parent cells treated with tg wasreduced fig 2a ii iv and vi however the expression was increased in the as3 and as4cells fig 2b ii iv and vi and 2c ii iv and vi at the protein level treatment with pd decreasedfoxa1 protein in the parent cells but the levels were elevated in the as3 cells tgpdreduced the expression of foxa1 in the urotsa parent cells but it increased the expression offoxa1in the as4 cells at the protein leveltreatment of the urotsa parent cells with tg pd or tgpd did not increase the mrnalevels of gata3 but there was an increase in the protein levels after treatment with tg andtgpd when compared to the dmso treated group fig 2a iii iv and vii in as3 and as4cells there was an additive reduction of gata3 protein by using the combined tgpd treatment fig 2b iv and vii and 2c iv and vii one 101371 pone0237976 august one 0cactivation of luminal pathway in basal bladder cancer one 101371 pone0237976 august one 0cactivation of luminal pathway in basal bladder cancerfig expression of luminal markers in urotsa parent and ast cells the urotsa parent aivii as3 bivii and as4 civii weretreated with either dmso control troglitizone tg μm pd153035 pd μm or tg and pd tgpd for hr real time rtpcranalysis was performed to verify gene expression a b ciiii western blot analysis was used to measure protein levels a b civ and theintegrated optical density iod of each protein band was calculated a b cvvii gene expression was normalized to βactin and gene andprotein are plotted as foldchange relative to the dmso control amplification was below detectable levels for pparÎ in dmso as3 so adelta cycle threshold δct value of was assigned which is higher than the highest delta ct detected for pparÎ in that cell linetriplicate measurements of gene and protein data were performed and are reported as mean ± sem ordinary oneway anova wasperformed followed by tukeys posthoc test bars with differing letters indicate significant differences p 101371 pone0237976g002effect of tg and pd on the phosphorylation and expression levels of egfrthe effect of tg and the egfr inhibitor pd was determined on the expression and phosphorylation of egfr in the urotsa parent and ast cells only the combination of tgpdreduced the expression of egfr mrna in the urotsa parent cells however all three treatments decreased the protein levels fig 3a i ii and iii there was no basal phosphorylation ofegfr in the urotsa parent cells and none of the treatments had any effect on the phosphorylation levels fig 3a ii the expression of egfr in the ast cells varied with a decrease inas3 cells and an increase in as4 cells fig 3b i ii and iv and fig 3c i ii and iv respectivelyboth the transformed cell lines had basal phosphorylation of the egfr pegfr and treatment with pd decreased the pegfr levels in as3 fig 3b ii and iii and as4 fig 3c ii andiii which indicates that the pd treatment was effectiveeffect of the pparÎ agonist and inhibition egfr phosphorylation on theexpression of keratinsstudies performed by varley have shown that inhibition of the egfr with simultaneous activation of pparÎ signaling switches normal human urothelial cells from a squamous metaplastic phenotype to a transitional differentiated state with the repression ofkrt14 and the upregulation of krt13 and krt20 therefore we wanted to determineif the urotsa parent and the ast cells would revert more from a basal phenotype to amore transitionalintermediate phenotype when treated with tg and pd the mrnaexpression data is shown in fig and the protein expression data is shown in fig for theurotsa parent cells there was a decrease in expression of krt1 krt5 and krt14 with alltreatments fig 4a i ii and vi and fig 5a i ii and iv the protein for krt1 was undetectedin the urotsa parent cells the expression of krt6 and krt16 increased with tg butdecreased with pd and tgpd fig 4a iii iv v and vii and fig 5a iii and v the krt6antibody does not distinguish between the krt6a krt6b and krt6c isoforms and recognizes protein made by these three genes thus it is not known which isoform is beingexpressed at the protein level there was a decrease in expression of krt13 in the urotsaparent cells fig 4a viii and fig 5a i and vi in the as3 cells the expression levels of thebasal krts with various treatments was similar to the urotsa parent cells fig 4b ivii andfig 5b ivi with the exception of krt1 protein which was expressed in the as3 cells andits expression decreased with tg and tgpd treatment in the as4 cells the expression ofthe krts was similar to as3 with the exception of krt16 fig 4c iviii treatment withtg decreased the expression of krt16 the protein levels for the all the krts was similarto the mrna level with the exception of krt5 krt13 and krt16 krt5 showed anincrease in expression with pd and tgpd treatment and krt16 which showed anincrease in expression with pd fig 5c ivii in the as3 and as4 cells krt13 geneexpression was reduced however the protein levels were elevated from all three treatmentsfig 4b viii fig 4c viii and fig 5b i vii fig 5c i vii one 101371 pone0237976 august one 0cactivation of luminal pathway in basal bladder cancerfig expression and phosphorylation of epidermal growth factor receptor the urotsa parent aiiii as3 biiv and as4 ciiv weretreated with either dmso control troglitizone tg μm pd153035 pd μm or tg and pd tgpd for hr real time rtpcranalysis was performed to verify gene expression a b ci western blot analysis was used to measure protein levels a b cii and the integratedoptical density iod of each protein band was calculated aiii b and ciii and iv phosphorylatedegfr was not detected in the urotsa parentcell line gene expression was normalized to βactin and gene and protein are plotted as foldchange relative to the dmso control triplicatemeasurements of gene and protein data were performed and are reported as mean ± sem ordinary oneway anova was performed followed bytukeys posthoc test bars with differing letters indicate significant differences p 101371 pone0237976g003effect of pparÎ activation and egfr inhibition on expression oftranscriptional factors p63 and tfap2a and the oncogene trim29associated with squamous differentiationrecently tfap2a has been implicated in the development of squamous differentiation inbasal cancers and activation of pparÎ is shown to represses the expression of tfap2a we therefore investigated the effects of pparÎ activation and egfr inhibition on the expression of tfap2a in urotsa parent and ast cells our results demonstrate that tg reducedtfap2a protein within the parent and as3 cells while the mrna and protein was elevatedin the as4 cells from tg exposure treatment with pd as well as tgpd decreased the one 101371 pone0237976 august one 0cactivation of luminal pathway in basal bladder cancer one 101371 pone0237976 august one 0cactivation of luminal pathway in basal bladder cancerfig gene expression of keratins the urotsa parent aiviii as3 biviii and as4 civiii were treated witheither dmso control troglitizone tg μm pd153035 pd μm or tg and pd tgpd for hr real timertpcr analysis was performed to verify gene expression a b civiii gene expression was normalized to βactin andare plotted as foldchange relative to the dmso control triplicate measurements of gene levels were performed and arereported as mean ± sem ordinary oneway anova was performed followed by tukeys posthoc test bars withdiffering letters indicate significant differences p 101371 pone0237976g004expression of tfap2a in the urotsa parent fig 6a i iv and v as3 fig 6b i iv and v andas4 cells fig 6c i iv and vtranscription factor p63 is another protein associated with human bladder cancersenriched in basalsquamous markers [ ] there was a decrease in the expression of p63 inthe urotsa parent cells with all treatments fig 6a ii iv and vi in the as3 cells the expression of p63 was low and treatment with tg increased its expression however treatment withpd and tgpd decreased its expression fig 6b ii iv and vi the expression of p63 in as4cells increased at the mrna level with tg and tgpd treatments however the protein levelswere decreased when compared to the dmso control fig 6c ii iv and vithe expression of trim29 a gene associated with the basal gene expression program was also determined in the urotsa parent and the as3transformed cells for the urotsaparent and as3 cells there was a decrease in the expression of trim29 in cells treated withpd and tgpd fig 6a and 6b iii iv and vii for as4 pd and tgpd treatments increasedtrim29 protein fig 6c iv and viiexpression of trim29 tfap2a and p63 within tumors formed fromurotsa as3 as4 and rt4 cellsurotsa as3 and as4 are considered to be of the basal molecular subtype while rt4 cellsare considered to be of the luminal molecular subtype of bladder cancer cells therefore wewanted to confirm the in vivo expression of trim29 tfap2a and p63 within the nondifferentiated basalsquamous areas of tumors originating from urotsa as3 and as4 cells incomparison to the expression in tumors originating from the luminal rt4 cells all three ofthese proteins were enriched within the nondifferentiated areas of tumors developed from theurotsa as3 and as4 cells fig 7a 7b 7d 7e 7g and 7h signs a lower expression wasobserved in the welldifferentiated areas of the urotsa as3 and as4 tumors fig 7a 7b7d 7e 7g and 7h� asterisks there was low to no staining in the rt4 cells for trim29tfap2a and p63 fig 7c 7f and 7idiscussionthe classification of uc into various subtypes has implications in the overall patient management of the disease with the basal subtype having a worse outcome when compared to theluminal subtype the molecular mechanisms involved in the development of these subtypesare not yet established however the role of some transcriptional factors is established withpparÎ playing an important role in the activation of the luminal specific genes [ ] andtfap2a playing a role in the development of the basal subtype of uc in addition studieswith normal human urothelial cells show that activation of pparÎ with an agonist along withinhibition of cellular proliferation via the egfr pathway can switch cells from a squamousmetaplastic phenotype to a more transitional differentiated phenotype combination therapiesusing egfr inhibitors and pparÎ agonists show promising results against some urothelialtumors in vivo as well as against other cancers based on these studies we sought todetermine if the urotsa parent and the as3transformed urotsa cell lines that are one 101371 pone0237976 august one 0cactivation of luminal pathway in basal bladder cancer one 101371 pone0237976 august one 0cactivation of luminal pathway in basal bladder cancerfig protein expression of keratins the urotsa parent aivi as3 bivii and as4 civii were treated with either dmso control troglitizone tg μm pd153035 pd μm or tg and pd tgpd for hr western blot analysis was used to measure protein levels ai bi ci and the integratedoptical density iod of each protein band was calculated aiivi b and ciivii protein levels are plotted as foldchange relative to the dmso controltriplicate measurements of protein data were performed and are reported as mean ± sem ordinary oneway anova was performed followed by tukeysposthoc test bars with differing letters indicate significant differences p 101371 pone0237976g005molecularly characterized as a basal subtype of bc could convert to a luminal transitionalcell type when treated with a pparÎ agonist andor an egfr inhibitor as this could affect theoverall outcome of the diseasethe urotsa parent cells when grown in serum expresses many genes that are associatedwith the basal subtype in this study treatment of these cells with the pparÎ agonist tginduced the expression of pparÎ as well as gata3 but not foxa1 in mortal human urothelial cells treatment with the pparÎ agonist shows an increase in the expression of the transcription factor foxa1 and this is contrary to what is seen in our study these differencescould be due to the cell type since we are using immortalized cells the as3transformedurotsa cells showed an induction in the expression of pparÎ and foxa1 when treated withtg however the expression of gata3 in these transformed lines was not consistent in bccell lines studies by others have shown that have shown that gata3 and foxa1 cooperatewith pparÎ activation to drive the differentiation of a basal bladder cancer subtype to a moredifferentiated luminal subtype other studies have also linked the expression of foxa1 tothe development of the luminal subtype of urothelial cancer [] in our studies treatmentwith tg did result in the differentiation of the as3transformed cells based upon morphological changes and induced the expression of pparÎ as well as foxa1 both of which are factorsknown to drive luminal differentiation of bladder cancer cell linessignaling via the pparÎ pathway is essential for the growth arrest and terminal differentiation of adipocytes and other normal epithelial [] and cancer cells [] whereassignaling via the egfr receptor plays a role in cell proliferation the keratins play an essentialrole in the differentiation of epithelial cells and different keratin profiles are expressed at different stages of differentiation the normal bladder has three different stages of differentiationand these stages are marked by the expression of krt14 krt5 and krt20 krt14 isexpressed in a subset of basal cells that are thought to play a role in regeneration as well astumorigenesis whereas other basal cells and intermediate cells in the normal bladderexpress krt5 the expression of krt20 is restricted to the most differentiated cells theumbrella cells these differentiation stages of the bladder are shared by bladder cancersand malignant transformation can occur in any of the different cell types of the bladder theexpression of krt14 is seen in the least of the differentiated tumors and its expression correlates to poor prognosis krt14 whereas the expression of krt20 is restricted to differentiated bladder cancers and its expression is associated with good prognosis a study doneby varley showed that normal human urothelial cells in culture express krt14 andlack the expression of krt13 and krt20 activation of pparÎ in these cells induced theexpression of krt13 and decreased the expression of krt14 this effect was significantlyenhanced when | 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 |
" mutations in the exonuclease domain of pole a dna polymerase associated with dna replicationand repair lead to cancers with ultrahigh mutation rates most studies focus on intestinal and uterine cancers withpole mutations these cancers exhibit a significant immune cell infiltrate and favorable prognosis we questionedwhether loss of function of other dna polymerases can cooperate to pole to generate the ultramutatorphenotypemethods we used cases and data from cancer types in the cancer genome atlas to investigate mutationfrequencies of different dna polymerases we tested whether tumor mutation burden patient outcomediseasefree survival and immune cell infiltration measured by estimate can be attributed to mutations in polqand polzrev3lresults thirty six percent of colorectal stomach and endometrial cancers with pole mutations carried additionalmutations in polq eq polzrev3l ez or both dna polymerases ezq the mutation burden in thesetumors was significantly greater compared to poleonly e mutant tumors p in addition eq ez and eqz mutant tumors possessed an increased frequency of mutations in the pole exonuclease domain p colorectal stomach and endometrial eq ez and eqz mutant tumors within tcga demonstrated diseasefree survival even if the pole mutations occurred outside the exonuclease domain p however immunescores in these tumors were related to microsatellite instability msi and not pole mutation status this suggeststhat the host immune response may not be the sole mechanism for prolonged diseasefree survival ofultramutated tumors in this cohort results in this study demonstrate that mutations in polq and rev3l in pole mutant tumors shouldundergo further investigation to determine whether polq and rev3l mutations contribute to the ultramutatorphenotype and favorable outcome of patients with pole mutant tumors correspondence beatriceknudsenpathutahedu1department of biomedical sciences cedarssinai medical center losangeles ca usa2samuel oschin cancer research institute socci cedarssinai medicalcenter los angeles ca 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 0chuang bmc medical genetics page of the analysis of thousands of cancers by the cancergenome atlas tcga consortium and academic institutions revealed a small group of cancers with mutationsin pole and an ultramutator phenotype [ ] multiplestudies have found significantly improved survival in patients with pole mutated endometrial cancers [ ]while the survival benefit was not as profound in polemutated colorectal carcinoma the pole geneencodes the catalytic subunit of dna polymerase epsilon which catalyzes the leading strand synthesis duringdna replication pole possesses highfidelity dnapolymerization proofreading and ²² exonuclease activities which promote accurate dna synthesis firstidentified and reported in of colorectal carcinomas[ ] pole mutations were also noted at frequencies of amongst uterine corpus endometrialcancers [ ] and in gastric adenocarcinoma mutations can be found across the entire pole genebut those in the pole exonuclease domain are mostprevalent in cancers with ultrahigh mutation rates mutmb these cancers exhibit higher mutationrates than microsatellite instable msi tumors associated with mismatch repair abnormalities in additionpole ultramutant cancers also possess a high frequency of ctoa transversions [ ] a tumor associated inflammatory response similar to thatin msitumors has been reported to occur early on duringdevelopment of pole mutated endometrial and colorectal cancers it is thought to be caused by neoantigens that are generated as a result of the high mutation burden [ ] and render pole mutant cancersresponsive to immunotherapy polymerase theta polq is a lowfidelity dna polymerase lacking a ² to ² exonuclease function theenzyme is involved in the alternative nonhomologousendjoining pathway altnhej which is a backupmechanism of double stranded dna break repair thispathway predominates in cancer cells when other dnarepair pathways are missing or when telomere ends aredeprotected [ ] the loss of polq sensitizes cellsto ionizing radiation and polqdeficient mice exhibit increased dna instability and genomic rearrangementssuggesting a role for polq as a guardian of the genome both overexpression and loss of polq increasemutation frequencies [ ] multiple structuralmotives in polq can interact with dna rad51 andbrca1 in addition polq forms a complex withparp1 in a pathway of synthetic lethality with brca1and is thus considered a therapeutic target [ ]however which domains in polq should be targetedremains to be determined rev3l rev3 like dna directed polymerase zetapolz catalytic subunit is involved in dna synthesisthat reads through damaged dna translesion dnasynthesis tls the high efficiency of polz bypassing a broad spectrum of dna lesions led to its recognition as a master tls polymerase polzrev3l has been linked to carcinogenesis in breastlung gliomas and gastric cancers and modulatescisplatin sensitivity []because studies describe overlapping functions andsynergy among the over dna polymerases [ ] we undertook an unbiased approach to identifymutations in polymerases within the tcga tumorcompendium this analysis revealed a greater frequencyof mutations in polq and polzrev3l compared toother polymerases of similar gene length therefore wepropose that polq and polzrev3l may cooperatewith pole in generating ultrahigh mutation ratesmethodsdata acquisitionthe data used in this study are based upon the wholeexome sequence data sets generated by the tcgaresearch network httpcancergenomenihgov thelocations and frequencies of somatic mutations msistatus and clinical stage and follow up information intcga provisional datasets were obtained from cbioportal httpwwwcbioportal [ ] up to supplementary table mutation data were obtainedby first selecting the query tab from cbioportal thecancer type specific data were chosen from the tcgapancancer atlas studies we entered the genes ofinterest pole polq and rev3l to retrieve mutationdata from the genes when the result page wasdisplayed we accessed the information underneath themutations tab we downloaded the number of mutations in each sample the annotation of protein aminoacid change and the type of mutation the survivalinformation for each case months after diagnosis wasobtained through the comparisonsurvival link functional domains of the proteins were provided by pfamdatabase data visualization for mutations wasperformed with mutationmapper in cbioportalcase selection criteriawe searched all cancer types in tcga for those thatpossess or more cases with pole mutations thisyielded tumor types that we named the pancandata set in this study we then determined the frequencyof mutations within additional polymerase within pancan we selected the three adenocarcinomas uterinecorpus endometrial adenocarcinoma colorectal adenocarcinoma and stomach adenocarcinoma with the highesttriple mutated dnapolymerase status for more detailed analysisfrequency of double or 0chuang bmc medical genetics page of studies performedkaplan meier survival plots were generated using clinicalfollowup data available within the tcga database todetermine the global mutational spectrum we classified types of nucleotide transitions or transversions thefrequency of each mutation type was calculateddigital images of all eq mutant tumors and representative cases of tumors with neither pole nor polqmutations and of msi tumors were assessed by one author jr for the amount of tumor immune infiltratethe combination of tumor infiltrating lymphocytes andthe peritumorallymphoid infiltrate was graded on ascale between and tumor associated lymphocyteswere graded as none minimal rare to per highpowered field hpf to per hpf and perhpf peritumoral lymphocytes were assessed at thedeepest advancing tumor front graded at low powernone minimal mild moderate andmarked these visual semiquantitative scores werecompared with the immune scores evaluated using estimate estimation of stromal and immune cells inmalignant tumor tissues using expression data estimate score were obtained from the website ofestimate at md anderson httpsbioinformaticsmdandersonestimatediseasehtml theplatformtype selected was rnaseqv2 data for colorectalstomach and endometrial cancer types were downloaded next we separated the colorectal stomach andendometrial cancer cases into quartiles defined by thehighest intermediate and lowest of mutational counts or msi status and calculated the immunescores for each groupstatistical data analysisall statistical analyses were conducted in r v313 plotswere generated using ggplot2 package in r datavisualization methods were described previously the horizontal lines in the boxplots represent the 1st2nd and 3rd quartiles and whiskers outside the boxshow the interquartile range the significance of thedifferences of data illustrated in the boxplots wascalculated using the wilcoxon ranksum tests the chisquare test was performed to test the significance ofdifferences in frequencies of all tables the significancein the kaplanmeier survival plot was calculated usingthe log rank test statistical significance was accepted atp resultsof the cancer types in the tcga database weidentified cancer types pancan with or morecases that possessed mutations in the pole proteincoding region fig 1a and supplementary table these cancer types contained cases with polemutations anywhere in the exome of these polemutant tumors carried mutations in one or more of the other dna polymerases we observed dna polymerases polq and polzrev3l to be most frequentlymutated fig 1b these two polymerases were mutatedin of tumors with pole mutations in fact thesetwo polymerases were even more commonly mutatedthan pole in our pancan cohort supplementary figure altogether cases with pole and polq mutations eq cases with pole and polzrev3lmutations ez and cases with pole polq andpolzrev3l eqz mutations were identified in thepancan cohort fig 1c mutations in the exonucleasedomain of pole are responsible for causing the ultramutator phenotype in colorectal and uterine corpuscancers [ ] in order to determine the contribution of polq and rev3l to the ultramutator phenotype we compared the mutation frequencies of tumorswith mutations in only pole to eq ez and eqzmutant tumors mutation frequencies in the cellulargenome increased in the following order no polemutations poleonly mutations anywhere with thepole exome eq ez eqz mutations fig 1dthe median mutation count of eqz tumors was morethan 10fold higher p than that of tumors withonly pole mutations e q and e z mutant tumorsalso displayed significantly higher mutation countscompared to eonly mutant tumors suggesting a contribution of mutationally altered polq or polzrev3l tothe overall cancer mutation rates next we determinedthe number of mutations in the exonuclease and polymerase domains of pole in the cancer types withinour pancan compendium fig 1e the percentage ofpole mutations in the exonuclease domain was greaterin eq ez and eqz mutant tumors compared topoleonly mutant tumors p in contrast thepercentage of mutations in the dna polymerase domainwas similar thus our data confirm the notion thatmutations in the exonuclease domain of pole areresponsible for ultrahigh mutation rates in additionmutations in polq and rev3l may further increasethe mutation burden however why mutations in polqand rev3l preferentially increase tumor mutationfrequencies remains elusive endometrialto further investigate a potential role of these mutantdna polymerases in the ultramutator phenotype wefocused on colorectalucec andstomach stad cancers these cancer types containthe highest numbers of tumors with eq ez and eqz amongst the cancer types included in pancanfig 1a supplementary table among these cancertypes we identified cases with eq cases with ezand cases with eqz mutations fig 2a in thesecancers the mutation burden in pole eq ez and e 0chuang bmc medical genetics page of fig cancer types pancan with poleqz mutations in tcga a number of cases with pole only eq ez and eqz mutations in cancertypes cohort referred to as pancan within tcga the xaxis shows the actual number of cases with pole green eq orange ez pink and eqz blue mutations the yaxis displays the cancer types uterine corpus endometrial carcinoma ucec stomach adenocarcinoma stadcolon and rectum adenocarcinoma skin cutaneous melanoma skcm lymphoid neoplasm diffuse large bcell lymphoma dlbc lungadenocarcinoma luad breast invasive carcinoma brca sarcoma sarc cervical squamous cell carcinoma and endocervical adenocarcinomacesc pancreatic adenocarcinoma paad lung squamous cell carcinoma lusc head and neck squamous cell carcinoma hnsc bladderurothelial carcinoma blca kidney renal clear cell carcinoma kirc and liver hepatocellular carcinoma lihc b case numbers with mutationsin polymerase genes the number of cases in pancan with mutations in the following polymerases is displayed on the yaxis dntt pola1polb pold1 polg polh poli polk poll polm poln polq rev1 rev3l c venn diagram displaying the number of cases in pancan withmutations in or pol genes d mutations per mb yaxis of pancan cases without pole mutations other or with pole eq ez and eqzxaxis mutations number of cases in each group are listed in parenthesis e mutation frequencies in pole exonuclease and polymerase domainsas a percentage of total number of mutations in the pole exome other refers mutations in the entire exome outside the exonuclease orpolymerase domains the cases are grouped by their polymerase mutation status on the yaxis and the number in parenthesis represents thetotal number of pole mutations within each group 0chuang bmc medical genetics page of fig poleqz mutations in colorectal endometrial ucec and stomach stad cancers a venn diagram displaying the number of caseswith mutations in or pol genes b mutation groups of cases without polymerase mutations other or with mutations in pole only eq ez and eqz the number of cases in each group is listed in parenthesis the pvalue for comparison of pole and eq groups is not significantp c number of cases with mutations in pole exonuclease domain in various mutation groups d percentages the ratio of transitions tiand transversions tv are shown on the yaxis for core stad and ucec the xaxis shows the mutation groupsqz mutant tumors paralleled the mutation burden inthe whole pancan cohort compare fig 2b and fig1c supplementary figure 2ad eqz mutant tumors demonstrated on average an 8fold increase inmutation frequencies compared to tumors with onlypole mutations amongst pole mutant tumors tumors carried mutations outside the pole exonuclease domain while tumors carried mutations withinthe exonuclease domain the frequency of poleexonuclease mutationsin fig 2cprovides a valid explanation forthe difference inmutation rates and potential association of poleexonuclease domain mutations with mutationsinpolq and polzrev3l which are the other twomost frequently mutated dna polymerases casesoverall exonuclease domain mutations were identifiedin cases of pole only mutant tumors eqcases ez cases and of eqz cases fig 2cstratified by cancer types pole exonuclease domainmutations occurred in colorectal endometrial and stomach tumors demonstrating cancertype specific frequencies supplementary figure 3a incontrast to the pole gene that demonstrates mutationalhotspots in the exonuclease domain mutational hotspotsin the polq gene are not associated with a functionalprotein domain supplementary figure 3b c dwhile rev3l does not reveal mutational hotspotsapproximately of mutations lead to truncated protein expression supplementary figure 3e f anothercharacteristic of pole mutant tumors are c to a and g 0chuang bmc medical genetics page of to t transversions we observed the greatest increaseof nucleotide transversions in cancers with eqz mutafig 2d consistent with the loss of poletionsexonuclease activity in these tumorssince mutations in pole confer increased disease freesurvival dfs in patients with uterine cancer even inthose patients with highgrade tumors [ ] we investigated the prognostic role of polq and rev3l mutations in pole mutanttumors kaplanmeier curveswere constructed for colorectal endometrial and stomach cancer cases with followup data fig 3a using thetcga annotations of dfs in individual patients nocancer recurrences were observed in the eq ez andeqz mutant groups pole exonuclease domain mutations were observed in cases in the good survivalgroup and case in the poor survival group consistentwith the expected long dfs periods of patients withpole exonuclease domain positive tumors in additionanalysisin the pancan cohort cases with mutations in pole outside the exonuclease domain were in the good survival group of those had concurrent mutations in polq or rev3l orin both polymerases fig 3b furthermore a kaplanmeierrevealedimproved dfs associated with mutations in polq andrev3l the favorable survival outcome was observed incolorectal endometrial and stomach cancers howeverno favorable outcome was observed in diffuse bcelllymphoma p these data provide preliminaryevidence of cancertype specificfavorable survivaloutcomes in tumors with pole mutations that arelocated outside the pole exonuclease domain if concurrent mutations in polq rev3l or in both polymerasesare presentcompared to microsatellite stable tumorsmssmicrosatellite instability msiin colorectal cancerconfers a better prognosis to determine whetherfig survival and clinical characteristics of patients with polymerase mutations colorectal endometrial ucec and stomach stad cancers akaplanmeier curves of diseasefree survival dfs for groups of patients pole only n median followup months green line eqn median followup months orange line ez n median followup months pink line and eqz n median followup months blue line tumors without mutations in pole polq or rev3l exomes grey line the overall pvalue is p individual pvalues eqz vrs pole p eqz vrs none p eq or ez or eqz vrs pole p b polymerase mutation analysis ofcases in the good survival group in panel a the red bar indicates cases with pole exonuclease mutations c cancer typespecific illustration ofmutation count pole polq and rev3l mutations microsatellite instability msi and tumor stage 0chuang bmc medical genetics page of the favorable outcome of eq ez and eqz mutantcancers can be explained by msi or tmn stage we examined the relationship between msi statustumorstage and polymerase mutations in colorectal endometrial and stomach cancers fig 3c and supplementaryfigure and supplementary table despite improveddfs rates the full range of tumor stages was observedamongst eq ez and eqz tumors p supplementary table 2a comparing the poleonly and eqz mutant cancers did not reveal a significant difference in tumor stage but differed in the frequency ofmsi cases p pole mutant tumors were morefrequent in the msi group than in themss group in addition the frequency ofmsi cases in pole mutant tumors differed between the cancer types p supplementary table 2b c although msi is enriched in samples with highmutation levels supplementary table 2d as expectedp were10fold higher mutation countsobserved in cancers with eqz mutations compared tomsi without eqz mutations supplementary figure these results suggest that mutations in eq ez and eqz confer a better prognosis independent of msi statusand tmn stage in colorectal endometrial and stomachadenocarcinomaswe next examined the amount ofthe cancerassociated immune infiltrate the immune scoreobtained through estimate corresponded tothe categorical score of the immune infiltrate derivedfrom digital he images supplementary fiure therefore we used the estimate immune scoresfor further analysis of colorectal endometrial andstomach cancers as shown in fig 4a a significantdifference was observed in the median immunescores between groups with lowintermediate andhigh mutation burden grouped based on mutationburden and not on e q z mutantseemethods and as expectedthe median immunescores increased with total mutation levels surprisingly the immune scores in eqz mutant tumorsdid not differ significantly from tumors with a lowlevel of mutationsfig 4b as expected msitumors possessed higher immune scores than msstumors p immunescores of msi and eqz mutation tumors weresimilar to those in the msi group and higher thanmss and eqz mutation tumors fig 4d withinthe group of tumors with pole exonuclease domainmutations mss tumors possessed lowerimmunescores than msi tumors but the difference was notsignificanttogether results in this tcga cohort demonstratethat the immune response is driven by msi ratherthan pole exonuclease domain mutationssupplementary fig 4c finallystatusp figurediscussionan analysis of dna polymerases in tumors withmutations in the pole revealed additional mutations inspecific polymerases most commonly in polq andpolzrev3l among the cancer types colorectaluterine and stomach cancer were mostfrequentlyafflicted by these mutations cancers with mutations inpole and polq eq pole and polzrev3l ezand in all polymerases eqz were associated withthe highest mutation burden and an excellent prognosisindependent of msi status and tumor stage mutationsin the exonuclease domain were observed in of eqz mutant tumors but only in of poleonly mutant tumors or in of eq ez tumorshowever despite harboring 10fold more mutationsthan msi tumors and 8fold more mutations than themutation frequencies associated with poleonly mutanttumors eqz mutant tumors did not display significantly more inflammationthe main result from that analysis is that patients withcolorectal stomach and endometrial cancers bearing eq ez and eqz mutations have disease free survival dfs at a median follow up time of months incontrast patients with tumors bearing mutations inpole only most of which outside the pole exonucleasedomain had a dfs of at follow up of monthsthe favorable dfs in eq ez and eqz mutated tumors occurred even in tumors with mutations in polethat are located outside the pole exonuclease domainthe contribution of mutations in pole to tmb casubstitutions and cancer type associations are describedin table of raynor using a larger resourceof cases and should be used to interpret the mutationsin the current study listed in supplementary figure as a whole the current study expands the spectrum ofpole mutant tumors with an excellent prognosis thefavorable prognosis included patients with high tumorstage which echoes prior studies demonstrating a favorable outcome of uterine tumors with pole exonucleasemutations despite adverse standard clinicopathologicindicators including high grade high stage and lymphovascular invasion [ ] while the high mutationfrequencies may cause an early growth advantage as tumors evolve they may succumb to high mutationburden as new mutations can no longer be tolerated andcause tumor cell death [ ] or increased sensitivityto therapeutic agentsthe prevailing hypothesis for the favorable prognosisof cancers displaying the hypermutator phenotype is theincreased attack by the immune system evidence insupport of this theory is the observation that tumorinfiltrating and peritumorallymphocytes are increased and that cytotoxic activities in cd8 and cd4are heightened in polelymphocyte populations 0chuang bmc medical genetics page of fig estimate immune scores by mutation frequency quartiles eqz mutation groups and msi in colorectal endometrial ucec and stomachstad cancers a estimate immune scores in cancers within high intermediate and low overall mutation quartiles b immune scores of sampleswith eq ez and eqz mutations compared to the low mutation quartile from panel a c immune scores in groups of cancers separated by msistatus d immune scores in msi and mss eqz cases compared to all other msi cases for each panel the number of cases within each group isincluded in parentheses on the xaxismutated endometrial cancers [] similar to hypermutated msi tumors this observation has led tothe hypothesis that immune checkpoint inhibitors maybe efficacious in pole ultramutated tumors ourresults question a direct relationship between mutationburden tumor immune response and pdl1 expressionalso raised in a larger study across cases from cancer types in tcga while we observed a concordance between the computational and histological assessments of the immune infiltrate the immune score intumors with eqz mutations depended on msi statusthis result suggests that the immune infiltrate attributable to mutations in eqz mutant tumors may be lessor that its composition may involve immune cells otherthan lymphocytes lesser cd8 and gammainterferongene expression signatures have also been observed ingastrointestinal tumors with a large single nucleotidevariantsnv burden that was attributed largely topole exonuclease mutations perhaps eqz mutations occur at a later point in tumor evolution whenimmunosuppressive factors already dominate we alsocannot rule out the possibility of increased numbers ofcytotoxic lymphocytes intermixed with eqz mutanttumor cells because computational methods and inspection of he images are not sensitive enough to detectsmall differencesinfiltrating lymphocytestils that may have large antitumoral effectsin tumora significant limitation of the study lies in the relatively small number of tumors this limitation cautionsthe generalization of results and seemingly novel insights 0chuang bmc medical genetics page of into the hypermutator phenotype studies by othergroups attributed hypermutator phenotype to specificmutations primarily within the pole exonucleasedomain given the proofreading function of the exonuclease domain it makes sense that mutations outsidethe domain have a lesser effect on tmb in agreementwith this concept our study reveals that compared totumors harboring only a mutation in pole polesinglemutant tumors of cases pole exonucleasedomain mutations are more common in tumors withboth double ez and eq and triple eqz dnapolymerase mutations of cases and doubleand triple mutant tumors have higher mutation countsthan pole single mutant tumors while it cannot befully excluded that polq and polzrev3l mutationsare bystander events in pole mutanttumors weobserve a higher tmb in cases with mutations in allthree polymerases supplementary figure 2a and bmechanistically polq and polz are thought to function in different repair processes polq in alternativemicrohomologymediated nonhomologous dna repair pathway and polz in translesion dna synthesishow these dna repair processes cooperate with thereplicative dna polymerase pole to prevent genomeinstability remains unknown this will be an importantsubjectthe mechanismfor further understanding ofunderlying the hypermutator phenotypesif validated in additional cohorts our findings may haveimportant clinicalimplications they build upon andexpand the previously well documented good prognosticimpact of pole exonuclease mutationsin uterinecancer that have generated intense interest in part dueto the paradox of a favorable prognosis in tumors withpathologic indicators of poor prognosis while in thisstudy prolonged dfs is observed in colorectal endometrial and stomach cancers with eqz mutations thisis not the case in other noncarcinoma cancer typeswithin tcga thus we find that the positive outcomeprediction is cancer type specific altogether resultsfrom this study provide a rationale for including polqandor polzrev3l mutations in clinical outcomestudies of tumors with pole mutations however future validation is required to confirm the concept that isrevealed in the current studycolorectal core stomach stad and endometrial ucec cancers byspecific polymerase mutated groups in tcga data sets supplementaryfigure locations of mutations in pole polq and rev3l exomes inindividual colorectal core stomach stad and uterine cancers ucecsupplementary figure relationships between mutation spectrumand mutation counts pole polq and rev3l exome mutations msi andtumor stage of individual cases supplementary figure mutationrates per mb yaxis of core stad and ucec cases with msi and eq ez and eqz xaxis mutations supplementary figure relationshipbetween pathology inflammation score and estimate immune scores forcore stad and ucec supplementary figure estimate immunescores in colorectal core endometrial ucec and stomach stadcancers supplementary table number of cases with pole polq zrev3l or multiple exome mutations in the pancan cohortsupplementary table contingency tables showing number of casesof colorectal endometrial and stomach cancers in each categoryabbreviationspole polymerase epsilon polq polymerase theta polzrev3l rev3 likedna directed polymerase zeta polz catalytic subunit tcga the cancergenome atlas msi micro satellite instability mss micro satellite stabilitytmb tumor mutation burden tnm tumor lymph node metastasis estimate estimation of stromal and immune cells in malignant tumor tissuesusing expression dataacknowledgementsnot applicableauthors contributionsall authors have read and approved the content of this manuscript fhstudy design and data analysis ht data interpretation jr data analysis andmanuscript writing bsk data interpretation and manuscript writingfundingthe prostate cancer foundation funded salaries of fh and bsk forcomputational analysis steven spielberg team science award andr01ca131255 bsk funded salaries for data analysis and paper writing of fhand bsk we also acknowledge the institutional support of salaries throughthe nih g20 rr030860 to the cedarssinai biobank and translational research core for salaries of bsk and fh the content of this publication doesnot necessarily reflect the views or policies of the department of health andhuman services nor does any mention of trade names commercial products or anizations imply any endorsement by the us governmentavailability of data and materialssequencing data can be obtained from national cancer institue gdc dataportal and are published in raw genomic and clinical data can befound at the nci genomic data commons httpsportalgdccancergovlegacyarchive the mc3 mutation annotation file can be accessed at httpsgdccancergovaboutdatapublicationsmc32017 and the processed datafiles can be viewed at httpsgdccancergovaboutdatapublicationspancanatlasethics approval and consent to participatenot applicableconsent for publicationnot applicablecompeting intereststhe authors do not declare any competing interestssupplementary informationsupplementary information accompanies this paper at httpsdoi101186s12881020010899additional file supplementary figure number of cases polemutations n and mutations in the exomes of dna polymerasegenes in pancan supplementary figure mutation counts forauthor details1department of biomedical sciences cedarssinai medical center losangeles ca usa 2samuel oschin cancer research institute soccicedarssinai medical center los angeles ca usa 3surgerycedarssinai medical center los angeles ca usa 4pathology andlaboratory medicine cedarssinai medical center los angeles ca usa 5department of pathology university of utah salt lake city ut usa 0chuang bmc medical genetics page of received january accepted july referenceskandoth c schultz n cherniack ad akbani r liu y | Colon_Cancer |
" tumor mutational burden tmb has both prognostic value in resected nonsmall cell lung cancernsclc patients and predictive value for immunotherapy response however tmb evaluation by wholeexomesequencing wes is expensive and timeconsuming hampering its application in clinical practice in our study weaimed to construct a mutational burden estimation model with a small set of genes that could precisely estimatewestmb and at the same time has prognostic and predictive value for nsclc patientsmethods tmb estimation model was trained based on genomic data from nsclc samples from the cancergenome atlas tcga validation was performed using three independent cohorts including rizvi cohort and ourown asian cohorts including earlystage and n latestage asian nsclc patients respectively tcga data wereobtained on september the two asian cohort studies were performed from september to march pearsons correlation coefficient was used to assess the performance of estimated tmb with westmb thekaplanmeier survival analysis was applied to evaluate the association of estimated tmb with diseasefree survivaldfs overall survival os and response to antiprogrammed death1 pd1 and antiprogrammed deathligand pdl1 therapyresults the estimation model consisted of only genes correlated well with westmb both in the training setof tcga cohort and validation set of rizvi cohort and our own asian cohort estimated tmb by the 23gene panelwas significantly associated with dfs and os in patients with earlystage nsclc and could serve as a predictivebiomarker for antipd1 and antipdl1 treatment responsecontinued on next page correspondence zhangli6mailsysueducn jie_969163comzlhuxi163com yanhua tian jiachen xu and qian chu contributed equally to this work5state key laboratory of oncology in south china collaborative innovationcenter for cancer medicine sun yatsen university cancer center eastdong feng road guangzhou guangdong china1state key laboratory of molecular oncology department of medicaloncology national cancer centernational clinical research center forcancercancer hospital chinese academy of medical sciences and pekingunion medical college panjiayuan south lane chaoyang districtbeijing 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 0ctian bmc medicine page of continued from previous pages the 23gene panel instead of wes or the currently used panelbased methods could be used toassess the westmb with a high relevance this customized targeted sequencing panel could be easily applied intoclinical practice to predict the immunotherapy response and prognosis of nsclckeywords tmb estimation 23gene panel prognostic and predictive value nonsmall cell lung cancertoimmuneinhibitorscheckpoint tumor mutational burden tmb commonly defined asthe number of nonsynonymous mutations has been proposed as a promising predictive biomarker for the responseicisimportantly this metric tightly correlates with overallsurvival os in resected nonsmall celllung cancernsclc patients in rizvi demonstratedthat an increased number of nonsynonymous mutationswere associated with improved objective response durable clinical benefit dcb and progressionfree survivalpfs in nsclc patients who received antiprogrameddeath pd1 therapy clinical studies have also revealed a significant correlation between tmb and objective response rate orr to icis in multiple tumortypes [] in addition devarakonda recently reported that high tmb was associated with a better survival prognosis in patients with resected nsclc andthe benefit of adjuvant chemotherapy was more pronounced in patients with low tmb the gold standards for tmb calculation are throughwholegenome sequencing wgs or wholeexome sequencing wes however several obstacles such as thehigh demand for quality and quantity of tissue samplesthe cost and time consumption and the unavailabilityfor translation to tmb evaluation by circulating tumordna ctdna in blood btmb hinder the clinicalapplication of these techniques as a result targetednext generation sequencing ngs of cancerrelatedgene panels cgp has been developed serving as surrogates for wes for tmb estimation to date the foodand drug administration fda has approved severalngs panels for tmb estimation eg foundationonecdx f1cdx and memorial sloan kettering cancercenters integrated mutation profiling of actionablecancer targets mskimpact which include about genes and cover over one megabase of codingdna [ ] recently many new ngs panels consistingof different numbers of genes have been developed andvalidated most of which were designed initially for guiding the use of target therapies these panels mainly include cancerrelated oncogenes and tumor suppressenes many of which do not contribute to or even negatively correlate with tmb thus are not accurate fortmb evaluation besides inclusion of these genes in anngs panel enlarges the panel size used for tmbis importantestimation and can lead to an inferior costeffective consequence itto note that cancer typespecific mutation load estimation models have proven tobe necessary because of the different mutation landscapes among varying tumor types although dnadamage repair ddr genes negatively predictive genesstk11 and keap1 and tmbassociated genes such asmuc16 pole pold1 and ttn have been included inthe ngs panels for tmb evaluation [] with theburgeoning developments in immunotherapy there is aneed for more specific panels that focus on tmb estimation for nsclcherein by using the cancer genome atlas tcgadatabase as a training set and multiple realworld cohorts as a validation set we constructed an optimizedtmb estimation model with the smallest number ofcarefully selected tmbassociated genes that could beused as both predictive markers for immunotherapy andprognosis biomarkers for resected nsclc patientsmethodspatient cohortsgenomic and clinical data for nsclc samples including lung adenocarcinoma luad and lungsquamous cell carcinoma lusc samples were downloaded from tcga database for the model constructionfor the validation of the model three independent cohorts were used including a previously published studythe rizvi cohort a surgery cohort composing of earlystage nsclc patients who underwent surgicaltreatment and a zs immunotherapy cohort composingof advanced nsclc patients who received ici treatment all the patients in the zs immunotherapy coreceived either antipd1 nivolumab n hortpembrolizumab n shr1210 n or antipdl1 atezolizumab n monotherapy agents there are patients who received durable clinical benefit dcbantipd1 n antipdl1 n and patientswith no durable benefit ndb antipd1 n antipdl1 n all three validation cohorts were used toevaluate the performance of the tmb estimation modeladditionally the surgery cohort was also used for survival validation in resected nsclc patients both therizvi and immunotherapy cohorts were also used forvalidation of ici outcome predictability in advancednsclc patients the clinical details for all enrolled 0ctian bmc medicine page of patients were collected the treatment efficacy for thosetreated with immunotherapy was assessed using response evaluation criteria in solid tumors recistversion with durable clinical benefit dcb definedas partial or stable disease lasting over months allprocedures were approved by the ethics committees ofthe national cancer center all patients provided written informed consentwholeexome sequencing and data processingwe performed wholeexome sequencing of samplesfrom two cohorts in the validation setincluding earlystage nsclc patients who underwent surgicaltreatment and advanced nsclc patients who received ici treatment for those earlystage nsclcpatients both tumor and matched normal samples wereobtained and subjected to wes briefly dna librarieswere prepared using the mgieasy exome capture v4probe set capture kit cat no with a capture region size of mb bgiseq instruments wereused for pairend sequencing à bp the data wereprocessed according to the manufacturers protocol the mean coverage was à and à in tumor andnormal samples respectivelyfor those advanced nsclc patients biopsy specimens were available for wes the genomic dna wasextracted using the qiaamp dna ffpe tissue kit andquantified using the dsdna hs assay kit thermofisher scientific usa libraries were constructed withthe kapa hyper prep kit kapa biosystems usa anillumina hiseq4000 platform was used for sequencingwith pe150 sequencing chemistry illumina usa the average coverage depth was Ãcandidate gene selectiongenomic data for nsclc samples from tcgawere used for candidate gene selection which were usedto construct the mutation load estimation model thecandidate genes were selected based on two criteria mutation frequency higher than or equal to and significant association with mutation load the mutationfrequency of a gene was calculated as the percentage ofpatients with mutation in the gene mutation loadassociated genes were defined as where the westmbwas significantly different between the patients with themutated gene and those with wildtype counterpartsadditional file table s1mutation estimation model constructionthe mutation estimation model construction was basedon tcga data in the training set in detail the first stepwas to build a mutation estimation model using thefewest genes which tightly associated with westmbin our study we constructed the estimation model bysimply randomly selecting a specified number of genesfrom allthe genes or tmbassociated genes andsummed the mutational number as the estimated tmbunder every given number of genes the procedure wasrepeated times resulting in random modelswe then calculated the pearson correlation coefficientr between the estimated and actual mutation load ofwestmb the results allowed us to select the modelwith highest r under the specified number of genes thenext step was to identify which of those best modelsunder the specified number of genes correlated with theclinical outcomes of overall survival os and diseasefree survival dfs the final step was to select a modelusing the fewest genes that tightly associate with thewestmb and have both prognostic value for thoseearlystage nsclc patients and predictive value forthose latestage nsclc patients who received icitreatmentrna expression difference between tmb high and lowgroupsto compare gene expression patterns we downloadedan mrna data set of nsclc patients from tcgadatabase mrna expression was analyzed using gene setenrichment analysis gsea httpsoftwarebroadinstitutegseaindexjsp we divided these patientsinto estimated high ¥ mutational counts and lowtmb groups mutational counts and identifiedwhether immunerelated gene signatures associated withtumor mutation status the genes found to be on theleading edge of the enrichment profile were subjected topathway analysis genes with expression over in morethan of the samples were included in the gseathe normalized enrichment score nes is generally theprimary statistic for examining gene set enrichmentresultsstatistical analysisthe mannwhitney u test was used to assess thedifferences in the mutation load between the twogroups the genes with kruskalwalliscorrected pvalues lower than were identified as the mutationloadassociated genes and selected as potential candidate genes survival analysis was performed using thekaplanmeier curves with a p value determined by alogrank test and the statistical tests were twosidedand considered statistically significant at p unless otherwise stated the analyses were performedusing graphpad prism version graphpad prismcorrelations between estimated mutation burden andwholeexome sequencingcalculated tmb were determinedcorrelation coefficient theanalyses were performed using r353by pearsons 0ctian bmc medicine page of resultscandidate gene selection for model constructionthe flowchart of the construction of estimation model isshown in fig s1 in additional file the somatic mutation data of cases of nsclc were downloaded fromtcga database as the training set tcga cohort including adenocarcinoma and squamous cell carcinoma subtypes of nsclc additional file table s2subsequently a mutation matrix including screened nonsynonymous mutations in genes was generatedfurthermore we identified genetic alterations in genes with mutation frequency ¥ in general nsclcpatients and significantly correlating with westmb pvalue range 695e to 452e these genes werethen used as candidate genes for the construction of thetmb estimation model additional file table s3construction of the tmb estimation modelgenes used for the tmb estimation model were randomly selected from the candidate genes and theserialrandom models theestimated tmb was defined as the sum of all nonsynonymous mutation counts of the selected genes undereach specified number of abstracted genes the procedure was repeated times thus resulting in separatecorrelations ofestimated tmb by these random models and westmbwere evaluated using the pearson correlation coefficientr as expected the correlations between the estimationmodels and westmb increased with the number ofgenes fig 1a b additional file fig s2a b compared with unselected genes in the range of genomicgenes the estimated tmb based on selected geneswas significantly more closely associated with westmb in terms of either the mean or the maximum rfig 1c d additional file fig s2c d the maximumr increased from with one gene included to greaterthan with genes included and then reached aplateau when the included gene number exceeded the r values were comparable though increased slowlyas the number increased fig 1b we asserted that rfig the correlation of westmb and tmb as estimated by different gene panels a b correlation is represented by the pearson correlationcoefficient r genes used for the mutation model construction were either from unselected genes a or from selected genes b that correlatewith westmb c d comparisons of mean c and maximum d r of estimated tmb and westmb using unselected genes or selected genes 0ctian bmc medicine page of greater than in the estimation models was acceptable as such we considered a model with this effectbut including the least number of genes an ideal modelfor clinical applicationin reference to previous reports that tmb is associated with prognosis in patients with resected nsclcsthe optimal tmb estimation model was further evaluated based on the correlation of estimated tmb with osand dfs in models with r over ultimately we constructed an estimated tmb model with only genesand r of p fig 2a additional file which was significantly associated with both os anddfs fig 2b c the cutoff value of the estimated tmbby the 23gene panel was defined as mutational countsthe median value of estimated tmb based on tcgadatabase additional file fig s3a b that were equalor over mutational counts as tmbhigh cases and lessthan mutational counts as tmblow ones these genesincluded unc13c hmcn1 znf536 kmt2d ush2axirp2 pcdh15 ahnak2 adgrl3 reln nf1 ttnadgrg4 cubn cacna1e mrc1 col11a1 nav3csmd1 apob csmd3 col22a1and epha5additional file table s4 the model yielded goodperformances in both subtypes of nsclc with correlations of for luad additional file fig s4aand for lusc additional file fig s4b theaverage cds length of these genes was 12k nucleotides 3k80k additional file table s4 and the totallength was 028m nucleotides which was considered tobe a great reduction of sequencing cost for mutationload estimation we concluded that the 23gene panel isthe ideal model based on tcga training setanalytic validation of the 23gene panel in asian resectednsclc patientsto validate the performance of the estimation model weconducted wes on chinese stage iaiiia nsclcpatients after radical pneumonectomy surgery cohortadditional file table s1 the correlation of 23genetmb with wes was r p fig 3a asshown in fig 3b tmbhigh ¥ mutational counts according to the 23gene panel associated with a betterdfs compared with those with tmblow logrank p besides a tendency towards improved os wasobserved in the patients with higher estimated tmbthough a statistical difference was not reached due tothe fact that most patients were still alive fig 3cperformance verification by comparing the 23gene panelwith other commercial panelsnext we compared the 23gene panel with two commercial panels based the earlystage nsclc data including f1cdx genes and mskimpact genes there are two overlap genes between the gene panel with f1cdx and mskimpact namelynf1 and epha5 the 23gene tmb has a tight correlation with the tmb estimated by f1cdx f1cdxtmbor mskimpact msktmb r and respectively both p fig 4a b in additionwhen the genes were added to the two commercialpanels the correlation of the incorporated panels withwestmb significantly increased from ci to ci p for f1cdx fig 4c d and from ci to ci p for mskimpact fig 4e f to further verify thespecificity of these 23gene panels we compared themwith other randomly selected gene panels from the genes the procedure was repeated timesresulting in the random pearson correlation coefficientsfrom to of f1cdx plus random genesand from to of msk plus random genes the performance of our 23gene model was better than of random models which indicated the irreplaceability of these genesfig tmb estimation model construction based on tcga data in the training set a the correlation of 23gene tmb with westmb is with an empirical p value of r of p b the overall survival is significantly higher in the tmbhigh group ¥ mutational counts n than in the tmblow group mutational counts n with logrank test p c the diseasefree survival is significantly higher in thetmbhigh group than in the tmblow group with logrank test p 0ctian bmc medicine page of fig validation of the tmb estimation model based on the earlystage nsclc patients in the validation set a the pearson correlationcoefficient of estimated tmb by the 23gene panel and westmb is with an empirical p value of r of p b the diseasefree survivalis higher in the estimated tmbhigh group ¥ mutational counts n than in the tmblow group mutational counts n with logrank test p c the overall survival is comparable in the two groups with logrank test p fig performance evaluation of the 23gene panel against commercially used gene panels a b the pearson correlation coefficient of 23genetmb with f1cdxtmb a and msktmb b c d the pearson correlation coefficient of westmb with f1cdxtmb c and incorporated panel of cancerassociated genes in f1cdx with 23gene panel f1cdx genetmb d e f the pearson correlation coefficient of westmb withmsktmb e and incorporated panel of cancerassociated genes in mskimpact with genepanel msk genetmb f 0ctian bmc medicine page of f1cdxbased on the survival data from our earlystagensclc patients significant correlations were observedbetween survival outcomes dfs and the tmb levelstratified withor mskimpact paneladditional file fig s5a c interestingly the genescould improve the association of these two commercialpanels with dfs additional file fig s5b d if the incorporated panels were used for analysis tmbhigh estimated by both of the two new panels f1cdx 23genepanel or mskimpact 23gene panel demonstratedimproved dfs compared with those of estimated tmblow under the cutoff values indicated in fig s3c and s5of additional file immuneregulatory gene expression signatures stratifiedby tmb level based on the 23gene panelto investigate the difference in immune status betweentmbhigh and tmblow estimated by the 23genepanel we analyzed immuneregulatory gene expressionsignatures based on the rnaseq data of nsclccases from tcga the gsea revealed a prominent enrichment of mrna signatures involved in the inflammainterferonα γ ifnα γtoryresponse tnfαresponse il6jakstat3 signaling and allograft rejection fig immunotherapy response prediction by the established23gene panelfinally we analyzed the performance of tmb estimatedby the 23gene panel in the prediction of response toicis using two independent nsclc cohorts in therizvi cohort the correlation between the tmb estimatedby the 23gene panel and wes was empirical pvalue of r fig 6a the estimated tmb was significantly different between the patients with durableclinical benefit dcb a partial or stable response lastingover months and no durable benefit ndb mannwhitney p fig 6b survival analysis was thenapplied for the comparison of the pfs between the patients n with tmbhigh ¥ counts and tmblow counts by the 23gene panel patients withtmbhigh demonstrated significantly improved pfscompared with those with tmblow vs months logrank p fig 6cto further validate the performance of the estimationmodel for response to icis we performed wes of fig gene expression differences between the estimated high tmb and low tmb groups af tmbassociated pathways such as inflammatoryresponse tnfα signaling via nfκb interferon α response il6jakstat3 signaling interferon γ response and allograft rejection nes normalizedenrichment score fdr false discovery rate 0ctian bmc medicine page of fig immunotherapy response estimation by the 23gene panel a the correlation of the estimated tmb with westmb using the rizvi datan b estimated tmb in tumors from patients with dcb n or with ndb n mannwhitney p c pfs in tumors withestimated tmbhigh n compared to tumors with tmblow n in patients in the rizvi cohort hr ci to logrank p d the correlation of estimated tmb with westmb using the latestage nsclc patient cohort n e estimated tmb in tumors frompatients with dcb n or with ndb n mannwhitney p f pfs in tumors with estimated tmbhigh n compared totumors with tmblow n in patients in the latestage nsclc patient cohort hr ci to logrank p advanced stage iiibiv nsclcs in another asian cohort zs immunotherapy cohort all of these patients received with antipd1 or antipdl1 treatmentthe r between the estimated and actual mutation burden was calculated to be empirical p value of r fig 6d the estimated tmb was significantlydifferent between the patients with dcb and ndbmannwhitney p fig 6e the pfs was associated with estimated tmb logrank p fig 6fdemonstrating that the estimated mutation burden derived from caucasian nsclcs from tcga could predict the immunotherapy treatment response quite wellin asian patients we further calculated the hr at different cutoff values in the zs immunotherapy cohort andfound the mutational counts in this cohort resultedthe best hr value additional file fig s6 as a resultwhen applied in clinical practice the cutoff value stillneeds to be further evaluated accordinglycomparison of the 23gene panel with previouslyreported tmbrelated genesmutations in ttn muc16 pole and pold1 havebeen previously reported to correlate with elevated tmblevels [] the frequencies ofthese genes innsclc based on cases from tcga were and respectively westmb was significantly different between the patients with these mutatedandthose with wildtypegenescounterpartsadditional file fig s7 however only muc16 mutations exhibit significant correlation with os and dfs intcga cohort additional file fig s7ac while theyfailed to confirm the results in our surgery cohortadditional file fig s8 notably none of these genemutations could predict the response or pfs in eitherthe rizvicohortadditional file fig s9cohort or ourimmunotherapydiscussionin the present study we developed a novel and optimaltmb estimation model composed of only geneswhich allowed precise estimation ofthe wesbasedtmb both in earlystage and latestage nsclc patientsimportantly our established 23gene panel can successfully predict the survival outcomes in both resectednsclcs and patients receiving icis in multiple validation cohorts to the best of our knowledge our tmbestimation model is both the first and the smallest paneldescribed to date which can be used as a biomarker tostratify patients not only after radical pneumonectomybut also with advanced nsclc receiving icisthe total cds length of the 23gene panel was 028mnucleotides with an average of 12k 3k80k the ttnis also included in our panel although it has the longestcds length of 81k the total length was acceptable when 0ctian bmc medicine page of ttn is included besides in a recent study ttn mutation was reported to be associated with tmb in solid tumors including nsclc and correlated with response toicis as a result the 23gene panel was consideredto be a great reduction of sequencing cost for mutationload estimationseveral cancerrelated genes have been previously reported to be associated with westmb in some cancertypes for example melanoma patients with lrp1b mutations exhibited a higher mutationalload than thosewith the wildtype gene li reported that mutations in muc16 are associated with tmb and survivaloutcomes in patients with gastric cancer twoddrrelated genes pole and pold1 were also shownto correlate well with westmb in pancancer types undoubtedly it would be ideal to utilize a singlegene to estimate tmb and effectively predict responseto immunotherapy however we found that singly allthese genes failed to correlate well with the efficacy oficis or survival outcomes after resection the correlationof any individual gene with westmb was moderatemean r these results indicate thatusing a single gene to estimate tmb is insufficienttheoretically the larger a ngs gene panel the closerthe estimated tmb is to the actual amount howeverthe costeffective balance for clinical usage must be considered in particular when tmb is detected using peripheral blood super sequencing depth eg à due to the low abundance of circulating tumordna will significantly drive up the cost to datetwo commercial gene panels f1cdx and mskimpact have been widely used for tmb estimation thesetwo panels demonstrate good performance in correlationwith westmb our established gene panel whichincludes a very limited number of genes demonstratedcomparable correlation coefficients with these two largepanelsindicating the promising reliability of a smallpanel as a surrogate for westmb notably the majority of genes used in our model were not included in thecurrently used commercial gene panels if the genes inour panel were incorporated into the big commercialgene panels the correlation coefficients with westmbincreased these results demonstrate that the geneswe have selected here may be used independently or ascomplement to the currently used gene panels specificfor nsclc inclusion of the genes should be considered in future ngs gene panelsrecently lyu developed a small gene panel with genes to estimate actual tmb derived from luads in tcga database the construction and validation cohorts used for lyu s 24gene panel weremainly from caucasian patients however our 23genepanel though also derived from tcga database wassuccessfully validated in multiple asian patient cohortsthese results suggest that our 23gene panel may bemore suitable to nsclc and applicably potent regardless of race and subtypes additional file fig s10similar with the findings of devarakonda weobserved that high tmb associated with improved os inresected nsclc patients in colon cancer patients withresected stage ii mismatch repair deficiency high tmbhas been utilized as a good prognostic biomarker indeed these results possess internal rationality both highneoepitope burden and intense til infiltration have been associated with favorable survival outcomes inearlystage lung cancer high tmb may reflect the immunogenicity in some degree which could mediate theshaping of tumorhost immune interactions taken together these and our findings suggest that quantifyinggenomic instability through tmb estimation can be usedto stratify patients so as to guide adjuvant treatmentowing to the lack of information on hlai it is difficultto judge whether the predictive value of our gene panel isdue to neoantigen generation derived from the includedgene mutations or if the estimated tmb based on the gene panel is simply a representative reflection of genomicinstability as an accompanying passenger the otherlimitation of our study is the small number of patientswho received the immunotherapy treatment thus a larger number of cases from a multicenter study are requiredfor the validation of the performance of the treatment response prediction in addition our validation cohorts wereretrospective a prospective study is necessary to translateour estimation model into clinical practice in addition totmb other features such as pdl1 expression microsatellite instability and neoantigen burden have emerged aspotential predictive biomarkers for icis [] howeverchallenges in defining cutoff valuesintertumoral andintratumoral heterogeneity and test platform uniformitieshave limited their clinical applications therefore future strategies that combine different predictive featuresmay be more effective biomarkers for the accurate prediction of cancer immunotherapy response but need tobe carefully integratedsin summary we have successfully constructed a noveltmb estimation model using only genes that can beused to estimate the westmb and stratify survivalprognosis after radical surgery and clinical outcomes ofici therapy in nsclc patients thus a customized panelfor the targeted sequencing of these selected genes instead of wholeexome sequencing can be designed or utilized as complementary genes included in the currentngs panels consequently by using our model the costeffectiveness may be considerably improved makingrealization of cancer immunotherapy response more accessible in standard clinical settings 0ctian bmc medicine page of supplementary informationsupplementary information accompanies this paper at httpsdoi101186s12916020016948competing interestsno potential conflicts of interest were disclosed by the authorsadditional file table s1 data sets used to calculate westmb forthe study cohorts table s2 characteristics of the patients included inthis study table s3 candidate genes and related information tables4 genes and the corresponding cds length fig s1 flowchart ofthe construction of estimation model fig s2 the correlation of westmb and tmb as estimated by different gene panels fig s3 forestplots of hrs for os and dfs in the tcga and earlystage nsclcpatients study cohort fig s4 the performance of 23gene based tmbestimation model for the luad and lusc subtypes of nsclc tcgadata fig s5 forest plots of hrs for dfs in the earlystage nsclcpatients study cohort fig s6 forest plots of hrs for pfs of the nsclc patients in zs immunotherapy cohort fig s7 westmb is shownbased on muc16 a ttn b and pole c mutation status fig s8 thecorrelation of muc16 mutation status with overall survival a anddiseasefree survival b based on the earlystage nsclc patients figs9 the correlation of muc16 ttn and pold1 mutation status withprogressionfree survival pfs based on the rizvi cohort and ourimmunotherapy cohort fig s10 comparison of predictive performanceof response to icis by our 23gene panel with lyus 24gene paneladditional file the correlation of estimation models with genenumber to with os and dfs in the training setacknowledgementswe thank all patients that were involved in this study we also thankguoqiang wang jing zhao and shangli cai the medical department 3dmedicines inc shanghai peoples republic of china for their contribution tothe st | Colon_Cancer |
" rectus sheath block rsb is known to attenuate postoperative pain and reduce perioperative opioidconsumption thus a retrospective study was performed to examine the effects of bilateral rectus sheath blockbrsb in cytoreductive surgery crs combined with hyperthermic intraperitoneal chemotherapy hipecmethods a total of patients undergoing crshipec at our hospital were included patient information andanaesthesiarelated indicators were collected from the electronic medical record emr system all subjects weredivided into the following two groups the g group general anaesthesia and the gr group rsb combined withgeneral anaesthesia patients in the gr group received ropivacaine for brsb before surgery the primaryoutcomes included the total amount of remifentanil and rocuronium the total consumption of dezocine aftersurgery the visual analogue scale vas score and the patientcontrolled intravenous analgesia pcia input dose at h t6 h t7 h t8 h t9 and h t10 after surgery other outcomes were also recorded such aspatient demographic data the intraoperative heart rate hr and mean arterial pressure map and postoperativecomplicationsresults compared with the g group the gr group showed a shorter time to tracheal extubation p adecreased total amount of remifentanil and rocuronium p and a reduced vas score pcia input dose andnumber of pcia boluses at h h and h after surgery p however at h and h after surgery therewere no differences in the vas score of pain at rest or during motion between the two groups p moreoverthe incidence of hypertension emergence agitation delayed recovery hypercapnia and nausea and vomiting waslower in the gr group than in the g group p there were no differences in the changes in map and hrduring the surgery between the two groups p no complications associated with nerve block occurred brsb could provide shortterm postoperative analgesia reduce perioperative opioid consumption andreduce the incidence of postoperative complications it is an effective and safe procedure in crshipeckeywords cytoreductive surgery hyperthermic intraperitoneal chemotherapy rectus sheath block generalanaesthesia analgesia correspondence trmzltz126comdepartment of anesthesiology beijing shijitan hospital capital medicaluniversity beijing china 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 bmc anesthesiology page of radical cytoreductive surgery crs combined withhyperthermic intraperitoneal chemotherapy hipec isconsidered a standard for the treatment of peritonealcancer such as rectal cancer ovarian cancer peritonealpseudomyxoma and peritoneal mesothelioma thistechnique could prolong the longterm survival of patients with a decreased recurrence rate although thepositive results of this treatment have been proven inprevious studies [] because of the large peritonealsurface area involved in this kind of surgery crshipecis time consuming and complex which presents agreat challenge for the anaesthesiologist in terms of perioperative managementdue to the stable respiratory and circulatory supportgeneral anaesthesia is the preferred choice in this surgery however long periods of general anaesthesia leadto drug accumulation in the body followed by increasedanaesthesiarelated complications including delayed recovery respiratory inhibition and cognitive dysfunction consequently exploring better anaesthesia methodsfor this surgery is still a major concerna new approach called ultrasoundguided bilateral rectus sheath block brsb has been proven to amelioratepostoperative pain and reduce the consumption of morphine [] nonetheless there have been no reportson the application of general anaesthesia combined withbrsb in patients undergoing crshipec based on theinformation presented above this retrospective observational study was conducted to examine the efficacy andsafety of brsb in patients treated with crs and hipecmethodssubjectsall patients who underwent crs and hipec at beijingshijitan hospital between august and december were retrieved from the institutional database inthis study the exclusion criteria were as follows laparoscopic surgery with crshipec intraoperativeblood loss volume greater than ml mechanicventilation required after surgery and use of analgesictechniques apart from brsb and general anaesthesiaaccording to this standard a total of patients wereincluded and divided into the following groups generalanaesthesia g group n and general anaesthesiacombined with posterior rsb gr group n anaesthesia methodgeneral anaesthesia was consistently induced in all patients with intravenous propofol mgkg sufentanil μgkg and rocuronium mgkg invasive arterialpressure and central venous pressure were monitored byradial artery puncture flotracvigileo® edwards lifesciences irvine ca usa and internaljugular veinsitetargeteffectpuncture respectively after anaesthesia induction anaesthesia was maintained with sevoflurane and remifenconcentration ngmltanilkeeping the bispectral index bis between and rocuronium mgkg wasintermittently used tomaintain muscle relaxation in the gr group before anaesthesia induction patients received brsb under ultrasound guidance the puncture site was placed at theouter edge of the bilateral rectus abdominis at the levelof the umbilicus fig a a total of ropivacaine ml was injected into each side the spindleshapedspread of ropivacaine was observed between the posterior sheath of the rectus abdominis and the rectus abdominis itself implying success of the procedure fig b c patientcontrolled intravenous analgesia pciawas applied in both groups after the surgery sufentanil μgkg palonosetron hydrochloride mg was diluted to ml the dose was mlh and asingle dose was mlh with a 15min lockout intervalafter the surgery all patients were sent to the surgicalintensive care unit sicu if the visual analogue scalevas score at rest after surgery was ¥ dezocine mgwas used as a rescue analgesicdata collectionall the indicators we needed were obtained from theemr system the records included patient demographicdata patient medical history american society of anesthesiologists asa grade and new york heart association nyha grade the hr and map were recordedat the time before brsb t1 the time of anaesthesiat2 the time of skin incision t3 the time of peritoneal thermochemotherapy t4 and the end of surgeryt5 in addition the duration of the surgery time totracheal extubation the time after skin closure totalamount of remifentanil and muscle relaxants total fluidvolume urine volume and the total volume of allogeneicerythrocytes and plasma infused during the surgery wereall recorded moreover after surgery the occurrence ofhypertension the systolic blood pressure dropped bymore than of baseline blood pressure beforeanesthesia or the sbp mmhg during surgery nausea and vomiting hypoxemia spo2 or pao2 mmhg hypercapnia paco2 mmhg and emergence agitation during the recovery period were recorded the recovery period was considered as the timefrom switching off inhalation anaesthetics remifentaniland muscle relaxant to recovery of the patients abilitiesto command movement orientation as well as conscious state when the recovery period of patients is beyond min it was considered as delayed recovery thevas score for pain at rest and during motion the pciainput dose and the number of boluses at h t6 ht7 h t8 h t9 and h t10 after surgery 0cwang bmc anesthesiology page of the incomplete datachemotherapy crshipec during the year from to in our hospital one hundred and six patientsreceived brsb seventeen patients were excluded because ofincluding patientsundergoing intraoperative haemorrhage blood ml and other patients received mechanic ventilationbecause of acute respiratory distress syndrome ardsallergic shock and cardiac insufficiency thus patients with brsb were eventually obtained finally patients without brsb were randomly selected to analysis in this study fig statistical analysisspss software was used for statistical analysisnormal distribution data were recorded as the mean ±standard deviation sd and analysed by independentsamples t test for comparison between the two groupsnonnormally distributed data are presented as themedian range and were analysed by kruskalwallistest chisquared test or fishers exact test was used forcategorical data a p value of was considered statistically significantresultscharacteristics of study populationin total patients were included in the study thebaseline demographic and surgical variables of patientsare presented in table there were no significant differences in age sex body mass index bmi basic diseases asa grade nyha grade total surgery time totalfluid volume urine volume total volume of allogeneicerythrocyte infusion or total volume of plasma p however the time to tracheal extubation was shorter inthe gr group than in the g group p the totalamount of both remifentanil and rocuronium used wasless in the gr group than in the g group p thus posterior rsb could reduce the use of remifentaniland rocuronium during surgerychanges in haemodynamic parametersthe changes in hr and map are presented in table there were no significant differences in hr or map atany point in time t1 to t5 between the two groupsp the results suggest that brsb did not affectthe haemodynamics ofthe patient undergoing crshipecpainrelated indicatorstable shows the postoperative vas score the pca input dose and the number of pca boluses at h t6 ht7 h t8 h t9 and h t10 after surgeryas well as the dose of dezocine used as a rescue analgesic from t6 to t8 compared with the g group thegr group showed significantly decreased vas scores offig ultrasoundguided brsbas well as the dose of dezocine used as a rescue analgesic were also recordedin addition brsbrelatedcomplications such as peritoneal punctureinternalan injury and systemic toxicity were all recordeda total of patients underwent cytoreductive surintraperitonealcombined withgeryhyperthermic 0cwang bmc anesthesiology page of fig flow chart showing patient consecutive enrolment and analysis abbreviations crshipec cytoreductive surgery and hyperthermicintraperitoneal chemotherapy ga general anesthesia brsb bilateral rectus sheath block ards acute respiratory distress syndrome vas visualanalogue scalepain at rest and during motion p however at h and h after surgery there were no significant differences in the vas scores of pain at rest and duringmotion between the two groups p from t6 tot10 the pcia input dose and the number of pca boluses were also obviously reduced in the gr group compared with the g group p in addition as arescue analgesic the dose of dezocine after surgery inthe gr group was significantly lower than that in the ggroup p postoperative adverse eventsadverse events that occurred in the sicu are presentedin table after surgery there were cases withhypertension cases of emergence agitation cases ofdelayed recovery cases of hypercapnia and cases ofnausea and vomiting in the g group fewer cases of allof these events occurred in the gr group p there were no differences in the incidence of hypoxemiabetween the two groups p there were no complications associated with nerve block in either group 0cwang bmc anesthesiology page of table demographic and surgical variables mean ± sdage ysex malefemalebmi kgm2medical historydiabetes mellitus n yesnohypertension n yesnocoronary heart disease n yesnoasa grade iiiiiinyha grade iiitotal surgery time mintime to tracheal extubation minremifentanil mgrocuronium mgtotal fluid volume mlurine volume mltotal volume of allogeneic erythrocyte infusion mltotal volume of plasma mlg group n ± ± ± ± ± ± ± ± ± ± gr group n ± ± ± ± ± ± ± ± ± ± p value asa american society of anesthesiologists bmi body mass index calculated as weight in kilograms divided by height in metres squared nyha new york heartassociation g general anaesthesia gr bilateral rectus sheath block combined with general anaesthesia before bilateral rectus sheath block t1 the time ofanaesthesia t2 the time of skin incision t3 the time of peritoneal thermochemotherapy t4 and the end of surgery t5discussionin this retrospective study we examined the efficacy andsafety of brsb combined with general anaesthesia in patients undergoing crshipec regarding efficacy theresults show that ultrasoundguided brsb significantlyreduced the total dose of remifentanil used during thesurgery and shortened the time to tracheal catheter extraction which is consistent with the findings of previous studies of other surgeries [ ] in addition rsbreduced the total dose of rocuronium in this studytable haemodynamic parameters in both groups mean ± sdindextimepointgn ± grn ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± pvaluemmhghrbpmt1t2t3t4t5t1t2t3t4t5map mean arterial pressure hr heart rate g general anaesthesia gr bilateralrectus sheath block combined with general anaesthesiawhich may be associated with the high concentration ofropivacaine used in the studyrsb also effectively relieved postoperative pain in thisstudy we found that the vas scores of pain at rest andduring motion were all lower in the gr group than inthe g group at h after surgery however at h and h after surgery there were no differences in the vasscores of pain at rest and during motion between thetwo groups suggesting that the analgesic effects of asingle brsb remained within h after surgery thisresult may be different from the findings of others cho reported that at h after surgerythere were no differences in the vas scores of painat rest and during motion between the rsb and nonrsb groups the discrepant results may be related todifferences in the concentration of ropivacaine andthe physical constitution of patients a high concentration can prolong the duration of action of a localanaesthetic in this study we selected not ropivacaine additionallythese patientsundergoing crshipec may have been adaptive topain furthermore compared with the control groupthe rsb group showed a reduced totalinfused doseof sufentanil as pcia number of pca boluses within h after surgery and total dose of dezocine used asa rescue analgesic after surgery these results furtherprove the role of rsb in providing shortterm postoperative analgesia 0cwang bmc anesthesiology page of table painrelated indicators in both groups median [range]vas score of pain at rest [median range]t6t7t8t9t10vas score of pain during motion [median range]t6t7t8t9t10total infused dose of pcia [ml median range]t6t7t8t9t10cumulative number of pcia boluses [median range]t6t7t8t9t10total dose of dezocine as a rescue analgesic mggn [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] ± grn [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] ± p value vas visual analogue scale pcia patientcontrolled intravenous analgesia g general anaesthesia gr bilateral rectus sheath block combined with generalanaesthesia the time at h after surgery t6 h after surgery t7 h after surgery t8 h after surgery t9 and h after surgery t10table postoperative adverse events in both groupsadverse eventsn gn grn hypertensionemergence agitation delayed recoveryhypoxemiahypercapnia nausea and vomiting peritoneal punctureinternal an injurysystemic toxicity g general anaesthesia gr bilateral rectus sheath block combined withgeneral anaesthesiawe also examined the safety of rsb during the surgery ultrasoundguided brsb had no significant effectson the haemodynamics of patients during surgery compared with general anaesthesia alone in terms of postoperative adverse events the results show that comparedwith the control group the rsb group showed a reducedincidence of hypertension emergence agitation delayedrecovery hypercapnia and nausea and vomiting whichmight be correlated with the decreased analgesic andmuscle relaxant doses no rsbrelated complications occurred in any patient these data indicate that rsbcould reduce the risk of complications associated withgeneral anaesthesia and is safe for patientsrsb an established technique has regained popularityin clinical applications [] previous studies havedemonstrated that this technique could achieve relaxation of the anterior abdominal wall [ ] bashandyreported that anterior branches of the t7t12 thoracicnerve and the l1 lumbar nerve travelled through thepvalue 0cwang bmc anesthesiology page of plane of the transverse abdominis muscle entered the rectus abdominis sheath and distributed on the surface of theskin the main process of rsb is to inject local anaesthetics between the rectus abdominis and the posteriorsheath of the rectus abdominis therefore rsb exerteda good effect in terms of perioperative analgesia for medianabdominal incisions a midline incision is required inthis kind of surgery thus based on these results rsbcould meet the need for analgesia in these patientsin addition for a long time epidural analgesia eawas thought to be an effective method for abdominalsurgery [] studies have proved that epidural analgesia could maintain a good analgesic effect and reduceperioperative opioid consumption including in this typeof surgery [ ] however the safety of ea in crshipec remains controversial especially regarding effectson coagulation and circulatory function coagulationdysfunction and profound fluid loss are the main characteristics of patients with peritoneal cancer [ ]which might limit the administration of eaalthough epidural catheter is standard of care insolankis guideline in our hospital epidural catheter in not the standard of care we performed generalanesthesia combined with epidural anesthesia in somepatients to reduce the consumption of intravenous drugsand provide perfect analgesia but coagulation dysfunction and profound fluid loss are the main characteristicsof patients with peritoneal cancer in our previous studywe found that the mean arterial pressure of patientsundergoing epidural anesthesia was difficult to be maintained in the surgery besides there were many patientswith coagulation dysfunction before surgery who werenot suitable for the epidural anesthesia these results wefound in clinical were similar with others researcheskajdi and colleagues reported a case of epidural haematoma in their study godden found that the incidence of hypotension in the ea group was obviouslyhigher than that in the rsb group consequentlyrsb could be a better choice than ea in crshipecadditionally there are some limitations to this studyfirst all the data of this study were collected from theemr system as this was a retrospective study the findings are not as persuasive as those of a randomized controlled study we plan to conduct prospective studies toexplore the comprehensive influence of rsb in this surgery second we only examined the application of brsbestablished with a single injection which provides only ashortterm analgesic effect the efficacy of continuousanalgesia with brs catheters in crshipec remains unclear and needs further exploration third in our the results are initially presented according to the different aspects the primary outcome of this study is thetotal consumption of remifentanil during the surgeryother indicators were belonged to second outcomessin brsb could provide good postoperativeanalgesia reduce perioperative opioid consumption andreduce the incidence of postoperative complicationsthis is an easily applicable and safe procedure in crshipecabbreviationsrsb rectus sheath block brsb bilateral rectus sheath blockcrs cytoreductive surgery hipec hyperthermic intraperitonealchemotherapy emr electronic medical record vas visual analogue scalepcia patientcontrolled intravenous analgesia hr heart rate map meanarterial pressure bis bispectral index sicu surgical intensive care unitasa american society of anesthesiologists nyha new york heartassociation spo2 pulse oximetry paco2 partial pressure of carbon dioxidepao2 oxygen partial pressure bmi body mass indexacknowledgementsi would like to express my heartfelt thanks to the staff of the informationdata center of beijing shijitan hospital affiliated to capital medical universityauthors contributionswsh study design data collection writing paper lpf gt data collectionand data analysis gl coordinated the study and manuscript revision ltzstudy design and manuscript revision all authors read and approved thefinal manuscript all authors ensure the accuracy of the manuscript andagree to take personal responsibility for their contributionsfundingno fundingavailability of data and materialsthe datasets used andor analyzed during the current study are availablefrom the corresponding author on reasonable requestethics approval and consent to participatethis study was approved by the ethics committee of beijing shijitan hospitalaffiliated to capital medical university approval code research ethicsno69 this study is retrospective only anonymous data sources were usedand informed consent was not requiredconsent for publicationnot applicablecompeting intereststhe authors declare that they have no competing interestsreceived january accepted july referencesklaver ce musters gd bemelman wa punt cj verwaal vj dijkgraaf mgaalbers ag van der bilt jd boerma d bremers aj adjuvanthyperthermic intraperitoneal chemotherapy hipec in patients with coloncancer at high risk of peritoneal carcinomatosis the colopec randomizedmulticentre trial bmc cancer 201515undefined428van oudheusden tr braam hj nienhuijs sw wiezer mj van ramshorst bluyer md lemmens ve de hingh ih cytoreduction and hyperthermicintraperitoneal chemotherapy a feasible and effective option for colorectalcancer patients after emergency surgery in the presence of peritonealcarcinomatosis ann surg oncol passot g vaudoyer d villeneuve l kepenekian v beaujard ac bakrin ncotte e gilly fn glehen o what made hyperthermic intraperitonealchemotherapy an effective curative treatment for peritoneal surfacemalignancy a 25year experience with procedures j surg oncolarjonasánchez a barrios p boldoroda e camps b carrascocampos jmartÃn vc garcÃafadrique a gutiérrezcalvo a morales r ortegapérez g hipect4 multicentre randomized clinical trial to evaluate safety andefficacy of hyperthermic intraperitoneal chemotherapy hipec with 0cwang bmc anesthesiology page of huepenbecker sp cusworth se kuroki lm lu p samen cd woolfolk cdeterding r wan l helsten dl bottros m continuous epiduralinfusion in gynecologic oncology patients undergoing exploratorylaparotomy the new standard for decreased postoperative pain and opioiduse gynecol oncol teoh da hutton mj else s walker a lee a mack la epidural analgesia aprospective analysis of perioperative coagulation in cytoreductive surgeryand hyperthermic intraperitoneal chemotherapy am j surg schmidt c creutzenberg m piso p hobbhahn j bucher m perioperativeanaesthetic management of cytoreductive surgery with hyperthermicintraperitoneal chemotherapy anaesthesia kajdi me beckschimmer b held u kofmehl r lehmann k ganter mtanaesthesia in patients undergoing cytoreductive surgery withhyperthermic intraperitoneal chemotherapy retrospective analysis of asingle centre threeyear experience world j surg oncol solanki sl mukherjee s agarwal v thota rs balakrishnan k shah sb desain garg r ambulkar rp bhorkar nm society of oncoanaesthesia andperioperative care consensus guidelines for perioperative management ofpatients for cytoreductive surgery and hyperthermic intraperitonealchemotherapy crshipec indian j anaesth godden ar marshall mj grice as daniels ir ultrasonography guidedrectus sheath catheters versus epidural analgesia for open colorectal cancersurgery in a single centre ann r coll surg engl publishers notespringer nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliationsmitomycin c used during surgery for treatment of locally advancedcolorectal carcinoma bmc cancer baratti d kusamura s iusco d gimondi s pietrantonio f milione mguaglio m bonomi s grassi a virzì s hyperthermic intraperitonealchemotherapy hipec at the time of primary curative surgery in patientswith colorectal cancer at high risk for metachronous peritoneal metastasesann surg oncol chua tc robertson g liauw w farrell r yan td morris dl intraoperativehyperthermic intraperitoneal chemotherapy after cytoreductive surgery inovarian cancer peritoneal carcinomatosis systematic review of currentresults j cancer res clin oncol li y zhou yf liang h wang hq hao jh zhu zg wan ds qin lx cui szji jf chinese expert consensus on cytoreductive surgery andhyperthermic intraperitoneal chemotherapy for peritoneal malignanciesworld j gastroenterol willschke h bosenberg a marhofer p johnston s kettner sc wanzel o kaprals ultrasonographyguided rectus sheath block in paediatric anaesthesiaanew approach to an old technique br j anaesth azemati s khosravi mb an assessment of the value of rectus sheath blockfor postlaparoscopic pain in gynecologic surgery j minim invasive gynecol dingeman rs barus lm chung hk clendenin dj lee cs tracy s johnsonvm dennett kv zurakowski d chen c ultrasonographyguided bilateralrectus sheath block vs local anesthetic infiltration after pediatric umbilicalhernia repair a prospective randomized clinical trial jama surg relland lm tobias jd martin d veneziano g beltran rj mckee c bhalla tultrasoundguided rectus sheath block caudal analgesia or surgical siteinfiltration for pediatric umbilical herniorrhaphy a prospective doubleblinded randomized comparison of three regional anesthetic techniques jpain res 201710undefined2629 xu l hu z shen j pm mq efficacy of usguided transversus abdominisplane block and rectus sheath block with ropivacaine anddexmedetomidine in elderly highrisk patients minerva anestesiol cho s kim yj jeong k moon hs ultrasoundguided bilateral rectus sheathblock reduces early postoperative pain after laparoscopic gynecologicsurgery a randomized study j anesth li t ye q wu d li j yu j doseresponse studies of ropivacaine in bloodflow of upper extremity after supraclavicular block a doubleblindrandomized controlled study bmc anesthesiol bell jc rylah bg chambers rw peet h mohamed f moran bjperioperative management of patients undergoing cytoreductive surgerycombined with heated intraperitoneal chemotherapy for peritoneal surfacemalignancy a multiinstitutional experience ann surg oncol landmann a visoiu m malek mm development of a novel technique forbilateral rectus sheath nerve blocks under laparoscopicguidance j pediatrsurg kumar a wilson ga engelhardt te ultrasound guided rectus sheathblockade compared to perioperative local anesthetic infiltration in infantsundergoing supraumbilical pyloromyotomy saudi journal of anaesthesia bashandy gm elkholy ah reducing postoperative opioid consumption byadding an ultrasoundguided rectus sheath block to multimodal analgesiafor abdominal cancer surgery with midline incision anesthesiol pain med201443e18263 dowidar aerm ezz haa shama aae eloraby ma postoperative analgesiaof ultrasound guided rectus sheath catheters versus continuous woundcatheters for colorectal surgery a randomized clinical trial egypt journalof anaesth karaarslan e topal a avci o tuncer uzun s research on the efficacy of therectus sheath block method agri piccioni f casiraghi c fumagalli l kusamura s baratti d deraco m arientif langer m epidural analgesia for cytoreductive surgery withperitonectomy and heated intraperitoneal chemotherapy int j surg 16pt a99 vesterandersen m lundstrøm lh møller mh the association betweenepidural analgesia and mortality in emergency abdominal surgery apopulationbased cohort study acta anaesthesiol scand httpsdoi101111aas13461 0c" | Colon_Cancer |
biochemically interleukin6 belongs to the class of fourhelical cytokines the cytokine can be synthesised and secreted by many cells it acts via a cell surfaceexpressed interleukin6 receptor which is not signalling competent this receptor when complexed with interleukin6 associates with the signalling receptor glycoprotein kda gp130 which becomes dimerised and initiates intracellular signalling via the janus kinasesignal transducer and activator of transcription and rat sarcoma proto oncogenemitogenactivated protein kinasephosphoinositide3 kinase pathways physiologically interleukin6 is involved in the regulation of haematopoiesis and the coordination of the innate and acquired immune systems additionally interleukin6 plays an important role in the regulation of metabolism in neural development and survival and in the development and maintenance of various cancers although interleukin6 is mostly regarded as a proinflammatory cytokine there are numerous examples of protective and regenerative functions of this cytokine this review will explain the molecular mechanisms of the in part opposing activities of the cytokine interleukin6keywords gp130 sgp130fc il6 il6r sil6r transsignalling adam17invited reviewersversion aug faculty reviews are review s written by the prestigious members of faculty opinions the s are commissioned and peer reviewed before publication to ensure that the final published version is comprehensive and accessible the reviewers who approved the final version are listed with their names and affiliations elke roeb justus liebig university giessen germany hana alg¼l technical university of munich munich germany jacqueline bromberg department of medicine memorial sloan kettering cancer center new york usaany comments on the can be found at the end of the a0page of 0cf1000research 9faculty rev1013 last updated aug corresponding author stefan rosejohn rosejohnbiochemunikieldeauthor roles rosejohn s conceptualization data curation formal analysis funding acquisition investigation methodology project administration resources supervision writing original draft preparation writing review editingcompeting interests stefan rosejohn has acted as a consultant and speaker for abbvie amgen janssen chugai roche genentech roche pfizer eli lilly and sanofi he also declares that he is an inventor on patents owned by conaris research institute which develops the sgp130fc protein olamkicept and he has stock ownership in conarisgrant information the work of stefan rosejohn has been supported by grants of the deutsche forschungsgemeinschaft bonn germany under the grant numbers crc841 project c1 and crc877 project a1 and by the german excellence cluster inflammation at interfaces the funders had no role in study design data collection and analysis decision to publish or preparation of the manuscriptcopyright rosejohn s this is an open access distributed under the terms of the creative commons attribution license which permits unrestricted use distribution and reproduction in any medium provided the original work is properly citedhow to cite this rosejohn s interleukin6 signalling in health and disease [version peer review approved] f1000research 9faculty rev1013 1012688f1000research260581first published aug 9faculty rev1013 1012688f1000research260581 a0page of 0cintroductioninterleukin6 il6 is considered one of the most prominent proinflammatory cytokines1 blockade of il6 by the neutralising monoclonal antibody tocilizumab has been approved in more than countries for the treatment of patients with autoimmune disorders such as rheumatoid arthritis2 additionally the cytokine storm sometimes encountered when cancer patients are treated with chimeric antigen receptor car tcells3 could be effectively treated with the antibody tocilizumab leading to us food and drug administration fda approval of the drug for this condition even more recently it has been recognised that many patients experience a similar cytokine storm upon infection with sarscov2 covid19 virus4 and that these patients could also be treated with tocilizumab5 these new data led to a rekindled general interest in the cytokine il6il6 was initially discovered and cloned in the kishimoto laboratory as a bcell stimulatory factor6 immediately after the molecular cloning it was evident that il6 was identical to hepatocyte stimulating factor7 hybridomaplasmacytoma growth factor8 interferon 29 and kda protein10 this already indicated the pleiotropic nature of the cytokine later on it was also recognised that il6 shows profound activities in the brain1112 in the regulation of metabolism1314 in the response of the body to exercise15 and in the development and maintenance of various cancers16this review gives a short overview of the complex biology of il6 and explains how one cytokine can have extremely different biologic effects on different cells and in different physiologic states of the human body17the interleukin6 receptor complexthe fourhelical cytokine il6 figure on cells binds to a membranebound il6 receptor il6r and the complex of il6 and il6r associates with a second receptor protein glycoprotein kda gp130 which dimerises and initiates intracellular signalling via the janus kinase jaksignal transducer and activator of transcription stat and rat sarcoma proto oncogene rasmitogenactivated protein kinase and phosphoinositide3 kinase pathways figure importantly il6 exhibits only a measurable affinity to the il6r but not to gp130 and the il6r does not bind on its own to gp130 it is only the complex of il6 and il6r that binds to gp130 and induces its dimerisation figure all cells in the body express gp130 but only a few cells such as hepatocytes and some leukocytes express il6r it follows that cells that express only gp130 but not il6r cannot be stimulated by il61noteworthy gp130 is a component of the receptor complexes of the socalled gp130 cytokine family which besides il6 comprises il11 ciliary neurotrophic factor cntf cardiotrophin1 ct1 cardiotrophinlike cytokine clc leukaemia inhibitory factor lif oncostatin m osm and il27 for details please refer to recent reviews1920it has however been noticed that the membranebound il6r can be cleaved by the membranebound metalloprotease a f1000research 9faculty rev1013 last updated aug figure fourhelical topology of the interleukin6 il6 protein il6 belongs to the family of fourhelical cytokines the figure shows the four helices with the connecting loops the ab and the cd loops are long enough to reach the length of a helix whereas the bc loop is short consequently il6 has an upupdowndown topology meaning that helices a and b point upwards whereas helices c and d point downwards this topology is common to most cytokines such as il2 il4 il7 il11 il15 leukaemia inhibitory factor oncostatin m growth hormone leptin and many othersdisintegrin and metalloprotease adam17 to generate a soluble il6r sil6r21 to a minor extent the humanbut not the murinesil6r can be generated by translation from a differentially spliced mrna22 intriguingly the sil6r can still bind il6 and the complex of il6 and sil6r can associate with gp130 and induce signalling even on cells that lack the membranebound il6r23 this process has been named il6 transsignalling figure strikingly following this paradigm il6 can in the presence of sil6r stimulate any cell in the body since all cells express gp13017interestingly most il6rexpressing cells including hepatocytes express far more gp130 than il6r molecules therefore stimulation of such cells with il6 alone will only lead to engagement of few gp130 molecules whereas stimulation with the complex of il6 and sil6r will stimulate all cellular gp130 proteins a threshold for a given response might not be reached with il6 stimulation but only with stimulation of all gp130 molecules via il6 transsignalling this might be an explanation for the observed differences in signalling between transsignalling and classical signalling that lead to different phenotypes25molecular tools to elucidate the functions of interleukin6the concept of transsignalling has been corroborated by the use of two designer proteins the first such protein consists of il6 covalently fused to the sil6r via a flexible peptide linker which allowed the placement of il6 il6 page of 0cf1000research 9faculty rev1013 last updated aug figure stimulation of target cells by interleukin6 il6 il6 orange first binds to the il6 receptor il6r red the complex of il6 and il6r associates with glycoprotein kda gp130 blue which dimerises and leads to intracellular signalling it is important to note that il6 and il6r alone exhibit no measurable affinity to gp130 only the complex of il6 and il6r binds to and activates gp130 therefore il6 cannot stimulate cells that do not express il6r signalling occurs via the signal transducer and activator of transcription stat 1stat3 yamaguchi sarcoma viral oncogene homolog yesyesassociated protein yap phosphoinositide3 kinase pi3kakt and rat sarcoma proto oncogene rasmitogenactivated protein kinase mapk pathways jak janus kinaseat the correct distance to reach the il6 binding site of the sil6r this protein was called hyperil6 figure 3a26 this protein was shown to stimulate gp130expressing cells in vitro and in vivo and it was shown that liver regeneration27 stimulation of neural cells28 and expansion of hematopoietic cells29 was far more efficient in the presence of hyperil6 as compared to il6 alone30turned out while hyperil6 demonstrated only the biologic potential of il6 transsignalling these experiments did not prove that this process occurred in vivo a second soluble protein was designed which consisted of the entire extracellular portion of gp130 covalently fused to the fc region of human igg1 figure 3b the resulting protein named soluble gp130fc sgp130fc to exhibit similar properties as membranebound gp130 it did not bind il6 or il6r alone but it bound with high affinity the complex of il6 and sil6r3132 consequently the sgp130 protein in vitro and in vivo specifically inhibited il6 transsignalling without compromising il6 signalling via the membranebound il6r ie classic signalling32 the sgp130fc protein could be used to define il6mediated biologic responses which were dependent on classic or transsignalling this was accomplished by comparing the treatment of animals with sgp130fc or with neutralising antibodies against il6 or il6r which blocked all il6 signalling figure 3c d using animal models of human inflammatory diseases or inflammationassociated cancer it turned out inflammationassociated cancers were mainly driven by il6 transsignalling whereas regenerative and protective activities of il6 were mediated by classic il6 signalling via the membranebound il6r figure that autoimmune disorders and levels in physiologic and pathophysiologic functions of interleukin6under homeostatic conditions il6 the circulation are as low as pgml but during inflammatory states these levels can rise more than 1000fold and under extreme conditions leading to sepsis il6 levels in the µgml range have been reported33 il6 is produced by myeloid cells upon tolllike receptor stimulation together with the cytokines il1 and tumor necrosis factor α tnfα which via a feedforward loop lead to an immense amplification of il6 production during inflammatory conditions34 there is perhaps no other protein in the human body whose level can go up by six orders of magnitude this lets us conclude that il6 is the major alarm signal to infection inflammation and possibly cancer35the human body in response in however under normal conditions il6 plays an important role in ancellular homeostasis mice in which the il6 gene has been ablated il6 knockout mice become obese late in life13 cannot regenerate their liver upon hepatectomy36 and show no signs of osteoporosis upon ovariectomy37 indicating roles for il6 in body weight regulation liver physiology and bone metabolism in pathophysiologic states however there are marked differences between il6 knockout mice and wildtype mice il6 knockout mice are completely protected in animal models of rheumatoid arthritis38 and multiple sclerosis39 indicating a key role for il6 in these autoimmune disorderswith the help of the sgp130fc protein and of neutralising monoclonal antibodies it was possible to selectively block il6 transsignalling or to block all il6 signalling respectively page of 0cf1000research 9faculty rev1013 last updated aug tumour progression was induced by tumourinfiltrating myeloid cells which stimulated the neoplastic cells via il6 transsignalling47 selective blockade of this pathway by the sgp130fc protein blocked progression of pancreatic intraepithelial neoplasias to pancreatic ductal adenocarcinomas47 indicating a prominent role for il6 transsignalling in the development of pancreatic cancer in the murine apcmin model of colon cancer it was established that the genetic deletion of adam17 which is responsible for generating not only sil6r but also soluble tnfα and soluble ligands of the epidermal growth factor receptor egfr resulted in completely abrogated tumour development16 moreover the formation of neoplasias stimulated adam17 on macrophages leading to egfr ligand cleavage and subsequent egfr stimulation these macrophages now produced il6 and sil6r which led to the outgrowth of the tumours again selective blockade of the il6 transsignalling pathway by the sgp130fc protein blocked tumour development in the apcmin model and an additional mouse model of colon cancer16 this was highly reminiscent of a study in liver cancer in which it was shown that the egfr expressed in macrophages but not egfr in hepatocytes was involved in the development of hepatocellular carcinoma48 apparently macrophage activation may be an important step in the initiation and progression of tumours via the il6 transsignalling pathway20 figure therapeutic targeting of interleukin6 activitytherapeutic targeting of the proinflammatory cytokine tnfα was introduced as an efficient strategy to treat patients with autoimmune disorders such as rheumatoid arthritis and inflammatory bowel disease49 subsequently blockade of the biologic activity of the cytokine il6 was shown to be an efficient treatment for patients with rheumatoid arthritis and other autoimmune diseases2 and it was shown that blocking il6 activity was more efficient than blocking tnfα in a monotherapy trial50 blockade of il6 activity with the il6r neutralising monoclonal antibody tocilizumab was also highly effective in the treatment of patients with car t cellinduced severe cytokine release syndrome51 in patients with severe covid19 disease the administration of tocilizumab resulted in a marked improvement of the condition in the majority of patients the fever subsided creactive protein decreased and oxygen intake could be lowered no obvious adverse reactions were observed these preliminary data indicated that tocilizumab is a candidate for effective treatment of covid19 patients552 interestingly treatment of covid19 patients with the il6r neutralising monoclonal antibody sarilumab resulted in no significant difference in clinical improvement and mortality53summarythe discovery that the proinflammatory activities of il6 are mediated by il6 transsignalling whereas the protective and regenerative activities of il6 rely on classic signalling via the membranebound il6r suggested that the sgp130fc protein might be an ideal candidate for a more specific mode of cytokine blockade as opposed to global cytokine inhibition20 it was shown in appropriate animal models that blockade of il6 transsignalling was indeed superior to global il6 blockade in a bone healing model4445 in a sepsis model42 in abdominal page of figure designer proteins to probe for modes of interleukin il6 signalling a hyperil6 is a fusion protein between il6 and soluble il6 receptor sil6r b sgp130fc is a fusion protein of the extracellular portion of glycoprotein kda gp130 and the constant part of a human immunoglobulin g1 igg1 antibody c il6 can signal via the membranebound il6r classical signalling and via the sil6r transsignalling hyperil6 can be used to mimic il6 transsignalling b the sg130fc protein does not interfere with classical il6 signalling but it specifically blocks il6 transsignallingusing this approach it was shown that classic il6 signalling via the membranebound il6r was responsible for the defence of the body against bacteria4041 intestinal regeneration upon polymicrobial sepsis42 prevention of aortic rupture in animal models of abdominal aortic aneurysm43 and healing of bone fractures4445 important processes are severely compromised under blockade of global il6 activity46 it has been hypothesised that the same might apply for the treatment of covid19 patients46 figure indicating that these besides being the major alarm signal in the human body il6 plays a dominant role in various types of cancer one important reason could be that il6 via stimulation of the stat3 pathway is a prominent growth factor of many cancer cells the following scenario has been worked out in pancreatic cancer47 it was noted that in the krasg12d model the massive activation of the stat3 pathway which led to 0cf1000research 9faculty rev1013 last updated aug figure pro and antiinflammatory activities of interleukin6 il6 left antiinflammatory and protective activities of the cytokine il6 are associated with signalling via the membranebound il6 receptor il6r right proinflammatory activities of the cytokine il6 are associated with signalling via the soluble il6r sil6r the membranebound metalloprotease a disintegrin and metalloprotease adam17 orchestrates the pro and antiinflammatory activities of il6 treg regulatory t cellin patients with is presently ongoing aortic aneurysm models43 and in bacterial infection models4041 the sgp130fc protein was expressed and purified according to gmp regulations phase i clinical trials were successfully performed with healthy individuals and a phase ii clinical trial inflammatory bowel disease54 the future will tell whether this elegant therapeutic approach which was successfully tested in many animal models leads to a novel paradigm in cytokineblocking therapies in patients with autoimmune disorders46 similarly blockade of transsignalling while leaving classical signalling intact may prove to be beneficial for patients experiencing cytokine storms from covid19 or car tcell therapies finally we suggest that malignancies promoted by high levels of transsignalling could be contained by this therapeutic modalityinterleukin6 il6r abbreviationsadam17 a disintegrin and metalloprotease egfr epidermal growth factor receptor gp130 glycoprotein kda il6 interleukin6 receptor ras rat sarcoma proto oncogene sgp130fc soluble gp130fc fusion protein which under the name of olamkicept is in phase ii clinical trials sil6r soluble il6r stat signal transducer and activator of transcription tnfα tumor necrosis factor α yap yesassociated protein yes yamaguchi sarcoma viral oncogene homologacknowledgementsi thank all past and current colleagues of our laboratory for many helpful discussionsreferencesfaculty opinions recommended kishimoto t interleukin6 from basic science to medicine40 years in immunology annu rev immunol pubmed abstract publisher full text tanaka t narazaki m ogata a et al a new era for the treatment of inflammatory autoimmune diseases by interleukin6 blockade strategy semin immunol pubmed abstract publisher full text faculty opinions recommendation teachey dt lacey sf shaw pa et al identification of predictive biomarkers for cytokine release syndrome after chimeric antigen receptor tcell therapy for acute lymphoblastic leukemia cancer discov pubmed abstract publisher full text free full text page of 0c moore jb june ch cytokine release syndrome in severe covid19 science pubmed abstract publisher full text faculty opinions recommendation xu x han m li t et al effective treatment of severe covid19 patients with tocilizumab proc natl acad sci u s a pubmed abstract publisher full text free full text faculty opinions recommendation hirano t taga t yamasaki k et al molecular cloning of the cdnas for interleukin6b cell stimulatory factor and its receptor ann n y acad sci discussion pubmed abstract publisher full text gauldie j richards c harnish d et al interferon beta 2bcell stimulatory factor type shares identity with monocytederived hepatocytestimulating factor and regulates the major acute phase protein response in liver cells proc natl acad sci u s a pubmed abstract publisher full text free full text brakenhoff jp de groot er evers rf et al molecular cloning and expression of hybridoma growth factor in escherichia coli j immunol pubmed abstract zilberstein a ruggieri r korn jh et al structure and expression of cdna and genes for human interferonbeta2 a distinct species inducible by growthstimulatory cytokines embo j pubmed abstract free full text haegeman g content j volckaert g et al structural analysis of the sequence coding for an inducible 26kda protein in human fibroblasts eur j biochem pubmed abstract publisher full text rothaug m beckerpauly c rosejohn s the role of interleukin6 signaling in nervous tissue biochim biophys acta pt a pubmed abstract publisher full text willis ef macdonald kpa nguyen qh et al repopulating microglia promote brain repair in an il6dependent manner cell 833846e16 pubmed abstract publisher full text wallenius v wallenius k ahr©n b et al interleukin6deficient mice develop matureonset obesity nat med pubmed abstract publisher full text findeisen m allen tl henstridge dc et al treatment of type diabetes with the designer cytokine ic7fc nature pubmed abstract publisher full text faculty opinions recommendation pedersen bk febbraio ma muscles exercise and obesity skeletal muscle as a secretory an nat rev endocrinol pubmed abstract publisher full text faculty opinions recommendation schmidt s schumacher n schwarz j et al adam17 is required for egfrinduced intestinal tumors via il6 transsignaling j exp med pubmed abstract publisher full text free full text rosejohn s the biology of interleukin6 in the 21st century semin immunol pubmed abstract publisher full text schaper f rosejohn s interleukin6 biology signaling and strategies of blockade cytokine growth factor rev pubmed abstract publisher full text jones sa jenkins bj recent insights into targeting the il6 cytokine family in inflammatory diseases and cancer nat rev immunol pubmed abstract publisher full text faculty opinions recommendation garbers c heink s korn t et al interleukin6 designing specific therapeutics for a complex cytokine nat rev drug discov pubmed abstract publisher full text m¼llberg j schooltink h stoyan t et al the soluble interleukin6 receptor is generated by shedding eur j immunol pubmed abstract publisher full text lust ja donovan ka kline mp et al isolation of an mrna encoding a soluble form of the human interleukin6 receptor cytokine pubmed abstract publisher full text mackiewicz a schooltink h heinrich pc et al complex of soluble human il6receptoril6 upregulates expression of acutephase proteins j immunol pubmed abstract rosejohn s heinrich pc soluble receptors for cytokines and growth factors generation and biological function biochem j 300pt pubmed abstract publisher full text free full text rosejohn s the soluble interleukin receptor advanced therapeutic options in inflammation clin pharmacol ther pubmed abstract publisher full text fischer m goldschmitt j peschel c et al i a bioactive designer cytokine for human hematopoietic progenitor cell expansion nat biotechnol f1000research 9faculty rev1013 last updated aug pubmed abstract publisher full text galun e zeira e pappo o et al liver regeneration induced by a designer human il6sil6r fusion protein reverses severe hepatocellular injury faseb j pubmed abstract publisher full text m¤rz p otten u rosejohn s neural activities of il6type cytokines often depend on soluble cytokine receptors eur j neurosci pubmed abstract publisher full text audet j miller cl rosejohn s et al distinct role of gp130 activation in promoting selfrenewal divisions by mitogenically stimulated murine hematopoietic stem cells proc natl acad sci u s a pubmed abstract publisher full text free full text rosejohn s interleukin6 family cytokines cold spring harb perspect biol a028415 pubmed abstract publisher full text free full text horsten u schmitzvan de leur h m¼llberg j et al the membrane distal half of gp130 is responsible for the formation of a ternary complex with il6 and the il6 receptor febs lett pubmed abstract publisher full text jostock t m¼llberg j ozbek s et al soluble gp130 is the natural inhibitor of soluble interleukin6 receptor transsignaling responses eur j biochem pubmed abstract publisher full text waage a brandtzaeg p halstensen a et al the complex pattern of cytokines in serum from patients with meningococcal septic shock association between interleukin interleukin and fatal outcome j exp med pubmed abstract publisher full text free full text tanaka t narazaki m kishimoto t il6 in inflammation immunity and disease cold spring harb perspect biol a016295 pubmed abstract publisher full text free full text faculty opinions recommendation rosejohn s il6 transsignaling via the soluble il6 receptor importance for the proinflammatory activities of il6 int j biol sci pubmed abstract publisher full text free full text cressman de greenbaum le deangelis ra et al liver failure and defective hepatocyte regeneration in interleukin6deficient mice science pubmed abstract publisher full text poli v balena r fattori e et al interleukin6 deficient mice are protected from bone loss caused by estrogen depletion embo j pubmed abstract publisher full text free full text alonzi t fattori e lazzaro d et al interleukin is required for the development of collageninduced arthritis j exp med pubmed abstract publisher full text free full text okuda y sakoda s bernard cc et al il6deficient mice are resistant to the induction of experimental autoimmune encephalomyelitis provoked by myelin oligodendrocyte glycoprotein int immunol pubmed abstract publisher full text hoge j yan i j¤nner n et al il6 controls the innate immune response against listeria monocytogenes via classical il6 signaling j immunol pubmed abstract publisher full text sodenkamp j waetzig gh scheller j et al therapeutic targeting of interleukin6 transsignaling does not affect the outcome of experimental tuberculosis immunobiology pubmed abstract publisher full text barkhausen t tschernig t rosenstiel p et al selective blockade of interleukin6 transsignaling improves survival in a murine polymicrobial sepsis model crit care med pubmed abstract publisher full text paige e cl©ment m lareyre f et al interleukin6 receptor signaling and abdominal aortic aneurysm growth rates circ genom precis med e002413 pubmed abstract publisher full text free full text kaiser k prystaz k vikman a et al pharmacological inhibition of il6 transsignaling improves compromised fracture healing after severe trauma naunyn schmiedebergs arch pharmacol pubmed abstract publisher full text free full text prystaz k kaiser k kovtun a et al distinct effects of il6 classic and transsignaling in bone fracture healing am j pathol pubmed abstract publisher full text magro g sarscov2 and covid19 is interleukin6 il6 the culprit lesion of ards onset what is there besides tocilizumab sgp130fc cytokine x pubmed abstract publisher full text free full text faculty opinions recommendation lesina m kurkowski mu ludes k et al stat3socs3 activation by il6 transsignaling promotes progression of pancreatic intraepithelial page of 0cf1000research 9faculty rev1013 last updated aug neoplasia and development of pancreatic cancer cancer cell pubmed abstract publisher full text faculty opinions recommendation lanaya h natarajan a komposch k et al egfr has a tumourpromoting role in liver macrophages during hepatocellular carcinoma formation nat cell biol pubmed abstract publisher full text free full text faculty opinions recommendation sacre sm andreakos e taylor p et al molecular therapeutic targets in rheumatoid arthritis expert rev mol med pubmed abstract publisher full text gabay c emery p van vollenhoven r et al tocilizumab monotherapy versus adalimumab monotherapy for treatment of rheumatoid arthritis adacta a randomised doubleblind controlled phase trial lancet pubmed abstract publisher full text faculty opinions recommendation le rq li l yuan w et al fda approval summary tocilizumab for treatment of chimeric antigen receptor t cellinduced severe or lifethreatening cytokine release syndrome oncologist pubmed abstract publisher full text free full text faculty opinions recommendation campochiaro c dellatorre e cavalli g et al efficacy and safety of tocilizumab in severe covid19 patients a singlecentre retrospective cohort study eur j intern med pubmed abstract publisher full text free full text faculty opinions recommendation dellatorre e campochiaro c cavalli g et al interleukin6 blockade with sarilumab in severe covid19 pneumonia with systemic hyperinflammation an openlabel cohort study ann rheum dis pubmed abstract publisher full text faculty opinions recommendation safety and efficacy of tj301 iv in participants with active ulcerative colitis clinicaltrials reference sourcepage of 0cf1000research 9faculty rev1013 last updated aug open peer reviewcurrent peer review status editorial note on the review processfaculty reviews are review s written by the prestigious members of faculty opinions the s are commissioned and peer reviewed before publication to ensure that the final published version is comprehensive and accessible the reviewers who approved the final version are listed with their names and affiliationsthe reviewers who approved this areversion jacqueline bromberg department of medicine memorial sloan kettering cancer center new york ny usa competing interests no competing interests were disclosedhana alg¼l comprehensive cancer center munich university hospital klinikum rechts der isar mildredscheelchair of tumor metabolism technical university of munich munich | Colon_Cancer |
" recent impressive advances in cancer immunotherapy have been largely derived from cellular immunity the role of humoral immunity in carcinogenesis has been less understood based on our previous observations we hypothesize that an immunoglobulin subtype igg4 plays an essential role in cancer immune evasionmethods the distribution abundance actions properties and possible mechanisms of igg4 were investigated with human cancer samples and animal tumor models with an extensive array of techniques both in vitro and in vivoresults in a cohort of patients with esophageal cancer we found that igg4 containing b lymphocytes and igg4 concentration were significantly increased in cancer tissue and igg4 concentrations increased in serum of patients with cancer both were positively related to increased cancer malignancy and poor prognoses that is more igg4 appeared to associate with more aggressive cancer growth we further found that igg4 regardless of its antigen specificity inhibited the classic immune reactions of antibody dependent cell mediated cytotoxicity antibody dependent cellular phagocytosis and complement dependent cytotoxicity against cancer cells in vitro and these effects were obtained through its fc fragment reacting to the fc fragments of cancer specific igg1 that has been bound to cancer antigens we also found that igg4 competed with igg1 in reacting to fc receptors of immune effector cells therefore locally increased igg4 in cancer microenvironment should inhibit antibody mediated anticancer responses and help cancer to evade local immune attack and indirectly promote cancer growth this hypothesis was verified in three different immune potent mouse models we found that local application of igg4 significantly accelerated growth of inoculated breast and colorectal cancers and carcinogen induced skin papilloma we also tested the antibody drug for cancer immunotherapy nivolumab which was igg4 in nature with a stabilizing s228p mutation and found that it significantly promoted cancer growth in mice this may provide an explanation to the newly appeared hyperprogressive disease sometimes associated with cancer immunotherapy there appears to be a previously unrecognized immune evasion mechanism with igg4 playing an essential role in cancer microenvironment with implications in cancer diagnosis and immunotherapyintroductionwhile new immune therapy for cancer focuses mostly on manipulating cellular immunity humoral immunity also holds great promise for cancer therapy1 igg4 is a unique antibody that has the lowest concentration among igg subtypes in healthy individuals and its function has not been well understood3 igg4 was regarded as a blocking antibody because of its reduced ability to trigger effector immune reactions6 therefore whatever molecules igg4 reacts to the subsequent immune reaction was subdued8 igg4 has a unique structure of fab arm exchange fae in which the two heavy and light chains of two different antibodies with different specificities are exchanged resulting in an asymmetric bispecific antibody with reduced ability to bind to antigen and to form immune complexes9 another unique feature of igg4 is that it can react to other iggs via its fc fragment and the significance of this property has not been well understood evidence suggests that fae and fc fc reactivity may involve the same molecular structure on igg4 molecule10 davies et al11 resolved the crystal structure of igg4 fc fragment revealing a unique molecular conformation supporting its fc binding property recent interests in igg4 related diseases unveiled a wide range of pathologies with a common phenomenon of often increased igg4 concentration in the serum and igg4 postive plasma cells in the affected ans accompanied by local inflammation and fibrosis but its pathogenic mechanism is still poorly understood13to cite wang a0h xu a0q zhao a0c et a0al an immune evasion mechanism with igg4 playing an essential role in cancer and implication for immunotherapy for immunotherapy of cancer 20208e000661 101136jitc2020000661 º additional material is published online only to view please visit the online http dx jitc hw qx cz and zz are joint first authorsaccepted july authors or their employers re use permitted under cc by nc no commercial re use see rights and permissions published by bmjfor numbered affiliations see end of correspondence toprofessor jiang gu qq comwang a0h et a0al j immunother cancer 20208e000661 101136jitc2020000661 0copen access the possible functions of igg4 in cancer and also in the immune system have not been well elucidated increases of igg4 positive plasma cells were reported in gastrocarcinoma16 extrahepatic cholangiocarcinoma17 and melanoma18 the above studies were performed on limited number of cases without convincing explanation of mechanism or significance wu 19 reported that serum igg4igg ratio could predict recurrence of hepatocellular carcinoma after surgery the most extensive study on igg4 and cancer was performed by karagiannis 20 who investigated the possible effect of cancer specific igg4 in inhibiting cancer immunity in melanomas and suggested competition between cancer specific igg4 and igg1 in binding to cancer antigens as the cause for the inhibition a recent report raised the concept of cancer educated b cells and toxic igg produced by these cells in facilitating lymph node metastasis for breast cancer in a mouse model21 we performed a multidimensional investigation of igg4 in a wide array of patients with cancer and tissues with both in vitro and in vivo experiments extensive new evidence led us to hypothesize that there is a potent humoral immune editing mechanism in cancer microenvironment with igg4 playing an essential role we propose that fc fc reaction could be the basic mechanism of this immune inhibition we validated this in three immune potent animal models this property was also found applicable to cancer immunotherapy drug nivolumab which was igg4 in nature our study points to a so far little appreciated mechanism of immune evasion in cancermaterials and methodskey resourcesdetailed information about key resources including antibodies biological samples chemicals assay kits cell lines and software are shown in online supplementary table experimental model and subject detailspatients and healthy volunteerswe collected tissue and blood samples from over patients with cancer in shantou university affiliated tumor hospital and the east guangdong provincial pathological consultation center details of the human samples are shown in online supplementary table immunohistochemistrydetails of the protocols and the antibodies are shown in online supplementary datasds techniquethe expressions of igg1 igg2 igg3 and igg4 in esophageal cancer were detected at the histological level the stain decolorize stain sds technology22 was performed on the same slide sequentially with four different antibodies to demonstrate the four antigens the distribution pattern abundance and relationship of the four antigens were processed with an image software photoshop to achieve an integrated figure the proximity of different cell types on the tissue sections was measured with an image analyzing software image pro plus v60 details of the protocol are presented in the online supplementary dataimmunofluorescence double stainingtwo antibodies from different species were incubated on the same tissue section primary antibodies were detected with goat antimouse 555antirabbit igg or goat antimouse 488antirabbit igg alexa fluor life sciences and immunoreactivity was visualized with a fluorescence microscope antigens were detected and demonstrated with two fluorescence signals in red and green and the blue signal of ' diamidino2 phenylindole dapi as for cell nuclei images were acquired with the evos fl fluorescence microscope life technologies usaigg4 immunohistochemistryto verify that igg4 could react to igg1 that had been immobilized to tissue sections we used human pancreas and brain and antibodies to insulin glucagon and neurofilament as models detailed protocol of this experiment is shown in the online supplementary dataimmunoglobulin preparationsfab and fc fragments of igg igg1 and igg4 were prepared with papain digestion in the presence of cysteine iggs were cleaved at a position above the hinge region by papain at °c for hours after purification with protein a affinity chromatography pure fab and fc fragments were isolated with elution buffer igg fc fragment was washed down from protein a column after centrifugation and concentration measurement igg preparations were collected and stored at °c before usewestern blotigg subclasses were resolved on sodium dodecyl sulfate polyacrylamide gels under reducing conditions and then transferred onto nitrocellulose membranes ge healthcare life sciences the membranes were blocked for hour with bovine serum albumin bsa in tris buffered saline with tween20 ph and then incubated with primary antibody biotin labeling kit anaspec overnight at °c it was then incubated with secondary antibody for hour at room temperature finally reaction density was measured with odyssey western at nm and nm detection channelserum igg4 and igg assessmentsera samples collected from patients with esophageal cancer and healthy volunteers were sent to golden field medical test company guangzhou china and roche immune turbidimetry method was used to wang a0h et a0al j immunother cancer 20208e000661 101136jitc2020000661 0cmeasure serum igg4 and total igg concentrations all serum samples were stored at °c freezer immediately before analysis all quantitative data were treated statisticallymeasurement of igg4 concentrations in tumor and tumoradjacent normal tissues with elisaconcentrations of igg4 in pairs of esophageal cancer and adjacent normal tissues cm from the edge of the cancer mass were measured with elisa detailed protocol of this experiment is shown in the online supplementary datacell culture for fc receptor binding assaysu937 cell line was bought from procell life science technology china cl0239 and cells were cultured in rpmi gibco c22400500bt supplemented with fetal bovine serum gibco and uml penicillin streptomycin gibco at Ã106ml in ml cell culture bottleprotein preparationsdetails of the protocols for protein preparation separation and papain digestion are shown in online supplementary datafluorescence activating cell sorter facs for fc receptor assaysdetails of the relevant protocols are shown in online supplementary dataadcc adcp and cdc teststhe classic antibody dependent cell mediated cytotoxicity adcc antibody dependent cellular phagocytosis adcp and complement dependent cytotoxicity cdc tests were performed based on previously reported protocols23 non specific igg4 instead of cancer specific igg4 was used to inhibit these reactions igg1 was used as control details of the protocols are shown in online supplementary dataanimal modelsbreast cancer and colon cancer modelsbalbc mice were obtained from vital river technical beijing china mice aged between and weeks and weighed ± g were used in all experiments all mice were inoculated with 4t1 mouse breast cancer cells or ct26 mouse colon cancer cells subcutaneously under the left forearm à 4t1 cells per mouse to build cancer models the mice were divided into different groups and were treated with igg1 or igg4 derived from intravenous igg ivig ivig without igg4 nivolumab and fc of nivolumab respectively details of the protocols are shown in online supplementary datacarcinogeninduced skin tumor modelwe employed a well established carcinogen induced skin tumor model to study the effect of igg4 and ivig without igg4 in comparison with controls phosphate buffered open accesssaline pbs detailed protocol is shown in online supplementary dataquantification and statistical analysisdata were analyzed in graphpad prism all reported p values were derived from two sided comparisons with values of less than considered to be statistically significantresultsigg4 was significantly increased in the serum of patients with cancer and this increase was related to cancer stage and patient prognosiswe first measured the concentrations of igg1 igg2 igg3 and igg4 in sera of patients with esophageal cancer n82 igg4 was significantly increased in patients with cancer in comparison with normal healthy subjects n70 the concentration of igg4 was increased from about to the ratio of igg4iggtotal was also significantly increased the statistical significance of both reached p00001 the increase of igg4 was also positively correlated to the stages of cancer with late stage cancers having more obvious increases higher igg4 serum concentrations were associated with worse prognosis figure 1adigg4containing lymphocytes and igg4 concentration were significantly increased in cancer microenvironment and this increase was associated with cancer cell infiltrationigg4 positive lymphocytes were significantly increased in cancer microenvironment in comparison with tissue more distant to the cancer mass igg4 positive lymphocytes were barely detectable in tumor adjacent normal tissue figure 1h on the same tissue sections employing the sds technique22 to simultaneously visualize the four subtypes of igg with four distinct colors we found that one plasma cell only contained one igg subtype that is igg1 igg2 igg3 and igg4 were all contained in their own plasma cell populations separately figure 1i the marked increase of igg4 containing plasma cells in comparison with other subtypes was clearly visualized on cancer tissue sections igg4 positive plasma cells appeared to accumulate more in tissues with extensive cancer cell infiltration than in other areas figure 1eh in addition igg4 positive cells are often in close proximity to cells containing igg1 and igg2 but not to igg3 this phenomenon was not seen among other igg subtypes apart from igg4 quantitative data of the proximity of different cell types are presented in online supplementary figure s2 with the multiple immunostaining method we also demonstrated that igg1 containing and igg4 containing lymphocytes were distinct from cd3 positive t lymphocytes in the same region of the cancer tissue figure 1j in addition we measured the concentrations of igg4 in cancer tissue and cancer adjacent normal tissue n46 pairs the average concentration of igg4 in cancer tissue was about four times higher than that in the adjacent wang a0h et a0al j immunother cancer 20208e000661 101136jitc2020000661 0copen access figure significant increase of igg4 and igg4iggtotal in serum and igg4 positive b lymphocytes in esophageal cancer a igg4 in serum of esophageal squamous cell cancer escc n82 was significantly higher when compared with healthy controls n70 p00001 b igg4iggtotal ratio in escc n82 was significantly higher than that in matching healthy adults n70 p00001 c igg4 in stage
³ n18 was significantly higher than those in stages
° and
± n16 p001 d igg4iggtotal in serum of stage iv escc n18 was significantly higher than those in stages
° and
± n16 p005 e scatter diagram of igg4 positive cell numbers in cancer cancer adjacent normal tissue adjacent and normal tissues igg4 positive lymphocytes in and around the esophageal cancer mass n110 are significantly more abundant than those in the adjacent normal tissue n60 and normal lymphoid tissues n63 p0001 for both increases of igg4 positive lymphocytes were most abundant in areas of cancer cell proliferation f the increase of igg4 positive cell numbers was related to the prognoses of the patients more igg4 positive cells were associated with worse outcome p005 g igg4 concentration in cancer tissue n46 was significantly higher than that in adjacent normal tissue n46 p001 h immunohistochemistry of igg4 in esophageal cancer tissues from left to right are igg4 in cancer tissues cancer adjacent tissue and normal lymphoid tissue tonsil it clearly demonstrates that igg4 positive lymphocytes red were markedly increased left in comparison with normal lymphoid tissue right and with tumor adjacent normal tissue middle scale bar µm i demonstration of four subpopulations of igg containing plasma cells with multiple immunostaining sds method in cancer each subclass has its own distribution pattern and one plasma cell only produces one subclass of igg igg1 yellow igg2 green igg3 purple igg4 red j on the same tissue section a triple immunostaining was performed with the sds method to demonstrate the distribution and relationship among cd3 positive t cells yellow igg1 positive b cells greens and igg4 positive b cells red each cell type has its own distribution and no overlap between different cell types is observed sds stain decolorize stainwang a0h et a0al j immunother cancer 20208e000661 101136jitc2020000661 0cnormal tissue and the difference between these two groups was statistically significant p001 figure 1gigg1 extracted from patients with cancer reacted to cancer cells of the same patients but igg4 extracted from the same patients did notwe extracted igg1 and igg4 from the serum of patients with esophageal cancer with breast cancer and with colon cancer with respective specific antibody columns we then labeled the antibodies with biotin and tested the reactivity of these extracted antibodies to cancer tissue sections of the same patients in all cases igg1 reacted to cancer cells from the same patients but igg4 did not figure 2a it appeared that the increased igg4 in cancer microenvironment and in the patients serum was not reactive to cancer antigens while igg1 extracted from the same patients reacted to the cancer antigensigg4 reacted to cancerspecific igg1 that was bound to cancer cellsalthough igg4 extracted from patients with cancer did not react to cancer antigen it did react to cancer specific igg1 that was bound to cancer antigen on tissue sections as shown in figure 2b when cancer specific igg1 that was not labeled with biotin was applied to cancer tissue sections followed by biotin labeled igg4 the cancer cells became positive while when the same biotin labeled igg4 was applied to the same cancer tissue section without prior application of igg1 it did not react this reaction of igg4 to cancer specific igg1 was not via the antigen specific variable region of igg4 as such igg4 was neither specifically against igg1 nor was it specifically against cancer antigen with its antigen recognizing fab variable region as shown in figure 2a the only explanation was that igg4 reacted to igg1 through its fc region this was validated by subsequent experiments with western blot analysis as shown in figure 3aein western blot igg4 was found to react to other iggs igg1 igg2 igg3 and igg4 via an fcfc mechanism and this reaction was across species but igg4 did not react to other ig subtypes igm ige iga or igdto test if and how igg4 could react to igg1 we performed western blot with igg4 from different sources extracted from patients with cancer from normal adults and commercially purchased igg4 was found to react to igg1 igg2 igg3 and igg4 at the molecular weight mw of about kd and this reaction was not seen when igg1 igg2 or igg3 was used as the antibody and igg4 as the target molecule running on the gel the above phenomenon was observed in western blot of both reducing and non reducing conditions figure 3a online supplementary figure s3 however human igg4 did not react to human igm iga igd or ige online supplementary figure s4 nevertheless we found that this reaction was across species that is human igg4 reacted to iggs of human mouse rabbit and goat online supplementary figure s5 we open accessigg4 extracted from a patient with cancer reacted figure to cancer bound igg1 and blocked antibody mediated cancer immunity a upper panel these photos serve as an example of the reactivity of igg1 and igg4 extracted from patients with cancer igg1 from the serum of a patient with breast cancer was labeled with biotin and stained a frozen cancer tissue section of the same patient cancer cells were positively stained by igg1 left the cancer cells were confirmed by their characteristic histopathology of he staining middle igg4 from serum of the same patient labeled with biotin and applied on the same cancer on a consecutive section was completely negative right lower panel another breast cancer positively stained by igg1 from the patients serum left the cancer cells were identified by positive immunostaining of cytokeratin ck on a consecutive section middle igg4 from the same patient was not reactive to the same cancer on a consecutive section right b the upper panel illustrates the principle of the experimental reactions and the middle and lower panels show staining results from two patients with breast cancer left igg1 from a patient with cancer positively reacted to frozen cancer tissue of the same patient brown cells middle igg4 from the same patient with cancer applied to consecutive sections but did not react to the same cancer right however when unlabeled igg1 was applied to the same cancer tissue section followed by biotin labeled igg4 the cancer cells were positively stained brown cellsfurther digested human igg4 and igg1 into fab and fc fragments with papain it was found that it was the fc fragment of igg4 reacting to fc of igg1 figure 3be this reaction was easy to occur as only min incubation wang a0h et a0al j immunother cancer 20208e000661 101136jitc2020000661 0copen access igg4 reacted to igg1 in western blot and tissue figure sections in an fc fc fashion a in western blot non cancer specific igg4 from a patient with breast cancer reacted to igg1 and igg4 from the same patient with cancer right panel arrows however when igg1 and igg4 were run on the gel and biotin labeled igg1 was used as the primary antibody no band was seen left panel these are the same antibodies used in figure a02ab providing support to explain the reaction between igg4 and igg1 seen on cancer tissue b western blot demonstrated that igg4 reacted with igg1 igg2 igg3 and igg4 c igg4 reacted with igg fc fragment but not with fab arm d igg4 reacted with igg1 fc fragment but not with fab arm e biotin labeled igg4 fc fragment reacted to igg1 and igg4 fc fragments but not with igg1 or igg4 fab biotin labeled igg4 fab did not react to igg1 or igg4 fc or fab these results demonstrate that it is the fc region of igg4 that reacted to fc of igg1resulted in a clear band therefore the positive reaction obtained by sequential applications with cancer specific igg1 followed by non cancer specific igg4 on cancer tissue figure 2b had to take place between the fc of igg4 and the fc of igg1 as shown in figure the fcfc reaction between igg4 and igg1 bound to tissue sections was further tested and validated with a number of antibodies and tissue types apart from cancerfollowing the same logic we tested the reactivity between the fc fragments of igg4 and igg1 already demonstrated in western blot on tissue sections we used igg1 primary antibodies to insulin and glucagon in normal human pancreas and antibody to neurofilament in human brain for this test the same principle was established with these normal tissues detailed results and figures are shown in online supplementary figure s6igg4 competed with igg1 to bind to fc receptors of pbmc and macrophageswe performed immunoglobulin and fc receptor binding assays with peripheral blood monocytes pbmc and with a human monocyte cell line u937 procell life science technology china cl0239 igg1 and igg4 were extracted from the serum of patients with cancer and pbmcs were isolated from the same human subjects the extracted and purchased igg1 and igg4 were labeled with biotin or fitc in the igg1 and igg4 binding assay we found that igg1 and igg4 competed with one another in binding to pbmc and this reaction could be completely blocked by fc receptor blocker this competition was concentration dependent that is as the concentration of igg4 increased more igg1 bound to pbmc was replaced the reverse was also true that is igg1 could also replace igg4 in this competition assay this phenomenon was demonstrated on cytospin slides of pbmc preparation online supplementary figure s7in addition flow cytometry was performed to examine the binding properties of igg1 and igg4 to fc receptors of monocytes the ability of igg1 and igg4 to bind to all three subtypes of fc gamma receptor fcγrfcγr
° cd64 fcγr
± cd32 and fcγr
² cd16was examined with corresponding antibodies we found that igg4 could compete with igg1 in binding to the fc receptor of monocytes u937 we further found that the binding force of igg1 was about twice as strong as that of igg4 for individual receptor subtypes igg1 could bind to all three receptor subtypes that is fcγr
° cd64 fcγr
± cd32 fcγr
² cd16 in contrast igg4 could only bind to fcγr
° cd64 although their binding sites were different igg4 could completely block igg1 we also found that it was necessary for a relatively high concentration of igg4 to be present in the solution in order to compete with igg1 in binding to fc receptors online supplementary figure s8igg4 inhibited the classic immune reactions of adcc adcp and cdc mediated via cancerspecific igg1 and effector immune cellscomplementswe first verified that non cancer specific igg4 indeed reacted to igg1 cetuximab used in adcc adcp and cdc figure 4a we then found that igg4 inhibited adcc elicited cytotoxicity with cancer specific igg1 antibody and pbmc figure 4b the igg4 used in our test was not directed against cancer antigen or to lymphocytes non cancer specific igg1 could also inhibit adcc but to a much lesser extent also reached statistical significance we obtained evidence to show that inhibition of adcc appeared to take place at the site of the cancer specific antibody that is igg4 reacted to the igg1 antibody bound to cancer cells figure 2b and the site of immune effector fc receptors the latter effect could wang a0h et a0al j immunother cancer 20208e000661 101136jitc2020000661 0copen accessfigure non cancer specific igg4 inhibited classic adcc adcp and cdc reactions against cancer but had no direct effect on cancer cell growth a on western blot the chimeric antibody cetuximab igg1 against egfr was run on the gel and igg4 and igg1 at concentrations of and µgml were used as the primary antibodies igg4 reacted to cetuximab at a concentration dependent manner but igg1 did not react b left in a classic adcc experiment cetuximab igg1 was incubated with an egfr expressing lung cancer cell line a549 and then with pbmc from normal healthy adult cancer cell activity was significantly reduced n12 non cancer specific igg1 and hsa were used as controls showing that they had no direct effect on the cancer cells n12 middle when non cancer specific igg4 was added to the mixture the effect of cetuximab was significantly reversed demonstrating an inhibitory effect of igg4 in adcc n12 non cancer specific igg1 had a much smaller but also significant effect in inhibiting adcc action n12 right when fc receptor blocker was incubated with pbmc the effect of cytotoxicity was blocked ce adcp was performed with a lung cancer cell line a549 expressing egfr as the targets human peripheral monocyte derived macrophages as the effector cells and the antibody cetuximab igg1 against egfr as the mediating antibody the tumor cells were stained with cfda se fluorescence probes green macrophages derived from pbmc were stained with dii fluorescent probes orange blue fluorescence is the nuclei stained with dapi d higher magnification of c the orange colored macrophages were in close contact with green tumor cells tumor debris ingested by macrophages appeared yellow in the cytoplasm of macrophages e bar chart showing the effect of adcp and its inhibition by igg4 f left in µgml rituximab mediated adcp model giemsa staining results of phagocytosis of raji cells by macrophages after the addition of µgml igg1 and igg4 respectively right igg4 significantly inhibited rituximab mediated adcp in phagocytosis by macrophage but igg1 could not inhibit the adcp effect scale bar30 µm p005 p001 p0001 g in a classic cdc experiment cetuximab anti egfr antibody was incubated with an egfr expressing lung cancer cell line a549 and then with complement co from serum of a normal healthy adult the cancer cell activity was significantly reduced h when non cancer specific igg4 was added to the mixture in the above cdc experiment the effect of cetuximab was significantly reversed i igg4 and igg1 were incubated with kyse150 for hours and no effect on cell growth was found adcc antibody dependent cell mediated cytotoxicity adcp antibody dependent cellular phagocytosis cdc complement dependent cytotoxicity cfse da carboxyfluorescein diacetate succinimidyl ester dapi ' diamidino2 phenylindole egfr epidermal growth factor receptor hsa human serum albumin pbmc peripheral blood mononuclear cellwang a0h et a0al j immunother cancer 20208e000661 101136jitc2020000661 0copen access be abolished with the addition of fc receptor blocker human trustain fcx biolegend china to the pbmcwe also performed an adcp experiment employing human monocyte derived macrophages and esophageal cancer cells cetuximab igg1 was used as the mediating antibody this was performed employing a coculture and cell counting method and fitc labeled antibody flow cytometry two models were employed and both methods showed that non cancer specific igg4 was able to reduce the effect of adcp mediated by cancer specific igg1 figure 4cfin a classic cdc assay we used cancer cell line a549 atcc usa c4215 as the target cancer cells cetuximab as the cancer specific igg1 mediating antibody and human plasma as the source of complements and demonstrated the destructive effect on cancer cells we then used non cancer specific igg4 or igg1 to inhibit the effect we found that the cdc effect was partially inhibited by non cancer specific igg4 but not by igg1 figure 4ghfor comparison we added igg4 or igg1 at various concentrations in cancer cell culture for different periods of time and found no direct effect of these proteins on cancer cell growth figure 4iigg4 including nivolumab significantly accelerated breast cancer cell and colon cancer cell growth in two immune potent mouse models in vivothe above results point to a mechanism that igg4 plays an important role in local immune evasion by blocking immune responses mediated by cancer specific igg antibodies to further examine this mechanism mediated by such antibodies we performed in vivo studies to verify this hypothesis with immune competent mouse models in one model we injected non cancer specific igg4 into a location where breast cancer cells were inoculated subcutaneously in this group of mice cancer cell growth was significantly increased resulting in a much larger cancer mass by days in comparison with other groups injections of pbs or igg without igg4 figure 5ab as there is no direct effect of igg4 on cancer cell growth figure 4i these results unequivocally confirmed that igg4 can inhibit local immune reaction and thereby promote cancer growth in vivo through immune evasionin a separate but similar experiment of a colon cancer mouse model we injected antibody to programmed cell death1 pd1 nivolumab which is a widely used drug in cancer immune therapy and is also an igg4 isotype with s228p mutation which replaces a serine residu | Colon_Cancer |
" immune checkpoint inhibitors that block programmed cell death1 pd1 and programmed cell death ligand1 pd l1 have improved outcomes for many cancer subtypes but do exhibit toxicity in the form of immune related adverse eventsobjective the aim of this study was to investigate the emerging toxicities of pd1 and pd l1 inhibitors including acute or reactivation of tuberculosis tb and atypical mycobacterial infection amimethods this study was completed as a retrospective review using the us food and drug administration adverse events reporting system faers for incidence of tb and ami due to pd1 and pd l1 inhibitors compared with other fda food and drug administration approved drugs the statistical methods included disproportionality signal analysis using the reporting or ror to compare cases the wald ci was reported to assess the precision of the rorresults out of the adverse events aes reported to faers for all drugs between january and march aes were due to the five fda approved pd1pd l1 inhibitors seventy two cases of tb were due to pd1pd l1 inhibitors specifically cases due to nivolumab due to pembrolizumab due to atezolizumab and due to durvalumab there were cases of ami due to nivolumab due to pembrolizumab and each due to durvalumab and atezolizumab avelumab was not attributed to any ae of tb or ami from analysis of the faers database the calculated ror for tb due to pd1pd l1 inhibitors was ci to p00001 and for ami was ci to p00001 pd1pd l1 inhibitors used in the treatment of cancer subtypes is associated with increased tb and ami risk although this complication is rare clinicians using pd1pd l1 inhibitors should be aware of the risksintroductionimmune checkpoint inhibitors icis that block programmed cell death1 pd1 and programmed cell death ligand1 pd l1 have transformed care for many cancer subtypes and have improved outcomes for patients with pd l1 overexpression1 through blockade of the pd1pdl1 axis the t lymphocyte mediated response against tumour cells is enhanced resulting in accelerated immune mediated destruction of key questionswhat is already known about this subject º case reports and case series suggest programmedcell death1programmedcell death ligand1 pd1pd l1 inhibitors are associated with acute tuberculosis tb or reactivation of tbwhat does this study add º this is the first large systemic effort to quantify the risk of tb due to pd1pd l1 inhibitors through retrospective analysis of faers food and drug administration adverse events reporting system a pharmacovigilance database pd1pd l1 inhibitors were not only associated with increased risk of tb compared with other drugs but atypicalmycobacterial infection as wellhow might this impact on clinical practice º although this complication is rare clinicians using pd1pd l1 inhibitors should be aware of thiscancer cells however facilitating immune mediated activation is not benign and patients receiving icis are known to exhibit unique toxicities that result in an damage known as immune related adverse events iraes3 the most common iraes with pd1 and pd l1 inhibitors are fatigue pruritus and diarrhoea4 some iraes can be fatal with pneumonitis hepatitis neurotoxicity and most commonly myocarditis reported5 while counterintuitive when the mechanism of action is considered an emerging and increasingly reported toxicity of pd1 and pd l1 inhibitors is acute tuberculosis tb and reactivation of tb6 the first case of tb due to the pd1 inhibitor was described in a patient with relapsed hodgkins lymphoma who developed pulmonary tb following treatment with pembrolizumab7 since then there have been other case reports of tb following initiation of pd1 or pd l1 inhibitors that make the development of tb a relevant concern8 in a preclinical mouse study pd1 deficient mice were found to be highly susceptible to tb with reduced survival compared with wild type mice12 however anand a0k et a0al esmo open 20205e000866 101136esmoopen2020000866 0copen accesstable adverse events of tb and ami due to pd1pdl1 inhibitors from january to march in faerstotal aes due to all drugs in faerstotal aes due to pd1pdl1 inhibitorstotal aes due to pd1 inhibitorstotal aes of tb in faerstotal aes of tb due to pd1pdl1 inhibitorstotal aes of ami in faerstotal aes of ami due to pd1pdl1 inhibitorsror calculation ror for tb due to pd1pdl1 inhibitors versus full database ror for ami due to pd1pdl1 inhibitors versus full database ci to ci to aes adverse events ami atypical mycobacterial infection faers food and drug administration adverse events reporting system pd1 programmed cell death1 pd l1 programmed cell death ligand1 ror reporting or tb tuberculosisthere is no current risk estimate describing the potential risk of developing tb or atypical mycobacterial infection ami from pd1 and pd l1 inhibitors in this study we retrospectively reviewed the us food and drug administration adverse events reporting system faers a pharmacovigilance database for the risk of tb and ami due to pd1 and pdl1 inhibitors compared with other fda foodand drug administration approved drugsmethodsthis study is a retrospective analysis that used data queries from the faers pharmacovigilance monitoring database faers is a public database that contains nearly million adverse event ae reports medication error reports and product quality complaints reported by healthcare professionals manufacturers and consumers from around the world since these reports are managed by fda and evaluated by clinical reviewers in the center for drug evaluation and research and the center for biologics evaluation and research date in each event report where applicable include individual case identification numbers for reference the suspected pharmaceutical agent treatment indication adverse reactions nature of the event ie serious outcomes eg hospitalised death other outcomes sex male female or unknown age weight event date initial fda receipt date latest fda receipt date pharmaceutical company reporter eg healthcare professional consumer pharmaceutical company unknown concomitant medications latest manufacturer received date country where the event occurred and manufacturer control number individual names and date of birth are excluded from these liststhe present study involved data queries of the faers database between january and march for aes secondary to pd1 inhibitors namely pembrolizumab and nivolumab and pd l1 inhibitors namely atezolizumab durvalumab and avelumab in all aes due to above five drugs we then searched for three aes specifically tuberculosis pulmonary tuberculosis and atypical mycobacterial infection tuberculosis and pulmonary tuberculosis were grouped together for analysis all other events that were reported in patients with tb or ami were characterised into subcategories including pulmonary infectious endocrine gastrointestinal hepatobiliary dermatological cardiac haematological neurological vascular infusion related rheumatological and otherstb and ami cases among patients treated with pd1 and pd l1 inhibitors were compared with all reported tb and ami events in the database due to other drugs by conducting a disproportionality signal analysis based on the reporting or ror the ror is a measure of the magnitude of association between an exposure to a pharmaceutical agent and the odds of a specific outcome occurring in the setting of an elevated ror it can be conferred that there is an elevated risk of an adverse event occurring with a specific medication the wald ci was used to assess the precision of the ror when lower limit of ror and ci did not cross ror was considered significant13 the likelihood of association between pd1pd l1 inhibitors and tbami were investigated using two sided Ï2 or fishers exact tests as warranted all analyses were conducted using sas sas institute inc cary north carolina usa and statistical significance was defined as p005resultsbetween january to march a total of adverse events report cases were generated in faers out of ae there were associated with the approved five pd1pd l1 inhibitors the majority of aes were reported with nivolumab and pembrolizumab at in faers there were reports of tb with any drug of which were reported with pd1pdl1 inhibitors the ror for tb due to pd1pd l1 inhibitors was elevated at ci to p00001 for ami there were reports associated with all drugs of which were due to pd1pd l1 inhibitors the anand a0k et a0al esmo open 20205e000866 101136esmoopen2020000866 0ctable details of tb ae due to pd1pdl1 inhibitorstable continuedopen access serious nivolumab pembrolizumab atezolizumab durvalumab lung cancer gastric cancer head and neck cancer hodgkins lymphoma malignant melanoma colon cancer neuroendocrine carcinoma ovarian carcinoma pancreatic carcinoma plasma cell myeloma renal cell carcinoma transitional cell carcinoma unknowntotal number of tb aespd1pdl1 inhibitor used indication for pd1pdl1 use type of reaction sex median age years range min maxoutcome reporter year initial report received region of origin of ae died hospitalised life threatening other outcome asia europe americas africa australia healthcare professional consumer male female n54 continuedsuspected drug pd1pdl1 inhibitor pd1pdl1 inhibitor ¥ aes adverse events pd1 programmed cell death1 pd l1 programmed cell death ligand1 tb tuberculosisror for ami due to pd1pd l1 inhibitors was elevated at ci to p00001 table out of cases of tb due to pd1pd l1 inhibitors were due to nivolumab followed by due to pembrolizumab and due to atezolizumab and durvalumab respectively there were no cases reported with avelumab the most common indication for which pd1pd l1 inhibitor was used was lung cancer median age of the whole cohort was years eighty per cent of the patients were men and were women out of cases cases had a reported outcome of death the most common region of origin in which tb was reported was asia sixteen cases had pd1pd l1 inhibitors plus one of more non checkpoint inhibitor drug listed as a suspect drug leading to ae table out of cases of ami due to pd1pd l1 inhibitors were due to nivolumab followed by due to pembrolizumab and each due to durvalumab and atezolizumab no report of ami attributable to avelumab was found the most common reason for use of pd1pd l1 inhibitor was lung cancer median age of the entire cohort was years seventy three per cent of patients were men and were women out of cases patient died the most common region in which ami was reported was asia one case had pd1pd l1 inhibitors plus one of more non checkpoint inhibitor drug listed as a suspect drug leading to ae table patients who had tb due to pd1pd l1 inhibitors also had additional reported pulmonary complications in of cases followed by other infectious complications in of cases similarly patients who had ami attributed to use of pd1pd l1 inhibitors had pulmonary complications in of cases followed by endocrine dermatological and others in of the cases table discussionin this retrospective pharmacovigilance database review pd1pd l1 inhibitors had a statistically significant positive signal with tb and ami with a proportion of these events associated with mortality nivolumab had the highest frequency of reported tb and ami whereas avelumab had no reported events most commonly affected patients were receiving treatment for lung cancer and the most commonly reported country of origin was japananand a0k et a0al esmo open 20205e000866 101136esmoopen2020000866 0copen accesstb has a high disease burden worldwide with the highest disease associated mortality of any infectious agent in there were million new cases globally and million reported deaths14 amis are estimated to occur in approximately to per persons with an increasing incidence in developed countries15 there is growing evidence that patients receiving icis can develop tb reaction while on treatment6 to date there are reported cases of tb secondary to icis none of which were attributed cytotoxic t lymphocyte associated protein ctla4 inhibitors median time to diagnosis from ici initiation was months range to months17 the mechanism by which a pd1pd l1 inhibitor results in tb is not clear in a murine study pd1 knockout mice had decreased survival compared with wild type mice following exposure to mycobacterium tuberculosis furthermore pd1 inhibition is needed to prevent cd4 t cells from promoting development of tb18 pd1 inhibition mitigates over production of interferon gamma ifnγ which is important for host resistance to tb19increased risk of tb and ami is also found in patients on tumor necrosis factor tnf alpha inhibitors and janus kinase jak inhibitor ruxolitinib20 patients treated with infliximab a tnf alpha inhibitor were and times more likely to develop tb and ami respectively22 in patients prior to start tnf alpha inhibitors screening for latent tb is recommended20 if the patient is found to have latent tb treatment with isoniazid is recommended as it substantially reduces the risk of developing tb reactivation23 however a recent study suggests that pd1 inhibition induced tb reactivation is actually driven by tnf alpha and use of tnf alpha inhibitor could reverse this complication24 there are currently no recommended screening guidelines for latent tb prior to starting pd1pd l1 inhibitors a single institution study in germany found that of patients had positive test for quantiferon gold tb plus qgt prior to starting icis however none of the patients who had a positive qgt test developed tb while on treatment with icis6 of the cases of tb reported in literature due to icis treatment with icis was stopped in all cases tb treatment was initiated and seven cases had re initiation of icis out of seven who had re initiation of ici five had response to therapy one had progression and in one case response was not available17 as tb reactivation may lead to treatment interruptions or discontinuation standardised recommendations for tb screening in patients with planned ici should be considered with substantiation of results from the current study in prospective studiesthis is the first study using faers to demonstrate the potential risk of developing tb and ami in pd1pd l1 inhibitor treated patients as pd1pd l1 inhibitors use becomes more prevalent on a global scale including regions with an elevated prevalence of latent tb clinicians need to consider the risk benefit and economic impacts of screening for latent tb and treatment initiation if the patient is positive these questions cannot be table details of ami ae due to pd1pdl1 inhibitors serious male female nivolumab pembrolizumab atezolizumab durvalumab lung cancer head and neck cancer malignant melanoma unknowntotal number of ami aespd1pdl1 inhibitor used indication for pd1pdl1 use type of reaction sex median age years range min maxoutcome reporter healthcare professionalyear initial report received region of origin of ae suspected drug died hospitalised life threatening other outcome pd1pdl1 inhibitor pd1pdl1 inhibitor ¥ asia europe americas n10 aes adverse events ami atypical mycobacterial infection pd1 programmed cell death1 pd l1 programmed cell death ligand1answered in this observational signal analysis and future prospective research studies should be conducted if a patient develops tb or ami while on treatment with pd1pd l1 inhibitors permanent discontinuation of therapy should be avoided if there is clear clinical benefit from ici and multidisciplinary discussions regarding treatment delay should be conducted with the treating oncologist and infectious disease specialists a majority anand a0k et a0al esmo open 20205e000866 101136esmoopen2020000866 0copen accesstable other aes grouped into major an systems in patients treated with pd1pdl1 inhibitorstb death events n13tb alive events n59tb totaln72ami death events n1ami alive events n12ami totaln13pulmonaryinfectiousendocrinegastrointestinalhepatobiliarycardiachaematologicaldermatologicalneurologicalvascularinfusion relatedrheumatologicalothers aes adverse events ami atypical mycobacterial infection pd1 programmed cell death1 pd l1 programmed cell death ligand1 tb tuberculosisof patients in whom tb or ami have reported are those with lung cancer it is worth pointing out that especially in patients with lung cancer there is significant difficulty in differentiating immune pneumonitis or radiation pneumonitis true disease progression or infectious causes prospective studies of iraes should include testing for tb or ami in diagnostic work upfrom pseudoprogression this study has limitations this analysis was a retrospective study of reported events in faers and as such baseline characteristics including presence of latent tb was not known moreover the actual incidence of tb or ami due to pd1pd l1 inhibitors cannot be determined because faers reports patients with aes not total number of patients taking the medication furthermore it is likely that not all cases of tb that occur in the clinical setting are reported within faers as such there are similar limitations in ror estimate ae reporting for a drug may be influenced by extent of use publicity and bias25 although the use of disproportionality analysis through pharmacovigilance databases to determine the increased risk of aes secondary to particular drug has been shown in various settings25 it is critical that any hypothesis generated by using pharmacovigilance databases are validated through prospective studiespd1pd l1 inhibitors used in treatment for cancer is associated with increased risk of tb and ami the most common drug in faers attributed to tb and ami is nivolumab in this study lung cancer was the most common indication for which use of pd1pd l1 inhibitor leads to tb or ami although this complication is rare clinicians using icis should be aware of this anand a0k et a0al esmo open 20205e000866 101136esmoopen2020000866possibility currently there is no additional data available to support or refute the need to screen patients for latent tb prior to initiation of icis prospective studies are needed to address these questions as well as indications to initiate prophylactic therapytwitter vivek subbiah viveksubbiahcontributors conceptualisation ka gs and spi data collection and analysis ka gs je and spi statistics je first draft preparation ka gs and spi review and final approval all authorsfunding the authors have not declared a specific grant for this research from any funding agency in the public commercial or not for profit sectorscompeting interests vs and spi coi can be found onlinepatient consent for publication not requiredprovenance and peer review not commissioned externally peer revieweddata availability statement data are available in a public open access repository all data relevant to the study are included in the article or uploaded as supplementary information faers used for this study is public databaseopen access this is an open access article distributed in accordance with the creative commons attribution non commercial cc by nc license which permits others to distribute remix adapt build upon this work non commercially and license their derivative works on different terms provided the original work is properly cited any changes made are indicated and the use is non commercial see a0http creativecommons licenses by nc orcid idskartik a0anand http orcid vivek a0subbiah http orcid references coit dg thompson ja albertini mr et a0al cutaneous melanoma version nccn clinical practice guidelines in oncology j natl compr canc netw ettinger ds wood de aggarwal c et a0al nccn guidelines insights non small cell lung cancer version j natl compr canc netw 0copen access postow ma sidlow r hellmann md immune related adverse events associated with immune checkpoint blockade n engl j med wang y zhou s yang f et a0al treatment related adverse events of pd1 and pd l1 inhibitors in clinical trials a systematic review and meta analysis jama oncol wang dy salem j e cohen jv et a0al fatal toxic effects associated with immune checkpoint inhibitors a systematic review and meta analysis jama oncol langan ea graetz v allerheiligen j et a0al immune checkpoint inhibitors and tuberculosis an old disease in a new context lancet oncol 202021e55 lee jjx chan a tang t tuberculosis reactivation in a patient receiving anti programmed death1 pd1 inhibitor for relapsed hodgkin's lymphoma acta oncol fujita k terashima t mio t anti pd1 antibody treatment and the development of acute pulmonary tuberculosis j thorac oncol chu y c fang k c chen h c et a0al pericardial tamponade caused by a hypersensitivity response to tuberculosis reactivation after anti pd1 treatment in a patient with advanced pulmonary adenocarcinoma j thorac oncol 201712e111 anastasopoulou a ziogas dc samarkos m et a0al reactivation of tuberculosis in cancer patients following administration of immune checkpoint inhibitors current evidence and clinical practice recommendations j immunother cancer picchi h mateus c chouaid c et a0al infectious complications associated with the use of immune checkpoint inhibitors in oncology reactivation of tuberculosis after anti pd1 treatment clin microbiol infect lázár molnár e chen b sweeney ka et a0al programmed death1 pd1 deficient mice are extraordinarily sensitive to tuberculosis proc natl acad sci u s a rothman kj lanes s sacks st the reporting odds ratio and its advantages over the proportional reporting ratio pharmacoepidemiol drug saf anization wh global tuberculosis report cassidy pm hedberg k saulson a et a0al nontuberculous mycobacterial disease prevalence and risk factors a changing epidemiology clin infect dis 200949e124 prevots dr shaw pa strickland d et a0al nontuberculous mycobacterial lung disease prevalence at four integrated health care delivery systems am j respir crit care med zaemes j kim c immune checkpoint inhibitor use and tuberculosis a systematic review of the literature eur j cancer barber dl mayer barber kd feng cg et a0al cd4 t cells promote rather than control tuberculosis in the absence of pd1 mediated inhibition j immunol sakai s kauffman kd sallin ma et a0al cd4 t cell derived ifnγ plays a minimal role in control of pulmonary mycobacterium tuberculosis infection and must be actively repressed by pd1 to prevent lethal disease plos pathog 201612e1005667 solovic i sester m gomez reino jj et a0al the risk of tuberculosis related to tumour necrosis factor antagonist therapies a tbnet consensus statement eur respir j anand k burns ea ensor j et a0al mycobacterial infections with ruxolitinib a retrospective pharmacovigilance review clin lymphoma myeloma leuk winthrop kl baxter r liu l et a0al mycobacterial diseases and antitumour necrosis factor therapy in usa ann rheum dis gómez reino jj carmona l ángel descalzo m et a0al risk of tuberculosis in patients treated with tumor necrosis factor antagonists due to incomplete prevention of reactivation of latent infection arthritis rheum tezera lb bielecka mk ogongo p et a0al anti pd1 immunotherapy leads to tuberculosis reactivation via dysregulation of tnfα elife 20209e52668 anand k ensor j trachtenberg b et a0al osimertinib induced cardiotoxicity a retrospective review of the fda adverse events reporting system faers jacc cardiooncology anand k ensor j pingali sr et a0al t cell lymphoma secondary to checkpoint inhibitor therapy j immunother cancer 20208e000104anand a0k et a0al esmo open 20205e000866 101136esmoopen2020000866 0c" | Colon_Cancer |
"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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contribute to genomic instabilityin human cells int j mol sci winters ze p53 pathways involving g2 checkpoint regulators and the roleof their subcellular localisation j r coll surg edinb yang j jing l liu cj bai ww zhu sc 53bp1 regulates cell cycle arrest inesophageal cancer model eur rev med pharmacol sci guo j wu g bao j hao w lu j chen x cucurbitacin b induced atmmediated dna damage causes g2m cell cycle arrest in a rosdependentmanner plos one yan y cao pt greer pm nagengast es kolb rh mumby mc cowan khprotein phosphatase 2a has an essential role in the activation of gammairradiationinduced g2m checkpoint response oncogene zhu t matsuzawa s mizuno y kamibayashi c mumby mc andjelkovic nhemmings ba onoe k kikuchi k the interconversion of proteinphosphatase 2a between pp2a1 and pp2a0 during retinoic acidinducedgranulocytic differentiation and a modification on the catalytic subunit in sphase of hl60 cells arch biochem biophys kim lh hong st choi kw protein phosphatase 2a interacts 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 |
craigpattersontelecaretoj rajani r perry m the reality of medical work the case for a new perspectiveon telemedicine virtual real bf01421807 hoek pd schers hj bronkhorst em vissers kcp hasselaar jgj the eï¬ectof weekly specialist palliative care teleconsultations in patients with advancedcancer a randomized clinical trial bmc med s1291601708669 ministero della salute telemedicina linee diavailabledocumentazionep6_2_2_1jsplinguaitalianoid2129 onlineatindirizzo nazionalihttpwwwsalutegovitportaleaccessed april aiom indicazioni aiom cipomo su covid19 per loncologia wwwaiomitwpcontentuploads202003accessedavailable20200313_covid19_indicazioni_aiomcipomocomupdfapril onlineat cox a lucas g marcu a piano m grosvenor w mold f cancer survivors experience with telehealth a systematic review andthematic synthesis j med internet res 19e11 102196jmir rogante m giacomozzi c grigioni m kairy d telemedicine in palliativecare a review of systematic reviews ann ist super sanita 104415ann_16_03_16 kruse cs krowski n rodriguez b tran l vela j brooks mtelehealth and patient satisfaction a systematic review and narrativeanalysis bmj open 7e016242 101136bmjopen2017 holzner b giesinger jm pinggera j zugal s sch¶pf f oberguggenbergeras the computerbased health evaluation software ches aelectronic patientreported outcome monitoring bmcsoftwaremedinform decis makfor gutteling jj busschbach jj de man ra darlington as logistic feasibilityin clinical practiceof health related quality ofresults of a prospective study in a large population of chronic liverpatients health qual life outcomes life measurement taenzer p bultz bd carlson le speca m degagne t olson k impact of computerized quality of life screening on physician behaviourand patient satisfaction in lung cancer outpatients psychooncology wright ep selby pj crawford m gillibrand a johnston c perren tj feasibility and compliance of automated measurement of quality of life inoncology practice j clin oncol 101200jco200311 jazieh ar alenazi th alhejazi a alofoncoloncologypatientssaï¬f al olayaninfected with coronavirus101200go20a outcomejcoglobtelemed flodgren ginteractivehealthsandrevrachas afarmer ajtelemedicinecareoutcomes2015cd002098eï¬ectsoninzitari mprofessionalshepperdpracticesyst10100214651858cd0databasecochrane ye z hong y wu x hong d zhang y dong x[management of a colon cancer patientdisease ] zhejiang da xue xue bao yi xue banalinfected with corona viruset ogorman ld hogenbirkin northern ontario astelemedicineunitstohealthcare telemed j e health 101089tmj20a measure of potentialjc driving distanceaccessto perri fc ottaiano acancertranslionna f longo f della vittoria scarpati g de angelisagainst head and necktherapy101016jtranon2019immunebiological mechanismsoncolresponseandimplicationonaletfrontiers in oncology wwwfrontiersinaugust volume 0ccrispo covid19 emergency and postemergency hisada y mackman n cancerassociated pathways and biomarkers of venousthrombosis blood 101182blood20170374 xu x lai y hua zc apoptosis and apoptotic body disease message andtherapeutic target potentials biosci rep 39bsr20180992 bsr20180992 zhao z bai h duan j wang j recommendations ofandlungtreatmentcancermedicalforepidemic thorac cancerpatientsindividualizedevents managementof covid19 commonduringadversetheoutbreakconï¬ict of interest the authors declare that the research was conducted in theabsence of any commercial or ï¬nancial relationships that could be construed as apotential conï¬ict of interestcopyright crispo montagnese perri grimaldi bimonte augustin amorecelentano di napoli cascella and pignata this is an openaccess distributedunder the terms of the creative commons attribution license cc by the usedistribution or reproduction in other forums is permitted provided the originalauthors and the copyright owners are credited and that the original publicationin this is cited in accordance with accepted academic practice no usedistribution or reproduction is permitted which does not comply with these termsfrontiers in oncology wwwfrontiersinaugust volume 0c' | Colon_Cancer |
" lung carcinoma is a prominent cause of mortality among patients with cancer previous studies have reported the vital role of long noncoding rnas lncrnas in the malignant progression of lung cancer lncrna rp11284f219 was originally identified to be expressed in lung carcinoma but its specific function remains unknown therefore the present study aimed to elucidate the role of lncrna rp11284f219 in lung carcinoma progression the expression of rp11284f219 in lung cell lines and tissues was measured using reverse transcriptionquantitative pcr the endogenous expression of rp11284f219 was silenced using rna interference and cell viabilities were measured with a cell counting kit assay the invasion and apoptosis of cells were determined via transwell assays and flow cytometry respectively the protein expression levels were measured by western blotting an increased expression of rp11284f219 was identified in both lung carcinoma tissues and cells knockdown of rp11284f219 in lung carcinoma cells inhibited cell proliferation and invasion but promoted cell apoptosis the present study identified the existence of a direct interaction between rp11284f219 and microrna mirnamir6273p mechanistically it was demonstrated that rp11284f219 promoted the proliferation and invasiveness of lung carcinoma cells in part via the regulation of mir6273p furthermore cell division cycle and apoptosis regulator ccar1 was identified as a target gene of mir6273p the in vivo tumor growth assay also demonstrated that the knockdown of rp11284f219 suppressed tumor growth upregulated mir6273p and downregulated correspondence to dr yuan wang department of medical imaging the first affiliated hospital of xi'an jiaotong university west yanta road xi'an shaanxi pr chinaemail wangyuan8003126comabbreviations ccar1 cell division cycle and apoptosis regulator nsclc nonsmall cell lung cancer sclc small cell lung cancerkey words rp11284f219 lung carcinoma proliferation invasion microrna6273p ccarccar1 in the xenograft model of nude mice thus the present findings indicated the tumor promoting functions of rp11284f219 in the progression of lung carcinoma and provided a novel lncrnamirna axis as a target for the management of lung cancerintroductionpulmonary malignancies including lung and bronchus cancer rank first and second among different cancer types in terms of mortality and morbidity respectively in both men and women furthermore of lung cancer cases are categorized as nonsmall cell lung cancer nsclc while the remaining are classified as sclc although diagnostic methods and therapeutic strategies based on traditional surgical excision chemotherapy and chest radiotherapy have continuously improved the prognosis of lung carcinoma remains at for an overall 5year survival therefore an increased understanding of the malignant progression and studies on novel therapeutic targets for the improved management of this disease are essentiallong noncoding rnas lncrnas are nucleotides in length and have little or no protein coding capacity the mechanisms via which lncrnas regulate gene expression are diverse and include regulating the transcription of target genes functioning as transcriptional precursors of small rnas generating different splice variants via regulating mrna splicing patterns modulating protein activity and subcellular localization and scaffolding for the assembly of multiple component complexes in recent years previous studies have reported that various human cancer types exhibit lncrnas dysfunction and these lncrnas are involved in different aspects of pathogenesis such as the proliferation metastasis and apoptosis of tumor cells in lung cancer lncrna metastasisassociated lung adenocarcinoma transcript is found to be upregulated in patients with advanced lung adenocarcinoma and may serve as a prognostic marker to predict the survival outcome of patients with cancer lncrna hox transcript antisense rna is also highly expressed in lung cancer and it enhances the aggressiveness of lymph node metastasis and indicates a short diseasefree survival in patients with nsclc furthermore studies have shown that the expression of lncrna urothelial carcinomaassociated 0cli rp11284f219 promotes lung carcinoma proliferation and invasionis significantly upregulated in nsclc and may induce resistance to treatment of egfrtyrosine kinase inhibitors by activating the aktmtor pathway lncrna rp11284f219 was primarily discovered in a pancancer transcriptomic analysis lncrna rp11284f219 exists as a cluster of three annotated lncrnas rp11284f219107 antisense to brevican which is a proteoglycan linked to invasiveness in glioma but lacks expression in squamous cell lung carcinomas however the specific function and the underlying mechanism of rp11284f219 in lung carcinoma remain unknownto the best of our knowledge the present study demonstrated for the first time that lncrna rp11284f219 was significantly upregulated in lung carcinoma tissues and cell lines and was involved in the carcinogenesis of lung cancer together with microrna mirnamir6273p and cell division cycle and apoptosis regulator ccar1 the regulatory axis of rp11284f219mir3pccar1 exists both in the lung carcinoma cells in vitro and in the tumor growth model in vivo the present study aimed to investigate rp11284f219 function in lung carcinoma and demonstrate the molecular mechanism underlying the regulation process via the rp11284f219mir3pccar1 axismaterials and methodstissue samples and cell lines between may and jan paired tumor and adjacent healthy tissues were isolated from patients with lung carcinoma age range years nine male patients four female patients who were diagnosed and treated in first affiliated hospital of xi'an jiaotong university the samples were dissected during the surgery and immediately flashfrozen in liquid nitrogen and transferred to Ëc storage for further extraction of both rna and protein all the tissue samples were obtained with written informed consent from the patients the protocol was approved by the first affiliated hospital of xi'an jiaotong university approval no a normal lung epithelial cell line beas2b and lung carcinoma cell lines ncih460 ncih1299 and a549 were purchased from american type culture collection atcc and cultured according to the atcc guidelines 293t cells were purchased from procell life sciencetechnology co ltd and cultured in dmem supplemented with fbs cat no atcc and 1x penicillinstreptomycin thermo fisher scientific inc beas2b cells were cultured in bronchial epithelial growth medium begm cat no cc clonetics corporation according to the manufacturer's instructions ncih460 and ncih1299 cells were cultured in rpmi medium cat no atcc and a549 cells in f12k medium cat no atcc supplemented with fbs cat no atcc and 1x penicillinstreptomycin thermo fisher scientific inc all cells were culture at Ëc with co2rna extraction and reverse transcriptionquantitative pcr rtqpcr total rna from both tissue samples and cell lines were extracted using trizol® reagent invitrogen thermo fisher scientific inc for each sample ng total rna was reverse transcribed to synthesize the firststrand cdna using the primescript rt reagent kit takara bio inccdna samples were diluted times to perform the rtqpcr using sybr premix ex taq takara bio inc on a cfx96 realtime pcr detection system biorad laboratories inc expression levels of mrnas lncrnas and mirnas were normalized to gapdh the primers used for rtqpcr analyses were as follows gapdh forward 'aac gac ccc ttc att gac c' and reverse 'tcc acg aca tac tca gca cc' rp11284f219 forward 'agg att ggc act cac ttc gg' and reverse 'tct ctc acc acg tct ggt ct' and ccar1 forward 'ctg atg gct agc cct agt atg ga' and reverse 'tgc ctt tca tgc cca cta aaa ' the temperature protocol used to perform rt was Ëc for h followed by Ëc for min thermal conditions of pcr reactions were initial denaturation at Ëc for min followed by cycles for sec at Ëc and sec at Ëc the mrna expression levels were determined using the 2δδcq method oligonucleotides and cell transfection the small interfering rna sirna synthetic negative control sinc rp11284f219 sirnas sirp11284f219 mirnc mir3p mimics and mir3p inhibitor were purchased from shanghai genepharma co ltdall primer sequence information is presented in table i at a density of 2x105 cellswell the cells were plated in 6well plates h before transfection and were transfected at confluency all of the oligonucleotides were transfected at a final concentration of nm using lipofectamine® reagent invitrogen thermo fisher scientific inc according to the manufacturer's instruction cells were collected at h posttransfection for subsequent experimentscell counting kit cck assay and edu labeling of proliferating cells a cck was used for cell proliferation assay the cells were seeded into well plates 2x103 cellswell and observed for and days or indicated time points following the manufacturer's instructions dojindo molecular technologies inc the optical density was measured at nm using a spectrophotometer thermo fisher scientific incfor the edu assay cells were incubated with µm edu cat no ab219801 abcam for h at Ëc and fixed with formaldehyde at room temperature for min after a brief washing with pbs click reagent was added into each well and incubated in the dark for min at room temperature followed by pbs washing the cells were stained with µgml dapi at room temperature for min images were captured using a fluorescence microscope nikon corporation and measured using adobe photoshop software adobe systems inc the edu labeled cells were analyzed with moflo astrios beckmancoulter inc magnification x200transwell assay and flow cytometry measurement of cell apoptosis transwell assays were performed with a coating of matrigel bd biosciences mixed with culture medium mixed at ratio at Ëc for h a total of 1x105 cells in µl serumfree medium were added to the upper layer of the transwell chambers µm pore size corning inc and cultured for h the lower chamber contained the culture medium with fbs the migrated cells were fixed with 0concology reports table i sequence of sirnas and mirna mimics and inhibitorsoligonucleotides sinc sirp11284f219 mirnc mir3p mimics mir3p inhibitor mir microrna sirna small interfering rna nc negative controlsequence ''uucuccgaacgugucacguttuauuggcaccaaggauagcucguuaaucggcuauaauacgcucuuuucuuugagacucacuucuuuucuuugagacucacu paraformaldehyde for min at room temperature stained with crystal violet for min at room temperature and images of six randomly selected fields in each well were captured under a light microscope magnification x200cellular apoptosis was detected using the apoptosis detection kit cat no kgf001 nanjing keygen biotech co ltd according to the manufacturer's instructions cells were stained with fluorescein isothiocyanateconjugated annexin v and pi after incubated for min at Ëc in the dark µl 1x binding buffer was added to each tube and stained cells were analyzed using bd facs canto ii flow cytometry facs calibur bd biosciences data were analyzed using flowjo software version tree star incluciferase reporter assay the rp11284f219 wildtype wt or mutant mut 'untranslated region 'utr and ccar1 wt or mut 'utr sequences were cloned into the pmirglo plasmid youbio httpwwwyoubiocn cat no vt1439 the vectors µgml were cotransfected with mirnc or mir6273p mimic nm and renilla plasmids ngwell used as an internal control into cells seeded in a 48well plate 1x104well using lipofectamine® reagent invitrogen thermo fisher scientific inc cell lysates were collected at h after transfection and the luciferase activities were detected with the dualluciferase reporter assay system promega corporation according to the manufacturer's instructionswestern blotting cell were lysed using ripa lysis buffer sigmaaldrich merck kgaa and protein concentrations were assessed with the bca protein assay kit according to the manufacturer's instructions beyotime institute of biotechnology shanghai china equal amounts µg of cell protein lysates were loaded and separated by sdspage transferred to a pvdf membrane and blocked with nonfat milk at room temperature for h the membranes were then incubated with ccar1 primary antibody cat no ab70243 abcam overnight at Ëc followed by incubation with goat antimouse or goat antirabbit igghorseradish peroxidase conjugate secondary antibodies cat no ab205718 abcam at room temperature for h gapdh cat no ab181602 abcam was used as loading control the signals were detected using the ecl system protein simple according to the manufacturer's instructionsin vivo tumorigenicity analysis in mice male balbc nude mice age weeks weight g were obtained from beijing vital river laboratory animal technology co ltd and housed at a room temperature of Ëc with a h lightdark cycle the mice were maintained in an individually ventilated cage system under specific pathogenfree conditions temperature Ëc humidity and fed with sterile food and water free access to evaluate the effect of rp11284f219 knockdown on the growth of lung carcinoma in vivo 5x106 sinc or sirp11284f219 treated ncih1299 cells in µl serumfree medium were subcutaneously injected into each mouse n5 per group under anesthesia which was induced by isoflurane and maintained by isoflurane flow rate 1lmin the animals were monitored daily and the following criteria for humane endpoint was used severe tumor burden mm in diameter difficulty breathing significant bodyweight loss and clinical signs such as prostration hypothermia and significant abdominal distension tumors were measured on days and and the volumes were calculated using the formula a x b22 [the largest diameter a and the smallest diameter b] then weeks after inoculation the mice were euthanized by co2 inhalation co2 flow rate of cage volume and the death of animals were confirmed by cessation of heartbeat the xenografts were imaged and weighedthe total rna was then extracted from the xenografts as aforementioned animal care and study were approved by the institutional animal care and use committee of the first affiliated hospital of xi'an jiaotong university approval no target prediction potential target mirnas of rp11284f219 were predicted using lncbase v2 httpcarolinaimisathena innovationgrdiana_toolswebindexphprlncbasev2index the target genes of mir3p were predicted using three bioinformatics algorithms targetscanv72 httpwwwtargetscanorgvert_72 and mirdb httpwwwmirdborgmininghtmlstatistics analysis data were analyzed using the graphpad prism software graphpad software inc and presented as the mean ± sd from ¥ independent experiments a twotailed unpaired student's ttest or oneway anova with tukey's posthoc analysis were performed to evaluate the statistical significance p005 was considered to indicate a statistically significant difference 0cli rp11284f219 promotes lung carcinoma proliferation and invasionfigure rp11284f219 expression is upregulated in lc tissues and cell lines a expression of rp11284f219 in lc tissues in comparison with adjacent healthy tissues was analyzed using rtqpcr p0001 vs adjacent tissues n13 b expression of rp11284f219 in human lung carcinoma cell lines ncih460 ncih1299 and a549 compared with normal human lung epithelial cell line beas2b was analyzed using rtqpcr p005 p0001 vs beas2b n3 lc lung carcinoma rtqpcr reverse transcriptionquantitative pcrresultsexpression of rp11284f219 is upregulated in lung carcinoma to investigate the potential role of rp11284f219 in lung carcinoma its expression was analyzed in tissue samples and matched adjacent healthy tissues from patients with lung carcinoma the results demonstrated that the expression of rp11284f219 was significantly upregulated in tumor tissues compared with healthy tissues fig 1a the expression of rp11284f219 was also analyzed in human lung carcinoma cell lines ncih460 ncih1299 and a549 and normal human lung epithelial cell line beas2b consistent with the findings in the tissue samples the expression of rp11284f219 was significantly increased in carcinoma cell lines compared with the normal epithelial cell line fig 1b these results indicated that rp11284f219 may serve an oncogenic role in lung carcinomaknockdown of rp11284f219 exerts antioncogenic effects in lung carcinoma cells to study the specific role of rp11284f219 in lung carcinoma cells rp11284f219 sirna was transfected into ncih1299 and ncih460 cells fig 2a after transfection the proliferation of these cells was measured using cck and edu assays fig 2bd the results suggested that knocking down rp11284f219 significantly reduced the proliferation of lung carcinoma cells compared with the nc group fig 2bd the invasiveness of sirp11284f219 transfected cells also significantly decreased as indicated by the data from the transwell assay fig 2f to further validate the invasive capability a rtqpcr assay was performed to detect the expression levels of invasionrelated genes and the results identified that both mmp2 and mmp9 were significantly decreased when rp11284f219 was downregulated fig s1the results of flow cytometry measurement based apoptosis assay suggested that cells transfected with sirp11284f219 had a higher apoptotic rate compared with the sinc transfected group fig 2e these data demonstrated the antitumor effects of rp11284f219 knockdown in lung carcinoma cells indicating an oncogenic role of rp11284f219rp11284f219 directly interacts with mir3p based on the prediction of the online tool lncbase v2 from diana prediction module httpcarolinaimisathenainnovationgrdiana_toolswebindexphprlncbasev2index which was used to identify the downstream mirnas of rp11284f219 the first five mirnas in the output list were tested among the predicted potential targets it was found that mir6273p had the most significant upregulation in ncih1299 cells transfected with sirp11284f219 fig s2using sequence alignment it was identified that mir3p was partially complementary with the 'utr of rp11284f219 fig 3a subsequently 293t cells were transfected with the pmirglorp11284f219wt or mut vector containing the wt or mut sequence of rp11284f219 'utr with or without mir3p mimics results from the luciferase reporter assay suggested that mir6273p mimics significantly decrease the signal of rp11284f219wt transfected cells but not the rp11284f219mut transfected cells indicating a direct interaction between the two noncoding rnas fig 3a furthermore transfection of sirp11284f219 into ncih1299 and ncih460 cells resulted in the suppression of endogenous rp11284f219 leading to a significant increase in mir3p expression fig 3b thus these findings suggested an inhibitory effect of rp11284f219 on the expression of mir3p in lung carcinoma cellsthe expression of mir3p was detected in both lung carcinoma tissues and cell lines it was demonstrated that mir3p was significantly downregulated in carcinoma tissues fig 3c and ncih460 ncih1299 and a549 cells fig 3d compared with healthy tissues and cells collectively these data suggested a direct interaction between rp11284f219 and mir6273p in which rp11284f219 suppresses the expression of mir3prp11284f219 regulates the proliferation and invasiveness of lung carcinoma cells via mir3p to rescue the antitumor effects of sirp11284f219 in lung carcinoma cells the mir3p inhibitor which specifically downregulates the expression of mir3p was transfected into ncih1299 and ncih460 cells fig 4a the results from the cck and edu assays demonstrated that treatment with sirp11284f219 0concology reports figure rp11284f219 knockdown inhibits lung carcinoma cell proliferation and invasion and promotes cell apoptosis a rp11284f219 knockdown was achieved via rp11284f219 sirna and the knockdown efficiency was verified using reverse transcriptionquantitative pcr n3 cell counting kit assay was performed to measure the proliferation of b ncih1299 and c ncih460 cells after transfection with sirp11284f219 compared with the sinc group n5 d an edu assay was performed to measure the proliferation of ncih1299 and ncih460 cells after transfection with sinc and sirp11284f219 magnification x200 e flow cytometry analysis was performed to determine the effects of rp11284f219 knockdown on apoptotic rates in ncih1299 and ncih460 cells n3 f transwell assay was performed to determine the effects of rp11284f219 knockdown on ncih1299 and ncih460 cell invasion n3 magnification x200 p005 p001 vs control group nc negative control sirna small interfering rna od optical density and mirnc significantly decrease the proliferation of both ncih1299 and ncih460 cells fig 4bd however the administration of mir3p inhibitor partially reversed the antiproliferative effect of sirp11284f219 indicating that rp11284f219 regulates the proliferation of lung carcinoma cells partially via mir6273p fig 4bd in addition the 0cli rp11284f219 promotes lung carcinoma proliferation and invasionfigure rp11284f219 directly interacts with mir3p a binding site between rp11284f219 and mir3p that was identified using the diana tools and a luciferase reporter assay was conducted in pmirglorp11284f219wt or mut treated cells in the presence of mir6273p mimics or mirnc n3 p005 vs mirnc b expression of mir3p in ncih1299 and ncih460 cells transfected with sirp11284f219 was analyzed using rtqpcr p001 vs sinc n3 mir3p expression in c lc tissues and d ncih460 ncih1299 and a549 cells compared with adjacent healthy tissues and normal lung epithelial cells was analyzed using rtqpcr n3 p005 p001 vs adjacent tissue or beas2b cells nc negative control sirna small interfering rna wt wildtype mut mutant mir microrna lc lung carcinoma mir3p inhibitor restored the reduction in the number of ncih1299 and ncih460 cells that migrated through the transwell membrane induced by sirp11284f219 treatment fig 4f these data indicated the participation of mir6273p in the rp11284f219mediated invasive effectthe qpcr assay results identified that both mmp2 and mmp9 expression levels were restored in rp11284f219downregulated cells when mir6273p was inhibited compared with the mirnc group fig s3 in addition transfection with mir3p inhibitor also diminished the proapoptosis effect of sirp11284f219 in both ncih1299 and ncih460 cells fig 4e therefore it was suggested that rp11284f219 promoted the proliferation and invasion as well as suppressed the apoptosis of lung carcinoma cells by inhibiting the expression of mir3prp11284f219 regulates ccar1 via targeting mir3p to further evaluate how rp11284f219 exerts an oncogenic role via mir3p the publicly available algorithms of targetscan httpwwwtargetscanorg and mirdb were used which identified ccar1 as a potential target for mir6273p fig 5a in order to validate this prediction mir6273p mimic was transfected into cells and the transfection efficiency was assessed the results demonstrated that transfection of mir6273p mimic increased the expression of mir3p by times compared with cells transfected with mirnc fig s4after validating the upregulation of mir6273p mimic a ccar1wt vector was constructed which contained the wt binding site between mir3p and the ccar1 'utr and ccar1mut vector containing the mut sequence fig 5a the results from luciferase reporter assays indicated that compared with the mirnc group the mir6273p mimic significantly decreased the luciferase activity of ccar1wt treated cells but not the ccar1mut treated cells suggesting a direct binding of mir3p to the 'utr of ccar1 fig 5b increased expression levels of ccar1 were present in the lung carcinoma tissues compared with the adjacent healthy tissues fig 5c moreover a significant decrease in both mrna and protein expression levels of ccar1 was detected upon transfecting ncih1299 and ncih460 cells with mir6273p mimics fig 5d and e thus ccar1 may be a direct target of mir3p in lung carcinoma cells and tissuesrp11284f219 knockdown inhibits tumor growth and the expression of ccar1 in vivo in order to investigate the effect of rp11284f219 on in vivo tumorigenicity ncih1299 cells were transfected with sinc or sirp11284f219 and injected into the nude mice after weeks a significantly 0concology reports figure rp11284f219 regulates proliferation and invasiveness in lung carcinoma cells via mir3p a expression of mir3p in ncih1299 and ncih460 cells transfected with mirnc or mir3p inhibitor was detected using rtqpcr analysis n3 p005 vs mirnc cell counting kit assay was performed in b ncih1299 and c ncih460 cells stably transfected with sirp11284f219 in the presence of mirnc or mir3p inhibitor n5 d edu assay was performed in ncih1299 and ncih460 cells stably transfected with sirp11284f219 in the presence of mirnc or mir3p inhibitor magnification x200 e flow cytometry analysis was performed in ncih1299 and ncih460 cells stably transfected with sirp11284f219 in the presence of mirnc or mir3p inhibitor n3 f transwell assay was performed in ncih1299 and ncih460 cells stably transfected with sirp11284f219 in the presence of mirnc or mir3p inhibitor magnification x200 n3 p005 vs sinc nc negative control sirna small interfering rna od optical density mir microrna 0cli rp11284f219 promotes lung carcinoma proliferation and invasionfigure mir3p directly targets ccar1 a bioinformatic analysis of the predicted binding sites between the ccar1 'untranslated region and mir3p b luciferase reporter assay in ccar1wt or ccar1mut treated cells in the presence of mirnc or mir3p mimic n3 p005 vs mirnc c ccar1 expression in lc tissues compared with adjacent healthy tissues was analyzed using rtqpcr n13 p001 vs adjacent tissue expression of ccar1 in ncih1299 and ncih460 cells transfected with mirnc or mir3p mimics was detected using d rtqpcr and e western blotting n3 p005 vs mirnc mir microrna nc negative control wt wildtype mut mutant rtqpcr reverse transcriptionquantitative pcr ccar1 cell division cycle and apoptosis regulator lc lung carcinoma slower proliferative rate of the tumors was observed in the sirp11284f219 group compared with the sinc group fig 6a and b furthermore the tumor volume and weight were significantly decreased in the sirp11284f219 group compared with the control group fig 6a and b rtqpcr analysis also demonstrated that compared with the sinc group the tumors in the sirp11284f219 group expressed higher levels of mir6273p fig 6c and lower levels of ccar1 fig 6d providing further evidence to the existence of the rp11284f219mir3pccar1 regulatory axis in lung carcinoma tumor tissuesdiscussionthe present study investigated the function of rp11284f219 in lung carcinoma it was initially found that rp11284f219 was significantly upregulated in both lung cancer tissues and cell lines following the deduction of a potential oncogenic role of this lncrna sirp11284f219 was transfected into ncih460 and ncih1299 cells and it was demonstrated that knockdown of rp11284f219 inhibited the proliferation and invasion while promoting apoptosis of lung carcinoma cells in the mechanistic studies using online prediction tools and in vitro assays the results indicated that mir3p directly interacts with rp11284f219 by binding to its 'utrthe function of mir627 was initially reported in colorectal cancer crc padi found that when upregulated by calcitriol mir627 targets the histone demethylase jumonji domain containing 1a to increase methylation of histone h3k9 and suppresses the proliferative factors of crc cells thus inhibiting the proliferation of crc both in vitro and in vivo moreover in crc sun discovered the role of mir in vitamin denhanced efficacy of irinotecan via inhibition of the cytochrome p450 enzymemediated intratumoral drug metabolism mir is also reported to be a potential noninvasive diagnostic marker in gastric and breast cancer types in pulmonary diseases mir627 is downregulated in patients with chronic obstructive pulmonary disease and targets the highmobility group box protein to inhibit its expression thus improving transforming growth factorβ1induced pulmonary fibrosis the present results demonstrated the inhibitory effect of rp11284f219 on the expression of mir3p in addition it was identified that the mir3p inhibitor can neutralize the antitumor effects of rp11284f219 knockdown indicating that rp11284f219 promotes the proliferation and invasiveness of lung carcinoma cells partially by regulating mir3p this antitumor role of mir6273p under the regulation of rp11284f219 in lung carcinoma tissues and cells is in accordance with the previous aforementioned findings on human crc gastric and breast cancer types 0concology reports figure rp11284f219 knockdown inhibits tumor growth in vivo a macroscopic image of xenografted tumors b tumor volume in nude mice injected with ncih1299 cells transfected with sinc or sirp11284f219 measured over weeks n5 c weight of tumors in nude mice at weeks after injection of ncih1299 cells transfected with sinc or sirp11284f219 n5 expression levels of d mir3p and e ccar1 in the tumor tissues from nude mice injected with ncih1299 cells transfected with sinc or sirp11284f219 for weeks were detected using reverse transcriptionquantitative pcr n5 p005 p001 p0001 vs sinc mir microrna nc negative control sh short hairpin rna ccar1 cell division cycle and apoptosis regulator using the publicly available rna interaction prediction algorithms the current study identified that ccar1 which was initially shown as the target gene of mir6273p is also regulated by rp11284f219 furthermore the regulatory axis of rp11284f219mir3pccar1 exists in the lung carcinoma cells both in vitro and in vivo in the tumor growth model the interaction between rp11284f219 and mir3p and the interaction between mir3p and ccar1 were demonstrated by the dualluciferase assay although this method has been used to validate rnarna interactions in previous studies other assays such as rna pulldown and rna binding protein immunoprecipitation that would provide more direct evidence for the rnarna and rnaprotein interactions should be performedccar1 was initially reported as a protein essential for cancer cell apoptosis induced by retinoids or chemotherapeutics such as adriamycin and etoposide subsequently kim et al revealed that this protein functions as a transcriptional coactivator of nuclear receptors in human breast cancer cells as ccar1 interacts and cooperates with the coactivators of estrogen receptor signaling it promotes the estrogendependent proliferation of cancer cells in crc cells ou reported that ccar1 can be recruited by βcatenin to act as a coactivator for the transcriptional activation of lymphoid enhancer binding factor ccar1 is essential for the expression of wnt target genes as well as the neoplastic transformation of crc cells in gastric cancer cells researchers have revealed the cooperation between ccar1 and βcatenin which leads to the promotion of the proliferation and migration of cancer cells in lung cancer ccar1 was reported to be an effector of doxorubicininduced apoptosis moreover muthu demonstrated that certain chemical compounds that bind with ccar1 can increase the expression of ccar1 and induce apoptosis however a contradictory conclusion was reported in a recent study which observed that ccar1 was promoted by serine and arginine rich splicing factor which is activated by glucose intake and further enhanced tumorigenesis by increasing the glucose consumption rate corroborating this finding in the current study via the targeting of mir3p the expr | Colon_Cancer |
"it is well understood that the level of molecular oxygen o2 in tissue is a very important factor impacting both physiology and pathological processes as well as responsiveness to some treatments data on o2 in tissue could be effectively utilized to enhance precision medicine however the nature of the data that can be obtained using existing clinically applicable techniques is often misunderstood and this can confound the effective use of the information attempts to make clinical measurements of o2 in tissues will inevitably provide data that are aggregated over time and space and therefore will not fully represent the inherent heterogeneity of o2 in tissues additionally the nature of existing techniques to measure o2 may result in uneven sampling of the volume of interest and therefore may not provide accurate information on the average o2 in the measured volume by recognizing the potential limitations of the o2 measurements one can focus on the important and useful information that can be obtained from these techniques the most valuable clinical characterizations of oxygen are likely to be derived from a series of measurements that provide data about factors that can change levels of o2 which then can be exploited both diagnostically and therapeutically the clinical utility of such data ultimately needs to be verified by careful studies of outcomes related to the measured changes in levels of o2k e y w o r d sclinical measures of oxygen oxygen in tissues partial pressure1department of radiology dartmouth medical school hanover nh usa2department of medicine section of radiation oncology dartmouthhitchcock medical center lebanon nh usa3thayer school of engineering dartmouth college hanover nh usa4department radiation and cellular oncology university of chicago chicago il usa5department of surgery dartmouthhitchcock medical center lebanon nh usa6louvain drug research institute universit catholique de louvain brussels belgium7department radiation oncology university medical center university of freiburg freiburg germany8german cancer center consortium dktk partner site freiburg german cancer research center dkfz heidelberg germanycorrespondenceann barry flood clinepr llc river road lyme nh usaemail annbarryflooddartmouthedufunding informationmajor funding for this work was from the national institutes of health national cancer institute ppg grant p01ca190193 r01 ca p30 ca023108 and national institute of biomedical imaging and bioengineering p41 this is an open access under the terms of the creative commons attribution license which permits use distribution and reproduction in any medium provided the original work is properly cited the authors physiological reports published by wiley periodicals llc on behalf of the physiological society and the american physiological societyphysiological reports 20208e14541 1014814phy214541 wileyonlinelibrarycom phy2 of 0c of introduction the overall goal of this review is to facilitate clinically effective use of measurements of molecular oxygen o2 in tissues with the explicit intent of improving clinical care that is improving the accuracy and effectiveness of diagnoses treatments and prognoses for individual patients this review focusses especially on improving personalized medicine and outcomes of care by carefully considering the basis and validity of clinical measurements of o2 in tissues and how those measurements can be used to advance diagnosis and therapy while measurements of o2 in tissues have been recognized as an important factor in the clinical evaluation and treatment of many diseases especially cancer busk overgaard horsman a0 and pathologies involving ischemia such as in peripheral vascular disease and wound healing insufficient attention often has been paid to the meaning of the values that have been obtained note this review is derived in part from a series of recent papers on this topic flood et a0al a0 swartz flood swartz vaupel instead all too often when a measurement technique has indicated that the level of o2 in a given tissue is x that is is some specific quantitative number for the o2 in the tissue researchers and clinicians alike assume that x is a reliable accurate representation of the true oxygenation status of the tissue this approach ignores the complexity and dynamics of o2 in living biological systems the reality is that any o2 measurement has been taken at only one point in time of a distribution in the subvolume that was interrogated by the method while the o2 is in fact varying with time and across space in the tissue and is unlikely to be uniform in the volume that is being interrogatedin this review we focus on the biologicalclinical meaningfulness of o2 measurements made in living anisms while recognizing that tissue o2 is in constant flux we emphasize that to obtain maximum clinical utility of the measurements it is necessary to consider the goal of the measurements and the limitations of the data that are obtained we particularly focus on the clinical value of making repeated measurements of o2 especially in association with strategiesevents that potentially change o2 levels what are oxygen levels physical concepts and terminology for in tissues reporting on oxygen levels in tissuesthe level of molecular oxygen that is o2 is usually reported as partial pressure of oxygen po2 or concentration of oxygen [o2] or co2 these terms have physically rigorous meanings that can usefully be extended to describe gases such as o2 that are dissolved in liquids or solids including tissues partial pressure is the pressure exerted by oxygen in a mixture of gases while concentration is the content of oxygen in the gas mixture or solid partial pressure is commonly expressed in mmhg and these units are sometimes referred to as torr or kpa si unit used in the eu while concentration of o2 is commonly expressed in ml of o2 per a0ml for example in bloodhowever the solubility of oxygen varies greatly in different media bennett swartz brown koenig a0 jordan et a0al a0 and this affects the relationship between po2 and [o2] the transport of o2 across lipid membranes is known to depend on both diffusion and solubility in the bilayer and to be affected by changes in the physical state and by the lipid composition especially the content of cholesterol and unsaturated fatty acids for example because o2 partitions preferentially into lipophilic media such as membranes the solubility of o2 in membranes is about four times greater than in aqueous solutions m¶ller et a0 al a0 this difference has significant consequences for physical and chemical interactions involving molecular oxygen in biological systems because these interactions depend on the number of oxygen molecules that are present and their rate of diffusiondoes it matter clinically to know whether the technique is reporting [o2] or po2 while these measures are not identical there is a known relationship between them according to the ideal gas law po2 is directly proportional to concentration assuming the volume and temperature are constant that ispv nrtwhere p a0 a0pressure po2 v a0 a0volume n a0 a0amount of substance [o2] r a0 a0ideal gas constant t a0 a0temperatureit is less straightforward in biological systems if the solubility of oxygen in each component of tissue is known and this is not always fairly readily derived experimentally and po2 can be measured then it is relatively straightforward to calculate [o2] conversely if the component in which [o2] is measured and the solubility of o2 in that compartment is sufficiently known then it should be feasible to determine po2 however it often is not feasible to measure these parameters readilybecause of the biological complexities in assessing o2 in tissues each reported measure of o2 level in a tissue can be better considered as an average value average is used here in its more colloquial usage rather than as a statistically defined term because different techniques output their measurement of o2 using differing methods pertinent to that technique each technique gathers information from a particular volume of tissue irrespective of whether that volume is well characterized which we refer to hereafter as the interrogated swartz 0cvolume the sampling of data within the interrogated volume is then used to produce a measure based on a sort of average o2 within that volume characterizing that average measure is made difficult both by the imprecision of knowing the exact volume queried but also because the detection of o2 in that volume may be affected by factors such as its distance from the detector or for optical techniques different rates of scattering that also may not be well characterized hence we conclude that it is important to bear in mind that the measurements of o2 in vivo are fundamentally based on a sort of average within the interrogated volumebecause of all of these challenges in obtaining precise measurements of relevant parameters necessary to assess whether the data obtained by any technique is truly measuring either po2 or [o2] and because of the imprecisions of knowing the volume being assessed and the tissues within that volume it is more realistic to acknowledge the complexity of these issues for in vivo measurements in tissues by using a less precise term for measures of molecular oxygen in tissues such as o2 levels which is the convention we follow in this review we also argue that while it is important to recognize the biological imprecisions in these measures there are still many clinically viable uses of this information such as assessing change in o2 levelsnote too within this paper while focusing on the uncertainties due to sampling issues of each technique and variations due to biological factors we are not taking into consideration further uncertainties due to inevitable instrumental noise variations in the placement of the detector etc expected levels of o2 in tissues table a0 presents some illustrative data on the o2 levels in various tissues both in normal states and as altered by some diseases these measures are presented here as reported in the literature the first column presents the median po2 obtained using the eppendorf electrode or comparable polarographic techniques to measure o2 levels in patients also presented are two other indications of o2 levels the hypoxic fraction the percentage of measurements in a given type of tissue that is below a defined hypoxic level in this case a0mmhg and the range of po2 values found experimentally these data illustrate both the variation in median o2 levels between types of tissues and how they may vary with physiology or disease for example intertissue in general the median o2 levels are lower in skeletal muscle and heart compared to the spleen intratissue the median o2 in skeletal muscle at rest is higher than with exercise while in contrast there is almost no variation between the normal spleen and with hodgkin's disease the data in column illustrate the wide range that any given measurement can have in the same type of tissue that is almost all tissues range from of to a0mmhg even when their median value is quite different see again spleen vs bone however these very high values in the upper range may include experimental artifacts due to the measurement being taken within or very close to an arteriole for example a vessel feeding the microcirculatory bedtable a0 presents the same types of information for a more detailed analysis of changes in an important pathology cancer where o2 levels have been an especially important focus for informing clinical treatment and prognosis to give the reader a sense of how well supported the numbers are the data in table a0 have been ordered by the number of patients included in each rowof interest here all seven cancer types with at least patients studied have a fairly consistent and fairly hypoxic median o2 level a0mmhg similarly all but soft tissue sarcoma have a similar hypoxic fraction sarcoma appears to be about half that glioma appears to be an outlier on the range glioma had no patient whose o2 level was above while all others as was true for the tissues in table a0 have at least one measurement in the upper 80s or 90s these occasional high readings are not surprising since it is plausible that randomly some readings will have been obtained in or very near to arteriolesfeeding microvessels in contrast to the first seven cancers the types of cancers with fewer than patients appear to be more varied in their o2 levels but this is possibly due to being based on few patientsnevertheless even though as noted elsewhere in this review the data presented are not unconditionally absolute values of o2 levels as they are sometimes referred to eg koch a0 macnab gagnon gagnon minchinton fry a0 nevertheless as argued in this review they can provide very useful data as long as their limitations are recognized by researchers and clinicians heterogeneity of distributions of levels of oxygen in tissues examples and causesheterogeneity of distributions of oxygen values in the tissues of interest exists over many dimensions including time and space and over a wide range of scales harrison vaupel a0 tables a0 and focus on intertissue and intratissue variation in overall levels of o2 figures a0 and illustrate heterogeneity using more refined data points to illustrate the skewed nature of the data particularly for malignancies the data in figure a0 are based on multiple measurements made in a series of patients using a computerized polarographic microsensor technique which enables direct assessment of the o2 levels with an o2sensitive needle electrode subject to the limitations of providing true absolute values as discussed swartz 0c of table oxygenation status of anstissuesantissuekidneycortexouter medullainner medullaliverpancreasspleennormalin hypersplenismhodgkin's diseasemyocardiumsubepicardialsubendocardialmucosaoralrectallarge bowelbreastnormalfibrocystic diseaseprostateuterine cervixsubcutisbonecorticalhematopoietic marrowadipose marrowskeletal musclerestingexercisehypovolemic shockpaodskinthermoneutral conditionscritical limb ischemialimbs venous diseasebraingray matterwhite mattermedian po2 mmhghf po2 range mmhgreferencesnananananananag¼nther aum¼ller kunke vaupel and thews samesamekallinowski and buhr 1995akoong vaupel wendling thom and fischer wendling vaupel fischer and br¼nner samewinbury howe and weiss moss for bothkallinowski and buhr 1995asamesamevaupel schlenger knoop and h¶ckel vaupel and harrison samevaupel and kelleher h¶ckel schlenger knoop and vaupel samespencer et a0al a0samesamelandgraf and ehrly jung kessler pindur sternitzky and franke harrison and vaupel landgraf schultehuermann vallbracht and ehrly carreau et a0al a0harrison and vaupel clyne ramsden chant and webster vaupel samecontinuesswartz 0c of table continuedantissueretinamedian po2 mmhghf napo2 range mmhgreferenceshogeboom van buggenum van der heijde tangelder and reichertthoen linsenmeier and zhang white adipose tissuenonobeseobesenanapasarica et a0al a0 hodson lempesis van meijel manolopoulos and goossens abbreviations arterial hf25 hypoxic fraction ¡ fraction of po2 values ¤ a0mmhg na information not available paod peripheral arterial occlusive diseaseelsewhere in the paper oxygen was measured along several electrode tracks in each individual during a given measurement session from near the tumor surface up to tissue depths of a0 a0 a0mm in breast cancers and in cancers of the uterine cervix each row in figure a0 presents a type of tissue breast and uterine cervix with comparisons of o2 levels made in normal tissue green versus malignancies prior to treatment of these ans red the summary measures median po2 values parallel to data reported in tables a0 and are included in the text boxesnote that the data in figure a0 are not normally distributed the distribution of o2 levels made in normal breast tissue is the closest approximation to a normal distribution comparing the two distributions for malignancies we see that breast cancer is more highly skewed than cervical cancer although they have the same median thus using a single measure such as the median could overlook potentially important clinical informationsimilarly comparing the distributions for the normal tissues the normal cervix had a substantial number of o2 measurements within a hypoxic range defined as ¤ a0 mmhg while there were no hypoxic measurements in the normal breast while figure a0 does not differentiate the measurements made per patient it illustrates why several authors report the hypoxic fraction when trying to capture a meaningful overall number to characterize a tissue finally these data underscore why it is important to understand what is well captured by a given measure of o2 leveland what is missed or obscuredanother example of heterogeneity is presented in figure a0 these o2 measurements were taken in a breast cancer patient using epr oximetry with carlo erba ink as the o2 sensor flood et a0al a0 jeong et a0al a0 the data are presented as line widths because the epr oximetry technique using india ink as a sensor undoubtably gathers data from volumes too large to have homogenous oxygen levels however because this sensor remains in the same place in the tissue it still can provide very useful indications of changes over time andor the impact of interventions such as breathing enriched oxygen the data were taken in 30min sessions during which epr spectra were collected continuously but the period was divided into three 10min periods differing by the gas mixture the patient was breathing room air red baseline o2 delivered by a nonrebreather mask green followed by again breathing room air blue recovery the sessions were repeated approximately weekly throughout the period of radiation therapy thus mimicking a clinical course of radiation therapy although there was no attempt to impact therapy in this studythe data for this particular patient illustrate that the o2 levels responded to the patient's breathing an hyperoxic gas mixture and then returned rapidly to the baseline level after the 10min period in this patient there appeared to be some variation across the weeks of treatment with the final levels for each period baseline o2 and recovery all being slightly higher than at the beginning of radiation therapy based on nonoverlapping standard deviations of the first and last measurements causes of heterogeneity in normal tissuesthere are spatial variances in oxygen levels in normal tissues due to the longitudinal gradient in oxygen as the blood passes through the microcirculatory bed decreasing from the arterial inlet to the outlet of the microvessels erickson after the oxygen leaves the microvascular networks the partial pressure of o2 decreases due to radial gradients that is o2 diffuses through the tissues as it gets further from the vessels due to o2 being consumed by the cells as a result there are variations in o2 levels from cell to cell according to their distance from the microvessel within the cells o2 decreases in a microspatially complex manner as it is intracellularly consumed with most of the consumption occurring in the mitochondria there is growing evidence that diffusion of o2 into the cell may be constrained that is that o2 does not freely and rapidly flow into cells across the membrane and therefore there are gradients from outside to inside of cells khan et a0al a0 kurokawa et a0al a0 pias a0these variations of o2 levels that is gradients between and within cells cannot currently be measured as detailed below in discussing temporal variations even if such measurements of spatial heterogeneity could be made they would still be inadequate to understand the full complexity of heterogeneity of o2 for example in some normal tissues there is additional significant macroscopic heterogeneity of o2 swartz 0c of table pretherapeutic oxygenation status of human tumorstumor type ordered by no of patientscervix cancerhead and neck cancerprostate cancersoft tissue sarcomabreast cancerglioblastomavulvar cancermedian po2 mmhgno of patientsrectal cancerlung cancermalignant melanoma metastaticnonhodgkin's lymphomapancreas cancerbrain metastasesnahf po2 range mmhgliver metastasesrenal cell carcinomagall bladder cancerbile duct cancerabbreviations hf25 hypoxic fraction ¡ fraction of po2 values ¤ a0mmhg na information not availablenanananareferencesvaupel et a0al a0vaupel vaupel data synopsesthese ref apply to allabove the linevaupel thews mayer h¶ckel and h¶ckel vaupel mayer and h¶ckel stone et a0al a0kallinowski and buhr 1995a mattern kallinowski herfarth and volm falk ward and bleehen le et a0al a0lartigau et a0al a0powell et a0al a0koong graffman bjork ederoth and ihse rampling cruickshank lewis fitzsimmons and workman kallinowski and buhr 1995a 1995blawrentschuk et a0al a0graffman et a0al a0graffman et a0al a0over space because of their physiology as a consequence of substantial differences in vascularity blood flow and oxygen consumption eg macroscopic heterogeneity between gray and white matter of the brain between subepicardial and subendocardial layers of the myocardium and between renal cortex and renal inner medullathere also are temporal changes in o2 levels in normal tissues griffith a0 moreover the supply of o2 can vary periodically due to rhythmic changes in microcirculatory blood flow which is reflected at all levels from the inflow arteries to the microcirculation finally within the microcirculation there are variations in microvascular flow due to regional regulation in response to varying metabolic demands kimura et a0al a0 important differences in o2 solubility across tissues affecting the relationship between po2 and [o2] were discussed earlier impact of pathology on heterogeneity of o2 levelsthe presence of pathology often significantly increases the amount and extent of oxygen heterogeneity both spatially and temporally vaupel harrison vaupel mayer a0 the presence of pathology often impacts the structuremorphology of the vessels in tumors there often is a significant amount of neoangiogenesis which results in much less ordered and less functional blood vessels busk et a0al a0 the resulting vessels are much less efficient in delivering blood and also tend to be much more prone to leak leakage from these vessels can cause increases in the interstitial pressure which can reduce the effectiveness of the microcirculation due to reduction of the perfusion pressure within the tumor capillaries fukumura duda munn jain a0pathological changes also can result in altered consumption of o2 in malignant tumors o2 consumption is likely to decrease due to poor oxygen delivery andor because of a switch to glycolysis due to metabolic reprogramming ie the warburg effect as a consequence of hif1α overexpression upregulation of oncogenes downregulation of suppressor genes and activation of certain signaling pathways vaupel multhoff a0 vaupel schmidberger mayer a0 pathology can also impact the integrity of the blood vessels for example tumor growth may physically impinge on the integrity of the blood vessels and the swartz 0c of figure distributions of multiple o2 levels made in patients with normal versus malignant tissue breast and cervix figure adapted from vaupel mayer a02017b p figure repeated o2 level measurementsa during each measurement session and over a0days breast cancer patient measured in skin and superficial breast tissue within the radiation field during a course of radiation therapy figure adapted from flood et a0al a0 p afor carlo erba ink epr line width increases with increasing o2 level but the relationship is nonlinear and can be impacted by several factors therefore the data are given here as line widthmetabolic abnormalities in diabetes can impact the structure of blood vessels causing either microangiopathy andor macroangiopathy the results of these processes can produce very significant local variations in the availability of fully functional vascular structures resulting in locally hypoxic regionsswartz 0c of pathology also can impact temporal changes of oxygen and the response to treatment the presence of pathology especially cancer eg acute and cycling hypoxia in cancers and peripheral vascular disease can result in significantly greater variability in o2 levels braun lanzen dewhirst a0 these include shortterm changes especially associated with the structural abnormalities of the microcirculation resulting in increased local variability in flow and longterm changes that develop over time such as those due to disease progression and responses to therapy baudelet et a0al a0 kimura et a0al a0 konerding fait gaumann a0 matsumoto there also is a potential for pathologies to interrelate with each other for example anemic hypoxia can develop in tumors due to the underlying systemic anemia of the patient vaupel mayer a0in addition to these underlying effects of pathology on tissue oxygen levels any applied therapeutic interventions are very likely to induce changes for example cell killing due to radiation or chemotherapy will alter oxygen consumption patterns these same therapies will also affect the o2 supplying vasculature via both antiangiogenic effects andpossiblynormalization of vessel structure jain a0 the effects of therapies will generally vary both spatially and temporally reiterating the complexity of meaningfully characterizing tissue oxygen levels analysis of the ability of clinically available techniques to directly measure levels of o2 in tissues andor resolve the heterogeneity of o2 distributions in tissuesalthough many techniques are often thought to measure actual o2 in tissues only a few actually have the potential to make o2 measurements directly in the tissues of interest springett swartz a0 tatum techniques that can potentially assess o2 directly in tissues include epr epel et a0al a0 swartz et a0al a0 swartz the eppendorf electrode vaupel h¶ckel mayer a0 some optical methods based on direct measurements of target molecules in tissues for example phosphorescence quenching of optical sensors placed directly in tissues or as part of a physical probe such as the oxylite wen et a0al a0 and nmr relaxation techniques colliez et a0al a0two other types of measurements assess o2 in the vascular system blood gases do this directly while optical methods that measure both hemoglobin saturation and total hemoglobin especially near infrared spectroscopy [nirs] scheeren schober schwarte a0 provide a plausible link to the po2 in the bloodhowever the techniques most often used clinically to characterize tissue oxygenation do not in fact measure o2 directly instead they measure indirect parameters that can be plausibly linked to actual o2 levels but only under appropriatedefined circumstances this latter group of techniques includes positron emission tomography pet imaging of glucose derivatives neveu et a0al a0 pet imaging of drugs that localize in hypoxic tissues tran laser doppler flow measures of metabolites that may be affected by o2 levels for example lactate and redox intermediates and several magnetic resonance imagingnuclear magnetic resonance mrinmr blood oxygenation level dependent bold imaging baudelet gallez a0 and mri egeland et a0al a0 note if their basis is understood and the data considered accordingly these can all provide clinically and physiologically useful information even though they do not provide direct information on the amount of o2 in the tissues direct measures of o2 in targeted tissues that potentially can be used in human subjectsthese are techniques that while they have the capability of providing direct quantitative measurements of o2 in homogeneous media cannot provide such data in tissues in vivo because the volumes that they sense are larger than the volumes of homogeneity of o2 in actively metabolizing tissues consequently all in vivo measurements of o2 are inherently averages of the actual oxygen content in that volume even neglecting the need to include measures of heterogeneity inside cells based on the usual volume of cells and assuming that differences are sought for aggregates of ¤ cells for a measurement of heterogeneity sensed within a a0mm diameter volume the spatial resolution needed to appropriately characterize o2 levels in this volume becomes million voxels the measuring techniques may not even provide a welldefined averaged po2 value within the volume that they sense for example sensors for the signal that is being measuredin the next sections we review the characteristics of each technique that can directly measure o2 levels in tissues we also briefly remark on the volumes they measure and how the measures obtained can be useful clinically see also ortezprado dunn vasconez castillo visco epr oximetry using appropriate particulate paramagnetic materials epr oximetry can provide direct measurements of o2 that is the epr signal is directly proportional to the amount of o2 epel bowman mailer halpern a0 swartz vaupel swartz 0cthe because each multisite sensor senses a volume that is much larger than capillary networks these techniques provide a volume averaged sampling of all compartments within the tissues the time resolution of the techniques can be milliseconds or shorterthe measured parameter of an epr spectrum that indicates the amount of o2 present is the line width of the observed resonance peak there usually is a fixed relationship between the line width and the amount of o2 with the relationship being specific for each type of paramagnetic material for example carbon charcoal or phthalocyanine particulates using particulate oximetric materials measurements can be continuous over any span of time and can be repeated indefinitely see example in figure a0 the method requires that the sensing material be injected or implanted in one or more regions of interest but thereafter all measurements can be made entirely noninvasively importantly the measurements can be carried out in a clinical setting and can fit into the workflow needed for patient careinitial clinical epr measurements of oxygen in tissues have used india ink as the oxygen sensor swartz et a0al a0 the carbon ps are the components that respond to oxygen lan beghein charlier gallez a0 after injection of a0µl of the suspension through a small needle the carbon ps disperse nonuniformly through the local region as small extracellular aggregates they are often engulfed by macrophages the resulting epr spectra in the region probed by the resonator ie the surface coil used for signal detection are a composite of the oxygendependent line widths from each of the ps in reality because of the relatively broad lines from the india ink ps the range of oxygen levels that are likely to be present in the tissue and the limited number of ps in each subregion it is a challenge to resolve directly even the major groups of similar line widths therefore using the observed line width to provide a quantitative measure of oxygen would seem to have modest utility in itselfthe other method of clinical epr oximetry is based on the use of microcrystalline probes eg lipc lincbuo encapsulated in biocompatible polymers swartz et a0al a0 clinical measurements currently are being performed using the oxychip which consists of oxygen sensitive microcrystals of lithium octanbutoxynaphthalocyanine lincbuo embedded in polydimethylsiloxane pdms hou khan gohain kuppusamy kuppusamy a0 hou et a0al a0 jarvis et a0al a0 the dimensions currently used in humans are cylinders that are a0 mm long with a diameter of a0mm the epr signal from the sensor oxychip reflects the po2 within the pdms which itself reflects an average of the po2 in contact with the external surface of the cylinder the dimensions of the oxy | 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 |
"carcinogenesis is a process of somatic evolution previous models of stem and transient amplifying cells in epithelial proliferating units like colonic crypts showed that intermediate numbers of stem cells in a crypt should optimally prevent progression to cancer if a stem cell population is too small it is easy for a mutator mutation to drift to fixation if it is too large it is easy for selection to drive cell fitness enhancing carcinogenic mutations to fixation here we show that a multiscale microsimulation that captures both withincrypt and betweencrypt evolutionary dynamics leads to a different epithelial tissues are metapopulations of crypts we measured time to initiation of a neoplasm implemented as inactivation of both alleles of a tumor suppressor gene in our model time to initiation is dependent on the spread of mutator clones in the crypts the proportion of selectively beneficial and deleterious mutations in somatic cells is unknown and so was explored with a parameter when the majority of nonneutral mutations are deleterious the fitness of mutator clones tends to decline when crypts are maintained by few stem cells intercrypt competition tends to remove crypts with fixed mutators when there are many stem cells within a crypt there is virtually no crypt turnover but mutator clones are suppressed by withincrypt competition if the majority of nonneutral mutations are beneficial to the clone then these results are reversed and intermediatesized crypts provide the most protection against initiation these results highlight the need to understand the dynamics of turnover and the mechanisms that control homeostasis both at the level of stem cells within proliferative units and at the tissue level of competing proliferative units determining the distribution of fitness effects of somatic mutations will also be crucial to understanding the dynamics of tumor initiation and progressionk e y w o r d scancer evolution initiation metapopulation dynamics neoplastic progression simulationmajor findings competition between epithelial units such as colonic crypts tends to suppress initiation of neoplasms by suppressing mutator clones this suppression of initiation is enhanced when crypts have few stem cells and so are likely to go extinct due to stochastic fluctuations in stem cell numbers this is an open access under the terms of the creative commons attribution license which permits use distribution and reproduction in any medium provided the original work is properly cited the authors evolutionary applications published by john wiley sons ltdevolutionary applications wileyonlinelibrarycom eva 2003 2003 0c 2003 2003 2003 2002 2003 2003introductionthe anization of a population into spatially distinct subpopulations can have a dramatic effect on the evolution of that metapopulation hanski gaggiotti this has implications for both the evolution of anisms and for the effect of tissue architecture on somatic evolution and tissue health in multicellular anisms epithelia are typically divided into subpopulations of tissue stem cells along with the transient amplifying cells and differentiated cells that they produce these subpopulations go by different names in different tissues such as crypts in the intestine or more generally epithelial proliferative units cairns first recognized that the division of stem cells into subpopulations such as crypts acts as a tumor suppressor cairns a mutant stem cell with a reproductive or survival advantage may take over a crypt but is generally constrained from expanding beyond that subpopulation unless it breaches the crypt via a process known as crypt fission which tends to duplicate the mutant crypt cell population however by establishing a population size barrier the mutant clone has to overcome the subpopulation structure of the tissue limits the probability that that clone will acquire further carcinogenic mutations yet clones of mutant stem cells can be observed at scales spanning many crypt diameters especially in compromised tissues such as ulcerative colitis and barrett's esophagus maley salk a fundamental question is therefore how the cryptlevel metapopulation dynamics affect the accumulation of somatic mutations during carcinogenesishere we explore the evolutionary dynamics of mutant stem populations that lead to tumor initiation that is the breach of the crypt barrier allowing clonal expansions of crypts across a tissue as well as of mutant stem cells within and out of a crypt while there may be multiple pathways to tumor initiation it has been shown that the inactivation of a single tumor suppressor gene tsg such as the adenomatous polyposis coli apc gene in colon is sufficient to abrogate crypt homeostasis leading to the formation of aberrant crypts and nascent adenomas humphries wright using an agentbased microsimulation model for both stem cell turnover within a crypt and for the cryptpopulation tissuelevel dynamics we study the role of pretumor evolution in tumor initiation this exploration allows for the selection of mutant crypts across the tissue prior to the inactivation of the tumor suppressor genea form of premalignant field cancerizationwhile the stem cell in which the tumor suppressor is inactivated can proliferate beyond the limit of a single crypt due to crypt bifurcationthe evolution of somatic cells is a complex multiscale process depending on the nature of somatic mutations which may either increase or decrease cell fitness stem cell divisions and differentiation or apoptosis as well as subpopulation eg crypt division and extinction rates there is considerable evidence that carcinogenesis involves both an increase in the rate of epigenetic lesions bielas loeb rubin true loeb breivik ji king weisenberger et al and expansions of clones with a relative fitness advantage over their competitor cells cannataro gaffney townsend maley pepper findlay kassen spencer maley vermeulen williams however it continues to be unclear whether the mutator phenotype is a preinitiation phenomenon in carcinogenesis or is more likely to occur during tumor progression in barrett's esophagus another cryptstructured precancer we found evidence that genomic instability precedes genome doubling and transformation martinez the frequency of deleterious versus beneficial mutations in somatic cells is also unknown though the large number of genes in metazoans devoted to differentiation apoptosis and cell cycle control suggests that the frequency of deleterious mutations may be lower in somatic evolution than anismal evolution rajagopalan nowak vogelstein lengauer recent analysis of somatic mutations in cancer found no evidence of purifying selection except in a few essential genes and strong evidence of positive selection with large selective effects williams suggesting that beneficial mutations are more common than deleterious mutations in somatic evolution martincorena although a definitive answer to these questions can only come from further experimental data a theoretical exploration that recognizes the roles of metapopulation dynamics the mutator phenotype and the proportion of deleterious to advantageous mutations in the process of tumor initiation is called for such an exploration will help the identification of factors that drive the tumor initiation processour model integrates previous efforts to characterize the stem cell dynamics within a crypt cannataro mckinley st mary cannataro mckinley st mary frank iwasa nowak komarova komarova cheng loeffler birke winton potten meineke potten loeffler michor frank may iwasa nowak nowak et al pepper sprouffske maley with models of the dynamics of crypt populations cannataro et al chao eck brash maley luebeck kostadinov maley kuhner loeffler bratke paulus li potten totafurno bjerknes cheng mathematical studies of the stem cell population in the crypt niche suggest that epigenetic alterations that increase the rate of genetic lesions mutator mutations and reduce the fitness of stem cells will tend to drift to fixation if the stem cell population is small whereas carcinogenic mutations that increase the proliferation or survival of a stem cell will tend to spread if the stem cell population is large cannataro komarova michor assuming that most nonneutral somatic mutations are deleterious the accumulation of deleterious mutations may lead to senescence of the intestine over time cannataro however competition between crypts of different fitnesses may significantly change the dynamics of the establishment of a mutator clone through a metapopulation dynamic our in silico experiments suggest that there may have been selection at the level of the anism to minimize the number of stem cells within each subpopulation of birtwell 0c 2003 2002 2003 2003its structured epithelium so as to reduce the probability of tumor suppressor gene inactivation and the initiation of carcinogenesisproliferation rate decreased stem cell loss and caused the mutator phenotypethe following equations and assumptions govern the model 2003 2003methodswe implemented a multiscale model of epithelial tissue architecture with stem cells subdivided into crypts under homeostatic control we examined the time required until the two alleles of a tumor suppressor gene tsg were inactivated in at least one stem cell to represent tumor initiation the model was run at least times for every parameter setting crypts were arranged in a flat hexagonal tissue similar to that observed in colon and contained a population of stem cells as well as an implicitly modeled transient amplifying compartment stem cells divided both symmetrically and asymmetrically symmetric division resulted in two daughter stem cells each having the opportunity during the division event synthesis to acquire a mutation asymmetric division did not result in any new stem cells but did provide an opportunity for stem cell mutation stem cell loss due to cell death or differentiation and stem cell gain due to division events were modeled as a stochastic birthdeath process with parameters that were functions of the stem cell fitness and of homeostatic feedback effects in response to deviations of the crypt cell population from its normal target level equations and a flow chart of the model algorithm is shown in figure s1homeostasis operated at two spatial scales within a crypt if the stem cell population dropped below the target level stem cell division rates increased by a parameterized amount equation if the population rose above the target level stem cell loss rates increased equation the level of homeostatic feedback was proportional to the degree of deviation away from the target equilibrium level equations and we also introduced a mechanism for homeostasis on the hexagonal lattice of crypts if all the stem cells in a crypt died the inhibition on stem cell population growth was released from the neighboring crypts when the stem cell population of a neighbor reached twice the equilibrium level we modeled crypt bifurcation by allocating half of its stem cells to a new crypt in the location of the dead neighborwe included both beneficial mutations that increased the division probability or the survival probability of stem cells as well as deleterious mutations that decreased them these can accumulate indefinitely and affect fitness multiplicatively equation we also implemented a genetic instability mutation that increased the clone's mutation rate 100fold bielas herr kennedy knowels schultz preston ji king the frequency of each mutation type those that changed the cell's fitness the proportion of nonneutral mutations that were deleterious as well as the rate of tsg inactivation was set by parameters each mutation affected proliferation survival or mutation rate parameters by a constant factor half of the deleterious mutations decreased stem cell proliferation and half decreased their survival increased cell loss for the beneficial mutations increased the cell's 2003 2003equationsequation time to stem cell losslet t be a random exponential deviate with distribution function fr t and rate parameter r the time to cell loss due to apoptosis or differentiation is the minimum of the time to cell loss due to background cell death or differentiation and the time to cell loss due to crypt feedbacktcell_loss min 01t1 ¼ fbackground_loss t2 ¼ ffeedback_loss 01 equation homeostatic crypt feedback by differentiationwhen the stem cell population within a crypt expands beyond the homeostatic target level kcrypt\\_size the crypt provides homeostatic feedback via a change in the rate of stem cell loss with a rate parameter equal to the base stem cell loss rate multiplied by the crypt feedback multiplier the crypt feedback rate multiplier is used to calculate the time to stem cell loss due to crypt homeostatic feedback the crypt feedback multiplier is equal to raised to the nth power where n is the excess number of stem cells above the crypt size divided by the kcrypt\\_deviation parameter here kcrypt\\_deviation and kcrypt\\_size is a parameter that we varied across experimentsrfeedback\\_cell_loss rbase_cell_loss2max0ncellskcrypt_sizekcrypt_deviationequation homeostatic crypt feedback by proliferationwhen the stem cell population of a crypt drops below kcrypt\\_size the division rate of the remaining stem cells is increased by a factor that depends on the difference between the current number of stem cells ncellsand kcrypt\\_sizerfeedback_division rbase_division2max0kcrypt_sizencellskcrypt_deviationequation fitness mutation effectskï¬tness is a constant factor representing the effect of a single beneficial mutation on fitness as a first approximation we assume that there are many possible mutations that increase and decrease the fitness of a somatic clone by approximately the same amount and so the effect of n beneficial mutations nbeneï¬cial on stem cell fitness is the constant fitness effect raised to the nth power the effect of n deleterious mutations ndeleterious of small effect is just the inverse of kï¬tness raised to the nth power there is a separate mï¬tness calculated for the division probability and the survival probability of a cell because beneficial and deleterious mutations may affect either of those probabilitiesmï¬tness 01kï¬tness 01nbeneï¬cial 03 kï¬tness 03ndeleteriousbirtwell 0c 2003 2003 2003 2002f i g u r e 2003plots of cumulative hazard functions using the kaplanmeier estimator the tissue was x crypts with stem cells per crypt in panels a through d each colored line represents the function for a specific proportion of deleterious mutations a baseline experiment with default parameter values table b mutation rate reduced to 01x of baseline c mutator phenotype reduced to 01x of baseline d each colored line represents a different number of cells per crypt all proportions of deleterious mutations were included 2003 2003assumptionscrypts consist of stem cells and of transient amplifying cellscrypt density is fixed that is the tissue contains a fixed number of crypts arranged on a hexagonal griddrops below the target level the division rate of each stem cell in the crypt is increased when the number of stem cells grows above the target level the cell loss rate of each stem cell in the crypt is increasedcrypts divide to fill vacant slots left by adjacent crypts that have the number of cells in a crypt transient amplifying compartment gone extinct due to loss of the constituent stem cellsis fixedcrypts attempt to maintain a stable population of stem cells through homeostatic feedback when the number of stem cells the extinction of an adjacent crypt suppresses the homeostatic apoptotic signals allowing the stem cell populations in neighboring crypts to expand once that extinct crypt is replaced the normal birtwell 0cta b l e 2003baseline simulation parameterssimulationmaximum simulation duration in daysstem celldivision rate rbase\\_divisionratio of asymmetric divisions to symmetric divisionsstem cell loss rate rbase\\_cell\\_lossmutation rate per stem cell divisionmutation rate maximumtumor suppressor gene mutation ratetumor suppressor gene mutation rate maximumnumber of transient amplifying cells associated with each stem cellcell division minimum time in dayscell loss minimum time in dayscrypttarget equilibrium level of number of stem cells in a crypt kcrypt\\_sizestandard deviation from equilibrium level the crypt uses this value to determine its level of effect on the cell loss and division rates of the stem cells kcrypt\\_deviationbifurcation threshold factor below which a crypt will not bifurcatecrypt cell loss effect multipliercrypt division effect multipliermultiplier to the division effect for each dead neighbor cryptcanceruncontrolled cell proliferation threshold if a crypt has this threshold times the equilibrium number of stem cells it is considered to be experiencing uncontrolled stem cellnumber of tumor suppressor gene hits that mean cancermutation 2003 2002 2003 2003 years x x x also tested x x default varied from percent of nonneutral mutations that are deleteriousthe factor affecting the loss rate of the stem cell from a beneficial mutation kï¬tnessthe factor affecting the division rate of the stem cell from a beneficial mutation kï¬tnessthe factor affecting the cell loss rate of the stem cell from a deleterious mutation kï¬tnessthe factor affecting the division rate of the stem cell from a deleterious mutation kï¬tnessthe factor affecting the mutation rate of the stem cell from a mutator mutation also tested homeostatic controls on stem cell numbers of neighboring crypts are restoredcrypt division is triggered by an expansion of the stem cell population of a crypt to twice its homeostatic level as hypothesized by garcia park novelli and wright as long as there is an empty slot adjacent to the enlarged crypta stochastic birthdeath process governs the scheduling of division and cell loss eventsfitness mutations affect in a multiplicative fashion the rate parameters of the birthdeath processthere is a single mutator phenotype that requires only a single mutator mutation additional mutator mutations have no effect on the mutation ratethe loss of the first allele of the tsg has no effect on stem cell fitness 2003 2003results 2003 2003tsg inactivation depends on the emergence of a mutatorat baseline for comparison our tissue was a 5x5 hexagonal lattice of crypts each crypt having stem cells stem cell loss and symmetric division rates were balanced mutations were acquired stochastically with probabilities defined by proportions starting with deleterious mutations beneficial mutations and mutator mutations and ranging in increments to deleterious beneficial and mutator beneficial versus mutator the incidence of tsg inactivation decreased as the proportion of deleterious mutations increased figure 1a table birtwell 0c 2003 2003 2003 2002reducing the base mutation rate to of baseline we observed a marked decrease in tsg inactivation figure 1b as expected similarly reducing the effect of the mutator mutation to 10x the baseline mutation rate instead of 100x produced a significant decrease in tsg inactivation figure 1c we found that the vast majority of tsg inactivation occurred in stem cells that had previously acquired the mutator phenotype figure s2 this assumes that the mutator phenotype can be caused by a single mutation that is otherwise neutral eg overexpression of dna polymerase beta canitrot or a dominantnegative mutation in p53 de vries though this assumption is easily relaxed 2003 2003proportion of deleterious mutations negatively correlates with tsg inactivationnot surprisingly we found that the proportion of deleterious mutations and the incidence of tsg inactivation were negatively correlated figure cell divisions per time remained roughly constant across all proportions of mutations however as the proportion of deleterious mutations decreased the cost of being a mutator also decreased because it accumulated less mutational burden of deterious mutations this resulted in an increased emergence of crypts with fixed mutator stem cell populations conversely across all experiments we observed progressively less tsg inactivation as the proportion of deleterious mutations approached our maximum of turnover levels we observed a reduction in the average number of stem cells per crypt figure s3 the implemented homeostatic control was unable to maintain the target stem cell population size in the face of high turnover rates essentially there is a lag between depletion of the stem cell pool due to cell death and differentiation and replenishment provided by an increase in stem cell division rates with higher levels of cell loss the simulated crypts spend more time further below the target homeostatic number of stem cells as a result there were fewer total stem cells in the simulation and therefore fewer mutations per time allowing less chance for mutator acquisition and tsg inactivation this may or may not be realistic second as the turnover level increased above our baseline the number of mutator crypts present in the tissue at any given time decreased figure since increased turnover should lead to increased opportunities for mutator mutations to arise the decline in mutator crypts was a surprise however the loss of mutator crypts is due to intercrypt competition as described below increased turnover led to increased stochastic fluctuations in stem cell numbers and thereby increased crypt extinction events the reduction in stem cell numbers and mutator crypts combined to produce a reduction in the overall incidence of tsg inactivation as turnover rates increased above baseline if in reality homeostatic control of stem cell numbers prevents increased crypt extinctions with increased stem cell turnover this result would likely not hold however all things being equal increased cell turnover would be expected to increase stem cell number fluctuations 2003 2003increased stem cell turnover initially increased and then decreased tsg inactivationwe modulated stem cell turnover by varying cell loss and symmetric division rates in unison at lower turnover levels we found that increased cell turnover increased tsg inactivation however at turnover levels 2x and 5x our baseline level the incidence of tsg inactivation declined figure due to two factors first at higher 2003 2003the number of stem cells per crypt had a varying effect on tsg inactivationin general fewer stem cells per crypt reduced the rate of tsg inactivation even though the total number of stem cells in the tissue was held constant figure 1d however there is a tradeoff between tsg inactivation and tissue death at very low stem cells per crypt with only one stem cell per crypt there is no opportunity for homeostatic signals within a crypt to compensate for stem cell loss in f i g u r e 2003this graph represents the proportion of crypts at the end of the run that contained a population of stem cells with the mutator mutation fixed each bar corresponds to a specific proportion of deleterious mutations the proportion of mutator crypts correlates with the risk of tumor initiation as seen in figure birtwell 0cbase cell loss divison rate base cell loss divison rate 2003 2002 2003 2003base cell loss divison rate base cell loss divison rate f i g u r e 2003the effect of changes in stem cell turnover plots of cumulative hazard functions using the kaplanmeier estimator where each colored line represents the function for a specific proportion of deleterious mutations the baseline division and stem cell loss rates were as seen in figure initially turnover correlated positively with the risk of tumor initiation however at higher turnover rates the risk of tumor initiation decreased due to a reduction in the overall number of living stem cells and decreased incidence of mutator cryptsour model tissues with one stem cell per crypt were mostly unviable and died out before tsg inactivation or the predetermined simulation end timeat and cells per crypt we observed a reduction in the incidence of tsg inactivation figure 1d at all but the lowest proportions of deleterious mutations as was predicted by models of the crypt stem cell niche of single crypts komarova michor figure s4 as in other experiments along with a reduction in the incidence of tsg inactivation the frequency of fixed mutator crypts was reduced as well this shows that selection against mutator cells increases as the number of stem cells increases above some threshold in our model as long as the majority of nonneutral mutations are deleterious supporting the s by michor and komarova komarova michor et al when the stem cell populations are large it is very unlikely that a crypt will go extinct and so there is no intercrypt competition in this case the metapopulation dynamics are reduced to the single crypt dynamicsthe increased risk of tsg inactivation associated with increased stem cells per crypt appeared to plateau after approximately stem cells per crypt the total number of cells in the tissue remained constant as did the total stem cell divisions per time figure s5 the average number of mutations per time increased through stem cells per crypt but then reached a temporary plateau figure there was no statistically significant difference between the average number of mutations per time in the and stem cells per crypt cases as the number of stem cells per crypt increased beyond the average mutations per time decreased except when the proportion of deleterious mutations was the incidence of mutator crypts followed a similar trend figure birtwell 0c 2003 2003 2003 2002f i g u r e 2003this graph represents the proportion of crypts at the end of the run that contained a population of stem cells with the mutator mutation fixed as a function of turnover rate each bar corresponds to a specific proportion of deleterious mutations as turnover increased mutator crypts became more rare at higher proportions of deleterious mutationsf i g u r e 2003number of mutations per unit time as a function of cells per crypt and proportion of deleterious mutations where each bar represents a specific proportion of deleterious mutations at most proportions of deleterious mutations mutations per time peaked around stem cells per crypt at deleterious mutations mutations per time reached a minimum at cells per crypt and increased as the cells per crypt grew past we have chosen to explore the case where the total number of stem cells is kept constant assuming that a certain number of selfrenewing tissue stem cells might be required to maintain an epithelial tissue an alternative view is that a fixed number of epithelial units like the crypts might be required to maintain a tissue and that the number of stem cells per crypt could vary by changing the number of differentiated cells produced by each stem cell in this case the risk of tsg inactivation continues to increase with increasing number of stem cells per crypt figure s4 because we held the number of crypts constant but changed the number of cells per crypt in this case the total number of cells in this simulation also changed leading to a large evolving population size of stem cells and thus an increased chance for at least one cell to inactivate both alleles of the tsg 2003 2003partitioning the tissue into crypts imposed a metapopulation dynamicfitness levels measured by the difference between division and loss rates were greater in tissues with smaller crypts even as the total number of stem cells remained constant figure 6a counts of crypt births and crypt life span measurements showed that there was more crypt turnover in smaller crypts figures s6 and s7 this crypt turnover provided an opportunity for fitter phenotypes to spread more easily across the tissue crypt fitness peaked at cells per crypt where there was the most crypt turnover and bottomed out at and cells per crypt after cells per crypt we observed no crypt death and therefore no turnover crypt fitness began to increase again at and cells per crypt through natural selection of stem cells within larger crypts however fitness levels did not increase to the levels seen at cells per cryptthe metapopulation dynamic was observed in the clonal expansion of mutator crypts across the tissue we considered two crypts to have mutator phenotype agreement if they both have a fixed mutator mutation or neither has a fixed mutator we calculated mutator crypt agreement across spatial distance and found that overall agreement decreased as the cells per crypt increased figure 6b further at and cells per crypt closer crypts had increased mutator agreement suggesting clonal expansion of mutator crypts conversely at cells per crypt and above we found that spatial distance correlated less well with mutator agreement indicating that birtwell 0c 2003 2002 2003 2003f i g u r e 2003a crypt fitness measured by the difference between division and cell loss rates across cells per crypt and proportion of deleterious mutations the total number of stem cells remained constant across these experiments the crypt turnover at and cells per crypt allowed fit clones to spread across the tissue resulting in increased overall fitness b mutator agreement by distance class two crypts were in mutator agreement if they both had fixed mutator mutations or neither did overall agreement was higher in the smaller crypts suggesting that mutator clones were able to spread across the tissuewhen there was less crypt turnover mutator crypts arose de novo rather than through cryptlevel clonal expansion 2003 2003discussionin the colon the development of adenomatous polyps frequently involves the inactivation of the apc gatekeeper gene a member of the wntsignaling pathway which represses proliferation and facilitates orderly cell differentiation in the luminal part of the crypts barker goss groden as long as this gatekeeper gene is active mutant stem cell progeny with neoplastic potential is likely eliminated from the crypt and clonal expansion thus averted however when the gatekeeper gene is inactivated the brakes on mutant stem proliferation are removed and mutant cell progeny may undergo focal clonal expansion therefore we asked the question what are the factors that determine the risk of a tsggatekeeper inactivation leading to tumor initiation in a compartmentalized tissue a metapopulationour microsimulations indicate that the base mutation rate figure 1b and the increase in that rate for a mutator clone figure 1c are the main driving forces behind tsg inactivation and thus the initiation of carcinogenesis the proportion of deleterious mutations is important for its effect on selection of the mutator clone we find that if most mutations are assumed to be deleterious then a mutator clone quickly accumulates a large genetic load of deleterious mutations and tends to be driven to extinction in competition with nonmutator cells however if mutations are more likely to be beneficial then a mutator clone can spread more easily which leads to the early inactivation of both alleles of the tumor suppressor gene figure 1a similarly increasing turnover of the stem cells effectively increases the mutation rate and consequently the rate of initiation however when turnover is very high the homeostatic feedback signals cannot maintain the target number of stem cells and so the total stem cell population of the epithelium decreases which in turn decreases the chances for initiation figure since the rate of tsg inactivation is trivially relat | Colon_Cancer |
lenvatinib inhibits tyrosine kinases including vascular endothelial growth factor vegf receptor fibroblast growth factor receptor platelet derived growth factor receptor alpha ret proto oncogene and kit proto oncogene receptor tyrosine kinase we assessed the efficacy and safety of lenvatinib in patients with metastatic colorectal cancer after failure of standard chemotherapiespatients and methods this was an open label single centre single arm phase study eligible patients had unresectable metastatic colorectal adenocarcinoma refractory or intolerant to fluoropyrimidine irinotecan oxaliplatin trifluridinetipiracil anti vegf therapy and anti epidermal growth factor receptor therapy for tumours with wild type ras patients were treated with oral lenvatinib at mg one time a day in day cycles until disease progression or unacceptable toxicity the primary endpoint was centrally assessed disease control rate secondary endpoints included safety response rate progression free survival and overall survival the planned sample size was patients to expect a disease control rate of with a threshold disease control rate of one sided alpha of and power of results between october and january patients were enrolled and had received or ¥ lines of prior chemotherapy for metastatic disease respectively the median number of lenvatinib cycles was range the centrally assessed disease control rate was ci to one sided p00001 patients had a partial response and had a stable disease median progression free survival was months ci to median overall survival was months ci to the most common grade ¥ adverse events were hypertension thrombocytopenia increased alanine aminotransferase and anorexia eachs lenvatinib showed promising clinical activity and was tolerated in patients with metastatic colorectal cancer after failure of standard chemotherapiestrial registration number umin ctr umin000023446 and jamcct ctr jma iia00261introductionthe combination of cytotoxic chemotherapy with a molecular targeted agent has significantly key questionswhat is already known about this subject º no studies have previously reported the efficacy and safety of lenvatinib monotherapy in patients with metastatic colorectal cancer refractory to standard chemotherapieswhat does this study add º lenvatinib showed promising antitumour activity with acceptable toxicity for heavily pretreated patients with metastatic colorectal cancer refractory to standard chemotherapies º no unexpected safety signals were observed and toxicities were manageable with dose modification interruptions and supportive medicationshow might this impact on clinical practice º further prospective randomised studies are warranted to evaluate the efficacy of lenvatinib in patients with metastatic colorectal cancer refractory to standard chemotherapiesimproved the survival of patients with unresectable metastatic colorectal cancer1 from results of recent clinical trials trifluridinetipiracil and regorafenib are recognised as new treatment options for patients with metastatic colorectal cancer refractory or intolerant to standard therapies6 nevertheless the prognosis of patients which are refractory or intolerant to standard chemotherapies is poor and there are still an unmet medical needs for these patients especially for those who are in a good performance status and eligible for further therapieslenvatinib is an oral multitargeted tyrosine kinase inhibitor of the vascular endothelial growth factor receptor vegfr fibroblast growth factor receptors platelet derived growth factor receptor alpha ret and kit8 preclinical studies have shown that iwasa a0s et a0al esmo open 20205e000776 101136esmoopen2020000776 0copen accesslenvatinib not only interferes the interaction between cancer cells and endothelial cells but also inhibits tumour growth10 several phase trials of patients with solid tumours in the usa11 europe12 and japan13 showed that the optimum dosage of lenvatinib was mg one time a day in a day cyclea total of patients were enrolled in four phase studies of lenvatinib monotherapy of whom had colorectal cancer disease control rate dcr was achieved in out of patients including one with a partial response which continued for weeks mg two times a day for weeks of a week cycle grade palmar plantar erythrodysesthesia was reportedly much lower in of patients treated with lenvatinib for thyroid cancer in a japanese population of the select trial than that of reported in a japanese population of correct trial using regorafenib for metastatic colorectal cancer15 these results suggested that lenvatinib may have a potential for improving the outcomes of patients with unresectable metastatic colorectal cancer who have already received conventional chemotherapy with a fluoropyrimidine irinotecan and oxaliplatinwe conducted a single centre phase study to evaluate efficacy and safety in patients with metastatic colorectal cancer failing to standard therapiespatients and methodsstudy design and patientsthis study was a single arm phase study conducted at national cancer center hospital tokyo japan the inclusion criteria were histological diagnosis of colorectal adenocarcinoma excluding carcinoma of the appendix and the anal canal unresectable metastatic disease an eastern cooperative oncology group performance status of or an age of years no previous treatment with regorafenib or lenvatinib sufficient oral intake adequate an and bone marrow function at least one measurable lesion in accordance with the response evaluation criteria in solid tumors recist version refractory or intolerant to fluoropyrimidine irinotecan oxaliplatin therapy and antiepidermal growth factor receptor therapy for tumours with wild type ras and no systemic therapy for at least weeks weeks if any investigational drug had been administered before study enrolment the exclusion criteria were provided in the online supplementary materialtrifluridinetipiracil anti vegf all patients provided written informed consentprocedurespatients received lenvatinib at mg one time a day in day cycles orally until disease progression or unacceptable toxicity the dose was reduced to mg mg mg mg and mg if a patient had an intolerable grade or grade adverse event treatment was discontinued if a dose interruption was required for more than consecutive daystumour response was assessed by the independent radiological review committee based on the ct or mri performed at baseline every weeks for weeks and every weeks thereafter until confirmed objective disease progression safety assessments including laboratory tests were done at screening days and of cycle and days and of the subsequent cycles urinalysis thyroid function prothrombin time international normalized ratio pt inr and tumour markers both carcinoembryonic antigen and carbohydrate antigen were measured at screening and on day of each treatment cycle adverse events were recorded from the first day of the protocol treatment to days after the last dose of study medication and graded using the national cancer institute common terminology criteria for adverse events version blood sampling for biomarker analyses was done at baseline on days and and at the end of treatment plasma levels of angiopoietin2 were measured by the human angiopoietin2 quantikine elisa kit rd systems minneapolis usaoutcomesthe primary endpoint was centrally assessed dcr which was defined as the proportion of patients with a complete response partial response or stable disease persisting for more than weeks from the initiation of study treatment according to recist version a complete response and partial response were needed to be confirmedthe secondary endpoints were the objective response rate orr proportion of patients who had a complete response or partial response progression free survival pfs time from the enrolment until investigator assessed disease progression or death overall survival os time from the enrolment until death due to any cause and adverse events the incidence of adverse events was calculated based on the information of the worst grade of each adverse event experienced in each patient relative dose intensity which is unprespecified outcome was calculated as the proportion of the actual cumulative dose divided by planned cumulative dose mg times treatment daysstatistical analysisfor this single arm study the required sample size of patients provided power to reject the null hypothesis of dcr ¤ with expectation that of patients would have a disease control one sided α of considering the possibility of a few ineligible patients we planned to recruit patientsthe final analysis was planned approximately months after enrolment of the last patient we included all eligible patients in the efficacy analysis and all patients receiving a least one dose of lenvatinib in the safety analyses for the primary analysis binomial test was performed and the centrally assessed dcr was estimated with ci using the clopper and pearson method which corresponds to one sided α of we also estimated the investigator assessed dcr a supplementary analysis of the primary iwasa a0s et a0al esmo open 20205e000776 101136esmoopen2020000776 0ctable baseline patient characteristicscharacteristicstable continuedoverall n characteristicsoverall n open access median range continued intolerant wild type mutant ras mutational status braf mutational status wild type mutant unknownmsi status mss unkown there is an overlapping this number includes patients with the ras wild type and patient with mutant rasecog eastern cooperative oncology group egfr epidermal growth factor receptor msi microsatellite instability mss microsatellite stableendpoint and orr with cis using the same method we estimated the median time and month and year probability of os and pfs with the kaplan meier method the cis for the median time were calculated using brookmeyer and crowley method the cis of month and year survival probabilities were calculated based on the greenwoods formula hrs and cis were estimated by cox regression we did subgroup analyses divided by prespecified baseline patient and disease characteristic variables including ras status for dcr pfs and os we also did a prespecified exploratory analysis of potential predictive biomarkers in blood samples we did all analyses with sas v94resultspatient characteristicsbetween october and january patients with unresectable metastatic colorectal cancer were enrolled all patients were eligible and received the study medication table summarises the baseline characteristics of all enrolled patients the median number of previous lines of palliative chemotherapy was range and patients had received or ¥ prior lines of chemotherapy for metastatic disease respectively the data cut off date was january with median follow up of months iqr efficacythe centrally assessed dcr was ci to one sided p00001 two patients had a partial response and had a stable disease including unconfirmed pr table figure a total of patients had a reduction in target lesion size from baseline figure time on treatment for all patients is ¥ ¥ male female months ¥ months right sided colon left sided colorectum lung liver lymph node peritoneumage years sex ecog performance status primary site number of metastatic site metastatic an time from start of first line chemotherapy number of previous palliative chemotherapy previous chemotherapy and reason for discontinuation fluoropyrimidine refractory intolerantoxaliplatin irinotecan tas102 trifluridinetipiracil angiogenesis inhibitor anti egfr inhibitor refractory intolerant refractory intolerant refractory refractory intolerant refractory intolerantiwasa a0s et a0al esmo open 20205e000776 101136esmoopen2020000776 0copen accesstable best response to treatmentcomplete responsepartial responsestable diseaseprogressive diseasenot evaluabledisease control rate ciresponse rate cicentral assessmentn30 to to investigator assessmentn30 to to shown in online supplementary figures and events for pfs were recorded in all patients and median pfs was months ci to figure all deaths were recorded median os was months ci to with a month and year os of ci to and ci to figure safetypatients received the study treatment for four cycles at median range the median relative dose intensity was iqr dose interruptions and reductions were required in and patients respectively the major treatment related adverse events ¥ for dose reduction were proteinuria patients palmar plantar erythrodysesthesia patients diarrhoea patients hypertension patients fatigue patients and thrombocytopenia patients the reasons for treatment discontinuation of all patients were disease progression in patients and adverse events in patients gastrointestinal perforation and grade proteinuria in of each after treatment with lenvatinib patients received a subsequent treatment online supplementary table most patients only had mild grades adverse events table the most common grade ¥ adverse events were hypertension patients thrombocytopenia patients increased alanine aminotransferase and anorexia patients each no clear relationship was found between the incidence of lenvatinib associated adverse event of any grade and baseline body surface area online supplementary table serious adverse events occurred in four patients including figure waterfall plot analysis of maximum percentage change from baseline in measurable target lesions response evaluation criteria in solid tumors version central reviewiwasa a0s et a0al esmo open 20205e000776 101136esmoopen2020000776 0copen accessfigure kaplan meier curves of a progression free survival pfs by investigator assessment and b overall survival os in all patients n30five treatment associated events anorexia in two and gastrointestinal perforation central venous catheter related bloodstream infection caused by staphylococcus aureus and nausea in each one in each of four patients all patients recovered from these adverse eventssubgroup analysisin patients with wild type ras the median pfs was months ci to and that was months ci to in patients with mutant ras online supplementary figure in patients with wild type ras the median os was months ci ci to and months ci to in patients with mutant ras online supplementary figure plasma angiopoietin2 levels were decreased by lenvatinib treatment in almost all patients and increased at the iwasa a0s et a0al esmo open 20205e000776 101136esmoopen2020000776time of treatment discontinuation online supplementary table with a first quartile cut off point17 the eight patients with a first quartile or lower level of angiopoietin2 had a median os of months ci to months compared with months ci to in the patients with higher than a first quartile level of angiopoietin2 hr ci to online supplementary figure patients with a first quartile or less level of angiopoietin2 had a median pfs of months ci to compared with months ci to in the patients with more than a first quartile level of angiopoietin2 hr ci to online supplementary figure 0copen accesstable treatment related adverse events occurring in ¥ patients n30any gradegrade ¥treatment related adverse eventhypertensionproteinuriathrombocytopeniafatiguehypothyroidismweight losshoarsenesspalmar plantar erythrodysesthesia syndromeanorexiadiarrhoeamucositis oralserum ast increasedserum creatinine increasedast aspartate transaminase discussionpatients with metastatic colorectal cancer with disease progression after three or more lines of therapy have limited treatment options in this open label single arm phase study of patients with previously treated metastatic colorectal cancer lenvatinib demonstrated manageable toxic effects and promising antitumour activity a total of out of patients had disease control including with partial responses moreover patients experienced reduction in measurable tumour size the overall toxicity profiles were similar to that reported for lenvatinib across a spectrum of advanced malignant neoplasmstwo recent international phase studies reported that regorafenib or trifluridinetipiracil provided significant improvements in dcr pfs and os compared with placebo in patients with metastatic colorectal cancer after failure of standard chemotherapies dcr median pfs months median os months in the correct study and dcr median pfs months median os months in the recourse study6 interestingly the present single arm phase study of lenvatinib revealed favourable dcr and median pfs values in patients with metastatic colorectal cancer compared with those in the regorafenib or trifluridinetipiracil study moreover about half of the patients received post study treatment which led to a favourable osthe lenvatinib safety profile in this study was similar to the published safety profiles of lenvatinib for thyroid cancer and hepatocellular carcinoma in the japanese population18 moreover we found no unexpected or off target safety signals the most common adverse events were hypertension proteinuria thrombocytopenia and fatigue while the most case of grade or hypertension and proteinuria required treatment interruption and dose reduction while the target population for thyroid cancer or hepatocellular carcinoma that showed efficacy for lenvatinib was first line setting20 this study targeted patients receiving salvage line therapy most patients with metastatic colorectal cancer in the salvage line setting had grade or proteinuria and hypertension at baseline because of the long term prior treatment with anti vegfvegfr treatment whereas the occurrence of grade hypertension was significantly higher compared with that of regorafenib in a similar study population in the correct concur and consign trials7 it was manageable by dose reduction or interruption but it may be necessary to consider the starting dose in the future although palmar plantar erythrodysesthesia is a not life threatening toxicity these adverse events have a significant impact on treatment schedules and quality of life in treated patients grade ¥ palmar plantar erythrodysesthesia has been observed in and of patients treated with lenvatinib in this study and the select japanese population15 respectively while in patients treated with regorafenib in the correct japanese population16 to date the clear mechanism of palmar plantar erythrodysesthesia by vegf receptor tyrosine kinase inhibitors is not known but it has been reproduced that palmar plantar erythrodysesthesia by lenvatinib is well tolerated overall it is suggested that lenvatinib might be a favourable treatment option in terms of toxicitiesseveral preclinical studies demonstrated that vegf targeted treatment affects immune suppression by promoting the expansion of suppressive immune cell populations such as regulatory t cells and myeloid derived suppressor cells24 several clinical studies suggested that modulation of vegf mediated immune suppression via angiogenesis inhibition could potentially augment the immunotherapeutic activity of anti programmed cell death pd1 antibody26 regorafenib and nivolumab showed antitumour activity in patients with metastatic colorectal cancer including those with microsatellite stable tumours in a phase study28angiopoietin2 a relatively novel regulator of angiogenesis that acts through the tek tyrosine kinase endothelial tie2 receptor has been identified as a potential prognostic biomarker for some types of cancer although the baseline ang2 level was a predictive biomarker in patients with thyroid cancer in the select trial17 it did not become a reliable biomarker of lenvatinib response in this study prior treatment with anti vegfvegfr antibodies probably had an effect on baseline angiopoietin2 levels because the study population was refractory to standard treatment in this study the decrease in angiopoietin2 levels was observed after treatment therefore it may be an indicator of treatment responsethe limitations of our study include its small size which could limit the interpretation of the subgroup analyses iwasa a0s et a0al esmo open 20205e000776 101136esmoopen2020000776 0cand the absence of a comparison group however the level of clinical benefit in the form of confirmed responses observed in this study was remarkable in the historical context of other clinical trials done in heavily pretreated patients with metastatic colorectal cancer moreover most of the patients in our study had left sided tumours which were known to have a better prognosis compared with right sided tumoursin lenvatinib provided promising activity with prolonged survival relative to the anticipated median pfs in heavily pretreated patients with metastatic colorectal cancer the safety profile of lenvatinib was similar to that in other tumour types with no new safety signals recorded based on these findings further investigation of lenvatinib with anti pd1 antibody or other novel combinations with the potential to build on the benefit of lenvatinib is currently taking place nct03797326 and nct04008797acknowledgements the authors thank the patients and their families the members of the clinical research support office for their support with data collection and running the study and nai incorporated for editing a draft of this manuscriptcontributors all authors conceived and designed the study and drafted and revised the manuscript for publication si no hs yh at kk th nb and yy collected data ak go mk and kn analysed the data and managed data and study progress all authors interpreted the data and approved the final version of the manuscriptfunding the study was supported by the project promoting clinical trials for development of new drugs and medical devices japan medical association from the japan agency for medical research and development grant number jp18lk0201037 and by eisai cocompeting interests si has received research grants from eisai and merck biopharma th has received research grants from eisai and honoraria from merck serono yy has received honoraria from eisaipatient consent for publication not requiredethics approval the study was conducted in accordance with the declaration of helsinki and good clinical practice guidelines the study protocol was approved by the national cancer center institutional review board t4329provenance and peer review not commissioned externally peer revieweddata availability statement data are available upon reasonable request proposals should be directed to siwasa ncc go jp the data will be available for achieving aims in the approved proposalopen 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 idssatoru a0iwasa http orcid yasuhide a0yamada http orcid references saltz lb clarke s dÃaz rubio e et a0al bevacizumab in combination with oxaliplatin based chemotherapy as first line therapy in metastatic colorectal cancer a randomized phase iii study j clin oncol tabernero j yoshino t cohn al et a0al ramucirumab versus placebo in combination with second line folfiri in patients with metastatic colorectal carcinoma that progressed during or after first line therapy with bevacizumab oxaliplatin and a fluoropyrimidine raise a randomised double blind multicentre phase study lancet oncol open access van cutsem e tabernero j lakomy r et a0al addition of aflibercept to fluorouracil leucovorin and irinotecan improves survival in a phase iii randomized trial in patients with metastatic colorectal cancer previously treated with an oxaliplatin based regimen j clin oncol yamazaki k nagase m tamagawa h et a0al randomized phase iii study of bevacizumab plus folfiri and bevacizumab plus mfolfox6 as first line treatment 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breast tumor mda mb231 via inhibition of vascular endothelial growth factor receptor vegf r and vegf r3 kinase clin cancer res wiegering a korb d thalheimer a et a0al e7080 lenvatinib a multi targeted tyrosine kinase inhibitor demonstrates antitumor activities against colorectal cancer xenografts neoplasia hong ds kurzrock r wheler jj et a0al phase i dose escalation study of the multikinase inhibitor lenvatinib in patients with advanced solid tumors and in an expanded cohort of patients with melanoma clin cancer res boss ds glen h beijnen jh et a0al a phase i study of e7080 a multitargeted tyrosine kinase inhibitor in patients with advanced solid tumours br j cancer yamada k yamamoto n yamada y et a0al phase i dose escalation study and biomarker analysis of e7080 in patients with advanced solid tumors clin cancer res nakamichi s nokihara h yamamoto n et a0al a phase study of lenvatinib multiple receptor tyrosine kinase inhibitor in japanese patients with advanced solid tumors cancer 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versus sorafenib in first line treatment of patients with unresectable hepatocellular carcinoma a randomised phase non inferiority trial lancet schlumberger m tahara m wirth lj et a0al lenvatinib versus placebo in radioiodine refractory thyroid cancer n engl j med li j qin s xu r et a0al regorafenib plus best supportive care versus placebo plus best supportive care in asian patients with previously treated metastatic colorectal cancer concur a randomised double blind placebo controlled phase trial lancet oncol van cutsem e martinelli e cascinu s et a0al regorafenib for patients with metastatic colorectal cancer who progressed after standard therapy results of the large single arm open label phase iiib consign study oncologist iwasa a0s et a0al esmo open 20205e000776 101136esmoopen2020000776 0copen access ott pa hodi fs buchbinder ei inhibition of immune checkpoints and vascular endothelial growth factor as combination therapy for metastatic melanoma an overview of rationale preclinical evidence and initial clinical data front oncol kato y tabata k kimura t et a0al lenvatinib plus anti pd1 antibody combination treatment activates cd8 t cells through reduction of tumor associated macrophage and activation of the interferon pathway plos one 201914e0212513 taylor mh lee c h makker v et a0al phase ibii trial of lenvatinib plus pembrolizumab in patients with advanced renal cell carcinoma endometrial cancer and other selected advanced solid tumors j clin oncol makker v rasco d vogelzang nj et a0al lenvatinib plus pembrolizumab in patients with advanced endometrial cancer an interim analysis of a multicentre open label single arm phase trial lancet oncol fukuoka s hara h takahashi n et a0al regorafenib plus nivolumab in patients with advanced gastric gc or colorectal cancer crc an open label dose finding and dose expansion phase 1b trial regonivo epoc1603 jco iwasa a0s et a0al esmo open 20205e000776 101136esmoopen2020000776 0c' | Colon_Cancer |
objective cannabinoids are able to reduce tumor growth in xenograft models but their therapeutic potential as anticancer drugs in humans is unclear yet in vitro studies of the effect of cannabinoids on cancer cells are often carried out in absence of serum or in low serum concentration ie serum conditions that limit cellular growth and therefore can increase the response of cells to additional challenges such as the presence of cannabinoids however the tumor microenvironment can be teaming with growth factors in this study we assessed the viability and proliferation of cancer cells treated with cannabidiol in presence of a serum concentration that commonly sustains cell growth serumresults the results show that cannabidiol exerts a markedly different effect on the viability of the human ht cancer cell line when cultured in presence of serum in comparison to serum displaying a cytotoxic effect only in the former situation in presence of serum no inhibitory effect of cannabidiol on dna replication of ht cells was detected and a weak inhibition was observed for other cancer cell lines these results indicate that the effect of cannabidiol is cell contextdependent being modulated by the presence of growth factorskeywords paclitaxel colon cancer cannabidiol serumintroductionthe cannabis plant has a therapeutic potential to treat a wide range of diseases including cancer phytocannabinoids are being tested in a0vitro and in a0vivo for the potential to fight different types of cancer cannabis extracts have recently been described to exert a cytotoxic effect on human cancer cell lines however in a0 vitro cancer models present limitations which reduce their predictive validity one of these limitations is to reproduce the nutritional environment of the cells using cell culture media and growth factors many in a0 vitro cancer studies use historical culture media with fetal calf serum fcs however it is usual correspondence albertosainzcgmailcom gh medical barcelona spainfull list of author information is available at the end of the to eliminate or reduce fcs concentrations ie fcs from the media at the moment of drug exposure to avoid confounding effects of growth factors present in serum as in many studies testing the cytotoxic properties of cannabinoids in cancer cells [ ]the deprivation of survival factors from the media can sensitize cells to a subsequent challenge pirkmajer and chibalin showed that the effects of serum starvation in cell cultures are unpredictable according to eastman serum should be kept in cell cultures to avoid both false positive and negative results due to its effects on cell proliferation stipulating the importance of replicating anic conditions to obtain clinically valid resultsin the present study we analyzed the viability response of different cancer cell lines to cannabidiol cbd in presence of a standard concentration of serum in comparison to a low serum concentration 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 0csainzcort a0et a0al bmc res notes page of main textmaterials and a0methodsmaterialscbd was supplied by schibano pharma ag waldsch¶nengrund switzerland mccoys 5a medium alamarblue® ab invitrogen were bought leibovitzs l15 medium l15 and rpmi and from thermofisher scientific barcelona spain paclitaxel ²6diamidino2phenylindole dapi dimethyl sulfoxide lglutamine penicillinstreptomycin and fcs were bought from sigmaaldrich madrid spain cell proliferation reagent wst1 and 5bromo2²deoxyuridine brdu cell proliferation elisa kit were bought from roche sigmaaldrich madrid spain paclitaxel was dissolved in dimethyl sulfoxide and cbd was dissolved in methanol at a0mm and kept at a0°c for a maximum of a0 months when needed paclitaxel and cbd were diluted conveniently in the cell media at the indicated final concentrations cellular controls without cbd or paclitaxel contained cell media without additivescell cultureht29 cells ref htb38 and sw480 cells ref ccl were obtained from american type culture collection ags cells were kindly provided by miguel a pujana catalan institute of oncology idibell barcelona spain and were originally obtained from nuria sala catalan institute of oncology idibell barcelona spain human colon cancer ht29 cells and sw480 cells were maintained in mccoys 5a and l15 media respectively human gastric cancer ags cells kindly provided by francesca mateo catalan institute of oncology bellvitge institute for biomedical research lhospitalet del llobregat spain were maintained in rpmi medium all of the media was supplemented with penicillinstreptomycin and a0nm lglutamine a0h before treatment cells were plated in 96well plates at cellswell a0 h later wells in triplicates received cbd and paclitaxel all assays with sw480 and ags cells included fcs while the assays using ht29 cells included either or fcscell viability and a0proliferation assaysfor the viability and proliferation assay based on resazurin and its redoxmediated reduction we used ab and measured the fluorescence of the wells using a plate readerfor the viability and proliferation assay based on cleavage of tetrazolium salts by mitochondrial dehydrogenase we used wst1for the proliferation based on the measurement of dna synthesis we added brdu to cells and detected its incorporation into dna following manufacturer instructionsto assess cell viability dapi was added to the cell suspension a0 min before the analysis by flow cytometry dapi emits higher fluorescence when bound to dna dapi enters rapidly through altered cell membranes allowing the detection of damaged cells the cell population was selected by gating in a forward scatter vs side scatter dot plot excluding aggregates and cell debris samples were analyzed using a gallios flow cytometerstatistical analysisdata was analysed using ibm spss statistics and real statistics using excelwe used shapirowilk test to assess data normality and nonparametrical independent samples kruskalwallis test to identify significant differences between each experimental condition we used dunn test as a posthoc analysis to identify which groups show statistically significant differencesresultsviability and a0proliferation of a0ht cells with a0serum deprivation fcswhen human colon cancer ht29 cells were incubated in media with serum adding cbd at a0µm reduced cell viability as assessed via the resazurin method which is based on evaluating mitochondrial reductive capacity fig a0 1a interestingly when cbd concentrations were a0 µm cell viability increased during the first a0 h differences between or and a0 µm were statistically significant p and p at a0h the increasing viability with cbd a0 µm disappeared while the blocking effect of a0µm cbd was more pronounced fig a0 1a this suggests that cbd can induce mitochondrial stress as reported by others looking at the morphology of cells the treatment with a0µm cbd led to changes in cell form such as massive cellular detachment cell rounding and presence of wrinkled cells characteristic of dead cells fig a0 1b in fact analyzing the presence of dead cells using dapi dye we found an increased percentage in samples incubated with a0 µm cbd when compared to control cells fig a01c thus the loss of mitochondrial activity observed at cbd a0 µm correlated with cell death of note at longer incubation times ie a0days massive cellular death was also observable at a0µm cbd data not shown in summary a0µm cbd shows cytotoxic activity on ht29 cells cultured in fcs 0csainzcort a0et a0al bmc res notes page of fig a ht cells were incubated with fcs and different concentrations of cbd for and h cell viability was assessed by incubation with ab the mean sd of three assays are shown b morphology of ht cells incubated with or without μm cbd for h representative images are shown bar µm c ht cell viability according to dapi staining see the materials and methods section ht cells were incubated without top or with μm cbd bottom for h stained with dapi and immediately analyzed by flow cytometry the cursor identifies dapipositive cells dead cells showing a higher percentage in cbdtreated cells a representative experiment c is shown p viability and a0proliferation of a0ht cells in a0 fcscontrary to the drop in viability of cells in fcs cbd did not inhibit the viability of ht29 cells even after a0days in media containing fcs fig a02a b an apparent increase in ht29 cell viability was observed at a0µm cbd as assessed by ab or wst1 fig a0 suggesting mitochondrial stress we sought to find whether in these conditions cbd could show additive or synergistic antiproliferative effects with the therapeutic drug paclitaxel paclitaxel partially decreased the viability of ht29 cells according to ab measurement but not wst1 thus cbd at a0µm does not grossly affect the viability of ht29 cells after a0days culture in presence of serumto ascertain whether cbd had any effect on proliferation of ht29 cells we measured the incorporation of brdu into dna no changes in dna synthesis were observed after a0days of incubation of ht29 cells with any concentration of cbd fig a02c although paclitaxel in itself did inhibit dna synthesis cbd did not increase the effect of paclitaxel fig a02c in summary cbd up to a0µm do not decrease the viability nor the proliferation of ht29 cells cultured in fcs none of these results showed statistically significant differencesviability and a0proliferation of a0sw480 and a0ags cellsto know whether other cancer cell lines behaved similarly to ht29 showing little or no response to cbd when cultured in fcs we used sw480 another colon cancer cell line and ags a gastric cancer cell lineags cells did not show changes of viability by incubation with cbd up to a0µm though a0nm paclitaxel did decrease their viability fig a0 3a higher paclitaxel concentrations resulted in a severe decrease of ags cells viability data not shown so we used a0nm paclitaxel to observe potential effects of cbd the viability of sw480 cells with cbd and fcs showed a trend to decline fig a03c surprisingly and contrary to ht29 cells a0µm cbd did actually impair dna replication in ags and sw480 cells fig a03b d in fact the inhibition of dna replication was additive to that produced by paclitaxel the assessment of dna replication in sw480 cells 0csainzcort a0et a0al bmc res notes page of showed significant differences between the control sample and a0µm cbd without paclitaxel p any other statistic analysis did not show significant resultsin summary in presence of fcs and during a0days of culture cbd does not affect the viability of ht29 sw480 and ags cells though cbd at a0µm does impair the proliferation of ags and sw480 cellsdiscussionin this study we investigated the effects of cbd and its combination with paclitaxel on the viability of three different cancer cells ht29 sw480 and ags under two different concentrations of serum a standard appropriate for cell growth for ht29 sw480 and ags and a restrictive one of for ht29 only for ht29 cells cbd only reduces cell viability under low fcs with no effects on viability or dna replication when cells were in fcs however for sw480 and ags dna replication was impaired under a0µm cbd with serum moreover the inhibition of dna replication in sw480 and ags cells by cbd and paclitaxel had an additive effectat low cbd concentrations ht29 cells showed a trend towards increased cell viability though the differences were not significant different concentrations of cbd have previously been shown to have opposing effects on cells thus a0µm cbd induces proliferation of t leukemia cells but at higher concentration kills the cells a low concentration cbd increases mitochondrial ca2 augmenting mitochondrial metabolism and cell growth but at high concentration it leads to fig ht cells were incubated for days with fcs and different concentrations of cbd in absence or presence of nm paclitaxel a the viability was assessed by incubation with ab the mean sd are shown n b the viability was assessed by incubation with wst the mean sd are shown n c before harvesting cells were incubated with brdu for h which incorporated into dna and dna synthesis was quantified the mean sd are indicated n fig ags cells and sw480 cells were incubated for days with different concentrations of cbd in absence or presence of nm paclitaxel ags or nm paclitaxel sw480 a c cell viability was assessed by incubation with ab the mean sd of three ags and six sw480 assays are shown b d before harvesting cells were incubated for h with brdu which incorporated into dna and dna synthesis was quantitated the mean sd of three assays ags and assays sw480 are shown p 0csainzcort a0et a0al bmc res notes page of excessive mitochondrial ca2 mitochondrial dysfunction and cell death appropriate culturing conditions are essential for the survival and growth of cells in many studies cell culture conditions are not sufficiently detailed which is essential for study replication one possible solution to address the potential effect of serum could be using culture media without fcs so the media does not need to be altered during drug exposition in any case neither higher serum concentrations nor lower serum concentrations represent the proper microenvironment of a cancer cell in the human body and both approaches could be valid to test the effects of a drug on cell lines the tumor microenvironment is enriched with metabolites including lactate and adenosine [ ] which increases tumor growth and may modulate the therapeutic effect of a drug in tumors that are highly glycolytic increasing mitochondrial activity as exerted by cbd may add metabolic stress to cells forcing them to decreased growth the effect of a drug on cells can be assessed effectively if the experimental conditions of the treatment are the same as the growing conditions before the treatment once growing conditions and treatment conditions differ from more than one variable drug treatment then the resulting effects cannot be associated only to the treatment but to the combination of variableslimitationsour results did not show statistically significant differences with the exception of the assessment of viability of ht29 cells under cbd treatment and the assessment of dna replication of sw480 under a0µm cbd the lack of statistically significant results could be due to the small sample size n for most of the assays our study was also not able to replicate the strongly inhibitory effect of cbd shown in other studies where cannabinoids were tested against cancer cells cultured with fcs fcs contains many growth factors and nutrients and differences in the fcs source could substantially modify the viability proliferation and differentiation of cultured cells there are also other studies where cancer cells were cultured with fcs and treated with cbd or other synthetic cbdlike molecules the results of these studies showed that cbd a0μgml reduced the viability of cancer cells and also had effects on other survival variables [ ] the cell lines used in these studies being different to the ones used in our study could account for the different results observedabbreviationsab alamarblue brdu bromo²deoxyuridine cbd cannabidiol dapi ²diamidinophenylindole fcs fetal calf serumacknowledgementswe would like to thank manuel reina for his expert adviceauthors contributionsschibano pharma ag participated in the idea of the study as and ee designed the study as and ee acquired analyzed and interpreted the data cm provided technical assistance and carried out some experiments as and ee drafted the work all authors read and approved the final manuscriptfundingthis study was partially funded by schibano pharma ag waldsch¶nengrund switzerland and gh medical barcelona spain the design of the study was prepared by as ee and cm and approved by schibano pharma ag and gh medicalavailability 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 participatenot applicableconsent for publicationnot applicablecompeting interestsas was employee at gh medical while performing this projectauthor details gh medical barcelona spain celltecub department of cell biology physiology and immunology faculty of biology university of barcelona av diagonal barcelona spain received may accepted august references ackermann t tardito s cell culture medium formulation and its implications in cancer metabolism trends cancer https doi101016jtreca n201905004 brand a singer k koehl ge kolitzus m schoenhammer g thiel a matos c bruss c klobuch s peter k kastenberger m bogdan c schleicher u mackensen a ullrich e fichtnerfeigl s kesselring r mack m ritter u schmid m blank c dettmer k oefner pj hoffmann p walenta s geissler ek pouyssegur j villunger a steven a seliger b schreml s haferkamp s kohl e karrer s berneburg m herr w muellerklieser w renner k kreutz m ldhaassociated lactic acid production blunts tumor immunosurveillance by t and nk cells cell metab https 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k buta c cochrane b dirks wg fu j hickman jj hohensee c kolar r liebsch m pistollato f schulz m thieme d weber t wiest j winkler s gstraunthaler g fetal bovine serum fbs pastpresentfuture altex https doi1014573 altex wu hy huang ch lin yh wang cc jan tr cannabidiol induced apoptosis in human monocytes through mitochondrial permeability transition poremediated ros production free radical biol med https doi101016jfreer adbio med201806023publishers 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 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bcl9 and pygo are bcatenin cofactors that enhance the transcription of wnt targetgenes they have been proposed as therapeutic targets to diminish wnt signaling output inintestinal malignancies here we find that in colorectal cancer cells and in developing mouseforelimbs bcl9 proteins sustain the action of bcatenin in a largely pygoindependent mannerour genetic analyses implied that bcl9 necessitates other interaction partners in mediating itstranscriptional output we identified the transcription factor tbx3 as a candidate tissuespecificmember of the bcatenin transcriptional complex in developing forelimbs both tbx3 and bcl9occupy a large number of wntresponsive regulatory elements genomewide moreover mutationsin bcl9 affect the expression of tbx3 targets in vivo and modulation of tbx3 abundance impactson wnt target genes transcription in a bcatenin and tcflefdependent manner finally tbx3overexpression exacerbates the metastatic potential of wntdependent human colorectal cancercells our work implicates tbx3 as contextdependent component of the wntbcatenindependenttranscriptional complexintroductionthe wnt pathway is an evolutionarily conserved cell signaling cascade that acts as major drivingforce of several developmental processes as well as for the maintenance of the stem cell populations within adult tissues nusse and clevers deregulation of this signaling pathway resultsin a spectrum of consequences ranging from lethal developmental abnormalities to several forms ofaggressive cancer nusse and clevers most prominently colorectal cancer crc is initiatedby genetic mutations that constitutively activate wnt signaling kahn secreted wnt ligands trigger an intracellular biochemical cascade in the receiving cells that culminates in the calibrated expression of target genes mosimann this transcriptionalresponse is orchestrated by nuclear bcatenin that acts as a scaffold to buttress a host of cofactorsto cisregulatory elements occupied by the tcflef transcription factors valenta among the cofactors the two paralogs bcl9 and bcl9l referred to as bcl99l and pygo12proteins reside within the wntbcatenin transcriptional complex and their concerted action isrequired to efficiently activate wnttarget gene expression kramps parker for correspondencekonradbaslerimlsuzhch kbandreasmoorbsseethzchaemclaudiocantuliuse cc these authors contributedequally to this workpresent address ¡division ofmolecular pathology thenetherlands cancer instituteamsterdam netherlandscompeting interests theauthors declare that nocompeting interests existfunding see page received april accepted august published august reviewing editor roel nussestanford university unitedstatescopyright zimmerli this is distributed under theterms of the creative commonsattribution license whichpermits unrestricted use andredistribution provided that theoriginal author and source arecreditedzimmerli elife 20209e58123 107554elife58123 of 0cshort reportdevelopmental biologyfigure the intestinal epitheliumspecific recombination of pygo12 does not recapitulate the effects of deleting bcl99l a schematic representationof the wntbcatenin transcriptional complex with emphasis on the socalled chain of adaptors components bcatenin bcl99l and pygo wntfigure continued on next pagezimmerli elife 20209e58123 107554elife58123 of 0cshort reportfigure continueddevelopmental biologyresponsive element wre the homology domains hd13 of bcl99l are shown b epithelialspecific pygo12 deletion via vilcreert2 pygo12ko does not lead to any obvious histological or functional defect neither in the small intestine nor in the colon as seen by hematoxylin and eosinstaining left panels the proliferative compartment detected via ki67 right panels seems also unaffected also refer to the count in figure figuresupplement 1de c quantitative rtpcr detecting lgr5 mrna extracted from colonic epithelium of control black bcl99l blue or pygo12 redconditional mutants ko d weekold male mice were treated with five tamoxifen tam injections ip mgday for five consecutive days days later mice were treated with dextran sodium sulfate dss ad libitum in drinking water for days while of control mice n wereseverely affected or died due to the dss treatment red lines of conditional bcl99lko n mice performed poorly in this test deletion ofbcl99l increased significantly the death rate after dss treatment pvalue000013 in fishers exact test no difference between pygo12ko andcontrol mice could be measured of control mice n and of pygo12ko n were affected upon dss treatment pvalue05626 infishers exact test e weekold female mice were exposed to a single dose of the carcinogenic agent azoxymethane aom followed by daysof dss administration in the drinking water this regimen results in the emergence of dysplastic adenomas that are collected for rna extraction andanalysis of the indicated targets via rtpcr wnt target genes and genes expressed during epithelialtomesenchymal transition emt associated withcancer metastasisthe online version of this includes the following figure supplements for figure figure supplement efficient epithelialspecific pygo12 deletion does not lead to obvious defectsfigure supplement intestinal epitheliumspecific recombination of pygo12 does not recapitulate the effects of deletingbcl99l van tienen figure 1a during vertebrate development their requirement in thebcateninmediated transcription appears to be contextdependent cantu li and they also have evolved bcateninindependentfunctions cantu cantu curiously however bcl9 and pygo always seem to act as a duetkennedy importantly bcl99l and pygo proteins were found to significantly contribute to the malignanttraits typical of wntinduced crcs deka gay jiang mani mieszczanek moor talla and brembeck theseobservations provided impetus to consider the bcl9pygo axis as relevant targetable unit in crclyou mieszczanek talla and brembeck zimmerli however here we noticed an apparent divergence between the roles of bcl99l and pygo proteins we found that genetic abrogation of bcl99l in mouse crc cells results in broader consequences than pygo12 deletion suggesting that bcl9 function does not entirely depend on pygo12among the putative bcateninbcl9 interactors we identified the developmental transcription factortbx3 intriguingly we show that also during forelimb development bcl99l possess a pygoindependent role in this in vivo context tbx3 occupies bcateninbcl9 target loci genomewide andmutations in bcl99l affect the expression of tbx3 targets finally tbx3 modulates the expression ofwnt target genes in a bcatenin and tcflefdependent manner and increases the metastaticpotential of human crc cells when overexpressed we conclude that tbx3 can assist the wntbcatenin mediated transcription in selected developmental contexts and that this partnership could beaberrantly reactivated in some forms of wntdriven crcsresults and discussionwe induced intestinal epitheliumspecific recombination of pygo12 loxp alleles pygo12ko thatefficiently deleted these genes in the whole epithelium including the stem cells compartment figure figure supplement 1a and b consistently with recent reports mieszczanek talla and brembeck and similarly to deletion of bcl99l deka mani moor pygo12ko displayed no overt phenotypic defects figure 1b figure figure supplement 1ce we were surprised in noticing that the expression of lgr5 themost important intestinal stem cell marker and wnt target gene barker was heavilydownregulated upon loss of bcl99l but unaffected in pygo12ko figure 1c to address the functionality of the stem cell compartment in these two conditions we subjected both bcl99l andpygo12 compound mutants koregeneration by dss treatmentkim figure 1d while bcl99lko mice showed a defect in regeneration after insultdeka pygo12ko proved indifferent when compared to controllittermatesfigure 1d while we cannot exclude that pygo12 also contributes to the wntbcateninto a model ofintestinalzimmerli elife 20209e58123 107554elife58123 of 0cshort reportdevelopmental biologydependent transcriptional regulation our results highlight that the bcl99l function in the intestinalepithelium homeostasis and regeneration does not entirely depend on pygo12 this was surprising since bcl99l proteins were thought to act as mere bridge proteins that tethered pygo tothe bcatenin transcriptional complex figure 1a fiedler mosimann both bcl9 and pygo proteins have been implicated in colorectal carcinogenesis gay jiang mieszczanek talla and brembeck we tested if the consequence of the deletion of bcl99l and pygo12 genes was also different in the context of carcinogenesis specifically we looked at the contribution to gene expression in chemicallyinduced aomdsscolorectal tumors figure 1e as previously observed bcl99lko tumors exhibit a massive decreasein wnt target gene expression epithelialtomesenchymal transition emt and stemness traitsdeka moor which was not observed in pygo12ko tumors figure 1efigure figure supplement 2a and b this phenotypic difference is consistent with a recentstudy in which bcl99l but not pygo12 loss reduced the activation of wnt target genes induced byapc lossoffunction mieszczanek we interpret this as an independent validation ofour observation all these experiments open up the question of how bcl99l imposes its functionindependently of pygosurprisingly the intestinespecific deletion of the homology domain hd1 of bcl99lfigure 2a that was previously annotated to interact only with pygo12 cantu kramps i suppressed the metastatic phenotype of the aomdss tumors while deletion of pygo12 did not figure 2b and ii induced a strong downregulation of wnt target emt andstemness genes figure 2c figure figure supplement the discrepancy between the geneexpression changes induced by recombining pygo12 or deleting the hd1 domain of bcl99l impliesthat currently unknown proteins assist bcl99l function we set out to identify new candidate bcl9partners that might be responsible for the different phenotypes to this aim we performed a pulldown of tumor proteins expressing either a fulllength or a hd1deleted variant of bcl9 followedby mass spectrometry figure 2d among the proteins differentially pulled down by control but notby mutant bcl9 we detected tbx3 figure 2d and e and selected it for further validation the invivo deletion of the hd1 domain in bcl99ldhd1 embryos leads to severe forelimb malformationswhile pygo12ko embryonic forelimbs are unaffected figure 2falso see schwab limb development thus represents another context where bcl99l appear to act independently ofpygo of note tbx3 plays a fundamental role in the development of this structure frank we confirmed cytological vicinity between transfected tagged versions of bcl9 and tbx3 byproximity ligation assay pla figure figure supplement 2ab however overexpressionbasedin vitro coimmunoprecipitation experiments could not detect any stable interaction between thesetwo proteins suggesting absence of direct binding or a significantly lower affinity than that betweenbcl9 and pygo figure figure supplement 2c hence we aimed at testing the functional association between tbx3 and bcl9 in a more relevant in vivo context to this aim we collected ca forelimbs from dpc wildtype mouse embryos and subjected the crosslinked chromatin toimmunoprecipitation using antibodies against bcl9 salazar or tbx3 followed bydeepsequencing of the purified dna chipseq figure 3a by using stringent statistical parameters and filtering with irreproducible discovery rate idr we extracted a list of high confidencebcl9 and tbx3 peaks figure 3b and c surprisingly we discovered that bcl9 occupies a largefraction ca 23rd of the tbx3bound regions figure 3d suggestive of a role for tbx3 within thewntdependent transcriptional apparatus motif analysis of the common tbx3bcl9 target loci identified statistical prevalence for tcflef and homeobox transcription factor consensus sequencesbut not for any tbx transcription factor figure 3e this suggests that tbx3 interacts with the dnain these locations via affinity to the wntbcatenin cofactors rather than via direct contact with dnaaccordingly tbxspecific motifs were detected within the group of tbx3 exclusive peaks which donot display bcl9 binding figure figure supplement notably tbx3 and bcl9 occupancywas detected at virtually all previously described wntresponsiveelements wre within known wnttarget genes figure 3fto test whether the in vivo abrogation of the simultaneous interactions mediated by bcl99lwould influence the expression of genes associated with tbx3 peaks we set out to mutate the bcl99l interaction domains while leaving tbx3 protein unaffected we combined different bcl99l allelesin which the hd2 bcatenininteracting and hd1 pygonew cofactorinteracting motifs arezimmerli elife 20209e58123 107554elife58123 of 0cshort reportdevelopmental biologyfigure identification of tbx3 as a putative bcl9 cofactor a the deletion of the hd1 pygointeracting domain of bcl9 and bcl9l induces avariation in the chain of adaptors causing the loss of pygo association with the wntbcatenin transcriptional complex cantu bimmunofluorescence staining of tumors collected from control or conditional pygo12ko and bcl99lko mice prox1 red and dapi blue are shownin in the top panels vimentin green and laminin red in the bottom panels c quantitative rtpcr of selected groups of targets compare it withfigure continued on next pagezimmerli elife 20209e58123 107554elife58123 of 0cshort reportfigure continueddevelopmental biologythe same analysis of pygo12ko in figure 1e of rna extracted from control or bcl99ldhd1 tumors d experimental outline of the tumor proteinspulldown and massspectrometry tbx3 was identified among the proteins potentially interacting with bcl9 but not with bcl9dhd1 e the ipproteins analyzed by mass spectrometry were in parallel subjected to sds page electrophoresis and probed with an antitbx3 antibody upper panelthe expression of tbx3 in control compared to bcl99ldhd1 tumors n was evaluated via qrtpcr bottom panel to exclude that differentialpulldown was due to lost expression in mutant tumors f dpc bcl99ldhd1 embryos display forelimb malformations and absence of digitsemphasized by dashed white lines a characteristic tbx3mutant phenotype upper panels the limb defect is absent in pygo12ko embryosbottom panels underscoring that bcl99l act in this context independently of pygo12the online version of this includes the following figure supplements for figure figure supplement qrtpcr of target genes associated with intestinal stem cell functionfigure supplement cytological proximity of bcl9 and tbx3deleted cantu double heterozygous animals for the hd1 bcl9dhd1 bcl9ldhd1 orthe hd2 bcl9dhd2 bcl9ldhd2 deletions are viable and fertile the cross between them leads to atransheterozygous genetic configuration in which both domain deletions are present bcl9dhd1dhd2bcl9ldhd1dhd2 referred to as bcl99ld1d2 figure 1a as in these mice bcl99l retain both thehd1 and the hd2 domains in heterozygosity this allelic combination is a way of testing the consequences of abrogating the tripartite complex mediated by the two interacting motifs of bcl99lwithout causing a full lossoffunction of these proteins bcl99ld1d2 embryos also display forelimbmalformations the cause of which cannot be due to pygo figure 2f but must be caused by thefailure of recruiting the new hd1interacting partner by bcl99l onto the bcatenin transcriptionalcomplex we collected forelimbs from control and bcl99ld1d2 mutant embryos at and measured gene expression via rnaseq figure 3g we found a significant enrichment hypergeometrictest p14e6 of tbx3 targets among the genes differentially expressed in bcl99ld1d2 mutantsfigure 3h the enrichment was particularly significant when considering downregulated genes inbcl99ld1d2 mutants indicating that the bcl9tbx3 partnership sustains transcriptional activationfigure 3h of note the design of our experiment directly implicates that these tbx3 transcriptional targets are also bcatenindependent the overlap list includes several regulators of limbdevelopment such as meis2 capdevila irx3 li and eya2 grifone figure 3h heat map on the right despite being of correlative nature this analysis supportsa model in which bcl99l and tbx3 cooperate to the activation of target genesso far we have presented genetic evidence that bcl9 proteins require additional cofactors andthat tbx3 associates with the bcateninbcl9 bound regions on the genome possibly influencing theexpression of target genes however the similarity of genomic binding profiles between tbx3 andbcl9 might be due to their binding in different cells and the decreased expression of genes withnearby enhancers bound by bcl9 and tbx3 might imply a requirement for bcl99l but not necessarily for tbx3 we reasoned that our hypothesis in which bcl9 functionally tethers tbx3 to the bcatenin transcriptional complex raises several testable predictions that will be addressed belowfirst our model implies that tbx3 could impact on wnt target gene expression and its activityshould be dependent on the main constituents of the wntbcatenin transcriptional complex second if tbx3 is tethered by bcl9 to its targets mutations in bcl99l should influence the ability oftbx3 to physically associate with wres finally as for bcl9 tbx3 should be capable of enhancingthe metastatic potential of colorectal cancer cellsto test our first prediction implying a potential role of tbx3 in the transcription of wnt targetgenes we overexpressed it in hek293t cells and monitored the activation status of wnt signalingusing the transcriptional reporter supertopflash stf consistent with its role as repressor tbx3led to a moderate but significant transcriptional downregulation that was importantly specific tothe stf but not the control reporter plasmid figure 4a upon wnt signaling activation achievedvia gsk3 inhibition tbx3overexpressing cells exhibited a markedly increased reporter activity whencompared to control cells in particular at nonsaturating pathway stimulating conditions figure 4aleft panel importantly tbx3 proved transcriptionally incompetent on the stf if the cells carriedmutations in tcflef d4tcf or ctnnb1 encoding for bcatenin dbcat doumpas strongly supporting the notion of its cellautonomous involvement in the activation of canonical wnttarget gene transcription figure 4a central and right panels respectively endogenous wnt targets showed a similar expression behaviour to that of stf upon tbx3 overexpression figure zimmerli elife 20209e58123 107554elife58123 of 0cshort reportdevelopmental biologyfigure tbx3 and bcl9 occupy wnt responsive elements wre in vivo a artistic representation of the chipseq experimental outline bc barplots showing the genomic distribution of highconfidence bcl9 peaks b total and tbx3 peaks c total d overlap of the highconfidence peak groups between bcl9 and tbx3 e selected result entries from motif analysis performed on the bcl9tbx3 overlapping highconfidence peaks significant enrichment was found for tcflef and homeobox motifs no tbx consensus sequence was detected in this analysis ffigure continued on next pagezimmerli elife 20209e58123 107554elife58123 of 0cshort reportfigure continueddevelopmental biologyselect genomic tracks demonstrating occupancy of bcl9 and tbx3 within the wnt responsive element wre of known wnttarget genes axin2ccnd1 nkd1 and lef1 and genes important in limb morphogenesis hand1 and hand2 the scale of peak enrichment is indicated in the topleftcorner of each group of tracks in light blue the bcl9 salazar and in orange the tbx3 replicates and in green the control track igggenomic tracks are adapted for this figure upon visualization with igv integrative genomic viewer igv two independent replicates forbcl9 and tbx3 chipseq experiments are shown g volcano plot displays all the differentially expressed genes degs in developing forelimbs uponmutation of bcl99l bcl99ld1d2 vs ctrl degs were a total of p005 with upregulated and downregulated n of individualmouse embryos for each condition were used for this analysis h a significant portion of degs exhibited overlap with tbx3 chipseq peaksthe overlap with tbx3 chipseq peaks appeared statistically significant in particular when the downregulated genes were considered hierarchicalclustering of samples ctrl versus bcl99ld1d2 right panel based on genes overlapping between degs and genes annotated for tbx3 chipseqpeaks normalized rnaseq read counts wards clustering method euclidian distance annotation added for genes associated by gene ontology townt signaling fgf10 ptk7 kremen1 zfp703 bmp2 and gli3 and genes known as regulators of limb development meis2 irx3 and eya2the online version of this includes the following figure supplements for figure figure supplement overlap of the highconfidence bcl9 and tbx3 peaks in developing murine limbs reveals the existence of bcl9 exclusive oneexample displayed in the genomic tracks on the left and tbx3 exclusive peaks one example in the genomic track on the rightfigure supplement while our experiments show that tbx3 can influence the expression of wnttarget genes the mechanisms by which this occurs remain to be elucidatedwe then addressed our second hypothesis in which bcl99l are required for tethering tbx3onto wres we performed chip of tbx3 in hek293t cells followed by quantitative pcr to detectenrichment on the wre present in the axin2 promoter jho consistent with an effecton transcription in the absence as well as in the presence of chir99021 chir figure 4a tbx3 wasbound to this region both in off and in on conditions figure 4b we then exploited ahek293t clone devoid of both bcl9 and bcl9l db99l van tienen and tested iftbx3 was capable of physical association with the wre of note enrichment of tbx3 in db99 l cellswas dramatically reduced to background levels figure 4b while we cannot exclude that tbx3might act independently of bcl99l on several of its targets this observation supports the notionthat bcl99l are responsible for tbx3 recruitment on classical wres figure 4c this also suggeststhat the previously identified targets of both bcl9 and tbx3 figure 3df must display simultaneous cooccupancy of these two factors in agreement with the notion that bcl99l are themselvesrecruited by the tcfbcatenin axis the physical association of tbx3 with the axin2 promoter wasalso lost in d4tcf and dbcat cells figure 4bfinally we evaluated the effects of tbx3 overexpression oe on growth and metastatic potentialof hct116 human colorectal tumor cells a representative model of crc driven by activating mutations in ctnnb1 mouradov using a in vivo zebrafish xenograft model rouhi approximately labelled control or tbx3oe hct116 cells were implanted in theperivitelline space of hours postfertilization hpf zebrafish embryos figure 4d three days afterinjection tbx3oe cells displayed a marked increase in number in the caudal hematopoietic plexusfigure 4ef the main metastatic site for cells migrating from the perivitelline space rouhi of note tbx3oe hct116 cells maintained consistently high expression of tbx3 within fishembryos throughout the experiment and this was accompanied by increased wntbcatenindependent transcription as measured by axin2 expression figure 4g while this experiment does notallow to exclude that tbx3 might also act independently of bcl9bcatenin in this context it showsthat increased expression of tbx3 enhances proliferation and migratory capability of human crccells bearing constitutively active wnt signaling and this is associated with simultaneous enhancement of the wntbcatenindependent transcription figure 4gtaken together our experiments show that in specific developmental and disease contexts thetranscription factor tbx3 can take active part in the direct regulation of wnt target genes by functional interplay with the bcateninbcl9dependent transcriptional complex our study suggests anew paradigm in which tissuespecific cofactors might be the key to understand the spectrum ofpossible transcriptional outputs observed downstream of wntbcatenin signaling nakamura moreover tbx3 has been linked to different cancer types willmer our observations suggest that tbx3 or its downstream effectors could be considered as new relevant targetsto dampen crc progressionzimmerli elife 20209e58123 107554elife58123 of 0cshort reportdevelopmental biologyfigure tbx3 controls the expression of wnt target genes a bcatenintcf luciferase reporter stf assay in parental left bcatenin knockout dbcat center and tcf knockout d4tcf right hek293t cells cells were treated with the indicated concentration of chir or dmso overnightoverexpression of tbx3 oe black bars compared to control ev empty vector white bars showed that tbx3 acts as a repressor on a wnttcfpathway reporter but switches to activator upon pathway induction only significant pvalues p005 are indicated three independent experimentsfigure continued on next pagezimmerli elife 20209e58123 107554elife58123 of 0cshort reportfigure continueddevelopmental biologyn are shown note that the logarithmic scale on the yaxis of the histogram on the left is different from the linear scale of the central and middlepanels b chip followed by qpcr in hek293t cells treated with dmso wntoff or chir wnton enrichment was identified on axin2promoter and the downstream enhancer note that the enrichment on the enhancer is only present upon pathway stimulation we interpret this asevidence for the enhancer looping onto the promoter occurring when the wntdependent transcriptional regulation is active the data are normalizedto immunoprecipitation performed in cells transfected with an empty vector ev and presented as the mean ± standard deviation of independentexperiments the fold enrichment of tbx3flag on axin2 promoter and enhancer n is lost upon mutations in bcl99l db99l n cnttb1dbcat n and tcflef d4tcf n c schematic representation of the axin2 locus indicates the position of the primers used black arrowsto test the binding of tbx3 despite the apparent absence of direct physical interaction between tbx3 and bcl99l the data support a model of tbx3recruitment by bcl99l onto the bcatenintcf transcriptional complex d schematic diagram of the human crc zebrafish xenografts model parentaland tbx3 overexpressing hct116 colorectal tumor cells were harvested and labeled with dii dye red the stained cells were injected into theperivitelline space of day old zebrafish embryos zebrafish were visualized with fluorescent microscopy at day post injection dpi and three dpi andprimary tumor cell invasion and metastasis were counted e representative images of hct116 tumor invasion and dissemination at and dpi inzebrafish xenografts for both control and tbx3 overexpressing cells the red asterisks indicate the position of the primary tumor red arrowheadspoint at clusters of disseminatingmetastatic cells f scatter plot representing the quantification of primary tumor growth and metastasis after hct116xenograft horizontal bars represent the mean value only significant pvalues p005 are displayed g quantitative rtpcr confirmed continuedincreased expression of tbx3 while hct116 disseminate through zebrafish tissue and that this is accompanied by enhanced wntbcatenintranscription as seen by axin2 expression each datapoint represents the extraction of total rna from pools of zebrafish embryos figure legendof figure supplementsthe online version of this includes the following figure supplements for figure figure supplement axin2 and nkd1 are here considered as representative wnt transcriptional targetsmaterials and methodstreatment of mice and histological analyseshomeostasis weekold male and female mice bcl9floxfloxbcl9lfloxflox vilcreert2 and bcl9floxfloxbcl9lfloxflox no cre littermates pygo1floxfloxpygo2floxflox vilcreert2 and bcl9floxfloxbcl9lfloxflox no cre were treated with five tamoxifen injections ip mgday sigma for five consecutivedays and the small intestine and colon were analyzed at different time points thereafter mouseexperiments were performed in accordance with swiss guidelines and approved by the veterinarianoffice of vaud switzerlandinduction of dss colitis weekold male mice were treated with five tamoxifen injections ip mgday for five consecutive days days later dss mg mp biomedicals catno was administered ad libitum in the drinking water for daysinduction of tumors weekold female mice were treated with five tamoxifen injections ip mgday for five consecutive days days later they were injected ip with mgkg body weightdmh 2hcl nn dimethylhydrazine dihydrochloride after another days later dss was administered ad libitum in the drinking water for daysmice were monitored clinically for rectal bleeding prolapse and general signs of morbidityincluding hunched posture apathetic behavior and failure to groomthe relative body weight in was calculated as follows x weight at a certain dayweight atthe first day of dss treatment epithelial damage of dss treated mice was defined as percentage ofdistal colon devoid of epitheliumto determine proliferation rates mice were injected ip with mgkg brdu sigma hr priorto sacrifice small intestines and colons divided into three equal segments to be named proximalmiddle and distal colon were dissected flushed with cold pbs cut open longitudinally and fixed in paraformaldehyde for hr at rt and paraffin embedded sections mm were cut and used forhematoxylineosin and alcian blue staining and for immunohistochemistry the primary antibodiesused were rabbit antisynptophysin dako rabbit antilysozyme dako mouse antiki67 novocastra mouse antibrdu sigma antibcatenin bd pharmigenantiactive caspase cell signalingthe peroxidaseconjugated secondary antibodies used were mouse or rabbit envision dakoor mouse antirat hrp biosourcezimmerli elife 20209e58123 107554elife58123 of 0cshort reportdevelopmental biologyrealtime pcr genotypingto determine the deletion rate the intestinal epithelium was separated from the underlining musclethe intestine was dissected flushed with pbs cut open longitudinally and incubated in mm ethylenediamine tetraacetic acid edta and mm dithiothreitol dtt in pbs for hr at rt on arotor the tubes were shaken vigorously the muscle removed and the epithelium centrifuged andused for genomic dna extraction sybr green realtime pcr assays were performed on each sample analyzedchromatin immunoprecipitationforelimb buds were manually dissected from ca rjorlswiss outbred dpc mouse embryoschromatin immunoprecipitation was performed as previously described cantu brieflythe tissue was dissociated to a single cell suspension with collagenase 1mgml in pbs for hr at Ëc washed and crosslinked in ml pbs for min with the addition of mm ethylene glycolbissuccinimidyl succinatethermo scientific waltham ma usa for proteinprotein crosslinkingsalazar and formaldehyde for the last min of incubation to preserve dnaprotein interactions the reaction was blocked with glycine and the cells were subsequently lysed in ml hepes buffer sds tritonx m nacl mm edta mm egta mmhepes chromatin was sheared using covaris s2 covaris woburn ma usa for min with the following set up duty cycle max intensity max cyclesburst max mode power tracking the sonicated chromatin was diluted to sds and incubated overnight at Ëc with mg of antibcl9abcam ab37305 or antitbx3 santacruz sc17871 or igg and ml of protein ag magneticbeads upstate the beads were washed at Ëc with wash buffer sds deoxycholate triton x100 m nacl mm edta mm egta mm hepes wash buffer sds sodium deoxycholate triton x100 m nacl mm edta mm egta mmhepes wash buffer m licl sodium deoxycholate np40 mm edta mmegta mm hepes and finally twice with tris edta buffer the chromatin was eluted with sds m nahco3 decrosslinked by incubation at Ëc for hr with mm nacl extractedwith phenolchloroform and ethanol precipitated the immunoprecipitated dna was used as inputmaterial for dna deep sequencing the pull downs | Colon_Cancer |
ammation is an established risk factor for colorectal cancer we and others have shown that colorectal cancerpatients with elevated cysteinyl leukotriene receptor cyslt2r and 15hydroxyprostaglandin dehydrogenase15pgdh levels exhibit good prognoses however both cyslt2r and 15pgdh which act as tumour suppressorsare often suppressed in colorectal cancer we previously reported that leukotriene c4 ltc4induced differentiationin colon cancer via cyslt2r signalling here we investigated the involvement of hedgehog hhgli1 signallingwhich is often hyperactivated in colorectal cancer we found that the majority of colorectal cancer patients hadhighgli1 expression which was negatively correlated with cyslt2r 15pgdh and mucin2 and overall survivalcompared with the lowgli1 group ltc4induced 15pgdh downregulated both the mrna and protein expressionof gli1 in a protein kinase a pkadependent manner interestingly the ltc4induced increase in differentiationmarkers and reduction in wnt targets remained unaltered in gli1knockdown cells the restoration of gli1 in15pgdhknockdown cells did not ameliorate the ltc4induced effects indicating the importance of both 15pgdhand gli1 ltc4mediated reduction in the dclk1 and lgr5 stemness markers in colonospheres was abolished incells lacking 15pgdh or gli1 both dclk1 and lgr5 were highly increased in tumour tissue compared with thematched controls reduced mucin2 levels were observed both in zebraï¬sh xenografts with gli1knockdown cellsand in the cysltr2 colitisassociated colon cancer cac mouse model furthermore gli1 expression waspositively correlated with stemness and negatively correlated with differentiation in crc patients when comparingtumour and mucosal tissues in restoring 15pgdh expression via cyslt2r activation might beneï¬tcolorectal cancer patientsintroductioncolorectal cancer crc one of the most prevalentcancers in the world has a high metastatic efï¬cacy and alow 5year survival rate1 a nontargeted therapeuticapproach combined with late diagnosis leads to poorprognosis and treatment failure more than of crccorrespondence anita sj¶lander anitasjolandermedluse1cell and experimental pathology department of translational medicine lunduniversity sk¥ne university hospital malm¶ sweden2chemical biology therapeutics group department of experimental medicalscience lund university lund swedenfull list of author information is available at the end of the intracellular mechanismscases exhibit anomalous apcwntcatenin signallingwhich regulates the progression of crc by adopting differentthus affecting cancerstem cells and interactions with the tumour microenvironment hedgehog hh signalling which regulatesdifferentiation under physiological conditions has attracted attention because of its emerging role in the promotion and maintenance of crc2 in the untransformedcolon hh ligands are secreted by epithelial cells targetingmesenchymal cells as a classic paracrine hh signallingpathway to ensuring the proper size and location of thecryptvillus axis5 as also observed in other tissues6 the authors open access this is licensed under a creative commons attribution international license which permits use sharing adaptation distribution and reproductionin any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the creative commons license and indicate ifchanges were made the images or other third party material in this are included in the s creative commons license unless indicated otherwise in a credit line to the material ifmaterial is not included in the s creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this license visit httpcreativecommonslicensesby40oncogenesis 0csatapathy oncogenesis page of in crc abnormal hh signalling functions in a liganddependent manner and is activated in human cc celllines7 and xenograft models4 however the role of hhsignalling and its importance in cell survival in crc arenot well deï¬ned although some previous studies havefailed to derive a positive correlation between hh signalling and crc initiation and maintenance89 major bodiesof evidence point to a positive correlation347 moreoverprevious reports have suggested high activity ofthehhsmogli axis in crc cell survival and metastasiswhich is coordinated by either canonical signalling viasmo or a noncanonical mode of activation via therasmap kinase pathway47 within these pathways themost prominent factors are gliomaassociated oncogenehomologue gli and and the transcriptional regulators downstream of smo which keep the oncogenicpathway activecrc which is considered to be an ammationassociated cancer is greatly uenced by ammatorymediators such as leukotrienes and prostaglandins whichbelong to the gproteincoupled receptor family thecysteinyl leukotriene receptors cysltrs cyslt1r andcyslt2r are activated by binding with their highafï¬nityligands leukotriene d4 and c4 ltc4 respectively1011these proammatory lipid mediators are derived fromthe arachidonic acid pathway via 5lipoxygenase and theyplay crucial roles in pathological ammation such asthat observed in ammatory bowel disease we previously showed that elevated cyslt1r levels were associated with poor prognoses in crc patients whilepatients with high cyslt2r expression had better prognoses12 another important group of eicosanoids areprostaglandins pgs which are produced via the cox2pathway the upregulation of cox2 in crc increasesthe pge2 level which promotes cancer cell proliferationangiogenesis survival migration and invasion these areimportant hallmarks of cancer1314thetumoursuppressor15hydroxyprostaglandindehydrogenase 15pgdh is an enzyme responsible forthe degradation of pge2 into an inactive metabolite15 pgdh is abundantly expressed in normal colon mucosabut its expression is lost in crc cells1617leading todisease progression recently researchers have exploredthe efï¬cacy of 15pgdh as a potential antitumour agentagainst colon cancer18 in a recent study we established that 15pgdh is induced by ltc4 via cyslt2rsignalling by phosphorylating cjun nterminal kinaseand ap1 to induce 15pgdh promoter activity andfurther guide colon cancer cells toward redifferentiation20however the detailed mechanism underlying this phenomenon remains unclearin this study we elucidated the mechanism by whichltc4induced 15pgdh promotes differentiation incolon cancer cells through cyslt2r activation with theoncogenesisinvolvement of hhgli signalling we observed thatgli1 was involved in the regulation of the redifferentiation and reduction in stemness induced by ltc4 via pgdh in colon cancer cellsresultsgli1 expression is negatively correlated with cyslt2r pgdh and mucin2 expression in crc patientsto elucidate the regulatory activity of gli1 on theantitumorigenic proteins cyslt2r and 15pgdh and thedifferentiation marker mucin2 in colon cancer we used atissue microarray tma of primary crcs from patients22 after ihc analysis we found only ï¬ve patientswith negative gli1 staining patients with weak stainingintensity patients with moderate staining intensityand patients with strong staining intensity the mean ±standard deviation sd of the immunoreactive score irsfor gli1 expression was ± then we grouped thepatients with negative and weak staining intensity anddeï¬ned them as the lowgli1 expression group n and those with moderate and strong staining intensitywere deï¬ned as the highgli1 expression group n fig 1a seventy patients had missing or incomplete coresand were excluded from the ï¬nal analysisafter ihc evaluation of cyslt2r and 15pgdh weobserved that patients with highgli1 expression hadsignificantly lower levels of cyslt2r irs ± and15pgdh irs ± expression than those with lowgli1 expression fig 1bd furthermore there was asignificant negative correlation between gli1 and pgdh r p suggesting hyperactivatedhhgli signalling with suppressed 15pgdh fig 1ehowever no significant correlation was found betweengli1 and cyslt2r expression fig 1eexhibitedhighgli1on the other hand patients with lowgli1 expressionhad a significantly higher irs for cyslt2r ± and15pgdh ± expression fig 1c d which indicates the adverse effects of cyslt2r15pgdh axis activation on gli1 we also noticed that the majority ofpatientspatients and when stratiï¬ed according to the tumournodemetastasis tnm staging a significantly strongerassociation for the patients with tnm stages iii and ivwas found fig 1e in addition to the above observationstranscriptome data from a public database23 also suggested a significant negative correlation between gli1 andthe tumour suppressors cysltr2 fig 1f and hpgd15pgdh fig 1gexpressionimportantly we observed that patients with lowgli1expression had a lower risk of overall mortalityhazard ratio ci than patientswith highgli1 expression after adjusting for age andtnm stage fig 1h the unadjusted survival curve isprovided in supplementary fig s1a the median follow 0csatapathy oncogenesis page of fig see legend on next pageoncogenesis 0csatapathy oncogenesis page of see ï¬gure on previous pagefig gli1 expression exhibited a negative correlation with cyslt2r and 15pgdh expression in colorectal cancer patient tissuesa matched pair immunohistochemistry ihc images of gli1 cyslt2r and 15pgdh expression in patients with low and highgli1 expressionshown at magniï¬cation the staining immunoreactivity was quantiï¬ed by the mean immunoreactive score irs calculated according to thefollowing formula irs staining intensity of stained cells the mean irs for the groups of patients with low and highgli1 expression b theyaxis represents irs for gli1 in these patients c cyslt2r and d 15pgdh expression according to patients with low and highgli1 expression in crctissue e distribution of tumournodemetastasis tnm stages of crc according to low and highgli1 expression pairwise pearson correlationcoefï¬cient r between the expression of gli1 and that of cyslt2r and 15pgdh p value according to chisquare test xyscatter plots showing mrnalevels of f gli1 and cyslt2r and g gli1 and 15pgdh hpgd gene expression from a public database containing crc patients kaplanmeiercurves for overall survival adjusted for age and tnm stage for patients with h low and highgli1 expression and subgroups of patients with bothgli1 and i cyslt2r or j 15pgdh expression compared by the logrank test the last patient group served as the reference category k western blotanalysis showing the protein expression of cyslt2r 15pgdh and gli1 in matched pairs of six patients with normal n and tumour t areas kdaindicated on the left side of the immunoblots kda represents protein size markers graphical representation of the densitometricanalysis showing the relative protein expression for cyslt2r 15pgdh and gli1 in matched pairs of patient samples n from normal mucosa mwhite and tumour t black areas l qrtpcr analysis of gli1 in matched pairs of normal mucosa m white and tumour t black tissues from crcpatients n scale bar as indicated in the images data represent the mean ± sd p p p mannwhitney testup time was months years with total eventsthisrole of gli1 in coloncarcinogenesissuggests a prominentwe have previously shown that crc patients with lowcyslt2r andor low15pgdh expression have a poorprognosis1220 in this study we noted that patients withhighgli1 expression coupled with either lowcyslt2rexpression orlow15pgdh expression had poorerprognoses than patients with either lowgli1 and low15pgdh expression orlowgli1 and lowcyslt2rexpression fig 1i j western blot analysis of six matchedpairs of crc patients showed significantly higher proteinexpression of cyslt2r and 15pgdh in normal tissuethan in matched tumour tissue fig 1k howevergli1 showed elevated expression in tumour tissue compared with matched normal tissue fig 1kfurthermorethe mrna analysis ofthese pairedtumour tissues with matched mucosa tissues from crcpatients n showed significantly higher mrnaexpression of gli1 in the tumour tissue compared with itsmatched normal mucosa the normal mucosa as referenceset to and tumour tissue ± mean ±sem mannwhitney test p fig 1l furthermore the muc2 mucin2 mrna analysis of thesepaired tumour tissues t and matched normal mucosam showed significantly lower mrna expression ofmuc2 in the tumour tissue compared with its matchednormal mucosa the normal mucosa as reference set to and tumourtissue ± mean ± semmannwhitney test p fig 2a a representativematched pair of high and lowgli1 and the corresponding mucin2 is shown fig 2b we observed thatpatients with highgli1 expression had lower levels ofmucin2 expression than those with lowgli1 expressionfig 2c and by combining these data a significantnegative correlation between elevated expression of gli1and decreased expression of mucin2 in tumour tissueswas found fig 2d moreover grouping the patients intooncogenesismucinous n and nonmucinous n typesrevealed that of patients with nonmucinous status had highgli1 expression suggesting anegative correlation with mucin2expressing cells while of patients in the mucinous category showedhighgli1 expression fig 2d furthermore we found asignificant negative correlation between gli1 and thedifferentiation marker mucin2 expression in these crcpatients n fig 2e we found a better overallsurvival for patients with lowgli1 than those with highgli1 expression regardless of mucin2 expressionfig 2e taken together these results suggest that gli1expression is negatively correlated with cyslt2r pgdh and mucin2 expression but positively with themucinous status of the patientscyslt2r is essential for differentiation in a colitisassociated colon cancercacmouse modelto further validate the role of cyslt2r in promotingdifferentiation in crc we adopted an ammatorymouse model that was induced by azoxymethane aomand dextran sodium sulfate dss brieï¬y c57bl6nwildtype and cysltr2 mice were subjected to aomand two dss cycles24 fig 2f as described in thematerials and methods section we found that all miceformed polyps in the colon regardless the phenotype butcysltr2 mice n developed significantlargerpolyps ¥ mm p in the colon compared withtheir wildtype wt n littermates supplementaryfig s1b c this result indicates that cysltr2 micedevelop a more progressive disease supplementary figs1 shows a representative image of colon polypsfig s1d and a likewise representative haemotoxylinand eosinstained imageshowing premalignant areas aberrant cryptfoci and metaplasiaareas from both wt and cysltr2 mice colonfig s1ecolon tissue sections from wt mice showed abundantmucin2expressing cells compared with sections from 0csatapathy oncogenesis page of fig see legend on next pageoncogenesis 0csatapathy oncogenesis page of see ï¬gure on previous pagefig gli1 expression was negatively correlated with differentiation a qrtpcr analysis of mucin2 in matched pairs of normal mucosa mwhite and tumour t black tissues from crc patients n b immunohistochemistry ihc images for gli1 and mucin2 expression in matchedpaired tissue samples from colorectal cancer crc patients with low and highgli1 expression represented at magniï¬cation c quantiï¬cation ofthe staining immunoreactivity by the mean irs for mucin2 expression according to patients with low and highgli1 expression in crc tissued distribution of tumour type mucinous adenocarcinomas versus nonmucinous adenocarcinomas according to low and highgli1 expressionpairwise pearson correlation coefï¬cient r between the expression of gli1 and mucin2 p value according to chisquare test e kaplanmeier curvesfor overall survival adjusted for age and tnm stage subgroups of patients with both gli1 and mucin2 expression compared by the logrank test thehighgli1 and lowmucin2 patient group was set as the reference category f experimental schematic of the aomdss mouse model gli1expression exhibited a negative correlation with differentiation in the cysltr2 aomdss mouse model immunohistochemical evaluation showingthe protein expression of g mucin2 and h gli1 in wt and cysltr2 aomdsschallenged mice graph bars showing the irs scores for mucin2 andgli1 respectively compared between wt and cysltr2 n micegroup scale bar as indicated in the images data represent the mean ± sdp mannwhitney testcysltr2 mice ± n fig 2g gli1 exhibited aslightly higher but nonsignificant overall expression incysltr2 mouse tissue sections compared with wt tissuesections ± n fig 2h taken together theabove observations encouraged us to further investigatehhgli signalling using both in vitro and in vivo coloncancer model systems and to delineate the involvement ofcyslt2r and 15pgdh in promoting differentiationltc4induced 15pgdh downregulates gli1 in coloncancer cellswe determined whether hhglisignalling wasinvolved in the ltc4induced 15pgdhpromoted differentiation of cc cells20 interestingly ltc4 stimulationsignificantly downregulated gli1 expression at both themrna and protein levels compared with unstimulatedht29 and caco2 cells fig 3a b in addition immunoï¬uorescence analysis of gli1 expression revealed adecrease in nuclear gli1 upon ltc4 stimulation fig 3cto determine the mechanism of 15pgdhmediateddepletion of gli1 we examined the expression of proteinkinase a pka which is a known gli1 antagonist2526the activation of the pka αδ catalytic subunit incaco2 cells after stimulation by ltc4 was significantlyincreased as indicated by the levels of phosphorylatedpka threonine but remained unchanged in stimulated ht29 cells fig 3b moreover phosphorylation atserine on the catalytic subunit of pka wasincreased after ltc4 stimulation in ht29 cells but wasnot present in caco2 wholecell lysates fig 3b15pgdhspeciï¬c shrna shhpgd was employed toinvestigate the involvement of 15pgdh in the ltc4mediated downregulation of gli1 expression comparedwith cells transfected with control shrna shctrl cccells transfected with shhpgd showed no response toltc4 stimulation we observed that shrnamediatedknockdown of hpgd did not affect the expression of theintestinal differentiation markers cdx2 or cdhr2 or thewnt target axin2 supplementary fig s2ad for ht29cells and unaltered gli1 expression at both the mrnaoncogenesisand protein levels fig 3df see supplementary figs3af for caco2 cells similarly 15pgdh knockdownreduced the effect of ltc4 stimulation on pka activationin both ht29 and caco2 cells fig 3e supplementaryfig s3fto further conï¬rm the role of pka as an intermediatemolecule in ltc4induced 15pgdhmediated downregulation of gli1 we used h89 a pkaspeciï¬c inhibitor nm prior to stimulating the cells with ltc4 wefound that neither ht29 nor caco2 cells treated withthe pka inhibitor affected ltc4induced 15pgdhexpression at either the mrna or protein levels however no significant alteration was observed in either themrna or protein level of gli1 poststimulation withltc4 fig 3g h supplementary fig s4a b which wasalso supported by immunoï¬uorescence analysis fig 3isupplementary fig s4c these data indicate that pkaplays a role in the 15pgdhmediated downregulation ofgli1 in colon cancer cellsgli1 regulates 15pgdhpromoted differentiation in coloncancer cellsnext we determined the mechanism by which ltc4induced 15pgdh promoted differentiation in coloncancer cells20 based on the above evidence we investigated the effects of gli1 knockdown fig 4aj for ht29cells and supplementary fig s5ak for caco2 cells onhpgd 15pgdh expression we used shgli1 todetermine possible changes in the expression of pgdh however gli1 knockdown in these cells did notaffect ltc4induced 15pgdh expression at either themrna or protein level compared with the correspondingshctrltransfected cells these data indicate that theeffect of ltc4 signalling on 15pgdh expression isindependent of gli1 suggesting that gli1 is downstreamof 15pgdh we next validated the involvement of gli1in differentiation by testing the mrna expression of sisucraseisomaltase and muc2 mucin2 which arerepresentative intestinal differentiation markers followingltc4 stimulation the observed increases in the mrna 0csatapathy oncogenesis page of fig 15pgdh regulates the ltc4mediated downregulation of hhgli signalling in colon cancer cells a qrtpcr analysis of gli1 mrnaexpression in ht29 and caco2 cells with or without ltc4 stimulation for h b western blot analysis of 15pgdh gli1 and phosphopka αγ subunitand subunit levels in ht29 and caco2 cells with or without ltc4 stimulation αtubulin served as the loading control c immunoï¬uorescence analysisof gli1 in ht29 cells with or without ltc4 stimulation for h d qrtpcr analysis of ht29 cells transfected with control shrna shctrl or pgdhspeciï¬cshrna shhpgd with or without ltc4 stimulation for h e western blot analysis of ht29 cells transfected with control shrna shctrl or pgdhspeciï¬cshrna shhpgd blotted with antibodies against 15pgdh gli1 or phosphopka αγ subunit and subunit with or without ltc4 stimulation for hαtubulin served as the loading control f immunoï¬uorescence analysis of gli1 in ht29 cells transfected with control shrna shctrl or pgdhspeciï¬cshrna shhpgd with or without ltc4 stimulation for h g qrtpcr analysis of 15pgdh and gli1 in ht29 cells treated with the pka inhibitor h89 pkainh for h followed by ltc4 for h h western blot analysis showing the expression of 15pgdh gli1 and phosphopka αγ subunit and subunit inht29 cells treated with the pka inhibitor h89 pkainh for h followed by ltc4 for h i immunoï¬uorescence analysis of gli1 in ht29 cells treatedwith the pka inhibitor h89 pkainh for h followed by ltc4 for h hprt1 was used as the housekeeping gene for normalisation of the qrtpcr geneexpression data graphs represent the mean ± sem of data from to independent experiments p p oncogenesis 0csatapathy oncogenesis page of fig 15pgdh promotes differentiation in colon cancer via gli1 qrtpcr validation of gene expression in shctrl control shrna or shgli1gli1speciï¬c shrnatransfected ht29 cells with or without ltc4 stimulation for h marker of tumour suppression in a 15pgdh markers ofdifferentiation in b si in c mucin2 and in d cdhr2 marker of differentiation regulation as in e cdx2 markers of wnt activation in f axin2 andg cmyc marker of proliferation in h ccnd1 hprt1 was used as the housekeeping gene for normalization of the gene expression data i western blotanalysis of wholecell lysates for 15pgdh gli1 si cdx2 and cdhr2 expression in shctrl control shrna or shgli1 gli1speciï¬c shrnatransfected ht29 cells with or without ltc4 stimulation for h αtubulin served as the loading control j immunoï¬uorescence analysis of gli1 andmucin2 in cells with or without ltc4 stimulation for h and transfected with shctrl or shgli1 graphs represent the mean ± sem of data from threeindependent experiments p p p oncogenesis 0csatapathy oncogenesis page of and protein expression of si after ltc4 stimulation inboth ht29 fig 4b and caco2 cells supplementaryfig s5c were abolished in gli1knockdown cellsfig 4b supplementary fig s5c j furthermore similarresults were observed for the mrna expression of otherdifferentiation markers such as muc2 fig 4c supplementary fig s5d and for both the mrna and proteinexpression of cdhr2 and cdx2 fig 4d e supplementary fig s5e f j ltc4 induced a reduction inaxin2 a potential wnt signalling target fig 4f supplementary fig s5g furthermore myc and ccnd1mrna expression was also abolished in gli1knockdowncells exposed to ltc4 fig 4g h supplementary fig s5hi to further validate the above observations we performed western blot fig 4i and immunoï¬uorescencemicroscopy using double staining for gli1 and mucin2in shctrl and shgli1transfected cells and found thatmucin2 expression was downregulated in cells lackinggli1 fig 4j supplementary fig s5k these ï¬ndingssupport our hypothesis of a possible regulatory effect ofgli1 in ltc4induced 15pgdhpromoted differentiation in colon cancer cellsgli1 suppresses differentiation in the absence of 15pgdhwe next investigated the ability of gli1 to regulate pgdh by overexpressing gli1 using the pegfphgli1construct in combination with the simultaneous knockdown of hpgd with shhpgd supplementary figs s6ab s7a b we found significant downregulation in theltc4induced increase in si muc2 cdhr2 and cdx2mrna expression levels in ht29 cells fig 5ad andalso in caco2 cells supplementary fig s7cf thatoverexpressed gli1 and lacked 15pgdh compared withcells expressing the control vector shctrl we alsofound that the ltc4induced reduction in axin2 mycand ccnd1 mrna was abolished although we observedincreased basal levels of these mrnas fig 5eg supplementary fig s7gi we next investigated the proteinexpression of si by western blot fig 5h supplementaryfig s7j and mucin2 by immunoï¬uorescence microscopy fig 5i supplementary fig s7k the ltc4induced increase in si was abolished in hpgdknockdown and gli1overexpressing cells fig 5h supplementary fig s7j a similar pattern was found regardingmucin2 protein expression in these cells which wasobserved using immunoï¬uorescence microscopy fig 5isupplementary fig s7k finallythe ltc4inducedreduction in gli1 was abolished fig 5iltc4induced cyslt2r signalling downregulates gli1 in a15pgdhdependent mannerto determine whether ltc4 acts via cyslt2r as ltc4is the highafï¬nity ligand of cyslt2r we investigated thespeciï¬c involvement of cyslt2r signalling in the ltc4oncogenesisstableofknockdownmediated downregulation of gli1 in colon cancer cellswe ï¬rst constructed hct116 cells with doxycyclinedoxinduciblecyslt2rshcysltr227 we examined the mrna and proteinexpression levels of cyslt2r 15pgdh and gli1 withand without ltc4 stimulation in this cell line with doxinduction and compared them with the levels in cellsgrown in the absence of dox the mrna expression ofcysltr2 and hpgd wassignificantly upregulatedfig 6a b while gli1 mrna expression was downregulated in cells cultured without dox fig 6c howin the doxinduced cells gli1 gene expressioneverremained unaltered most likely due to the low mrnaexpression levels of cysltr2 and hpgd these observations were also reï¬ected in the levels of proteinexpression fig 6d supplementary fig s8acto further study the involvement of cyslt2r we treatedht29 and caco2 cells with the cyslt2rspeciï¬cantagonist ap100984 µm followed by ltc4 stimulation for h20 ap100984 treatment efï¬ciently blocked boththe mrna supplementary fig s8d e and proteinexpression supplementary fig s8f of cyslt2r as well asof its downstream signal 15pgdh in ht29 cells as well asin caco2 cells supplementary fig s8gj ap100984 pretreatment abolished the effect of ltc4 stimulation on gli1at both the mrna and protein levels fig 6e f supplementary fig s8i j taken together the above resultsshowed that cyslt2r signalling plays a role in the ltc4induced 15pgdhmediated downregulation of gli1ltc4induced 15pgdh expression reduces stemness incolonosphereswe extended our study to determine whether ltc4induced 15pgdh affected stemness in colon cancer cellsas well as the possible role of gli1 we created a 3dmodel of multicellular colonospheres derived from coloncancer cells fig 6g we observed that shctrltransfected ht29 or caco2 cellderived colonospheresshowed reduced numbers and sizes with ltc4 stimulation fig unlike the shctrl group shhpgdtransfected cellderived colonospheres showed increases innumber and size howeverthe absence of gli1 inshgli1transfected cellderived colonospheres resulted inno significant alteration in size or number even afterltc4 stimulation the mrna expression levels of thecolon cancerspeciï¬c stemness markers dclk1 lgr5and aldh1a1 were elevated in ht29 as well as in caco cellderived colonospheres and were downregulatedafter ltc4 stimulation fig 6i supplementary figs9ah the mrna and protein levels of the cancer stemcell markers dclk1 and aldh1a1 remained unchangedin both shhpgd and shgli1knockdown ht29 andcaco2 cellderived colonospheres even after ltc4 stimulation compared with their unstimulated counterparts 0csatapathy oncogenesis page of fig gli1 negatively regulates the differentiation and promotes the proliferation of colon cancer cells in the absence of 15pgdh ht29cells were either transfected with shctrl alone or cotransfected with shhpgd and pegfphgli1 followed by ltc4 stimulation for h qrtpcranalysis of the differentiation markers a si b mucin2 and c cdhr2 d the differentiation regulation marker cdx2 e the wnt activation marker axin2f the prooncogene cmyc and g the proliferation marker ccnd1 h western blot analysis of wholecell lysates for 15pgdh gli1 and si expressionαtubulin was used as the loading control i immunoï¬uorescence analysis of gli1 and mucin2 in unstimulated or ltc4stimulated cells transfectedwith shhpgd or shgli1 hprt1 was used as the housekeeping gene for normalization of the qrtpcr gene expression data graphs represent datafrom to independent experiments and represent the mean ± sem p p p fig 6ik supplementary fig s9ah gli1 gene andprotein expression in ht29 and caco2derived colonospheres was also downregulated after ltc4 stimulation we next analysed the mrna expression levels intumour and adjacent mucosa samples from pairedcrc patients which also suggested a significantly positivecorrelation between gli1 and the stemness markersdclk1 and lgr5 with elevated expression of all threegenes in tumour tissues figs 1l 6ln the normalmucosa as reference set to and the tumour tissue fordclk1 ± and lgr5 ± mean± sem respectively mannwhitney test p asexpected the expression level of dclk1 showed a negative correlation with cysltr2 and hpgd expressiononcogenesis 0csatapathy oncogenesis page of fig see legend on next pagefrom our previously published results see ref whilemuc2 had a positive correlation fig 2c20 this suggeststhat a poorly differentiated tumour could occur due toenriched stemnessltc4induced 15pgdh promotes differentiation inzebraï¬sh xenograftsnext we used the zebraï¬sh xenograft model28 to further evaluate and visualise the differentiationpromotingoncogenesis 0csatapathy oncogenesis page of see ï¬gure on previous pagefig ltc4induced 15pgdh expression negatively regulates gli1 via cyslt2r graphs showing qrtpcr analysis of a cysltr2 b 15pgdhand c gli1 mrna expression in hct116 cells with stable transfection of doxycyclineregulated shcysltr2 cultured with or without doxycycline µm treatment followed by ltc4 nm stimulation for h d western blot analysis of cyslt2r 15pgdh gli1 and si expression in wholecelllysates αtubulin served as the loading control e qrtpcr analysis showing mrna expression of gli1 and f western blot analysis of gli1 proteinexpression in ht29 cells stimulated with or without ltc4 and with or without ap100984 a cyslt2r antagonist hprt1 was used as the housekeepinggene and αtubulin was used as the loading control in the western blot assay g schematic illustration of colonosphere formation gli1 regulates theeffect of ltc4 on stemness in multicellular colonospheres the cells were cultured in ultralowattachment conditions on matrigel containing serumfree medium for days h representative images of colonospheres from ht29 cells transfected with shctrl shhpgd or shgli1 and stimulated ornot stimulated with ltc4 bar graphs showing the number of colonospheres formed per well and the size of colonospheres with or without ltc4stimulation and comparing the shctrl shhpgd and shgli1transfected groups qrtpcr analysis of the stemness markers i dclk1 j gli1 andm lgr5 in colonospheres derived from shctrl shhpgd or shgli1transfected ht29 cells with or without ltc4 stimulation for h k western blotanalysis showing the expression of dclk1 15pgdh and gli1 in transfected ht29 cells as indicated αtubulin served as the loading control qrtpcr analysis of l dclk1 and n lgr5 in matched pairs of mucosa m and tumour t tissues from crc patients n hprt1 was used as thehousekeeping gene for normalization of the qrtpcr gene expression data data represent the mean ± sem from to independent experimentsp p p role of 15pgdh in colon cancer transgenic zebraï¬sh tgï¬i1egfp embryos were injected with u | Colon_Cancer |
to probiotics general attitudes and functions since the first observation of probiotic bacteria by elie metchnikoff there have been several studies on the immunological effects of probiotics on the host immune system according to who and fao probiotics are defined as live microanisms which when administered in proper amounts confer a health benefit on the host among several genera of bacteria and yeasts that identified and defined as probiotics health benefits of lactobacillus and bifidobacterium on the host have been proved and are generally consumed as a part of fermented foods like those in dietary supplements there are some reports about probiotics potential in promoting health benefits by regulating allergic reactions [] protecting the hosts against bacterial and viral infection [] and even reducing the tumor growth in some cancer models [] the probioticsconferred health benefits are attributable to their effects on the immune system recognition and stimulation of immune system in the gut lumen is followed through three distinct pathways engulfment of probiotics by macrophages mfs or dendritic cells dcs present immediately below m cells specialized epithelial cells dcsdirected sampling and processing of probiotics in the gut lumen and direct stimulation of intestinal epithelial cells iecs by probiotics to secrete an array of cytokines modulating the immune functions of dcs t cells and b cells in the gutassociated lymphoid tissue galt briefly the regulatory effects of probiotics on host immune responses are followed through activation of the function of dendritic cells macrophages and t and b lymphocytes [ ] in addition probiotics have proved to modulate and regulate innate and adaptive immune responses partly through the activation of tolllike receptors tlrs as the role of the intestinal epithelium is to form a physiological barrier against pathogenic microbes and detrimental substances available in the intestinal lumen this monolayer is responsible for distinguishing between pathogens and commensal bacteria as well as regulation of intestinal immune responses it has been shown that probiotics can regulate immunomodulatory responses of intestinal epithelial cells fig one family of pattern recognition receptors prrs in the innate corresponding author email addresses a_ghaemipasteuracir ghaem_amiryahoocom a ghaemi these authors contributed equally to this work 101016jmicpath2020104452 received april received in revised form august accepted august microbialpathogenesis1482020104452availableonline18august2020088240102020elsevierltdallrightsreserved 0cm mahooti abbreviation covid19 coronavirus disease sarscov2 severe acute respiratory syndrome coronavirus who world health anization food and agriculture anization fao macrophages mfs dendritic cells dcs m cells microfold cells intestinal epithelial cells iecs galt gutassociated lymphoid tissue tlrs tolllike receptors pattern recognition receptors prrs pamps pathogenassociated molecular patterns transmembrane protein tp myd88 myeloid differentiation protein rtis respiratory tract infections human rhinovirus hrv influenza virus ifv rsv respiratory syncytial virus iav influenza a virus ifn α interferon α interferon ifn tumor necrosis factor α tnfα ifnÎ interferon gamma il1 interleukin interleukin il6 interleukin il4 il8 interleukin il10 interleukin il12 interleukin t helper type th1 th2 t helper type mips macrophage inflammatory proteins mcps monocyte chemoattractant proteins bronchoalveolar lavage bal nk cell natural killer cells epss exopolysaccharides iga immunoglobulin a igg immunoglobulin g secretory immunoglobulin a siga peyers patches pps tfh follicular helper t acot acylcoa thioesterase cyr61 cysteinerich angiogenic inducer early growth response egr1 fos protooncogene fos rsad2 radical sadenosyl methionine domain containing klrk1 killer cell lectin like receptor k1 ilc innate lymphoid cells mediastina lymph node mln bronchoalveolar lavage fluid balf ip10 interferoninducible protein pedv porcine epidemic diarrhea virus cfs cellfree supernatants cytopathic effect cpe dcpep dctargeting peptide coe core neutralizing epitope oas oligoadenylate synthetase interferonstimulated gene isg15 swi2snf2related crebbinding protein activator srcap protein pfu a plaqueforming unit mpiv1 murine parainfluenza virus crp creactive protein cxcl8 cxc motif chemokine ligand immune system are tolllike receptors which play a pivotal role in the linking of innate and adaptive immunity tlrs can specifically recognize pathogenassociated molecular patterns pamps and convey pathogen related molecular signals into cells by transmembrane tm protein afterward tlrmediated multistep signaling cascades are initiated leading to the activation of transcriptional pathways such as nfκb against the invader pathogens this signal transmission activates both immune system arms aimed at the pathogenic microanism through a cascade reaction which is severely dependent on signaling pathway directed by tolllike receptor tlr7 and myeloid differentiation protein myd88 interestingly it has been determined that tlr7 expression considerably reduces after influenza infection in this context wu revealed that after consumption of probiotics by neomycintreated mice the balance of intestinal flora restored and thereby tlr7 pathway upregulated this evidence presents promise for the regulatory role of probiotics in host innate and adaptive immune responses as underlying mechanisms for protection from viral infection pathology of influenza virus the most common respiratory virus infection influenza virus belonging to the orthomyxoviruses family is among viruses that cause respiratory tract infections rtis several human viruses can cause rti and due to hospitalizations medical costs sick leave and school or daycare absences viral respiratory diseases can pose a considerable social and economic burden human rhinovirus hrv enterovirus influenza virus ifv respiratory syncytial virus rsv and adenovirus are common etiological agents of acute respiratory disease influenza a virus iav initiates pulmonary inflammation and intensifies chronic lung diseases in response to the infiltration of inflammatory cells and augmentation of airway hyperresponsiveness the main target and host for iav is the bronchial epithelial cell which plays a key role in influenza pathogenesis infection occurs following h of influenza virus replication for the first cycle and then initial high titers of virus are shed during this period ifv infection can result in several symptoms like fever cough headache and pneumonia which may become immunologically incompetent while the induction of inflammatory cytokines by influenza infection is attributed to its systemic feature it is unlikely that the virus to be propagated outside the respiratory tract during an uncomplicated infection one of the key components of the influenza virus in pathogenesis is ha domain which is recognized by the hosts neutralizing antibodies the emerged ha is directed to the cell membrane in an infected host cell fastening to the cell membrane by means of a short transmembrane region at the cterminal and once this domain attached to terminal sialic acid residues on the cell it facilitates entry and fusion of the virus due to the acidification of host cells by proton pumps ha rearranges so that the highly conserved nterminal of ha2 is exposed this exposure leads to the fusion of viral membrane with cell membranes and thus activation of the replication complex despite all known clinical and pathogenesis descriptions of the influenza virus the mechanism through which influenza virus disease being developed has not precisely understood however it is thought that local nonimmune cells which release early cytokines are the cause of many of the clinical signs some cytokines including ifnα tnfα and il1 α and located at the site of infection are responsible microbialpathogenesis14820201044522 0cm mahooti for local inflammatory reactions as well as some systemic effects [ ] afterward il6 and many other chemotactic cytokines like the neutrophil attracting interleukin8 il8 macrophage inflammatory proteins mips and monocyte chemoattractant proteins mcps are rapidly produced fever excessive sleepiness and anorexia are attributed to the activation of ifnα tnfα il1 and il6 after influenza infection neutrophil and macrophage functions are stimulated by tnfα and il1 and both cytokines potently upregulate leukocyte adhesion molecules on the vascular endothelium therefore mediating the first indispensable step for sequestration of neutrophils and or macrophages into the respiratory tract a study by van reeth demonstrated that there is a correlation between bal fluid levels of some cytokines ifnα tnfα and il1 and virus titers neutrophil infiltration and influenza disease additionally lee showed that ifnα tnfα il1 and il6 all participate in nonspecific and specific antiviral immune responses immunomodulatory role of probiotics on influenza virus in the context of preclinical studies mm2 can significantly reduce influenzaelicited proinflammatory cytokines such as il6 and tnfα moreover a slightly elevated ifnα level in the balf indicated the impact of this probiotic on the enhancement of nk cell activity these results along with the reduction of pulmonary mrna levels of nk cell activators including proinflammatory cytokine il1 and chemokines mip2 and mcp1 suggest the modulating effect of this probiotic on influenza infection in another study continuous oral administration of lactobacillus plantarum 06cc2 led to an elevation in the production of ifnα and th1 cytokines il12 and ifnÎ and reduction in the production of tnfα and il6 cytokines in balf this probiotic could also control the number of total infiltrated cells such as macrophages and neutrophils in the balf of infected mice similarly nagai revealed that days after oral administration of the yogurt fermented with l bulgaricus oll1073r1 or its exopolysaccharides epss influenza virus infection ameliorated which attributed to the development of nk cell activity of splenocytes assessment of kimchiderived lactobacillus plantarum and leuconostoc mesenteroides has confirmed their effectiveness against lethal influenza viruses h1n1 and h7n9 by decreasing the sizes of viral plaques both in vitro and in vivo in addition it has been shown that lactococcal strains or their eps induced weight regain and also reduced viral titer in the lung of mice infected with influenza virus h1n1 starosila investigated the antiviral ability of bacillus subtilis and showed that after a single dose administration of the probiotic bacteria the survival rate of mice challenged with the ifv increased song assessed the impact of oral intake of lactobacillus rhamnosus m21 on lethally ifvinfected mice an increase in the level of ifnÎ and il12 and a decline in il4 level suggested that this probiotic can modulate some disease outcomes attributed to changes in cytokine profiles such as that happens in the lung after influenza infection in our very recent study we showed that bifidobacterium bifidum can increase the level of both th1 ifny and il12 and th2 il4 since the manifestation of probiotics impacts on several diseases from nonviral to viral ones [] several studies have surveyed the probiotic roles in immune responses of influenzainfected animal models it has been fully demonstrated that upon infection with influenza many cytokines such as il12 one of the mediators of th1 immuneresponse interferon ifnÎ representative of th1 cytokine il4 and il10 th2 cytokines il1α il1 il6 and tumor necrosis factor tnfα proinflammatory cytokines and ifnα and ifn are produced in the respiratory tract [] studies on ameliorating influenza infection as well as alleviating influenza symptoms have been trying to redress the imbalance attributed to runaway cytokines production namely cytokine storm after ifv infection kawahara demonstrated that probiotic bifidobacterium longum fig schematic presentation of possible mechanisms of probiotic immunomodulation effects in the intestine probiotics trigger immunomodulation through direct and indirect interaction with intestinal epithelial cells dendritic cells extend their dendrites between intestinal epithelial cells iecs and might directly sample and process probiotics in the gut lumen leading to activation of innate and adaptive immune responses dendritic cells present immediately below m cells engulf probiotics resulting in the maturation of dcs and may derive b cells into plasma cells additionally after the interaction of probiotics with macrophages and dendritic cells presented in lamina propria these cells are activated and induce nk cell activation which leads to ifnÎ elevation to defend against viruses upon the interaction of probiotics pamps with different types of tolllike receptors tlrs nuclear factorκb nfκbmediated antiviral gene expression is stimulated eventually active immune cells migrate to sites of infection through lymphatic and circulatory systems to defend against respiratory viruses microbialpathogenesis14820201044523 0canother study enterococcus faecalis1 has been proved to improve the body weight and feed conversion ratio of treated broilers and also significantly elevated the total igy serum level resulting in efficient modulation of the cecal microbiota and decrease in the mortality percentage of broilers an investigation on the possible effect of interaction between lactobacilli and chicken macrophages on eliciting antiviral responses against the aiv showed that certain probiotic species such as l acidophilus and l salivarius when administered as live bacteria either alone or in combination can induce an antiviral response in chicken macrophages in another study seo reported that live leuconostoc mesenteroides yml003 significantly restored the body weight and increased the ifnÎ levels in splenic cells of lowpathogenic aiv h9n2infected chickens examining the effectiveness of enterococcus faecium ncimb and zinc oxide in modulating the immune system of piglets in confronting with swine influenza virus siv revealed that the body weights of piglets fed with the probiotic and vaccinated with trivalent influenza vaccine significantly increased and noticeably higher h3n2specific antibodies were detected among them based on these considerations probiotics administration is effective in the secretion of high concentration of cytokines from immune cells located in the airway leading to the migration of immune cells to the lung space and thereby amelioration of influenza infection fig the probiotic effects on coronavirus infections m mahooti cytokines an increase in the level of total igg antibodies in pooled sera of treated mice and igg1 and igg2a isotypes demonstrated the efficacy of the probiotic in eliciting humoral immune responses and th1th2 responses respectively moreover it revealed that the level of inflammatory cytokines like il6 which increases upon influenza infection decreased in the probiotic group suggesting the ameliorating potential of this probiotic in influenzainfected mice based on the results of body weight changes survival rates and viral titre among treatment groups of different influenza viruses park showed that lactobacillus plantarum has antiinfluenza effects that are not virus type or straindependent revealing that regular intake of that probiotic can help to alleviate the influenza symptoms concerning the effect of longterm probiotic administration kiso orally injected lactobacillus pentosus b240 to mice for weeks and evaluated its inhibitory properties against influenza challenge assessment of different cytokineschemokines in the lungs of infected animals revealed that excluding il5 administration of that probiotic did not affect the immune system regarding cytokineschemokines secretion however ah1n1 pdm infected mice survived probably due to protecting effects of the probiotic by downregulation of acots acot1 acot2 and acot5 cyr61 egr1 and fos as well as upregulation of stfa1 and antiviral rsad2 genes in the lungs of uninfected mice in agreement with all aforementioned results harata revealed that oral administration of probiotics lactobacillus gg and l gasseri tmc035 in mice infected with a lethal dose of influenza ah1n1 pdm prompted the secretion of il12 il6 ifnÎ and iga from isolated pp cells in vitro however unlike lactobacillus gg the oral administration of l gasseri had no impact on the production of ifnÎ il6 as well as total iga in vivo proving the vital role of probiotic interaction with the component cells of galt in the protection against influenza the investigation of the effects of l casei strain shirota on aged mice showed that this probiotic can enhance not only the level of ifnÎ and tnfα but also pulmonary and spleen nk cells activity and thereby ameliorates ifv infection in another study oral administration of bifidobacterium longum bb536 could significantly reduce the loss of body weight inhibit viral proliferation in the lungs and improve the symptoms of influenzainfected mice which may be related to the decreased level of il6 belkacem observed that while administration of probiotic l paracasei induced significantly higher levels of proinflammatory cytokines in probioticfed influenza mice models this trend was reversed seven days upon influenza challenge except for il33 the number of all tissueresident or circulatory myeloid cells and b cells after the probiotic consumption and before viral infection increased and the probiotic administration generated more ifnÎproducing ilc1 mainly nk cells and th2 cells during the late phase of influenza infection additionally l paracasei peptidoglycans administration before influenza infection increased dendritic cells but did not affect other cell types and significantly reduced viral loads besides the effectiveness of oral administration of probiotics intranasal administration of lactobacillus pentosus spt84 to mice proved to induce the production of il12 and ifnÎ in mediastinal lymph node mln cells and il12 and ifnα in balf thereby improved the survival rates of mice reduced the ifv titer in balf and subsequently suppressed influenza infection in mice employing the novel sublingual route lee showed that in contrary to proinflammatory cytokines the level of il12 in the lung homogenates of mice treated with lactobacillus rhamnosus significantly increased in addition besides the increase in nk cell activities and antiinfluenza virus iga the expression of cd25 by both cd8 and cd4 lymphocytes highly increased in the lungs of mice these results recommend that compared to the traditional methods sublingual delivery is a more effective way for the administration of probiotics against seasonal and pandemic influenza regarding other animal models poorbaghi et al showed that microencapsulated lactobacillus acidophilus probiotic and its symbiotic form with inulin decreased faecal shedding of h9n2 avian influenza virus aiv in both nonvaccinated and vaccinated broiler chicks in the current outbreak of coronavirus disease covid19 reported from wuhan china has again gained global attention to taking a new measure that could work out as fast as possible against such an outbreak of viruses interestingly accumulated data obtained from clinical investigations on patients who suffered from severe covid19 in a hospital in wuhan demonstrated the presence of signs for cytokine storm especially among patients in severe stages of the disease particularly the levels of cytokines and chemokines involved in both th1 such as il1b ifnÎ and th2 il4 and il10 immune responses were promoted in studied patients moreover the levels of ip10 mcp mip1a and tnfα were in direct correlation with the severity of patients symptoms on the other hand it has been determined that the cytokine storm may lead to a rise in platelets and longer hospitalization of covid19 patients other studies also have revealed other aspects of virallydriven manipulation of immune responses by human coronaviruses [] therefore addressing the cytokine storm may be the key for the treatment of patients infected with sarscov2 while some reagents such as steroids can be considered as hyperinflammation suppressors their sideeffects impede to count on them as a trustworthy medicine for covid19 alternatively addressing the urgent need for standing against the increasing rate of morbidity and mortality related to the current pandemic requires employing previously approved therapies harnessing safety profiles probiotics as a safe available treatment with the ability to modulate immune responses and manipulate cytokines production have been considered to be studied against different strains of coronavirus in some studies soon moreover a clinical survey has reported an intestine microbiota imbalance in particular a decline in the level of probiotics such as lactobacillus and bifidobacterium among some covid19 patients which may result in secondary infection in response to bacterial translocation one report has shown that the recent outbreak of porcine epidemic diarrhea virus pedv can be prevented through the use of either cell free supernatants cfs or live lactic acid bacteria lab it demonstrated that probiotics though the precise mechanism is not clear could be effective against the pandemic strain of pedv in a strainspecific manner using cpe reduction assays that further confirmed by qualitative immunofluorescence in another investigation lactobacillus casei was used as a carrier for the dctargeting peptide dcpep fused with the pedv core neutralizing epitope coe antigen this survey demonstrated that this genetically engineered lactobacillus casei oral vaccine is able to induce systemic igg and mucosal siga antibody microbialpathogenesis14820201044524 0cm mahooti fig model of the interaction of active immune cells triggered by probiotics with respiratory viruses in the lung following virus infection immune cells in the airway such as dendritic cells and macrophages secrete cytokines to defend against viruses in a probioticreceived subject the high concentration of cytokines leads to the migration of immune cells to the lung space through the gutlung axis resulting in rapid recruitment of activated t and b cells in the lung that eventually promote upregulation of virusspecific immunoglobulins and cytokines in probioticreceived subject whereas in the absence of activated immune cells the respiratory virus can cause severe lung damage due to the lack of immediate immune responses responses in mice models there have been other articles using different types of probiotics for displaying the desired genes or antigens against pedv [] for instance liu demonstrated that their modified lactobacillus plantarum has the property to act like a strong antiviral agent against coronavirus infection in the intestinal porcine epithelial cell line probiotic impacts on other viral respiratory infections eguchi et al demonstrated that lactobacillus gasseri sbt2055 lg2055 when administered orally to mice before infection with a human rsv could suppress the virus titre in lung tissue homogenates rsv replication and the intensity of the symptoms moreover a decrease in the expression level of proinflammatory cytokines and an increase in the mrna level of ifn ifnÎ oas1a and isg15 in the mice lung upon probiotic administration are satisfactory evidence for antiviral properties of this probiotic also swi2snf2related creb binding protein activator protein srcap introduced as a candidate for the antiviral activity of lg2055 against rsv to investigate whether probiotics can control the inflammatory pathway and modulate the coagulation system upon respiratory viral infection rhamnosus crl1505 was orally administered in rsv or ifv mice models the results elucidated that this probiotic could successfully modulate tlr3triggered immune coagulation reaction in the lung upon viral infection and prevent exacerbated respiratory injuries notably this study substantiated the vital role of probioticprovoked secretion of il10 in taming the coagulation system after the viral attack additionally in a study conducted by tomosada nasal administration of lactobacillus rhamnosus crl1505 and crl1506 has proved to modulate elevated respiratory levels of the proinflammatory mediators caused by administration of the viral pathogenassociated molecular pattern polyic moreover a nasal administration of the probiotic prior to pfu of rsv challenge improved resistance against rsv infection considering the effect of probiotics on the parainfluenza virus there is only one study evaluating the antiviral effects of oral administration of lactococcus lactis subsp lactis jcm5805 in a mouse model of murine parainfluenza virus mpiv1 infection the probiotic administration resulted in a rise in the survival rate of treated mice without any weight loss and also a decline in the lung histopathology scores compared to the nontreated group which was attributed to the incorporation of jcm5805 into cd11c immune cells in pp and thereafter activation of pp pdcs and ultimately elevation of ifns expression it is of note that although no activated local pdcs were observed at lung upregulation in ifnsinduced antiviral factors in the lung may be due to the delivery of ifns from the intestine of jcm5805fed mice into the lung studies reporting the effects of probiotic bacterias on respiratory viruses have been demonstrated in table supplementary section clinical evidence of probiotic immunomodulation in a pilot study intake of lactobacillus brevis kb290 has shown to curtail the incidence of influenza infection among schoolchildren with no adverse effects associated with consuming the probioticcontaining drinks hu demonstrated that h7n9 ifv infection led to a decrease in intestinal microbial diversity and species richness among patients they observed that although administration of c butyricum probiotic was unable to alleviate the antibioticrelated disturbances in the gut microbiome of h7n9infected patients an increase in microbiota diversity and evenness gradually appeared through continuous administration of probiotics after antibiotic cessation additionally based on the evaluation of crp levels or bacteremia and pneumonia in the patients treated with probiotics the safety of probiotic administration was approved and no inflammatory effects were observed in another study conducted by wang the impact of lactobacillus rhamnosus gg administration on nursing home residents aged and older was assessed it revealed that probiotic administration reduced the risk of influenza and other respiratory viral infections among the elderly received probiotics compared to those receiving a placebo although not statistically significant the trial provided a microbialpathogenesis14820201044525 0ctable clinical studies reporting regulatory effect of probiotic bacteria on immune responses study design an openlabel parallelgroup trial main findings the risk of infection subjects schoolchildren years of age a retrospective study patients nursing home residents years of age and older a randomized doubleblind placebo controlled pilot trial probiotics used lactobacillus brevis kb290 kb290 days per week for weeks in the form of test drink containing à cfu clostridium butyricum three times per day at the dose of cfu tablet prior to h7n9 infection lactobacillus rhamnosus gg twice a day for months in the form of capsule containing cfu m mahooti framework to assess the effectiveness of probiotics in reducing respiratory infections among senescent individuals similarly it has been shown that there is no connection between intaking the yogurt fermented with probiotic lactobacillus delbrueckii ssp bulgaricus oll1073r1 and incidence rate of influenza in humans however the immunological study showed an increase in the level of ifnÎ in the probiotictreated group while there are some available treatments for hrv the most frequent cause of the common cold most of them have failed to be efficient in clinical trials due to their drawbacks in this regard probiotics have shown to prevent or treat common colds and upper respiratory infections several studies have revealed that the rhinovirusrelated common cold pathogenesis is associated to the innate inflammatory response to the virus therefore more attempts have done to incorporate probiotics to modulate immune responses consequently leading to balanced responses and optimal outcomes in combating viral infection in this regard an investigation on the impact of bifidobacterium animalis ssp lactis bl04 on healthy adults showed a modest modulation of innate immune host responses upon infection with hrv particularly reduction of cxcl8 response in the nasal lavage resulting in a decline in the rhinovirus replication approved by a decrease in virus shedding in the nasal secretions tapiovaara demonstrated that adults consumed juice enriched with live or heatinactivated l rhamnosus gg before intranasal inoculation of hrv showed no significant differences in nasopharyngeal hrv loads compared to the placebo group another survey has illustrated that consumption of probiotic is a good strategy to prevent viral rtis in the first year of life in a cohort of preterm infants the results showed that the probioticdriven change in microbiota leads to the induction of longlasting effects which can reduce the risk of viral rtis in agreement with that study it was demonstrated that live l rhamnosus gg might be more effective in reducing the rhinovirus infection rate than the inactivated form of the same strain respiratory syncytial virus from the paramyxoviridae family is considered as the major cause of lower respiratory tract infection in infants and children around the world and is becoming an important pathogen of the elderly although most children have experienced a first rsv infection by two years of age some cases suffering premature birth bronchopulmonary dysplasia immunodeficiency and congenital heart disease are vulnerable to symptoms worsening and hospitalization as well however the probiotic administration has proved to be effective in developing protection against virallyinduced inflammation besides there is a study demonstrating that while probiotic consumption significantly reduced the number of days with respiratory symptoms during the intervention no significant effect was neither observed on the occurrence of viruses in the nasopharynx nor on the symptoms during viral episodes among daycare children clinical studies reporting regulation of immune responses by probiotic bacterias have been presented in table conclusion bifidobacterium animalis daily for days in the | Colon_Cancer |
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sci hamberger b bak s plant p450s as versatile drivers for evolution of speciesspecific chemical diversity philos trans r soc feyereisen r insect molecular biology and biochemistry elsevier amsterdam senate l m similarities variations and evolution of cytochrome p450s in streptomyces versus mycobacterium sci rep 101038s4159 y mnguni f c more p450s are involved in secondary metabolite biosynthesis in streptomyces compared to bacillus cyanobacteria and mycobacterium int j mol sci 103390ijms1 parvez m molecular evolutionary dynamics of cytochrome p450 monooxygenases across kingdoms special focus on mycobacterial p450s sci rep 101038srep3 syed p r cytochrome p450 monooxygenase cyp139 family involved in the synthesis of secondary metabolites in mycobacterial species int j mol sci 103390ijms2 khumalo m j comprehensive analyses of cytochrome p450 monooxygenases and secondary metabolite biosynthetic gene clusters in cyanobacteria int j mol sci 103390ijms2 kanehisa m sato y kawashima m 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into secondary metabolism from a global analysis of prokaryotic biosynthetic gene clusters cell tran p n yen m r chiang c y lin h c chen p y detecting and prioritizing biosynthetic gene clusters for bioactive compounds in bacteria and fungi appl microbiol biotechnol s0025 z marchlerbauer a cddsparcle functional classification of proteins via subfamily domain architectures nucleic acids res d200d203 101093nargkw11 syed k mashele s s comparative analysis of p450 signature motifs exxr and cxg in the large and diverse kingdom of fungi identification of evolutionarily conserved amino acid patterns characteristic of p450 family one e95616 101371journ alpone00956 graham s e peterson j a how similar are p450s and what can their differences teach us arch biochem biophys katoh k kuma k toh h miyata t mafft version improvement in accuracy of multiple sequence alignment nucleic acids res 101093nargki19 boc a diallo a b makarenkov v trex a web server for inferring validating and visualizing 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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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 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makarov v havel jjlandscape determines sensitivity to pd1 blockade in mutationalnonsmall cell lung cancer science 101126scienceaaa1348 van allen em miao d schilling b shukla sa blank c zimmer l genomic correlates of response to ctla4 blockade in metastatic melanomascience 101126scienceaad0095 dudley me wunderlich jr robbins pf yang jc hwu p schwartzentruberafterregression and autoimmunity in patientsal cancerrepopulation withdjetclonal 101126science1076514lymphocytesantitumorscience dudley me yang jc sherry r hughes ms royal r kammula u adoptive cell therapy for patients with metastatic melanoma evaluation ofintensive myeloablative chemoradiation preparative regimens j clin oncol 101200jco2008165449 rosenberg sa yang jc sherry rm kammula us hughes ms phan gq durable complete responses in heavily pretreated patients with metastaticmelanoma using tcell transfer immunotherapy clin cancer res 10115810780432ccr110116 besser mj shapirafrommer rlevy d adoptive transfer ofpatients with metastatic melanomaitzhaki o treves aj zippel dbtumorltrating lymphocytes inintenttotreat analysis and eï¬cacyfrontiers in oncology wwwfrontiersinaugust volume 0cli and dingisolating neoantigenspeciï¬c t cellsafter failure to prior immunotherapies clin cancer res 10115810780432ccr130380 andersen r donia m ellebaek e borch th kongsted piversentz longlasting complete responses in patients with metastaticmelanoma after adoptive cell therapy with tumorltrating lymphocytesand an attenuated il2 regimen clin cancer res 10115810780432ccr151879 stevanovic s draper lm langhan mm campbell te kwong mlwunderlich jr complete regression of metastatic cervical cancer aftertreatment with human papillomavirustargeted tumorltrating t cells jclin oncol 101200jco2014589093 huang j elgamil m dudley me li yf rosenberg sa robbins pf tcells associated with tumor regression recognize frameshifted products ofthe cdkn2a tumor suppressor gene locus and a mutated hla class i geneproduct j immunol 104049jimmunol172106057 zhou jh dudley me rosenberg sa robbins pf persistence of multipletumorspeciï¬c tcell clones is associated with complete tumor regression ina melanoma patient receiving adoptive cell transfer therapy j immunother lu yc yao x li yf elgamil m dudley me yang jc mutatedppp1r3b is recognized by t cells used to treat a melanoma patientwho experienced a durable complete tumor regression j immunol 104049jimmunol1202830 robbins pf lu yc elgamil m li yf gross c gartner j mining exomic sequencing data to identify mutated antigens recognizedby adoptively transferred tumorreactive t cells nat med 101038nm3161 lu yc yao x crystaljs li yf elgamil m gross c eï¬cient identiï¬cation of mutated cancer antigens recognized by t cellsassociated with durable tumor regressions clin cancer res 10115810780432ccr140433 prickett td crystal js cohen cj pasetto a parkhurst mr gartner jj durable complete response from metastatic melanoma after transfer ofautologous t cells recognizing mutated tumor antigens cancer immunolres 10115823266066cir150215 stevanovic s pasetto a helman sr gartner jj prickett td howieb landscape ofin successfulimmunotherapy of virally induced epithelial cancer science 101126scienceaak9510immunogenic tumor antigens tran e turcotte s gros a robbins pf lu yc dudley me cancerimmunotherapy based on mutationspeciï¬c cd4t cells in a patient withepithelial cancer science 101126science1251102 yossef r tran e deniger dc gros a pasetto a parkhurst mr enhanced detection of neoantigenreactive t cells targeting unique andshared oncogenes for personalized cancer immunotherapy jci insight 101172jciinsight122467 vinay ds kwon bs role of 41bb in immune responses semin immunol 101006smim19980157 wattstthcelltnftnfrresponsesfamilyannuof 101146annurevimmunol23021704115839revimmunolmembersincostimulation cannons jl lau p ghumman b de benedette ma yagita h okumurak 41bb ligand induces cell division sustains survival and enhanceseï¬ector function of cd4 and cd8 t cells with similar eï¬cacy j immunol 104049jimmunol16731313 halstead es mueller ym altman jd katsikis pd in vivo stimulation ofcd137 broadens primary antiviral cd8 t cell responses nat immunol 101038ni798 wolï¬ m kuball j ho wy nguyen h manley tj bleakley m activationinduced expression of cd137 permits detection isolation andexpansion of the full repertoire of cd8 t cells responding to antigenwithout requiring knowledge of epitope speciï¬cities blood 101182blood200611056168 ye qral cd137song dg poussin m yamamoto t best a li csnaturallyetoccurring tumorreactive t cells in tumor clin cancer res 10115810780432ccr130945accuratelyidentiï¬esenrichesandfor parkhurst m gros a pasetto a prickett t crystaletalisolation of tcelltumorassociatedpmutatedlymphocytes based on cd137 expression clin cancer res 10115810780432ccr162680speciï¬callyfromreceptorsantigensjs robbinsreactive withtumorltrating seliktaroï¬r s merhavishoham eitzhaki o yunger s markel gschachter j selection of shared and neoantigenreactive t cells foradoptive cell therapy based on cd137 separation front immunol 103389ï¬mmu201701211 makkoukacancerchesterimmunotherapyccd137 101016jejca201509026kohrt herationaleforeurj canceranti chen l coinhibitory molecules of the b7cd28 family in the control oftcell immunity nat rev immunol 101038nri1349 greenwaldrjfreemangjsharpeahfamilyimmunol 101146annurevimmunol23021704115611revisitedannurevtheb7 tran e robbins pf lu yc prickett td gartner jj jia l tcelltransfer therapy targeting mutant kras in cancer new engl j med 101056nejmoa1609279 sharpe ah wherry ej ahmed r freeman gj the function of programmedcell death and its ligands in regulating autoimmunity and infection natimmunol 101038ni1443 zacharakis n chinnasamy h black m xu h lu yc zheng zl immune recognition of somatic mutations leading to completedurable regression in metastatic breast cancer nat med 101038s4159101800408 gros a robbins pf yao x li yf turcotte s tran e pd1identiï¬es the patientspeciï¬c cd8 tumorreactive repertoire ltratinghuman tumors 101172jcij clin invest stronen e toebes m kelderman s van buuren mm yang ww van rooijn targeting of cancer neoantigens with donorderived t cell receptorrepertoires science 101126scienceaaf2288 yarchoan m johnson ba lutz er laheru da jaï¬ee em targetingneoantigens to augment antitumour immunity nat rev cancer 101038nrc2016154 tran e robbins pf rosenberg sa final common pathway of human cancerimmunotherapy targeting random somatic mutations nat immunol 101038ni3682 schumacher tn schreiber rd neoantigens in cancer immunotherapyscience 101126scienceaaa4971 inozume t hanada ki wang qj ahmadzadeh m wunderlich jr rosenbergsa selection of cd8pd1 lymphocytes in fresh humanmelanomas enriches for tumorreactive t cells j immunother 101097cji0b013e3181fad2b0 ahmadzadeh m johnson la heemskerk b w | Colon_Cancer |
covid19 has had an impact on the provision of colorectal cancer care the aim of the crccovid study is to describe the changes in colorectal cancer services in the uk and usa in response to thepandemic and to understand the longterm impactmethods and analysis this study comprises phases phase is a survey of colorectal units that aims toevaluate adherences and deviations from the best practice guidelines during the covid19 pandemicphase is a monthly prospective data collection of service provision that aims to determine the impactof the service modiï¬cations on the longterm cancer speciï¬c outcomes compared to the national standards phase aims to predict costs attributable to the modiï¬cations of the crc services and additionalresources required to treat patients whose treatment has been affected by the pandemic phase aims tocompare the impact of covid19 on the nhs and usa model of healthcare in terms of service provisionand cost and to propose a standardised model of delivering colorectal cancer services for future outbreaksethics and dissemination this study is a service evaluation and does not require hra approval or ethicalapproval in the uk local service evaluation registration is required for each participating centre in theusa ethical approval was granted by the research and development committee the results of thisstudy will be disseminated to stakeholders submitted for peer review publications conference presentations and circulated via social mediaregistration details nil the authors published by elsevier ltd on behalf of surgical associates ltd this is an open access under the cc byncnd license httpcreativecommonslicensesbyncnd40 introductionthe covid19 pandemic has had a significant impact on theprovision of healthcare worldwide as of the 29th june covid19 has resulted in conï¬rmed cases and corresponding author at department of surgery and cancer imperial collegelondon chelsea and westminster and the royal marsden campus unitedkingdomemail address ckontovounisiosimperialacuk c kontovounisiosdeaths in the uk at a local level hospitals have been forcedto make a number of workforce modiï¬cations and changes toserviceprovision to combat the crisis and maintain standards ofcare for our patients facetoface consultations have beendissolved or minimized in favour of telephone or virtual clinicsprovision of investigations including ct scans and endoscopieshave been significantly reduced and all benign surgical procedurespostponed furthermore the treatment algorithm for conï¬rmed colorectal cancer cases has proved challenging101016jisjp20200700524683574 the authors published by elsevier ltd on behalf of surgical associates ltdthis is an open access under the cc byncnd license httpcreativecommonslicensesbyncnd40 0ca courtney international of surgery protocols in the uk over forty thousand patients are diagnosed with colorectal cancer each year deviation from nice colorectal cancercrc guidelines may lead to significantly poorer outcomes however the current model of cancer services delivery cannot be maintained because of both resource limitation and the potential risksto patients and staff during the pandemic there is a lack of highdependency beds which are being utilized for covid19 patientsthere is the risk of exposing colorectal cancer patients the majority of whom are elderly and have significant comorbidities to thevirus during their treatment within the hospital patients requiringneoadjuvant or adjuvant therapy are at particular risk finallystaff safety must also be considered particularly around aerosolgenerating procedures such as endoscopy and laparoscopic surgery intercollegiate general surgery guidance on covid19 outlinedgeneral principles on the provision of a safe surgical service duringthe pandemic however there has been no speciï¬c guidance todate on how to best modify colorectal cancer crc service provision during the pandemic in the absence of a national consensusthe onus is on individual hospital trusts and multidisciplinaryteams to make very challenging decisions about individual patientcare lack of a uniï¬ed approach may have important consequencesat patient and healthcare institution levelsdelay in cancer diagnosis or treatment due to service modiï¬cation is likely to create an increased demand in resources once thecrisis has passed predicting the economic impact and planningfor this is essentialhow hospitals approach the new constraints on crc care andallocate resources may vary between the uk and usa it is hopedthat gaining insights from both perspectives will improve the problem solving methods and analysis aims and objectivesthe aim of the crc covid study is to describe the changes incolorectal cancer services in the uk and usa in response to thecovid19 pandemic and to understand the longterm impactour primary and secondary objectives relevant to each phase ofthe study are listed in table study designthis is a multicentre service evaluation conducted through aresearch collaborative with the support of the crc covid steeringcommittee all colorectal units continuing to provide cancer services in the uk ireland and the usa have been invited to participate all study and recruitment information is available on thetable primary and secondary study objectiveswebsite crccovid this service evaluation has been endorsedby the royal college of surgeons of england rcsthis service evaluation will be carried out in phases fig phase uses a questionnaire to assess the modiï¬cationsadopted by each colorectal unit in order to continue provision ofthe colorectal cancer services during the covid19 pandemic ithas been developed using an iterative process after research ofall relevant guidelines to construct the standard against which services would be evaluatedthe following guidelines relevant to the management of colorectal cancer have been used as standards for this serviceevaluation nice guidelines colorectal cancer [ng151] nice guideline suspected cancer recognition and referral[ng12] association of coloproctology of great britain irelandacpgbi guidelines for the management of cancer of thecolon rectum and anus [] british society of gastroenterologyassociation of coloproctology of great britain and irelandpublic health england postpolypectomy and postcolorectal cancer resection surveillanceguidelines informal consultations with consultants nurse specialists andpatients have been used to develop the tool and then it has beenmodiï¬ed after clinician review for face validity ï¬ow and relevance the ï¬nal instrument comprises questionsphase investigates the provision of colorectal cancer servicesduring the covid19 pandemic by evaluating the performance ofeach unit against the national bowel cancer audit outcomes all centres participating in phase will be required to registerthis service evaluation as per local protocol prior to commencement of data collection on redcap this will be the responsibilityof the local leadphase of the study will develop a prediction model of the economic burden of the modiï¬cations in cancer service delivery thismodel will be designed jointly by two international businessschools based on previous publications and national statisticsphase will evaluate and compare the impact of the covid19pandemic on the nhs and the usa healthcare using data collectedduring phase and the predictive mode utilized in phase speciï¬cdifferences in modiï¬cations of crc services will be examined recruitmentphase survey has been distributed to all colorectal consultantsin the uk and ireland through personalised emails social mediaand the rcs the recruitment of colorectal cancer units in thephasephase phase phase phase primary objectiveevaluate adherences and deviations from best practiceguidelines on colorectal cancer during covid19pandemicative crc service deliverysecondary objectives 0f describe modiï¬cations to screening process for crc 0f describe modiï¬cations to preoperative intraoperative and postoper 0f demonstrate global effect of covid19 pandemic on crc service provi 0f outline consensus recommendations for sustainable modiï¬cations to 0f predict the impact of modiï¬cations on the incidence and prevalence of 0f plan adjustments to crc service provision after the end of pandemicsion irrespective of the type of healthcare systemdetermine the impact of crc service provision followingmodiï¬cations on longterm cancer speciï¬c outcomescompared to national standards 0f predict the costs attributable to modiï¬cations of crc services during covid19 pandemic 0f predict additional resources required to treat patients whose treatment has been affected by covid19 0f compare the impact of covid19 on the nhs and usa model of healthcare in terms of service provision and cost 0f propose a standardised model of delivering colorectal cancer services for future outbreaksdifferent crc stages in monthscrc services 0ca courtney international of surgery protocols fig phases of crc covidusa will use similar approach all units recruited into phase arerecruited into phase participation in phase is not mandatory inorder to participate in phase data collectionall surveys and data collection follow the gdpr requirementsand comply with caldicott principles individual patient identiï¬able data is not collected in this study study data is collectedand managed using redcap electronic data capture tool hostedat the kennedy institute of rheumatology at the university ofoxford redcap research electronic data capture is asecure webbased software platform designed to support datacapture for research studies providing an intuitive interfacefor validated data capture audit trails for tracking data manipulation and export procedures automated export procedures forseamless data downloads to common statistical packages and procedures for data integration and interoperability with externalsources after the close of the phase data collection period all data setswill be checked for missing data where possible centres will begiven an opportunity to rectify missing data centres where of data is missing will be excluded from data analysis and localleads will be notiï¬ed a nominated data validator will need toensure data accuracy prior to submission if during this processmajor discrepancy is identiï¬ed within the data set the centresdata will be excluded completely from the analysisfor details of the data collected in phase and phase pleaserefer to supplement data analysisphase responses to the survey pertaining to deviations fromdiagnostic and treatment protocols during the covid19 pandemicsee supplement will be converted to a numerical scale where will denote no deviation and will denote complete cessation ofservice provision the scores will be summarized using appropriatesummary statistics and analyzed using unsupervised learningkmeans and hierarchical clustering to identify clusters of homogeneous response to the pandemicphase every month participating centres will report theirdiagnostic and treatment activity see supplement to determine the impact of covid19 on colorectal cancer activity we willuse timeseries methods and data on historical activity and patientoutcomes to estimate a baseline of expected monthly activity that would have taken place in the absence of the pandemicthe baseline and a conï¬dence interval will be estimated atthe national regional and individual nhs trust level the baselineestimate will then be compared to the actual activity as reportedby publicly available data and data collected by this studythe difference between expected and actual activity will providean estimate of the reduction in activityto quantify the impact on patient outcomes associated with theestimated reduction in activity and deviation from standardizedcare protocols we will use estimates of disease progression available in peerreviewed literature [] a similar methodologywill be used to predict the impact of the pandemic on the incidenceand prevalence of different colorectal cancer stages in the following months under different scenarios predictions will be madeat the regional and national level and depending on data granularity at the trust levelphase will estimate the ï¬nancial costs of modiï¬cations to thecrc service provision due to the covid19 pandemic this willallow prediction of the expenditure and the additional resourcesrequired to resume routine services we will base this on the literature regarding the price of treatment at different disease stages and the information about the cost of resource utilizationconsultations diagnostic tests operating theatre time and hospital stay phase will compare the results from phases in theuk with those in the us discussioncancer care and maintaining high standards of diagnosis andtreatment has long been a priority of the nhs and internationalhealth care systems the pandemic has shifted this focus awayfrom the cancer services colorectal cancer patients are particularly 0ca courtney international of surgery protocols vulnerable to the disruption of their care as diagnosis throughendoscopy was stopped due to concerns about virus aerosolysation this study is important because it is the ï¬rst study to ask howindividual units had to modify their services and adapt to the newconstraintsin addition to describing the changes and understandingwhether different units had different approaches we wish to gofurther by understanding the effects of diagnostic and treatmentdelay by prospective data collection of cancer cases referrals diagnosis staging and treatment and comparing them to nationally collected audit data these data will allow us to model the economic impact of thedelay and what resources are required to restore cancer servicesto precovid19 standardsthe strengths of this study are in the multimodal approach tothe issues international collaboration and support from the royalcollege of surgeons our diverse team of management and business academics colorectal surgeons nurse specialists and patientadvisors enable us to have a range of approaches to collect andanalyse the datathe main limitation to the study is nonresponder or samplingbias as we require voluntary participation from colorectal teamswe will ensure that we adjust statistical analysis for any underrepresentation we expect that even with minimal participationuseful models can be generated to understand future resourcerequirements at an individual hospital level the methodologyemployed by other units will demonstrate the utility of the modelin summary this is a novel and important multiphase studythat is vital to understand how to best care for cancer patientsand ensure that the effects of the pandemic are mitigated ethics and disseminationthis study is a service evaluation and does not require hraapproval or ethical approval in the uk departmental approvalhas been granted by the university each participating centre mustseek local permission from their local audit department prior tocommencement of data collection in the usa ethical approvalwas granted by the research and development committeedata for phase will be submitted for publication as soon as theresults become available interim data analysis will be presented tothe royal college of surgeons covid19 research collaborativedata for other phases will be submitted for publication once thedata collection has been completed which is anticipated to be afterthe routine service provision resumes all data will be presented atnational and international conferences circumstances permitting guarantornone research registration numbernoneethical approvalnoneauthor contributionsac designed the study wrote the initial proposal drafted themanuscript based on the study proposal and is part of the auditadvisory groupamh ns nd ow sm sr gm nt mg td bs jee ad ptadvised on the study design and the protocol and is part of thesteering committeeck is a project piall authors read commented on and approved the study designthe protocol and the ï¬nal manuscriptfundingthis research received no speciï¬c grant from any fundingagency in the public commercial or notforproï¬t sectorsdeclaration of competing interestthe authors declare that they have no known competing ï¬nancial interests or personal relationships that could have appearedto inï¬uence the work reported in this paperreferences world health anization the united kingdom who coronovirus diseasecovid19 dashboard covid19whointregioneurocountrygbaccessed june covidsurg collaborative global guidance for surgical care during the covid pandemic br j surg a spinelli g pellino covid19 pandemic perspectives on an unfolding crisisbr j surg british society of gastroenterology endoscopy activity and covid19 bsgand jag guidance apr wwwbsgukcovid19adviceendoscopyactivityandcovid19bsgandjagguidance accessed may nhs england nhs improvement letter to chief executives of all nhs trustsand foundation trusts ccg accountable ofï¬cers gp practices and primary carenetworks and providers of community health services mar wwwenglandnhsukcoronaviruswpcontentuploadssites52202003urgentnextstepsonnhsresponsetocovid19lettersimonstevenspdfaccessed may research uk cancerincidencecancerbowelstatisticswwwcancerresearchukhealthprofessionalcancerstatisticsstatisticsbycancertypebowelcancerincidenceheadingzeroaccessed may royal college of surgeons of england updated intercollegiate general surgeryguidance on covid19 apr wwwrcsengacukcoronavirusjointguidanceforsurgeonsv2 accessed may national institute for health and care excellence nice colorectal cancernice guideline ng151 wwwniceukguidanceng151accessed accessed march national institute for health and care excellence nice suspected cancerrecognition and referral nice guideline ng12 wwwniceukguidanceng12 accessed accessed march c cunningham k leong s clark a plumb s taylor i geh s karandikar bmoran association of coloproctology of great britain ireland acpgbiguidelines for the management of cancer of the colon rectum and anus diagnosis investigations and screening colorectal dis suppl s gollins b moran r adams c cunningham s bach as myint a renehans karandikar v goh d prezzi g langman s ahmedzai i geh association ofcoloproctology of great britain ireland acpgbi guidelines for themanagement ofmultidisciplinary management colorectal dis suppl rectum and anusthe coloncancer of glangman m loughrey n shepherd p quirke association ofcoloproctology of great britain ireland acpgbi guidelines for themanagement of cancer of the colon rectum and anus pathologystandards and datasets colorectal dis suppl k leong j hartley s karandikar association of coloproctology of greatbritain ireland acpgbi guidelines for the management of cancer of thecolon rectum and anus follow uplifestyle and survivorshipcolorectal dis suppl b moran c cunningham t singh p sagar j bradbury i geh s karandikarassociation of coloproctology of great britain ireland acpgbi guidelinesfor the management of cancer of the colon rectum and anus surgicalmanagement colorectal dis suppl md rutter j east cj rees n cripps j docherty s dolwani pv kaye kjmonahan mr novelli a plumb bp saunders s thomasgibson djmtolan s whyte s bonnington a scope r wong b hibbert j marsh bmoores a cross l sharp british society of gastroenterologyassociation ofcoloproctology of great britain and irelandpublic health england postpolypectomy and postcolorectal cancer resection surveillance guidelines gut 0ca courtney international of surgery protocols healthcare quality improvement partnership hqip national bowel canceraudit annual report an audit of the care received by people with bowelcancer in england and wales v20 wwwnbocaukcontentuploads202001nboca2019v20pdf accessed june pa harris r taylor r thielke j payne n gonzalez jg conde researchelectronic data capture redcapa metadatadriven methodology andworkï¬ow process for providing translational research informatics support jbiomed inform pa harris r taylor bl minor v elliott m fernandez l oneal l mcleodg delacqua f delacqua j kirby sn duda re consortium the redcapconsortium building an international community of software platformpartners j biomed inform nhs england and nhs improvement cancer waiting times wwwenglandnhsukstatisticsstatisticalworkareascancerwaitingtimesaccessed june national bowel cancer audit nboca datagov weblink accessed junewwwnbocaukresourcesnbocadatagovweblink cancer research uk incisive health saving lives averting costs an analysis ofthe ï¬nancial implications of achieving earlier diagnosis of colorectal lung andovarian cancer wwwcancerresearchuksitesdefaultï¬lessaving_lives_averting_costspdf accessed may s sun f klebaner t tian a new model of time scheme for progression ofcolorectal cancer bmc syst biol suppl s2 j emery p vedsted new nice guidance on diagnosing cancer in generalpractice br j gen pract hb keshava je rosen mr deluzio aw kim fc detterbeck dj boffawhat if i do nothing the natural history of operable cancer of the alimentarytract eur j surg oncol yh lee pt kung yh wang wy kuo sl kao wc tsai effect of length oftime from diagnosis to treatment on colorectal cancer survival a populationbased study plos one e0210465 d roder cs karapetis i olver d keefe r padbury j moore r joshi dwattchow dl worthley cl miller c holden e buckley k powell dburanyitrevarton k fusco t price time from diagnosis to treatment ofcolorectal cancer in a south australian clinical registry cohort how it variesand relates to survival bmj open e031421 cancer research uk bowel cancersurvivalstatistics wwwcancerresearchukhealthprofessionalcancerstatisticsstatisticsbycancertypebowelcancersurvival accessed june kk turaga s girotra are we harming cancer patients by delaying theircancer surgery during the covid19 pandemic ann surg 0c' | Colon_Cancer |
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 |
" the incidence of thyroid carcinoma is increasing all over the world some studies have suggestedthat the change of adipokines expression can induce thyroid carcinoma however other studies have come to theopposite therefore we studied the relationship between adipokines and thyroid carcinomamethods databasespubmed cochrane library sinomed cnki wanfang and clinical trial registries weresearched a metaanalysis was then performed through a fixed or randomeffects model to calculate i values forheterogeneity analysisresults twentynine s were finally included for analysis the level of serum tumor necrosis factoralpha tnfα [standardized mean difference smd confidence interval ci to i2 p ]and the ratio of tnfα immunoreactivity in tissues [odds ratios or ci to i2 p ]in thyroid carcinoma are significantly higher than those in control the serum interleukin6 il6 in patients withthyroid carcinoma is higher than that in control smd ci to i2 p there is nosignificant difference of the ratio of il6 immunoreactivity in tissues between carcinoma and control or ci to i2 p the ratio of leptin immunoreactivity in tissues is significantly associated with therisk of thyroid carcinoma or ci to i2 p however after analyzing theexpression level of serum adiponectin in three studies no significant difference is found between thyroidcarcinoma and the control p s adipokines tnfα il6 and leptin show a strong relationship between elevated concentrations inserum andor tissue and thyroid carcinoma however the association between adiponectin and thyroid carcinomaneeds further researchkeywords thyroid carcinoma adipokines tnfα il6 leptin metaanalysis correspondence liaolinsdueducn cwc_llsdueducn junyu zhao and jing wen contributed equally to this work1department of endocrinology and metabology the first affiliated hospitalof shandong first medical university shandong provincial qianfoshanhospital jinan china5department of endocrinology and metabology qilu hospital of shandonguniversity cheeloo college of medicine shandong university jinan chinafull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0czhao bmc cancer page of thyroid carcinoma is the most common endocrine malignancy but mostly has good prognosis during the pastdecades a rising incidence of thyroid carcinoma worldwide has aroused the widespread attention of researchers[ ] someone supposed that the growing use of diagnostic imaging and fineneedle aspiration biopsy may bethe main reason but this may be only partial andcan not totally explain the increased incidence of microcarcinoma changes in the incidence of a cancer are notonly associated with increased detection and other unknown risk factors need further explore recently somescientists found that the incidence of thyroid carcinomahas increased along with a marked rise in obesity rateand accumulating evidence of an association betweenobesity and increased thyroid carcinoma risk has beenproposed [] various hypotheses have been supposedto interpret the relaitonship between obesity and thyroidcarcinoma including hyperinsulinemia upregulation ofaromatase activity chronic low grade inflammation altered immune response and dna damage caused byoxidative stress furthermore recent data supportingthe notion that a changed expression of adipokinescaused by obesity can affect the cell proliferation andeven induce a thyroid tumorigenesis [] adipose tissue is a specialized connective tissue composed of fatcells which releases a number of biologically active molecules called adipokines or adipocytokinesincludingleptin adiponectin resistin and many cytokines of theimmune system such as tumor necrosis factoralphatnfα interleukin6 il6 and complement factor dalso known as adipsin adipokines refer to various enzymes hormones cytokines growth factors proteinsand other biological active substances secreted by adipocytes including adiponectin leptin resistin and interleukin the concentration of adipokines such as tnfαil6 and leptin were significantly higher in obese subjects and the elevated levels was linked to obesity andeven positively correlated with body mass index []it is reported that adipokines took part in the biologicalprocesses of insulin sensitivity inflammation and proliferation [ ] which the proliferation have been recognizedthetumorigenesis and development at present many kindsof adipokines have been reported to be associated withthyroid carcinoma rehem ra suggested thatserum leptin levels were higher in welldeffierentiatedthyroid carcinoma patients and a significant drop aftersurgery another envidence showed that adiponectin related with tumor size however the opposite resultswere also found in other studies some researchesreported the expression of adipokines is lower in tumortissue than normal control [] it is clearly that certain confounders such as age sex ethnicity and alsoimportantfactorleadingtoasanheterogeneity in study size methodology and original ofsample should be considered when trying to analyze theassociation between adipokines and thyroid carcinomathese confunding factors above may be the cause of inconsistency results from different researches additionaly the association between adipokines and thyroidcarcinoma are still not well documented therfore theaim of this metaanalysis was to investigate the association between adipokines and thyroid carcinoma andpropose that adipokine as a risk factor for thyroidcarcinomamethodssearching progresswe conducted a search of all studies published until27th july regarding the association between adipokine and thyroid carcinoma eligible casecontrol studieswere found by searching the database of pubmedcochrane library sinomed cnki and wanfang and restricted to published results clinical trial register centers httpwwwclinicaltrialsgov were also searchedthe following search terms adipokine or leptin oradiponectin or resistin or tumor necrosis factoralpha or interleukin6 or complement factor d oradipocytokines or tumor necrosis factorα or tnfα or il6 or adipsin and thyroid cancer or thyroid neoplasm or thyroid tumor or thyroid carcinoma or differentiated thyroid carcinoma or dtc orpapillary thyroid carcinoma or thyroid carcinomapapillary or ptc or thyroid cancer follicular orftc or thyroid carcinoma anaplastic or atc orthyroid cancer medullary or mtc hand searchingwas used to identify appropriate studies including reference lists of eligible s and related previous reviews eligible studies met the following criteria published in english or chinese language studyassessed the association between adipokine and thyroidcarcinoma study designed as the casecontrol study study reported the expression of at least one adipokine either in blood or tissue studies were excluded ifany of the followings were identified insufficient information concerning adipokine or thyroid carcinomaoutcome cannot directly extract or calculate or and95ci the type of study was not a casecontrol designhave not fulltext animal trialsstudy selection and data extractiontwo reviewers screened the studies and extracted dataindependently any disagreement was resolved by discussion or consensus with a third senior reviewer dataincluded the followingfirst author publication yearcountry participant characteristics ie mean age sample size sex ration pathological type of thyroid carcinoma source of controls measured outcomes or the 0czhao bmc cancer page of scores were considered to be of high quality disagreements were resolved by reevaluating and discussing between two reviewersinsearchingthis metaanalysisresultssearch results and characteristics of included studies s regarding the association between adipokine and thyroid carcinoma were searched in therelated database and clinicaltrial websites afterscreening the title and abstracts s were selected for fulltext review finally studies were eligibleprogressincluded and excluded details are all shown in fig eighteen of these studies are published in chinese[ ] and the rest are published in english[] nineteen studies were conducted in chinatwo in india and two in turkey brazil greece iranitaly denmark and serbia each had one study totally there are patients with thyroid carcinomain the case group and controls including healthysubjects patients with benign thyroid diseases or normal thyroid tissue near carcinoma were included inthe control group the sample size ranges from to in the case group while to in the controlgroup all the thyroid carcinoma patients were confirmed by pathologically among these studiesfourteen studies reported papillary thyroid carcinomaptc eight studies reported differentiated thyroidcarcinoma dtcreported differentpathological types in one paper one study reportedmedullary thyroid carcinoma mtc and the restfour studies did not show the pathological detailsthe detailed characteristics ofincluded studies aresummarized in table three studiespercentage of samples show immunoreactivity for adipokines antibody both in the case and control groups thecalculation method is shown below take thyroid cancerfor example the number of samples obtained from thyroid carcinoma that show immunoreactivity for adipokines antibody divided by the total number of thyroidcarcinoma samplesstatistical analysisfor metaanalysis dichotomous outcomes were analyzedby using the odds ratios or computed using the mantelhaenszel method fixed or random models continuousvariables measured on the same scale expressed as a meanvalue and standard deviation were analyzed by usingweighted mean differences wmd otherwise standardized mean difference smd were used for different scaleall results were reported with confidence interval ci i2 was used to assess heterogeneity between studies and i2 values of and representing no lowmoderate and high heterogeneity respectively visual inspection of the funnel plot was done to assess publicationbias the analyses were performed by review manager cochrane collaboration united kingdom httpwwwcochranequality assessment and risk of biasthe methodological quality of casecontrol study wasassessed by the newcastleottawa scale nos supplement table which consists of the three parameterseight questions with nine possible scores selection exposure and comparability a study can be awarded amaximum of one score for each numbered item withinthe selection an exposure categories a maximum oftwo scores can be got for comparability a higher scoremeans better quality in methodology and five or morefig flow chart of the systematic search process 0czhao bmc cancer page of zhao jianqiang chinaptc ftc atcand mtcthyroid adenoma andnormal healthunknownunknowntable characteristic of included studiesfirst authoryearcountrypathologicaltype of thyroidcancersource of controlsl kayser denmark ptc and ftccao guangyao chinaunknownmtrovato italydtc andundifferentiatedcarcinomamultinodular goitersadenomas hashimotosthyroiditis hyperplasticglandsthyroid adenoma andnodular goiternormal thyroid tissues andbenign nodulesmelih akinci wang jingxia zhuangxiaoming yu xiao hou sen snezanazivancevicsimonovic xu xiaocheng xeniprovatopoulou turkeyptchealthy volunteerschinaptc and ftcnormal thyroid tissueschinaptc ftc andmtcthyroid adenoma andnormal healthchinaptcthyroid adenoma andnormal thyroid tissue nearcarcinomachinaptcthyroid adenomaserbiawdtchealthy subjectschinathyroidcarcinomagreeceptcthyroid adenomabenign thyroid disease andhealthy controlssun qinnuan chinaptcnormal thyroid tissue nearcarcinoma and healthycontrolschinaptcthyroid adenomachinaptcthyroid adenomamean age yearfemale outcome indexnumber ofparticipants ncases control casesunknowncontrolcontrolcasesunknownunknownunknownunknownunknowntnfα tissuetnfα tissueil6 tissueil6ãtnfαblood ± ±unknown leptinblood unknown tnfα tissue unknown il6ãtnfαunknownunknownbloodleptintissueunknown unknown leptin ± ± tissuetnfαblood ± ± ± ± ± ± ± il6blood il6blood tnfαbloodandtissue ±unknown unknown leptinunknownunknowntissueadiponectintissueunknown unknown adiponectintissue il6ãtnfαblood ± ± zhang zijie zhong xiuxiu zhang bo hu jinhua snezanazivancevicsimonovic yanlan fan chinadtcchinadtcnormal thyroid tissue nearcarcinomathyroid adenoma andhealthy controls ±serbiaptccontrol subjectsunknownunknownil6bloodchinathyroidcarcinomanodular goitre hashimotosthyroiditis follicular adenomaand adjacent nonneoplasticthyroid tissue samplesunknownunknownleptintissue 0czhao bmc cancer page of table characteristic of included studies continuedfirst authorsource of controlsyearcountrypathologicaltype of thyroidcancerchinathyroidcarcinomabenign thyroid disease andnormal thyroid tissue nearbenign thyroid diseasechinaptcthyroid adenomaturkeyptchealthy volunteersindiaptcindiaptcbenign thyroid diseases andhealthy individualsbenign thyroid diseases andhealthy individualsnumber ofparticipants ncases control cases ±mean age yearfemale outcome indexcontrol ±casescontrol tnfαtissue ± ±tnfα tissue il6bloodunknown unknown tnfαbloodunknown unknown il6bloodwangxinzheng song runbo kemal beksac toral pkobawala toral pkobawala raziyehabooshahab zhang bo zhouxiaodong ma xiaokai marianabonjiornomartins iranmtchealthy subjects ± ± leptinãadiponectinbloodchinadtcnormal thyroid tissue nearcarcinomaunknown unknown leptintissuechinadtchealthy subjects ± ±il6ãtnfαbloodchinaptcthyroid adenomaunknown unknown leptinbrazildtcbenign thyroid nodules andhealthy controls ±tissue il6blood ± ±chinail6 sun zhenhua tissuetnfα tumor necrosis factora dtc differentiated thyroid carcinoma il6 interleukin6 ptc papillary thyroid carcinoma ftc follicular thyroid carcinoma atcanaplastic thyroid carcinoma mtc medullary thyroid carcinoma wdtc welldifferentiated thyroid carcinoma fnac fine needle aspiration cytologynodular goiterptcquality of included studiesthe quality assessment of these studies is assessed bythe nos and the resultis shown in supplementaltable five or more scores are determined as highquality two studies conducted by cao g in and l kayser in only get two scoresshowing a poor quality in methodology the rest studies are assessed as high qualitytnfα and thyroid carcinomatwelve studies reported the expression of tnfα bothin patients with thyroid carcinoma and control subjects[ ] among these sevenstudies [ ] had tested the level ofserum tnfα two studies [ ] had tested the expression of tnfα in tissues and the ratio of tnfα immunoreactivity was tested in four studies [ ] firstly fixedeffect model is used to merge the smdvalues of serum tnfα level however a large heterogeneity is found by the heterogeneity analysis heterogeneity test chi2 p i2 and itmay be due to the different units differenttestingmethods in different researches or other unknown factors then randomeffect model to merge the smd isused and pooled effect size in favor of control group is ci to p fig 2a smdvalues of the expression of tnfα in tissues is mergedby fixedeffected model and the heterogeneity analysisshow a considerable heterogeneity heterogeneity testchi2 p i2 the different unitsand limited numbers of research may be the original ofheterogeneity so the pooled smd with randomeffectmodel of the expression of tnfα in tissues is ci to p fig 2b the pooled orwith fixedeffect model of the ratio of tnfα immunoreactivity in thyroid carcinoma tissues is ci to p however a significant heterogeneity is detected heterogeneity test chi2 p i2 the published by l kayser in with a poor quality in methodology may attributeto this high heterogeneity then randomeffect model ofpooled or is used and pooled effect size in favor of 0czhao bmc cancer page of fig forest plot of the tnfα level and the ratio of tnfα immunoreactivity in tissues in patients with thyroid carcinoma a level of serum tnfα b expression of tnfα in tissue c ratio of tnfα immunoreactivity in tissuecontrol group is ci to p fig 2c in level of serum tnfα and theratio of tnfα immunoreactivity in tissues of thyroidcarcinoma patients are significantly higher than controlsubjects which are without thyroid carcinomail6 and thyroid carcinomaamong the included studies reported the level ofserum il6 in patients with thyroid carcinoma and control subjects [ ] due to thelarge heterogeneity of the merged smd values of serumil6 level by the heterogeneity analysis heterogeneitytest chi2 p i2 randomeffectmodel was used to pooled the smd values and thepooled effect size in favor of control subjects is ci to p fig 3a which meansthat patients with thyroid carcinoma have a significantlyhigher level of serum il6 than control subjects twostudies reported the ratio of il6 immunoreactivity bothin thyroid carcinoma tissue and noncarcinoma tissue[ ] the pooled or of the limited two studies donot show an increased ratio of il6 immunoreactivity inthyroid carcinoma tissues or ci to p and a large heterogeneity always existsheterogeneity test chi2 p i2 fig3b thus the level of serum il6 is higher in patientswith thyroid carcinoma however it needs more clinicaldata to verify the relationship between the expression ofil6 and thyroid carcinoma tissueleptin and thyroid carcinomatwo studies reported the level of serum leptin [ ]and another five studies reported the ratio of leptin immunoreactivity in tissues [ ] because ofthe considerable heterogeneity of the pooled wmd ofserum leptin level heterogeneity test chi2 p i2 and pooled or of the ratio of leptinimmunoreactivity in tissues heterogeneity test chi2 p i2 by the heterogeneity analysis with fixedeffect model randomeffect model is further used to merge the values and analysis howeverthere is no association of higher level of serum leptin 0czhao bmc cancer page of fig forest plot of the il6 level and ratio of il6 immunoreactivity in tissue in patients with thyroid carcinoma a level of serum il6 b ratio ofil6 immunoreactivity in tissuefig forest plot of the leptin level and ratio of leptin immunoreactivity in tissuein patients with thyroid carcinoma a level of serum leptin bratio of leptin immunoreactivity in tissue 0czhao bmc cancer page of with risk of thyroid carcinoma wmd 95ci to fig 4a moreover the pooled or of theratio ofleptin immunoreactivity in tissues from fivestudies is 95ci to fig 4b whichmeans a high ratio of leptin immunoreactivity in tissueis significantly related to thyroid carcinomaadiponectin and thyroid carcinomathree studies reported the expression of adiponectin inthyroid carcinoma including serum and tissue [ ] and the result is summarized in table it could befound that the level of serum adiponectin is not staticallydifferent comparing thyroid carcinoma patients withcontrol subjects p interestinglyit was foundthat the expression of adiponectin in thyroid carcinomatissue is significantly lower than control tissue while theopposite result is found when comparing the ratio ofadiponectin immunoreactivity however there was onlyone study for each result and this may be the reasonwhy the two results are diametrically opposed thus itneeds more clinical studies to confirm in the futurepublication biasthe funnel plot was applied for assessing publicationbias of studies included in the three results includingtnfα fig 5a il6 fig 5b and leptin fig 5c infig 5a and fig 5b almost all studies lies inside the95cis with an even distribution around the verticalindicating no evident publication bias was obtainedthrough the visual distribution of funnel plot howevera potential publication bias was found in fig 5c whencomparing the ratio of leptin immunoreactivity in tissues and that might influence the result of this metaanalysisdiscussioncurrently obesity affects one third of population amongus adults and china has become a big country ofobesity with the incidence ranking first worldwide in theyear of nowadays increasing clinical and experimental studies and documented the closely relationship between malignancies including colon esophaguskidney liver breast endometrium pancreas and prostate as well as nonhodgkins lymphoma and multiplemyeloma and obesityoverweight which affect its occurrence development and prognosis [] becauseof the increasing incidence of thyroid carcinoma duringthe past decades lots of scientists focus on studying therisk factors of thyroid carcinoma it was found that theincidence of thyroid carcinoma has increased along witha marked rising rate of obesity [] furthermore obesity is an independent risk factor for thyroid carcinoma increased insulin resistance elevated serum cholesterol level and upregulated cox2 expression may be thetarget of the correlation between obesity and thyroidcarcinoma it is reported that people with higherbody mass index have a higher concentration of adipokines [] adipokines take part in the followingpathological and physiological processes such as insulinsensitivity inflammation and proliferation [ ] andthese are important in the process of tumorigenesis anddeveloping so adipokines may be one of the targetslinking obesity with thyroid cancer the metaanalysiswas based on previous published studies in previousstudies the analysis of adiponectin and thyroid cancermostly focused on tnf il6 leptin and adiponectinwhile few studies focused on other molecules includingil1 and il8 and we failed to combine statisticstherefore in this metaanalysis only tnf il6 leptinand adiponectin which are the most published adiponectin were analyzedtnfα produced by adipose tissue and inflammatorycells can lead to inflammatory response necrocytosisand assist other cytokines to kill tumor cells and improve the antitumor ability meanwhile tnfα plays animportant role in the process of inflammation insulinresistance diabetes and obesity a moderate amount oftnfα has a protective effect while an excessive amountwill cause damage which may lead to a resistant oftumor cells to tnfassociated apoptosisinduced ligandswhen the microenvironment of apoptosis is maladjustedtnfα has the ability to promote the production ofgranulocytecolony stimulating factor by thyroid fibroblasts which may play an important role in thyroidcancer moreover tnfα can stimulate the vasoactivemediators such as interleukin and prostaglandin and these mediators can promote the proliferation oftumor cells and significantly reduce the immune function tnfα can also induce an increased expression ofvascular endothelial growth factor vegf the laterof that can promote the proliferation of tumor cells andprovide conditions for tumors metastasistable summary of adiponectin expression in thyroid carcinomaserum adiponectin ratio of adiponectin immunoreactivity effect sizewmd or adiponectin in tissue ci confidence interval wmd weighted mean differences or odds ratioswmd 95ci pi2not applicable 0czhao bmc cancer page of fig funnel plots of a tnfα b il6 and c leptin revealed no significant publication bias se smd standard error of standardizedmean differencein surprisingly the results of clinical studies provide evidence for basic research simonovic sz evaluated cytokine profiles determined in supernatants obtained from whole blood cultures in patients with dtc before and days after radioactiveiodine 131itherapy and control subjects andfound that the expression of tnfα in dtc patients ishigher than control subjects and it showed a decreasedlevel after 131i therapy than those before therapy however no statistical difference found for the limited sample size another study conducted by kobawala tp with more patients patients with benign thyroiddisease ptc patients and healthy individuals determined the circulating levels of tnfα and it wasfound that the serum level of tnfα was significantlyhigher in ptc patients than benign thyroid disease patients and the later was also significantly higher thanhealthy individuals furthermore serum tnfα was reported to be a significant prognosticator for overall survival in ptc patients it is a pity thatopposite result wasreported in a casecontrol study that included dtccases and matched cancerfree cohort participantswhich found that tnfa was not associated with thyroidrisk in either gender based on current evidence our metaanalysis suggeststhat tnfα exhibit a strong association with thyroid carcinoma it may because that elevated tnfα may involved in the tumorigenesis and development of thyroidcancer another possible reason is that the tnfα decreased with tumor cells less resulted the activation ofthe immune system by thyroid carcinomathereforemore clincal studies and basic reseaches should be conducted in the futureil6 a multifunctional cytokine plays important rolesin different types of cells including tumor cells it is reported that elevated serum il6 level is closely related tothe tumorigenesis and development of a variety of tumors a strong positive association between theserum il6 and the progression and poor prognosis oftumors in patients with several types of tumor wasalready found [] serum il6 level in thyroid cancer has been evaluated in numerous studies including 0czhao bmc cancer page of in vivo and in vitro studies provatopoulou x found that serum il6 were significantly higher in malignant and benign thyroid diseases compared to healthycontrols however other studies show a different resultthat no significance different of il6 was found betweenthyroid cancer and nonthyroid cancer [ ] a limited sample size different inclusion criteriadifferent population characteristics or different pathological type of thyroid cancer may explain such a difference for in vitro research il6 was also found to beexpressed in thyroid cancer cell lines and a potential roleof il6 in ptc was confirmed indirectly the underlying mechanism may be the followingsbelow tumor cells including esophageal cancerlungcancer colorectal cancer and melanoma were foundhave the function of autocrine il6 which can affect thegrowth and proliferation of tumor cells and participatein the tumor growth and metastasis by acting on themembrane receptors also il6r was found associated with the characterization of thyroid nodules malignancy and tumor aggressivenessin additioniliopoulos d found that src nonsomatic tyrosine kinase family oncogene can induce the normal epithelial cell transformation by activating nfκb and thistransformation contributes to tumorigenesis il6 is considered as an important regulatory factor in this processanother possibility is that the activation of the immunesystem of patients with thyroid cancer leads to an increase in adikopines level in general the data above support that il6 is important for thyroid cancer but the detail mechanism remainto be further studyleptin a circulating hormone secreted by adipocytesexerts its biological effect by combing with its receptorwhich is mainly presented in the hypothalamus meanwhile gene of leptin receptor is also expressed in manyother tissues such as lung liver and kidney it is reported that obesity and overweight can lead to a highlevel of serum leptin which may because that obesity always accompanies with insulin resistance and hyperinsulinemia and insulin further enhance the expression ofleptin moreover leptin acts as a growth factor in a variety of human cellsincluding both normal cells andtumor cells which regulates the process of differentiation proliferation and apoptosis thus stimulate thetumorigenesis and development of tumors through mediatingpathway rhoalimk1cofilinpathway and mapkerk pathway kim wg evaluated the effect of dietinduced obesity on thyroid carcinogenesis in a mouse model that spontaneously develops thyroid cancer thrb pvpv pten mice and found that obesity increases the frequency of anaplasia of thyroid cancer and exacerbatesthyroid cancer progression that were mediated byjakstat3increased activation of the jak2 signaling transducerand activator of stat3 signaling pathway and inductionof stat3 target gene expression leptin is always reported a high expression on solid tumors and it isconfirmed that serum leptin levelis significantly increased in thyroid cancer mainly ptc while otherstudies showed a same results in cancer tissues [ ] yu xiao conducted a clinical studycomparing the level of serum leptin in ptc patientsincluding patients with lymph node metastasis and thyroid adenoma patients in dalian china and foundthat patients with lymph node metastasis have a higherlevel of leptin than those without lymph node metastasisleptin can induce the expression of vascular endothelialgrowth factor and promote neovascularization in tumortissue in addition it can also inhibit the apoptosisthrough bcl2 dependent mechanism meanwhile leptinreceptor exists in all thyroid cancer cells it is overexpressed in ptc and is involved in tumor invasion andlymph node metastasis [ ] thus leptin may be involved in the tumorigenesis and metastasis of thyroidcancer through a complex pathway and a monitoringmay have some significance due to the absence of directevidence elevated leptin levels can also be caused bythyroid carcinoma the cause and effect relationship between leptin and thyroid carcinoma are unclear now andneed further studiescompared to lean women overweightobese womenhad lower serum adiponectin levels and this differencehas statistical significance in addition adiponectinis negatively associated with a variety of benign and malignant tumors especially those associated with obesityand insulin resistance such as leukemia renal carcinoma gastric carcinoma and colon cancer moreover the association of adiponectin with potential tumorlimiting functions has been widely proposed otvos l jr tried in vitro experiments andproved that adiponectin can inhibit the metastasis ofcancer cells mitsiades n measured circulatingadiponectin levels in ptaients with ptc and found thatit is independently and inversely associated with the riskof thyroid cancer as the receptor that binds to adiponectin for biological effects adiponectin receptor hadbeen reported closely correlated with the developmentof ptc adiponectin receptor1 and are higher expression in ptc tissues than that in the surrounding normaltissues and this is thought to be associated with a betterprognosis however other studies have shown different results[ ] and more studies should be done furtherly tosupport the antitumor effect of adiponectin and thepositive correlation between the increased level of adiponectin in circulating blood and the prognosis of thyroid 0czhao bmc cancer page of neoplasms and provide new ideas for the prevention andtreatment of thyroid neoplasmsfrom the above a strong relationship between elevatedconcentrations of adipokines in serum andor tissueand thyroid cancer can be concluded and this may explain why increased incidence of obesity and thyroidcancer are consistent thus targeted drugs for adipokinemay be useful for the treatment of thyroid cancer in thefuturehowever some limitations in our metaanalysis shouldbe taken into account first some data were not normally distributed and were reported in the form of median and quartile and therefore these data werecalculated by formulas second due to | Colon_Cancer |
environmental exposure to arsenite as3 has a strong association with the development ofhuman urothelial cancer uc and is the 5th most common cancer in men and the 12th mostcommon cancer in women muscle invasive urothelial cancer miuc are grouped into basalor luminal molecular subtypes based on their gene expression profile the basal subtype ismore aggressive and can be associated with squamous differentiation characterized byhigh expression of keratins krt1 and and epidermal growth factor receptoregfr within the tumors the luminal subtype is less aggressive and is predominatelycharacterized by elevated gene expression of peroxisome proliferatoractivated receptamma pparÎ and forkhead box protein a1 foxa1 we have previously shown thatas3transformed urothelial cells ast exhibit a basal subtype of uc expressing genesassociated with squamous differentiation we hypothesized that the molecular subtype ofthe ast cells could be altered by inducing the expression of pparÎ andor inhibiting theproliferation of the cells nontransformed and ast cells were treated with troglitazonetg pparg agonist μm pd153035 pd an egfr inhibitor μm or a combination oftg and pd for days the results obtained demonstrate that treatment of the ast cellswith tg upregulated the expression of pparÎ and foxa1 whereas treatment with pddecreased the expression of some of the basal keratins however a combined treatment oftg and pd resulted in a consistent decrease of several proteins associated with the basalsubtype of bladder cancers krt1 krt14 krt16 p63 and tfap2a our data suggeststhat activation of pparÎ while inhibiting cell proliferation facilitates the regulation of genesinvolved in maintaining the luminal subtype of uc in vivo animal studies are needed toaddress the efficacy of using pparÎ agonists andor proliferation inhibitors to reduce tumradestage of miuca1111111111a1111111111a1111111111a1111111111a1111111111open accesscitation mehus aa bergum n knutson pshrestha s zhou xd garrett sh activation of pparÎ and inhibition of cellproliferation reduces key proteins associated withthe basal subtype of bladder cancer in as3transformed urotsa cells one e0237976 101371 pone0237976editor karl x chai university of central floridaunited statesreceived may accepted july published august copyright mehus 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 paper and its supporting informationfilesfunding seema somji und school of medicineand health sciences pilot grant das allundergraduate research and core facilities ndinbre idea program p20 gm103442 from thenational institute of general medical sciences nih one 101371 pone0237976 august one 0ccompeting interests the authors have declaredthat no competing interests existactivation of luminal pathway in basal bladder cancerintroductionbladder cancer bc is the ninth most common cancer diagnosed worldwide and in theamerican cancer society estimated that about new cases of bc would be identified inthe us and about deaths would occur from bladder cancer among bcs urothelialcell carcinomas uc are the most common being the second most diagnosed cancer of thegenitourinary tract behind prostate cancer [ ] it is the 5th most common cancer in menand the 12th most common cancer in women urothelial cancers are classified as muscle invasive miuc or nonmuscleinvasivenmiuc nonmuscleinvasive urothelial cancers have a lower tendency to progress whereasmiucs have a high rate of metastasis and a year survival rate of approximately bothmiuc and nmiuc have been subtyped into various groups with the basal and luminal subtype being the most prominent the luminal subtype of human uc includes the majority ofthe early stage noninvasive ucs and a significant number of miucs this subtype isenriched for papillary histology is less aggressive and has a more favorable patient outcome [ ] basal classified tumors have a poorer overall survival compared to luminal tumors they often exhibit squamous differentiation are aggressive and found exclusively inmiuc that metastasize and spread to distal ans about of miucs arise independent of the papillary pathway have poor outcomes and an overall year survival rate of environmental exposure to arsenite as3 has a strong association with the developmentof human uc the increased risk of uc correlates to the same endemic areas of the worldwhere populations have been identified with arsenicinduced skin cancer [] we havedeveloped a cell culture model of arsenicinduced urothelial cancer by exposing the immortalized nontumorigenic urothelial cell line urotsa to arsenite these transformed cell lines produce tumors in athymic mice that express genes for keratin krt and asignature pattern highly similar to the basal subtype of human miuc [ ] the tumorshave a histology similar to urothelialtransitional cell carcinomas with focal areas of squamousdifferentiation [ ] which is associated with poor prognosis [ ]the molecular mechanism driving a tumor towards a basalsquamous subtype is currentlyunknown in a recent study yamashita show that transcription factor activatingprotein alpha tfap2a is expressed at high levels in basalsquamous bladder cancerenriched in areas of squamous differentiation and is associated with increase lymph nodemetastasis and distant recurrence of the disease the study also shows that increased expression of tfap2a can facilitate the expression of other transcription factors such as tumor protein p63 tp63p63 also known as p63 which is known to be associated with the basalsubtype of uc palmbos demonstrated that p63 binds to the transcriptional regulatory regions of the gene ataxiatelangiectasia group d complementing gene atdc alsoknown as trim29 and krt14 thus increasing their expression the study further showedthat both krt14 and trim29 promote the invasion of the basal subtype of uc in vitro and invivo the luminal subtype of uc is associated with the expression of the transcriptional factorsforkhead box protein a1 foxa1 gata binding protein gata3 and peroxisome proliferatoractivated receptor gamma pparÎ [ ] the activation of pparÎ with an agonistcan represses the expression of tfap2a and inhibit squamous differentiation in vitro the exact role of pparÎ signaling in carcinogenesis is somewhat unclear however theexpression of pparÎ in bladder cancers is a favorable prognostic marker both in vivoand in vitro studies indicate that pparÎ ligands such as troglitizone can promote differentiation inhibit cellular proliferation induce autophagy and enhance apoptosis in bladder cancer one 101371 pone0237976 august one 0cactivation of luminal pathway in basal bladder cancer[] likewise suppressing cellular proliferation with epidermal growth factor receptoregfr inhibitors has been used preclinically to reduce basallike muscle invasive bladdertumor growth although the egfr inhibitors did not have the same efficacy in nonbasalliketumors the goal of this study was to determine if the activation of pparÎ and inhibition of cellproliferation in the urotsa parent and the as3transformed urotsa isolates would repressthe expression of genes involved in maintaining the basalsquamous type of bladder cancerand induce genes that were associated with the luminaldifferentiated state of bladder cancermaterials and methodsanimalsathymic nude ncrnunu mice purchased from envigo were used in these studies themice were housed four to a cage at Ëc under a 12hour lightdark cycle food and water wasavailable ad libitum mouse heterotransplants of the urotsa transformed cell lines as3 andas4 and the rt4 cell line were produced by subcutaneous injection at a dose of x cellsin the dorsal thoracic midline of athymic nude ncrnunu mice this study adhered to allrecommendation dictated in the guide for the care and use of laboratory animals of thenih tumor sizes were assessed weekly and the animals were sacrificed when the size of thetumor was approximately cm or when dictated by clinical conditions euthanizationwas done by co2 asphyxiation and conformed to the american veterinary medical association guideline on euthanasia death was confirmed by ascertaining cardiac and respiratoryarrest following which the ans and tumor were harvested care of taken to ensure thatthere was no distress to the animals during the procedure the protocol was approved by theuniversity of north dakota animal care committee iacuc16122ccell culturethe urotsa parent cells and two of the as3transformed isolates as3 and as4 were cultured in in dulbecos modified eagles medium dmem supplemented with vv fetalbovine serum as described previously the cells were subcultured at a ratio usingtrypsinedta and the cultures were fed fresh growth medium every three days the urotsaparent cell line has been authenticated using short tandem repeat str analysis the as3transformed isolates used in the current study have been previously characterized for its ability to form colonies in soft agar form spheroids when grown in ultralow attachment flasksand form tumors when injected subcutaneously in immunecompromised mice [ ]the as3 can also form tumors upon intraperitoneal injection for drug treatmentsurotsa parent and the as3transformed isolates as3 and as4 were grown to confluence inserum containing medium following which the cells were incubated with a serum freemedium consisting of a mixture of dmem and hamss f12 growth medium for h thecells were then exposed to either dimethyl sulfoxide dmso the drug vehicle troglitizonetg μm a pparÎ agonist pd153035 pd μm an epidermal growth factor receptoregfr inhibitor or a combination of tg and pd tgpd for and hours the concentrations of the drugs were chosen based on preliminary studiesvisualization of dapistained cellstoxic effects of tg and pd on the urotsa cells was determined by visualization of 406diamidino2phenylindole dapistained nuclei as described previously by this laboratory atthe indicated time points the cell monolayers were washed twice with phosphate buffered one 101371 pone0237976 august one 0cactivation of luminal pathway in basal bladder cancersaline pbs following which the cells were fixed for min with ethanol and rehydratedwith 1ml pbs the rehydrated cells were stained with 10μl dapi 10μgml in distilled waterrna isolation and realtime pcr analysistotal rna was isolated using tri reagent molecular research center as described previously the expression of various genes was assessed with realtime reverse transcriptionpolymerase chain reaction rtpcr using primers that were purchased commercially frombiorad laboratories the genes along with the catalog number of the primers are listed insupplemental material s1 table total rna μg was transcribed to cdna using theiscript cdna synthesis kit biorad laboratories the amplification of the cdna was performed using the itaq universal sybr green supermix biorad laboratories with μlcdna and μm primers in a total volume of μl in a cfx96 realtime detection systembiorad laboratories amplification was monitored by sybr green fluorescence cyclingparameters consisted of a s hotstart followed by cycles of denaturation at Ëc for sannealing at Ëc for s and extension at Ëc for s which gave optimal amplificationefficiency the resulting levels were normalized to βactin expression assessed by the sameassay the threshold cycles cts for βactin and the resulting delta cts for the target genes arereported in s2 tablewestern blot analysiswestern blot analysis was performed as described previously the cell pellets were dissolved in 1x radioimmunoassay precipitation assay ripa lysis buffer supplemented withpmsf protease inhibitor cocktail and sodium orthovandate santa cruz biotechnology thecell suspension was sonicated and the lysate was centrifuged to remove cellular debris proteinlysates were quantified using the pierce bca protein assay kit thermoscientific pierceprior to loading samples were reduced and denatured the protein extracts were separated ontgx anykd sdspolyacrylamide gels purchased from biorad laboratories and transferred toa μm hybondp polyvinylidene difluoride membrane using semidry transfer the blotswere blocked in trisbuffered saline tbs containing tween20 tbst and wvbovine serum albumin bsa for min at room temperature the membranes were probedovernight at Ëc with the primary antibody diluted in wv bovine serum albumin allantibodies were purchased from commercial suppliers and were validated against known positive and negative expressing cell lines by western analysis prior to use in experimental protocols the source of the antibody along with their catalog numbers are reported in s3 tableafter washing times for minutes each wash in tbst the membranes were incubated withthe antimouse or antirabbit secondary antibody for min at room temperaturethe blots were visualized using the clarity¢ western ecl blotting substrate bioradlaboratoriesimmunohistochemical stainingserial sections were cut at μm and immersed in preheated target retrieval solutiondako in a steamer for min the sections were allowed to cool to room temperature andimmersed into tbst for min the primary antibodies used in this study along with theirdilutions and catalogue numbers are listed in s3 table the primary antibodies were localizedusing dako peroxidase conjugated envision plus for rabbit or mouse primary antibodies atroom temperature for min liquid diaminobenzidine dako was used for visualizationcounter staining was performed for sec at room temperature using readytousehematoxylin dako slides were rinsed in distilled water dehydrated in graded ethanol one 101371 pone0237976 august one 0cactivation of luminal pathway in basal bladder cancercleared in xylene and coverslipped two pathologists judged the presence and degree ofimmunereactivity in the specimensstatistical analysisall experiments were performed in triplicate and the results are expressed as the mean ± semstatistical analyses were performed using graphpad prism1 software version using oneway anova with tukeys or dunnetts posthoc testing for gene expression statistics wererun on the delta cycle threshold δct values that were generated from normalization to βactin levels unless otherwise stated the level of significance was p005resultseffect of troglitizone and pd153035 on the viability and morphology ofurotsa parent and ast cellsthe urotsa parent and ast cells as3 and as4 were treated with either dmso controltroglitizone tg μm pd153035 pd μm or tg and pd tgpd for hr as seenin fig 1a1d there was no change in the morphology of the urotsa parent cells with varioustreatments there was a change in the morphology of the as3 and as4 cells when treatedwith tg fig 1f and 1j and tgpd fig 1h and 1l the cells looked more differentiatedformed mounds and resembled the intermediate cells of the bladder there was a decrease inthe number of urotsa parent cells treated with pd and tgpd when compared to the cellstreated with tg alone or with dmso fig 1m there was also a decrease in the number ofas4 cells when treated with tg and tgpd when compared to the dmso treated cells fig1o there was no significant decrease in the number of as3 cells in any of the treatmentgroups fig 1n an examination demonstrated the lack of dead cells in the treated groups andthe decrease in cell number compared to the dmso group could be due to lack of proliferationandor increased differentiation of cellseffect of pparÎ activation and egfr inhibition on the expression ofluminal genesthe transcription factors pparÎ foxa1 and gata3 play a role in the establishment of theluminal subtype of bladder cancer [ ] studies done by varley have shown thatagonistdependent activation of pparÎ with simultaneous inhibition of egfr phosphorylation in normal human urothelial cells increases the effectiveness of the pparÎ agonist in thepresent study we investigated the effect of the pparÎ agonist tg and an egfr inhibitor pdon the expression of luminal transcriptional factors in the urotsa parent cells and the astcells expression levels for the target genes in this study are reported for a hr hr and hr timecourse for the parent as3 and as4 cells s1 s2 and s3 figs respectively for simplicity the hr gene and protein levels are reported in the main body of the manuscripttreatment with tg increased the expression of pparÎ in the urotsa parent fig 2a i iv andv cell line a similar effect was seen in as3 fig 2b i iv and v and as4 fig 2c i iv and vcell lines treatment with pd did not induce the expression of pparÎ in the urotsa parentfig 2a iv and v or as3 fig 2b iv and v cells but there was a small increase in pparÎ protein in the as4 cells fig 2c iv and v treatment with both tg and pd tgpd increasedthe expression levels of pparÎ mrna in the urotsa parent cells but there was no increase inthe protein levels there was no increase in mrna expression in the as4 cells but there was aslight increase in the protein levels fig 2c i iv and v one 101371 pone0237976 august one 0cactivation of luminal pathway in basal bladder cancerfig morphology and viability of urotsa parent and ast cells the urotsa parent ad and ast cells as3 eh and as4 il were treated witheither dmso control troglitizone tg μm pd153035 pd μm or tg and pd tgpd for hr the measurements were performed in triplicatesand the values reported are mean percentage of control ± sem ordinary oneway anova was performed followed by tukeys posthoc test bars withdiffering letters indicate significant differences p 101371 pone0237976g001foxa1 gene and protein expression in the urotsa parent cells treated with tg wasreduced fig 2a ii iv and vi however the expression was increased in the as3 and as4cells fig 2b ii iv and vi and 2c ii iv and vi at the protein level treatment with pd decreasedfoxa1 protein in the parent cells but the levels were elevated in the as3 cells tgpdreduced the expression of foxa1 in the urotsa parent cells but it increased the expression offoxa1in the as4 cells at the protein leveltreatment of the urotsa parent cells with tg pd or tgpd did not increase the mrnalevels of gata3 but there was an increase in the protein levels after treatment with tg andtgpd when compared to the dmso treated group fig 2a iii iv and vii in as3 and as4cells there was an additive reduction of gata3 protein by using the combined tgpd treatment fig 2b iv and vii and 2c iv and vii one 101371 pone0237976 august one 0cactivation of luminal pathway in basal bladder cancer one 101371 pone0237976 august one 0cactivation of luminal pathway in basal bladder cancerfig expression of luminal markers in urotsa parent and ast cells the urotsa parent aivii as3 bivii and as4 civii weretreated with either dmso control troglitizone tg μm pd153035 pd μm or tg and pd tgpd for hr real time rtpcranalysis was performed to verify gene expression a b ciiii western blot analysis was used to measure protein levels a b civ and theintegrated optical density iod of each protein band was calculated a b cvvii gene expression was normalized to βactin and gene andprotein are plotted as foldchange relative to the dmso control amplification was below detectable levels for pparÎ in dmso as3 so adelta cycle threshold δct value of was assigned which is higher than the highest delta ct detected for pparÎ in that cell linetriplicate measurements of gene and protein data were performed and are reported as mean ± sem ordinary oneway anova wasperformed followed by tukeys posthoc test bars with differing letters indicate significant differences p 101371 pone0237976g002effect of tg and pd on the phosphorylation and expression levels of egfrthe effect of tg and the egfr inhibitor pd was determined on the expression and phosphorylation of egfr in the urotsa parent and ast cells only the combination of tgpdreduced the expression of egfr mrna in the urotsa parent cells however all three treatments decreased the protein levels fig 3a i ii and iii there was no basal phosphorylation ofegfr in the urotsa parent cells and none of the treatments had any effect on the phosphorylation levels fig 3a ii the expression of egfr in the ast cells varied with a decrease inas3 cells and an increase in as4 cells fig 3b i ii and iv and fig 3c i ii and iv respectivelyboth the transformed cell lines had basal phosphorylation of the egfr pegfr and treatment with pd decreased the pegfr levels in as3 fig 3b ii and iii and as4 fig 3c ii andiii which indicates that the pd treatment was effectiveeffect of the pparÎ agonist and inhibition egfr phosphorylation on theexpression of keratinsstudies performed by varley have shown that inhibition of the egfr with simultaneous activation of pparÎ signaling switches normal human urothelial cells from a squamous metaplastic phenotype to a transitional differentiated state with the repression ofkrt14 and the upregulation of krt13 and krt20 therefore we wanted to determineif the urotsa parent and the ast cells would revert more from a basal phenotype to amore transitionalintermediate phenotype when treated with tg and pd the mrnaexpression data is shown in fig and the protein expression data is shown in fig for theurotsa parent cells there was a decrease in expression of krt1 krt5 and krt14 with alltreatments fig 4a i ii and vi and fig 5a i ii and iv the protein for krt1 was undetectedin the urotsa parent cells the expression of krt6 and krt16 increased with tg butdecreased with pd and tgpd fig 4a iii iv v and vii and fig 5a iii and v the krt6antibody does not distinguish between the krt6a krt6b and krt6c isoforms and recognizes protein made by these three genes thus it is not known which isoform is beingexpressed at the protein level there was a decrease in expression of krt13 in the urotsaparent cells fig 4a viii and fig 5a i and vi in the as3 cells the expression levels of thebasal krts with various treatments was similar to the urotsa parent cells fig 4b ivii andfig 5b ivi with the exception of krt1 protein which was expressed in the as3 cells andits expression decreased with tg and tgpd treatment in the as4 cells the expression ofthe krts was similar to as3 with the exception of krt16 fig 4c iviii treatment withtg decreased the expression of krt16 the protein levels for the all the krts was similarto the mrna level with the exception of krt5 krt13 and krt16 krt5 showed anincrease in expression with pd and tgpd treatment and krt16 which showed anincrease in expression with pd fig 5c ivii in the as3 and as4 cells krt13 geneexpression was reduced however the protein levels were elevated from all three treatmentsfig 4b viii fig 4c viii and fig 5b i vii fig 5c i vii one 101371 pone0237976 august one 0cactivation of luminal pathway in basal bladder cancerfig expression and phosphorylation of epidermal growth factor receptor the urotsa parent aiiii as3 biiv and as4 ciiv weretreated with either dmso control troglitizone tg μm pd153035 pd μm or tg and pd tgpd for hr real time rtpcranalysis was performed to verify gene expression a b ci western blot analysis was used to measure protein levels a b cii and the integratedoptical density iod of each protein band was calculated aiii b and ciii and iv phosphorylatedegfr was not detected in the urotsa parentcell line gene expression was normalized to βactin and gene and protein are plotted as foldchange relative to the dmso control triplicatemeasurements of gene and protein data were performed and are reported as mean ± sem ordinary oneway anova was performed followed bytukeys posthoc test bars with differing letters indicate significant differences p 101371 pone0237976g003effect of pparÎ activation and egfr inhibition on expression oftranscriptional factors p63 and tfap2a and the oncogene trim29associated with squamous differentiationrecently tfap2a has been implicated in the development of squamous differentiation inbasal cancers and activation of pparÎ is shown to represses the expression of tfap2a we therefore investigated the effects of pparÎ activation and egfr inhibition on the expression of tfap2a in urotsa parent and ast cells our results demonstrate that tg reducedtfap2a protein within the parent and as3 cells while the mrna and protein was elevatedin the as4 cells from tg exposure treatment with pd as well as tgpd decreased the one 101371 pone0237976 august one 0cactivation of luminal pathway in basal bladder cancer one 101371 pone0237976 august one 0cactivation of luminal pathway in basal bladder cancerfig gene expression of keratins the urotsa parent aiviii as3 biviii and as4 civiii were treated witheither dmso control troglitizone tg μm pd153035 pd μm or tg and pd tgpd for hr real timertpcr analysis was performed to verify gene expression a b civiii gene expression was normalized to βactin andare plotted as foldchange relative to the dmso control triplicate measurements of gene levels were performed and arereported as mean ± sem ordinary oneway anova was performed followed by tukeys posthoc test bars withdiffering letters indicate significant differences p 101371 pone0237976g004expression of tfap2a in the urotsa parent fig 6a i iv and v as3 fig 6b i iv and v andas4 cells fig 6c i iv and vtranscription factor p63 is another protein associated with human bladder cancersenriched in basalsquamous markers [ ] there was a decrease in the expression of p63 inthe urotsa parent cells with all treatments fig 6a ii iv and vi in the as3 cells the expression of p63 was low and treatment with tg increased its expression however treatment withpd and tgpd decreased its expression fig 6b ii iv and vi the expression of p63 in as4cells increased at the mrna level with tg and tgpd treatments however the protein levelswere decreased when compared to the dmso control fig 6c ii iv and vithe expression of trim29 a gene associated with the basal gene expression program was also determined in the urotsa parent and the as3transformed cells for the urotsaparent and as3 cells there was a decrease in the expression of trim29 in cells treated withpd and tgpd fig 6a and 6b iii iv and vii for as4 pd and tgpd treatments increasedtrim29 protein fig 6c iv and viiexpression of trim29 tfap2a and p63 within tumors formed fromurotsa as3 as4 and rt4 cellsurotsa as3 and as4 are considered to be of the basal molecular subtype while rt4 cellsare considered to be of the luminal molecular subtype of bladder cancer cells therefore wewanted to confirm the in vivo expression of trim29 tfap2a and p63 within the nondifferentiated basalsquamous areas of tumors originating from urotsa as3 and as4 cells incomparison to the expression in tumors originating from the luminal rt4 cells all three ofthese proteins were enriched within the nondifferentiated areas of tumors developed from theurotsa as3 and as4 cells fig 7a 7b 7d 7e 7g and 7h signs a lower expression wasobserved in the welldifferentiated areas of the urotsa as3 and as4 tumors fig 7a 7b7d 7e 7g and 7h� asterisks there was low to no staining in the rt4 cells for trim29tfap2a and p63 fig 7c 7f and 7idiscussionthe classification of uc into various subtypes has implications in the overall patient management of the disease with the basal subtype having a worse outcome when compared to theluminal subtype the molecular mechanisms involved in the development of these subtypesare not yet established however the role of some transcriptional factors is established withpparÎ playing an important role in the activation of the luminal specific genes [ ] andtfap2a playing a role in the development of the basal subtype of uc in addition studieswith normal human urothelial cells show that activation of pparÎ with an agonist along withinhibition of cellular proliferation via the egfr pathway can switch cells from a squamousmetaplastic phenotype to a more transitional differentiated phenotype combination therapiesusing egfr inhibitors and pparÎ agonists show promising results against some urothelialtumors in vivo as well as against other cancers based on these studies we sought todetermine if the urotsa parent and the as3transformed urotsa cell lines that are one 101371 pone0237976 august one 0cactivation of luminal pathway in basal bladder cancer one 101371 pone0237976 august one 0cactivation of luminal pathway in basal bladder cancerfig protein expression of keratins the urotsa parent aivi as3 bivii and as4 civii were treated with either dmso control troglitizone tg μm pd153035 pd μm or tg and pd tgpd for hr western blot analysis was used to measure protein levels ai bi ci and the integratedoptical density iod of each protein band was calculated aiivi b and ciivii protein levels are plotted as foldchange relative to the dmso controltriplicate measurements of protein data were performed and are reported as mean ± sem ordinary oneway anova was performed followed by tukeysposthoc test bars with differing letters indicate significant differences p 101371 pone0237976g005molecularly characterized as a basal subtype of bc could convert to a luminal transitionalcell type when treated with a pparÎ agonist andor an egfr inhibitor as this could affect theoverall outcome of the diseasethe urotsa parent cells when grown in serum expresses many genes that are associatedwith the basal subtype in this study treatment of these cells with the pparÎ agonist tginduced the expression of pparÎ as well as gata3 but not foxa1 in mortal human urothelial cells treatment with the pparÎ agonist shows an increase in the expression of the transcription factor foxa1 and this is contrary to what is seen in our study these differencescould be due to the cell type since we are using immortalized cells the as3transformedurotsa cells showed an induction in the expression of pparÎ and foxa1 when treated withtg however the expression of gata3 in these transformed lines was not consistent in bccell lines studies by others have shown that have shown that gata3 and foxa1 cooperatewith pparÎ activation to drive the differentiation of a basal bladder cancer subtype to a moredifferentiated luminal subtype other studies have also linked the expression of foxa1 tothe development of the luminal subtype of urothelial cancer [] in our studies treatmentwith tg did result in the differentiation of the as3transformed cells based upon morphological changes and induced the expression of pparÎ as well as foxa1 both of which are factorsknown to drive luminal differentiation of bladder cancer cell linessignaling via the pparÎ pathway is essential for the growth arrest and terminal differentiation of adipocytes and other normal epithelial [] and cancer cells [] whereassignaling via the egfr receptor plays a role in cell proliferation the keratins play an essentialrole in the differentiation of epithelial cells and different keratin profiles are expressed at different stages of differentiation the normal bladder has three different stages of differentiationand these stages are marked by the expression of krt14 krt5 and krt20 krt14 isexpressed in a subset of basal cells that are thought to play a role in regeneration as well astumorigenesis whereas other basal cells and intermediate cells in the normal bladderexpress krt5 the expression of krt20 is restricted to the most differentiated cells theumbrella cells these differentiation stages of the bladder are shared by bladder cancersand malignant transformation can occur in any of the different cell types of the bladder theexpression of krt14 is seen in the least of the differentiated tumors and its expression correlates to poor prognosis krt14 whereas the expression of krt20 is restricted to differentiated bladder cancers and its expression is associated with good prognosis a study doneby varley showed that normal human urothelial cells in culture express krt14 andlack the expression of krt13 and krt20 activation of pparÎ in these cells induced theexpression of krt13 and decreased the expression of krt14 this effect was significantlyenhanced when | Colon_Cancer |
despite great advances in recent decades in screening diagnosisand curative surgery hepatocellular carcinoma hcc remainsthe second leading cause of cancerrelated mortality worldwidegrandhi siegel epidemiologicalevidence has conï¬rmed that the longterm outcomes of patientswith hcc have notimproved significantly with the rapiddevelopment of surgical techniques madduru moreimportantly because of the limitations of systematic statustumor position and the need to preserve liver function morethan of patients are not eligible for surgical treatment evenafter curative resection prognosis remains unsatisfactory becauseof a high incidence of postoperative recurrence colecchia the initiation and maintenance of hcc is a complexand regulated process involving the accumulation of numerousgenetic changes over decades niu erkekoglu these sequential alterations not only endow normal livercells with neoplastic ability enabling uncontrolled growth butalso provide potential therapeutic targets and biomarkers thusfurther understanding of the initiation and maintenance of hccat the molecular level is crucial to prolonging survival and makingindividual treatment decisionsthe exive development of highthroughput technologyhas provided powerful tools for the molecular study of cancerschmidt and hildebrandt rna sequencing rnaseq and microarraysthe most representative methods ofthis technology are mature enough for use in commercialapplications mantione zhang duringthe past decades the genomewide transcriptional analysis ofgene expression has become critically important to gain betterinsight into the biological processes of hcc and other typesof cancer jin xiong in additionto the aberrant expression of transcripts studies have focusedon diï¬erent molecular levels multilevel omicsincludingcopy number variation epigenetic modiï¬cations nucleotidepolymorphisms and dna methylation especially in hcclee lin evidence obtained from thesestudies clearly demonstrates that hcc is a disease causedby cumulative aberrations at diï¬erentlevels of molecularregulation thus only a highthroughput multiomics analysiscan decipher the complex biology of hcc many previousstudies despite promising results focused only on the aberrantregulation of expression and its biological eï¬ects howeverstructural transcript variation in hcc which is heavily shaped byalternative splicing as has until recently been less well studiedaccording to the manual genome annotation project harrow pruitt there are only about proteincoding genes this number is obviously inconsistent withthe overall cellular complexity which includes at least distinct proteincoding sequences harrow thisdiscrepancy between the numbers of transcripts and proteincoding genes in human cells indicates the existence of anadditional mechanism between the transcriptional and the posttranslational levels that increases the coding capacity of thegenome through the as process a single rna precursorcan be spliced via distinct arrangements to generate rnaslandscape of as in hccwith diï¬erent structures and functions biamonti song this may be one ofthe main causesof cellular complexity and proteome diversity experimentalstudies on the eï¬ects of individual as events suggest that asmay change the biological function of a protein by regulatingits stability controlling its location modifying the mutualinteractions of proteins and even adding or deleting activedomains brett yang these ï¬ndingssuggest that as well as expression abundance the balance ofdiï¬erent as events that result from the same rna precursormust be considered howeverthe latter consideration hasoften been neglected in previous studies in fact emergingdata from genomewide analyses feng kahles indicate that as occurs in more than ofmultiexon genes suggesting thatthe widespread existenceof as is the product of physiological processes rather thantranscription errorsin recent years the diagnostic and the therapeutic role ofas in many human diseases has attracted increasing attentionlargescale screening of as events has been performed usingexpressed sequence tag libraries although this approach isprone to a high rate of false positives venables exonjunction probes provide a higher experimental validation ratelapuk however this method has the disadvantageof being limited to known splice junctions owing to thelimited available techniques complicated mechanisms and hugenumbers involved transcriptomewide as dysregulation and itspotential associations with biological behavior in hcc haveremained uncharacterizedrnaseq not only supports the quantitative measurementof novel as events but also provides deeper coverage higheraccuracy and better resolution li y thus it maybe the most suitable of the currently available approaches foras study in recent years the cancer genome atlas tcgatomczak wang has accumulated a richand publicly available source of rnaseq data and correspondingclinical information this enables the analysis of as dysregulationin hcc at a genomewide level tcga includes rnaseqdata samples obtained from hcc patients together withtheir corresponding clinical information thereby facilitating theclinical analysis of hccrelated as events in a large cohorthowever without reliable and eï¬cient bioinformatical methodsthe advantages of rnaseq in as analysis cannot be fullyexploited spliceseq a recently developed bioinformatics toolcan exactly match rna reads with gene splice graphs and ishelpful for accurately calculating complex or lowfrequency asevents ryan there has been a lack of studies combining largescalernaseq data with the corresponding clinical information tocomprehensively analyze as at singleexon resolution howeverthis is very necessary especially in hcc thereforein thecurrent study we comprehensively analyzed wholegenome asin the tcga hcc cohort to screen out hccrelated asevents and further studied the relationships of these eventswith clinical outcomes our ï¬ndings suggest that certain hccrelated as events including nek2at and troptat havecritical roles in the progression and maintenance of hcc morefrontiers in genetics wwwfrontiersinaugust volume 0cxiong landscape of as in hccimportantly these hccrelated as events represent potentialnew therapeutic targetsmaterials and methodstumorlocationinvasioninformation ofdata curationclinicopathologicalthe hcc cohort andcorresponding rnaseq data were retrieved and downloadedfrom tcga1 to ensure appropriate protection of patientprivacy the tcga data were stratiï¬ed according to data typeand level conforming to the publishing guidelines formulatedby tcga wang then the rnaseq data andcorresponding clinicopathological information were mutuallypaired using the unique tcga barcodes only patients whomet the criteria listed below were included grandhi patients with corresponding rnaseq data siegel patients with complete clinicopathological informationincluding localsex age distalmetastasis pathological stage diï¬erentiation grade lymph nodemetastasis and survival information madduru histological diagnosis of hcc and colecchia survival for at least month after the primary pathologicaldiagnosis spliceseq was used to determine rna splicing patternsand produce as proï¬les for each hcc patient as previouslydescribed li y zhu each as eventwas quantiï¬ed using the percent spliced in psi value rangingfrom to a commonly used method to reï¬ect the abundanceof as events in order to remove the eï¬ects of splicing noiseand generate as reliable a set of as events as possible a seriesof strict ï¬lters average psi ¥ percentage of samples withpsi ¥ was applied to the detected as events the interactivesets between the seven types of as were quantitatively analyzedand the results were visualized in upset plots using upsetrversion conway circlize version wasused to generate circos plots to depict the parent genes and theiras events in chromosomes gu the details of thedesign of the present study are illustrated in supplementaryfigure s1 all the methods used in this study were in line withthe relevant guidelines and regulationsidentiï¬cation of deas and enrichmentanalysisto screen the diï¬erentially expressed alternative splicing deasevents between hcc and corresponding normal tissues the psivalue of each as event was determined in the tcga hcccohort hcc tissue samples and paired adjacent normaltissues a generalized linear model was applied to remove thebatch eï¬ects the deas were determined based on adjustedp adj p and associated log2 fold change fc values withadj p ¤ and log2fc ¥ representing as events thatwere downregulated and upregulated respectively biologicalfunction enrichment analysis was performed based on the deasparent genes gene ontology go and kyoto encyclopediaof genes and genomes kegg terms with false discoveryrate less than were considered to be significantly enrichedand were selected for further analysis enrichment analysis wasperformed using the clusterproï¬ler package version yu the parent genes of deas events were importedinto the string database and used to determine proteinprotein interactions ppis a relationship network was thengenerated using cytoscape version su clusteranalysis was conducted using the average linkage agglomerationalgorithm and correlation distance metricsestablishment of hccrelated splicingcorrelation networka total of splicing factors sfs supplementary table s1were identiï¬ed by comprehensive and handcurated screening ofthe literature all the sfs included in the current study had beenexperimentally validated in previous research giulietti and included heterogeneous nuclear ribonucleoproteinsproteins serineargininerich proteins and other proteinsbelonging to the celf fox khdrbs nova and elav familiesthe expression of each sf was obtained from the broadinstitute2 correlations between the psi values of deas and theexpression of sfs were analyzed by weighted gene coexpressionnetwork analysis version langfelder and horvath benjamini and hochberg correlation was used to adjust thepvalues the adjusted pvalues less than were consideredto indicate statistically significant diï¬erences cytoscape version was used to generate the correlation plotssurvival analysisall the included hcc patients were divided into two groupsbased on the psi value of each deas median cut and thetwo artiï¬cial categories were modeled as continuous variables toderive more easily interpretable hazard ratios based on overallsurvival os and diseasefree survival dfs cox regression wasperformed to evaluate the prognostic value of each deas eventlogrank test and kaplanmeier analysis were used to comparepatient survival between subgroups p was consideredas statistically significant the overall survivalrelated deaswere further analyzed in lasso regression to identity the mostpowerful prognostic markers finally a prognostic model wasconstructed for predicting the os in order to quantify the risk ofos a standard form of risk score rs for each colorectal cancercrc patient was calculated combine the levels of the psiι1 psii à lito divide the patients into the high or lowrisk group kaplanmeier curves were used to estimate the survival for patients in thetraining the testing and the validation sets between the highriskand the lowrisk groupspsii and lasso coeï¬cients li risk score pnfunctional experiment of cxcl12splicing variants in hccthe human hcc cell line hepg2 was obtained from the chineseacademy of sciences committee on type culture collectioncell bank shanghai china the cell line was cultured in 1httpsportalgdccancergov2httpgdacbroadinstitutefrontiers in genetics wwwfrontiersinaugust volume 0cxiong landscape of as in hccprimers was used asgibco carlsbad ca united states supplemented with fetal bovine serum fbs bi beit haemek israel at ¦c with co2 total cdna from tissues was obtained as described abovethe pcr reaction was carried out using the forward primer5cid48tgcccttcagattgttgcac3cid48 common for allisoforms and theisoformspeciï¬c reverse primers 5cid48gctaactggttagggtaatac3cid48 and5cid48gctagcttacaaagcgccagagcagagcgcactgcg3cid48for np_9546371and np_0010290581 respectively bactin ampliï¬ed usingthe forward 5cid48acactgtgcccatctagcagggg3cid48and reverse 5cid48atgatggagttgaaggtagtttcgtggat3cid48aloading control quantitative realtime pcr was performedin mx3005tm qpcr system with a mxpro qpcr software stratagene la jolla ca united states and sybr greendetection system the adherent hepg2 cells were transfectedwith the corresponding hiscxcl12 construct by the calciumphosphate method and cultured for h at h before collectingthem the cell supernatants were removed and when indicatedbrefeldin a was added to the fresh medium the collectedcells were left untreated or permeabilized with saponin andimmunolabeled with the his mab and a pegoat antiigsecondary antibody and analyzed by ï¬ow cytometry the cellinvasion assay was conducted using matrigelcoated chambers µm pore size corning costar corporation cambridgema united states in brief à cells were plated in theupper chamber coated with matrigel and supplemented withserumfree medium the lower chamber was ï¬lled with a culturemedium containing fbs incubation was carried out for h at ¦c following which the noninvasive cells were scrapedoï¬ with cotton swabs the cells that had successfully translocatedwere ï¬xed with paraformaldehyde stained with crystalviolet and ï¬nally counted using an inverted microscope mttassay colony formation assay and soft agar growth assay wereperformed according to our previously described methods zhou protein structure homology modeling analysis wasperformed as previously described by using the online serverswissmodel waterhouse evaluation of the relationship of asclusters with clinicopathologicalfeaturesbased on the identiï¬ed deas n the tcga hcc cohortin the current study was stratiï¬ed by an unsupervised consensusapproach consensus cluster plus version wilkerson andhayes the optimal number of clusters was determinedby integrating the results of the elbow method and gap statisticthe relationship between clinical outcomes and as clusterswas evaluated using logrank test and kaplanmeier curves asdescribed by xiong 2018aresultslandscape of as event proï¬les in hccto systematically characterize the as events and their clinicalsigniï¬cance in hcc we collected rnaseq libraries andcorresponding clinicalinformation from hcc patientsthe tumor tissues and paired adjacent normal tissues from patients were sequenced simultaneously the includedpatients comprised males and femalesamong which patients developed recurrence and died of hcc the median followup period was months range months the general characteristicsof these hcc patients are fully detailed in supplementarytable s2 rnaseq data were associated with the clinicalthe corresponding patient via the tcgainformation ofbarcodes there were patients with rnaseq data bothfrom tumor tissue and adjacent normal tissue according tothe recommended analysis approach described in a previouslypublished study ryan we identiï¬ed asevents from genes based on their splicing patterns theseas events could be roughly classiï¬ed into seven types alternatepromoter ap mutually exclusive exons me retained intronri exon skipping es alternate acceptor site aa alternateterminator at and alternate donor site ad figure 1ato quantify the detected as events psi values were calculatedthese values measure the proportion of each detected splicingvariation in all of the expressed isoforms the expression ofcertain isoforms was fairly low psi and most of the asevents could not be stably detected in all of the given samplesafter screening average psi ¥ percentage of sampleswith psi ¥ a total of as events from geneswere obtained we further compared the variance in quantityof as events and the genes involved between tumor adjacentpaired normal and unpaired tumor tissues for diï¬erent splicingpatterns there were no significant diï¬erences in quantityvariations however on average one gene might have nearlythree as events figure 1b left panel moreover only annotated genes in this study stably underwent as figure 1bright panel notably diï¬erent as patterns may occur for a singlegene thus upset plots were used to depict the intersectionsbetween as types as demonstrated in figure 1c most of theparent genes only occurred in one type of as event whereascertain parent genes contained up to four types of as eventabout of the parent genes contained two or more asevents the arrangements of diï¬erent as types and as eventsbetween diï¬erent exonsintrons may be the major reason fortranscriptome diversity in order to comprehensively depict asevent proï¬les in hcc circos plots were used to visualize therelationships among as events and the corresponding parentgenes in chromosomes figure 1didentiï¬cation of hccrelated deascomparing the variations in molecular components amongdiï¬erent pathological states using highthroughput techniquesis an eï¬ective way to screen key molecules this approachhas been widely used to identify diseaserelated molecules inprevious research xiong 2018ab it is reasonableto consider that significant diï¬erences in as events betweenprimary hcc tissues and adjacent normal tissues may be relevantto the initiation and maintenance of hcc in this study thetcga barcodes corresponding to tissue samples rnaseq data were analyzed from which as proï¬les of pairednormal and tumor tissues were ï¬nally extracted these pairedfrontiers in genetics wwwfrontiersinaugust volume 0cxiong landscape of as in hccfigure landscape of alternative splicing as events in hepatocellular carcinoma hcc a diagrammatic sketch of the seven types of as events in the presentstudy alternate acceptor site aa alternate terminator at alternate promoter ap exon skipping es mutually exclusive exons me alternate donor site adand retained intron ri b number of as events and the corresponding parent genes illustrated according to as type left panel the color bar represents asevents ï¬ltered by criteria the black bar represents the corresponding genes involved in as each as type was divided into four groups based on the tissue sourcen normal tissue t tumor tissue pt paired tumor tissue npt unpaired tumor tissue number of detected as events asrelated genes ï¬ltered as events and thecorresponding genes right panel c intersection of parent genes between different as types n in hcc one gene may incur up to four types ofalternative splicing d circos plots depicting the distribution and the detailed alteration of as events and their parent genes in chromosomesas proï¬les were used to identify deas eventually deaswere identiï¬ed from genes using a threshold of log2fc and adj p including aps ess ats risnine ads aas and one me figure 2a and supplementarytable s3 the top deas are listed in table notablythe proportion of as types between the ï¬ltered as and deaswas inconsistent the es events accounted for of ï¬lteredas but only of deas however the proportion of apevents rose from of ï¬ltered as to of deasfigure 2b these statistical ï¬ndings suggest that as is notthe result of transcription errors but a tightly regulated processmoreover based on the identiï¬ed deas the samples could beclearly separated into normal and tumor groups by unsupervisedhierarchical clustering figure 2c indicating that the deashad been reliably identiï¬ed the psi values of deas eventsin diï¬erent hcc patients are illustrated in figure 2c as amatrix heat map the changes in color gradient intuitively revealthe heterogeneity of hcc a splice graph which representssplice junctions as edges and exons as rectangular nodes wasused to visualize some of the identiï¬ed deas figure 2dfurthermore the diï¬erences in expression of these as eventsbetween primary hcc tissue and corresponding adjacent normaltissues are intuitively depicted in figure 2e taken together theseresults show that a significant variation of as occurred duringhcc initiation and maintenance indicating that the potentialrole of hccrelated as events requires further researchenrichment and interaction analysis ofdeasemerging evidence indicates that as could change a transcribedsequence directly with eï¬ects on expression abundance orprotein function thus the potential biological eï¬ects of deascould be determined by analyzing the corresponding proteinsas shown in supplementary figures s2ac speciï¬c go termsclosely related to liver metabolism including negative regulationof hydrolase activity sterol homeostasis anic acid catabolicprocess and acidic amino acid transport were significantlyenriched by the parent genes of deas in addition certain keggpathways known to be involved in hcc were enriched includingthe cgmppkg signaling pathway the nfκb signaling pathwaythe mrna surveillance pathway and the phosphatidylinositolfrontiers in genetics wwwfrontiersinaugust volume 0cxiong landscape of as in hccfigure identiï¬cation of hepatocellular carcinoma hccrelated aberrant alternative splicing as a differences in as events between paired hcc tissue andparacancerous tissue volcano plot of the differentially expressed alternative splicing deas identiï¬ed in hcc the blue and the red points represent the deas withstatistical signiï¬cance logfc ¥ adj p b proportions of different as types among ï¬ltered as and deas c heat map of the deas the horizontal axisshows the clustering information of samples divided into two major clusters adjacent normal tissue n and paired tumor tissue n the left longitudinalaxis shows the clustering information of deas the gradual change of color from green to red represents the alteration of expression of deas from low to highd splice graph of some representative deas the thin exon sections represent untranslated regions and the thick exon sections represent coding regions theexons are drawn to scale and the connecting arcs represent splice paths e differences in percent spliced in values of as events between hcc and pairedadjacent normal tissuessignaling system supplementary figure s2d these resultssuggest that the parent genes of deas are critical in the biologicalregulation of hcc thus aberrant splicing of the transcribedsequences could inï¬uence their translation and change thecharacteristics of the resulting proteins therefore it is essentialto study as events from the perspective of ppi networks basedon the deasrelated genes a ppi network was establishedrepresenting not only normal interactions but also the potentialimpact of as events figure correlation network of sfs andhccrelated asas events are primarily regulated by sfs which attach to themrna precursor and aï¬ect the selection of exons and thechoice of splicing site aberrant as events in tumor tissuemay be orchestrated by a limited number of sfs for thisreason we conjecture that a few key sfs potentially regulatea large proportion of hccrelated as events to validate thisconjecture we ï¬rst identiï¬ed sfs supplementary table s1by comprehensive and handcurated screening of the literatureall of which had been previously experimentally validatedgiulietti then the copy number variation somaticmutations and expression abundance of these sfs in eachhcc patient were investigated using cbioportal figure 4avisualization using oncoprint revealed that each of the sfs harbored at least three molecular alterations figure 4aleft panel the most frequently aï¬ected sf was khdrbs3in which molecular alterations were detected in cases partly owing to the above changes the expressionabundance of the sfs showed a significant heterogeneity atan individual level figure 4a right panel the expressionproï¬les of the sfs in diï¬erent cancer types also showedheterogeneous characteristics figure 4b more importantlythe expression of sfs also diï¬ered between paired normal andcancer tissues of the same hcc patient figure 4c nextcorrelation analyses were performed between the psi value ofeach deas event and the sfs according to the correlationcoeï¬cient ttest p r a splicing regulatorynetwork was established as shown in figure 5a sfs weresignificantly correlated with deas events among which were downregulated and were upregulated several diï¬erentas events in the network were regulated by a single sf insome cases an sf had the opposite regulatory eï¬ect on diï¬erentas events figure 5a we also found that the same bindingsite as event could be competitively bound by diï¬erent sfsthese observations explain at least in part why one gene cangenerate several diï¬erent isoforms representative correlationsfrontiers in genetics wwwfrontiersinaugust volume 0cxiong landscape of as in hcctable the top most different alternative splicing as eventssymbolas typeexonsfrom exonto exonmean nmean tlog fcadjusted pvaluedownregulatedmthfd2ligf2kif22gstz1serpinb5ppargfam3afip1l1tmem59tpm4cdh23mid1gnesamd12kif4afbxw7nek2padi4rnf115vti1aupregulatedrdm1nr1i3psphtmem145nr1i3gpr116piddnr1i3ano1cldn7sardhscp2znf331eno3pemtfn1tnfrsf10carhgef39igf2neil3apapadapatapaaatapapatapapatatapatatesatatatapatriesapriesapapesapatapesatapapat1393e1593e4948e9941e2008e4387e8636e1393e4574e2519e1528e2202e6860e1522e2379e7699e1780e1843e4211e2609e1994e8164e1036e7765e1089e3569e1065e2135e5986e1038e1611e1110e2586e7736e2117e1072e4524e1151e1829e8136ebetween sfs and speciï¬c as events were illustrated using dotplots figures 5bg for example the expression of esrp2 wassignificantly correlated with both es of ceacam1 figure 5cand at of epb41l5 figure 5fassociation of deas with prognosis ofhcc patientsa crossvalidation method was used to evaluate the accuracyof the survival data and the clinical information as shown insupplementary figure s3 stratifying patients according to thetnm stage resulted in separate kaplanmeier curves for bothos and dfs these results conï¬rmed that the survival dataset forthe tcga hcc cohort although it contained censored data wasappropriate and informative for use in further survival analysistheeï¬ect of each deas on survival was determined by coxregression analysis the hcc patients were divided into twogroups according to their psi value median cut of eachdeas event according to univariate analyses a total of to investigate the prognostic signiï¬cance of deasfrontiers in genetics wwwfrontiersinaugust volume 0cxiong landscape of as in hccfigure proteinprotein interaction ppi analysis of the identiï¬ed differentially expressed alternative splicing deas interactions of the parent genes affectedby deas these genes were used to construct an intricate ppi network comprising nodes and edges the genes are denoted as nodes in the graph andthe interactions between them are represented as edges the shape size and color of the nodes respectively represent alternative splicing type value of logfcand change patterndeas events were significantly correlated with dfsand deas events were significantly correlatedwith os supplementary table s4 among these prognosisrelated deas events deas events were correlated withboth os and dfs p figure shows some of thedeas events for which the pvalue for both os and dfs waslower than to demonstrate the capability of as eventsfor prognostic prediction we randomly selected two prognosisrelated deas events and used them to draw survival curvesas shown in figure according to the psi value median ofnek2at and troptat the hcc patients could be stratiï¬edto form significant kaplanmeier curves by both os and dfssurvival analysis additionally the deas events that significantlycorrelated with survival in the univariate analysis were furtherassessed by multivariate analysis as shown in supplementaryfigure s4 there were ï¬ve and six deas events that could berecognized as independent prognostic indicators for os and dfsrespectively these ï¬ndings conï¬rm that deas events possessnot only an important biological meaning but also a potentialclinical signiï¬canceconsidering the prognostic value of the aboveidentiï¬ed asa prognostic model integrating multias was established so thatit can be easily applied to clinical practice based on the survivalrelated deas a relative regression coeï¬cient was calculated bylasso analysis by forcing the sum of the absolute value ofthe regression coeï¬cients to be less than a ï¬xed value certaincoeï¬cients were shrunk exactly to zero and the most powerfulprognostic marker of all the hcc survivalassociated deaswas selected including four as supplementary figure 5acombining the relative expression levels of the as in themodels and the corresponding lasso coeï¬cients a riskscore was calculated for each patient obviously patientswith higher rs generally had a significantly worse overallsurvival than those with lower rs p supplementaryfigures 5bc as the majority of events occurred within years timedependent roc curves were used to assess theprognostic power based on os at and years respectivelysupplementary figure 5dclustering hcc patients using deasassociated with prognosisgiven our ï¬ndings of significant heterogeneity among deas atan individual level which could reï¬ect the diï¬erent outcomesfrontiers in genetics wwwfrontiersinaugust volume 0cxiong landscape of as in hccfigure multiomics analysis of the splicing factors sfs in hepatocellular carcinoma hcc a cbioportal analysis of the sfs in the cancer genomeatlas hcc patients oncoprint was used to produce a landscape of genomic alterations legend in sfs rows at the individual level columns inframe deletionstruncated mutations and missense mutations with known or unknown signiï¬cance are shown in orange blue green and gray respectively with onethird heightthe copy number variations are annotated with the full height ampliï¬cation is shown in red and copy number loss is in blue heat map matrix shows the variation insfs at expression level the expression abundance from high to low is represented by color gradient from red to blue b expression of the sfs in tumortypes heat map color gradient depicts the normalized expression of sfs between different tumor types c differential expression analysis of representative sf tia1in hcc the expression of tia1 in hcc tissue was significantly higher than that in normal liver tissueof patients with hcc we conjectured that there might existdistinct patterns of as among diï¬erent hcc patients thishypothesis was veriï¬ed using consensus unsupervised analysisbased on the deas the optimal number of clusters wasdetermined by combining the gap statistic and elbow method theoptimal balanced partition as suggested was k figure 8aaccordingly all the hcc patients were divided into four clustersas follows i n ii n iii n and iv n figures 8bc as illustrated bythe heat map the four clusters had a significant interconnectivityamong which cluster ii appeared as a wellindividualized clusterwhereas there was more classiï¬cation overlap among clustersi iii and iv figures 8bc kaplanmeier survival analysisand logrank test were used to evaluate the associations betweenprognosis and the as clusters as illustrated in figures 8dethe stratiï¬cation of hcc patients based on as clusters showeda significant correlation with distinct patterns of survival thevariation tendency that resulted in the as stratiï¬cation betweenfrontiers in genetics wwwfrontiersinaugust volume 0cxiong landscape of as in hccfigure speciï¬c regulatory network of hepatocellular carcinomarelated alternative splicing as events a correlation network of splicing factors sfs anddifferentially expressed alternative splicing the shape size and color of nodes respectively represent type as event or sf value of logfc and change patternupregulated or downregulated the breadth of the line represents the interaction strength bg representative dot plots of the correlations between theexpression of sfs and percent spliced in values of as eventsfigure prognostic value of differentially expressed alternative splicing deas in hepatocellular carcinoma part deas events simultaneously associated withoverall survival os and diseasefree survival dfs univariate analysis of deas for os and dfs respectively unadjusted hazard ratios boxes and conï¬denceintervals horizontal lines | 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 |
the bacteroides fragilis b fragilis produce biofilm for colonisation in the intestinal tract can cause a series of inflammatory reactions due to b fragilis toxin bft which can lead to chronic intestinal inflammation and tissue injury and play a crucial role leading to colorectal cancer crc the enterotoxigenic b fragilis etbf forms biofilm and produce toxin and play a role in crc whereas the nontoxigenic b fragilis ntbf does not produce toxin the etbf triggers the expression of cyclooxygenase cox2 that releases pge2 for inducing inflammation and control cell proliferation from chronic intestinal inflammation to cancer development it involves signal transducers and activators of transcription stat3 activation stat3 activates by the interaction between epithelial cells and bft thus regulatory tcell tregs will activates and reduce interleukin il2 amount as the level of il2 drops thelper th17 cells are generated leading to increase in il17 levels il17 is implicated in early intestinal inflammation and promotes cancer cell survival and proliferation and consequently triggers il6 production that activate stat3 pathway additionally bft degrades ecadherin hence alteration of signalling pathways can upregulate spermine oxidase leading to cell morphology and promote carcinogenesis and irreversible dna damage patient with familial adenomatous polyposis fap disease displays a high level of tumour load in the colon this disease is caused by germline mutation of the adenomatous polyposis coli apc gene that increases bacterial adherence to the mucosa layer mutatedapc gene genotype with etbf increases the chances of crc development therefore the colonisation of the etbf in the intestinal tract depicts tumour aetiology can result in risk of hostility and effect on human health keywords bacteroides fragilis colon cancer stat3 pathway bacteroides fragilis toxin inflammationintroductionbacteroides species are nonspore forming anaerobe and gramnegative bacteria there are more than different species of bacteroides these bacteria act as normal flora in the intestine to maintain healthy intestinal microflora in humans bacteroides fragilis b fragilis has two classes nontoxigenic b fragilis ntbf and enterotoxigenic b fragilis etbf the differences between ntbf and etbf are the presence of b fragilis toxin bft gene and its ability to produce biofilm bft product is a kda zincdependent metalloprotease toxin also known as fragilysin or bft bft plays an important role in intestinal inflammation and tissue injury by damaging the tight junction and increasing intestinal permeability furthermore it has been proven that tissue inflammation and injury promote cancer formation simultaneously the biofilm produced by b fragilis induces carcinogenesis fortunately only etbf encompasses bft and can produce biofilms hence ntbf does not harm the intestinal tract malays j med sci julaug wwwmjmsusmmy penerbit universiti sains malaysia this work is licensed under the terms of the creative commons attribution cc by httpcreativecommonslicensesby40 0cmalays j med sci julaug in the united states colorectal cancer crc is the third most common cancer in both genders it is also the second most common cancerrelated death especially for older patients who are ¥ years old in the american cancer society stated that there were new cases of crcs that led to the death of people moreover crc is the fourth leading cancer resulting in deaths worldwide inflammatory bowel disease ibd and genetic mutations are factors predisposing an individual towards colon cancer this indicates that crc has a high mortality rate microbes are capable in promoting cancer development through several routes such as activation of chronic inflammation alteration of tumour microenvironment and production of toxins that damage dna when there is chronic etbf colonisation in the intestine it stimulates chronic intestinal inflammation triggering signal transducers and activators of transcription stat3 activation which contributes to interleukin il17 production il17 is involved in colon inflammation bft produced by etbf causes the alteration of signalling pathways and production of reactive oxygen species ros that leads to dna damage and cleavage of ecadherin in the below review we have provided a general information regarding bft produced by etbf triggering crc developmentliterature reviewcolon cancer associated with microbespromote nutrition in the human gastrointestinal tract there are nearly trillion microbes out of which make up normal flora in the intestine meanwhile the normal flora is characterised into beneficial and harmful microbes beneficial microbes including production of vitamins in the intestine and prevent disease formation however harmful microbes produce carcinogenic toxin and substances in the intestine these harmful substances may cause cancer there are many types of bacteria that stimulate a variety of cancer formation through their respective site of inflammation eg bacteria such as enterococcus faecalis e faecalis colibactinproducing escherichia coli e coli and etbf are involved in crc development however the mechanisms between each bacterium in contributing to crc formation are different for instance e faecalis damages the dna through ros colibactinproducing e coli produces colibactin that damages the dna and etbf produces bft that contributes to inflammation and immunecell infiltration intestinal dysbiosis inflammation and colon cancergenetic normal flora is advantageous to a person as it maintains intestinal health and gut homeostasis however as the bacteria such as etbf in the gut undergoes dysbiosis it brings harmful effects to the person according to deng et al a correlation was observed between microbiota imbalance and cancer progression while liu et al claimed is associated with that crc development intestinal microecology disorder imbalance among microbiota leads to bacterial infection that can progress to chronic inflammation one of the main environmental risk factors contributing to crc development is chronic intestinal inflammation chronic inflammation alters cellular microenvironment enhances gene mutation inhibits apoptosis and induces neovascularisation and cell proliferation that causes precancerous conditions eventually leading cancer simultaneously chronic inflammation alterations that directly affect the stat3 pathway and promoting there are three stages involved in tumour development namely initiation promotion and progression during initiation and progression cancer cells and microbes interact both producing genetic inflammatoryimmunological factors that are responsible for their survival and replication in tumour progression tumour cells interact with the inflammatory cells in the tumour microenvironment these tumour cells inflammatoryimmunological factors to attract the inflammatory cells and the stromal cells simultaneously activate both stromal cells start to produce various soluble factors including growth factors and protease these soluble factors play an important role in facilitating the growth differentiation and survival of tumour cells hence it promotes tumour progression and promotion additionally cytokines or microbes promote cancer by changing genetic sequence during gene mutation epithelial cells inflammatory and activated carcinogenesis cytokines chemokines causes and secrete wwwmjmsusmmy 0creplicate rapidly and develop into a hyperplastic epithelium which progresses into adenomas and then towards adenocarcinomas both adenomas and adenocarcinomas affect the growth rate of colonic epithelial cells and improve the cells toleration towards apoptosis and abnormal cells escape from the immune cells furthermore these invade submucosa turning into cancer when the growth of malignant cells continues the tumour continues to spread in the colon thus carcinogenesis becomes more efficientadenocarcinomas begin to ibd is an example of chronic intestinal inflammation that is associated with etbf pathogenic bacteria are capable of stimulating infection inflammation and carcinogenesis whereas the relationship between ibd and crc is well established surprisingly patients with ibd show a high level of immunoglobulin ig g antibodies il6 vascular endothelial growth factor vegf and tumour necrosis factor tnf igg antibodies are responsible for killing bacteria moving into the intestinal lumen simultaneously il6 and vegf are responsible for stat3 activation ibd is also known as ulcerative colitis uc and crohns disease cd this chronic intestinal inflammation increases the risk of colitisassociated crc the probability of which depends on multiple casual factors including severity duration of inflammation in the intestine and gut microbiota imbalance patients with uc or cd have folds higher incidence of crc when compared to healthy individuals it is also stated that patients with uc and cd have and respectively higher risks of crc compared to a normal healthy person this indicates that patients with uc tend to be more susceptible to crc than those with cd furthermore it is evident that the large intestine tends to have a higher risk of crc compared to the small intestine which can be attributed to the higher amount of bacteria simultaneously people with ibd and crc have a higher quantity of etbf in the intestine or stool examination compared to healthy persons additionally etbf are biofilm producers they can reduce or redistribute ecadherin in the colonic epithelial cells trigger the production of il6 by epithelial cells activate stat3 pathway and enhance cells proliferation at the site of crypt epithelial in normal colon mucosa this shows that biofilms are associated with the risk of colon cancer development review article formation of colon cancercox enzymes involved in inflammation carcinogenesis and biomarkerchronic inflammation is a principal factor that contributes to carcinogenesis prostaglandin is a paracrine hormone that plays an important role in inflammation cyclooxygenase cox is the ratelimiting enzyme responsible for producing prostaglandins cox1 and cox2 are the isoforms of cox enzymes that break down arachidonic acid into prostaglandins cox2 plays an important role in maintaining environment for the development of cancer inflammation cox2 is normally expressed in epithelial and stromal cells and the expression level is increased in both inflammation and cancer due to the presence of proinflammatory cytokines additionally bft triggers colonic epithelial cells to express cox2 but not cox1 cox2 releases prostaglandin e2 pge2 that triggers pain and inflammation at the site of tissue injury simultaneously pge2 controls cell proliferation by binding at the cell receptor and activating oncogenic signalling pathways thus it is proven that cox2 plays an important role in carcinogenesis and cancer progression by promoting cell proliferation angiogenesis and cancer stem cell formation inhibiting cell apoptosis and heightening metastatic potential through producing pge2 the in certain studies it is stated that aspirin and nonsteroidal antiinflammatory drugs have the ability to inhibit the activity of cox enzyme which reduces inflammatory response thus it delays crc occurrence fortunately cox1 and cox2 act as biomarkers for screening purposes the biomarker is defined as any substance structure or process that is measurable in the body to determine the incidence of a disease it is commonly detected in circulation and body fluids cox1 is present in most cells thus it is not a specific biomarker however cox2 is only detected when is stimulated by trauma release of cytokines and stimulation of arachidonate metabolism by a toxin such as bft thus cox2 acts as a useful biomarker to detect cox2 is also a useful biomarker for colorectal carcinogenesis screening the level of cox2 biomarker in the blood is dependent upon epithelial cell proliferation apoptosis inhibition and neoangiogenesis patients with crc have high levels of cox2 compared to normal individuals indicating more aggressive growth rate and higher mortality rate this inflammatory responses inflammation the wwwmjmsusmmy 0cmalays j med sci julaug suggests that cox2 expression is correlated to the aggressiveness of growth rate and mortality rate etbf activates stat3immune is activated to eliminate regulation of etbf is associated with ibd due to the abnormal response to bacteria the systemic adaptive immune response foreign antigens in the body this action eventually reduces intestinal mucosal tolerance although immune cells kill foreign antigens neutrophils and th17 cells contribute to inflammation and tumourigenesis transcription factors are known as stat protein family comprising seven members each stat protein responds to its specific cytokines they play an important role in regulating immune responses by controlling th cell types generation for instance the activation and generation of th17 cells require transcription factor stat3 protein the roles of stat3 protein include promotion of cell proliferation cell survival inflammation cellular transformation metastasis of cancer blood vessel formation and tumourpromoting moreover stat3 intrinsic pathway for cancer inflammation it induces genes in tumour cells that are responsible for inflammation within a tumour cell it exhibits an overly expressed stat3 pathway inflammation is a major factors etbf has the ability to activate stat3 rapidly in both colonic epithelial cells and colonic mucosal immune cells through phosphorylation and nuclear translocation however stat3 activation first occurs in colonic mucosal immune cells followed by colonic epithelial cells to activate stat3 in immune cells epithelial cells should respond in the production of cytokines such as il6 il10 and il23 besides cytokines growth including vegf and fibroblast growth factor fgf2 are also involved in activating stat3 when etbf and bft first interact with colonic epithelial cells they stimulate early stat3 activation in colonic mucosal immune cells this stat3 activation continuously rises slowly until it reaches the peak level the peak indicates that etbf activates the immune system due to barrier dysfunction during etbfinduced colitis it activates both stat3 and th17 immune response in the colonic mucosa stat3 activation induces prooncogenic inflammatory pathways and increases the permeability of mucosa although stat3 activation is longterm and lasts for months it highly increases the chance of getting a tumour as a result of chronic inflammation additionally stat3 activation promotes the accumulation of tumour regulatory tcell tregs and blocks the generation of antitumour immune responses which give an adverse effect to the body this abnormal persistent stat3 activation increases the cancer cell tolerance prevents rejection of cancer by the immune system reduces the effectiveness of immunotherapy and enhances the effectiveness of oncogenesis activated stat3 predominantly detected in human cancers is constitutively activated and depicts its association with neoplasms patients with ibd tend to show stat3 activation and a high level of th17 cells and il17 the level of activated stat3 in patients with ibd and dysplasia is different from patients with ibd and without dysplasia patients with ibd and dysplasia show a higher level of activated stat3 compared to those without dysplasia simultaneously the level of activated stat3 increases together with the continuum of dysplasia to colitisassociated cancer it is clear that b fragilis can either be toxigenic or nontoxigenic the latter does not activate stat3 because it does not produce bft therefore ntbf does not contribute to colon cancer development but etbf does are tregs th17 and il17 good or badreduces intestinal il17 this process in a normal healthy condition tregs play an important role in inflammatory responses and immune homeostasis they express high levels of il2 receptor and produce endogenous il2 which inhibits the production of intestinal inflammation and prevents carcinogenesis however when etbf colonises a particular site of the colon it produces a large amount of bft damaging the intestinal mucosa to initiate etbftriggered colitis with the activation of the stat3 pathway this leads to direct contact between tregs and etbf and promotes tregs activation activated tregs lack the ability to produce endogenous il2 instead of producing endogenous il2 tregs consume exogenous il2 for their survival the consumption of exogenous il2 by tregs reduces the levels of exogenous il2 and produces an environment that favours the growth of th17 cells as the levels of il2 drop th17 cells are no longer inhibited and undergo expansion to produce a large quantity of naïve tcells this naïve subset of tcells then differentiates into th17 cells in excess wwwmjmsusmmy 0ccarcinogenesis this shows that colonisation of etbf promotes the accumulation of both tregs and th17 cells th17 cells start to produce large amounts of cytokines including tnf and il17 these cytokines promote cell survival and proliferation during injuries although th17 cells heal an injured site they turn into pathogenic th17 cells when deregulated these pathogenic th17 cells initiate chronic inflammatory condition il17 produced by pathogenic th17 cells are involved in an early inflammatory stage of the injuries it promotes tumour cell survival proliferation tumour neovascularisation and metastasis which allow additionally tumour cells and fibroblasts are stimulated by il17 to produce high amounts of angiogenic factors for angiogenesis il17 can activate stat3 pathway indirectly through il6 when il17 binds to il17 receptorbearing tumour cells it stimulates il6 production that is highly important for stat3 pathway activation as mentioned above this stat3 pathway activation contributes to several characteristics such as cancer proliferation antiapoptosis and angiogenesis that favour carcinogenesis in the colon this shows that there is a relationship between stat3 pathway and tregs in contributing to crc formation when etbf is accumulating in the intestinal tract as shown in figure to some extent stat5 and stat6 have been reported to be involved in inhibiting antitumour immunity when all stat3 and are activated together it highly enhances the tumourigenesis effect cleavage of ecadherin stimulate cell proliferationapart from inflammation bft alters the structure and function of colon epithelial cells by degrading ecadherin ecadherin is a 120kda glycoprotein that is the major structural protein in zonula adherens and is also known to be a tumour suppressor and zonula adherence protein this protein is responsible for the epithelial polarity in normal conditions the expression of ecadherin is linked to cellular functions including apoptosis and homotypic cellcell unfortunately when ecadherin interacts with bft in the intestinal epithelial cells it degrades ecadherin rapidly in an atpindependent manner this cleavage promotes colonic injury inflammation and loss of membraneassociation resulting in morphological enhances cellular metastatic potential it is proven that changes and adhesion review article formation of colon canceretbfbftintestinal lumenepithelial cells il2intestinal immune system th17tnfcell proliferation il17tregsinflammationcarcinogenesisil17 receptorbearing tumour cellsstat3 activationfigure the mechanism through abnormal systemof intestinal carcinogenesis immune factordependent the cleavage of ecadherin correlates with the changes of cell morphologies simultaneously degradation of ecadherin also promotes the binding of nuclear localisation of βcatenin and tcell transcriptional activator this binding promotes gene regulation and transcription additionally βcatenin plays an important role in wingless and int wnt signalling pathway promoting cell proliferation and epithelialmesenchymal transition and enhancing the expression of protooncogene in primary colorectal tumours cells in the centre of the tumour exhibit the presence of βcatenin and ecadherin however when the cells move away from the centre of the tumour they exhibit high amounts of nuclear βcatenin and the junction of ecadherin is lost important role ecadherin plays an in maintaining the morphology of cells there is a relationship with the ecadherin and the apical factin ring of the intestinal epithelial cells secretion when the loss of ecadherin increases the integrity of the apical factin is lost resulting in the increase in cell volume and chloride secretion and cell and epithelial barriers become wwwmjmsusmmy 0cmalays j med sci julaug more permeable this contributes to intestinal inflammation diarrhoea and colon carcinogenesisalteration of the signalling pathway of colorectal cancerbft is involved in many colonic epithelial cell signal transductions when bft disturbs or activates the signalling pathway it brings adverse effects to the body and can lead to colorectal tumourigenesis figure the colonic epithelial cell signal transduction transpires through the nuclear factor kappalightchainenhancer of activated bcells nfκb wnt and mitogenactivated protein kinase mapk signalling pathways bft can stimulate nfκb pathway in the intestinal epithelial cells with the expression of heme oxygenase1 ho1 and cytokines to induce mucosal inflammation this pathway has the ability to enhance the survival of neoplastic cells by preventing them from undergoing apoptosis leading to tumour formation furthermore in figure it shows that when nfκb of intestinal epithelial cells is activated for a long time it induces the activity of nitric oxide synthase that breaks down larginine to produce nitric oxide which can damage cellular dna wnt signalling pathway is important to maintain the structures of the intestinal epithelium however wnt signalling pathway contributes negatively and affects cells which are extremely important for colorectal carcinogenesis and progression as wnt signalling pathway is activated it weakens tight junctions and reduces cellular adhesion this allows the cancer cells to undergo migration and metastasis hence cancerous cells can migrate to another ans spermine oxidase is a catabolic enzyme that increases ros which can be upregulated by bft in normal conditions figure ros acts as an important mediator in multiple cell signalling pathways and immune response that is produced naturally within biological systems it consists of superoxide hydroxyl radical and hydrogen peroxide however as the amount of ros becomes excessive it imparts negative effects in the disruption of redox homeostasis figure this excessive ros induces oxidative stress it oxidises cellular components including dna lipids and proteins within the cells etbfbftbftepithelial cellactivation of signaling pathwaynfκbwntnitric oxide synthase reduce cell adhesion weaken tight junction cancer cell migratenitric oxidedamage cellular dnafigure the role of the signalling pathways when epithelial cells contact bftspermine oxidase smorelative oxygen species ros mediatorimmune responsemultiple cell signalling pathwaysfigure normal condition of the smo and ros that helps in immune response and cell signalling pathwayswwwmjmsusmmy 0conce the cellular components are oxidised it generates irreversible damage to host cells additionally ros plays an important role in the survival of cancer cells enhancing the effectiveness of carcinogenesis and aggravating cancer formed in the body spermine oxidase smo upregulated relative oxygen species ros mediatordnalipidproteinirreversible damagecarcinogenesisfigure the adverse effect of smo contacted bftfamilial adenomatous polyposisfactors contributes the combination of both genetic and environmental to crc formation it is estimated that of crc development is due to genetic predisposition wherein nearly of all crcs are attributed to familial adenomatous polyposis fap fap is an autosomal dominant inherited disorder that describes the development of numerous colorectal adenomatous polyps these polyps are able to develop in the teenagers colon meanwhile the number of polyps formed in the colon depends on the age of a person which means the number of polyps is directly proportional to the age of a person if these polyps are not removed from the colon they may transform from benign to malignant developing crc the source of fap disease is mainly due in the adenomatous to germline mutation polyposis coli apc gene this apc mutation occurs due to frameshifts insertions or deletions that may introduce a premature stop codon during the halfway through the transcription process these earlyintroduced premature stop codons in the gene sequence lead to incompletetruncated apc protein formation thus the normal function of apc protein is lost eventually facilitating carcinogenesis review article formation of colon canceradditionally germline mutations along with somatic mutations of the normal allele or loss of the normal allele lead to inactivation of apc once apc is inactivated it precisely commences carcinogenesis in normal conditions apc pathway acts as a gatekeeper controlling a part of wnt signalling pathway unfortunately when apc is mutated the function of apc pathway is lost or inactivated this inactivation of the apc pathway results in the activation of wnt signalling pathway this characteristic is mainly found in crc moreover apc mutation has the ability to alter bacteriahost epithelial interaction where it allows the bacteria to attach onto the mucosa if a person has the apcmutated gene and is exposed to etbf the chances of developing crc are high concurrently high amount of tumour load is displayed in the persons colon conclusionthe human gastrointestinal tract contains its own bacterial flora that benefit humans daily b fragilis is one of them and consists of two classes namely ntbf and etbf the differences between both the classes is the presence of bft etbf is able to produce bft that can disrupt the intestinal environment and promotes inflammation simultaneously bft degrade ecadherin and causes inflammation ibd is a chronic intestinal inflammation associated with etbf and can induce crc however patients with cd have lower risk of developing crc as compared to those with uc patients with ibd exhibit stat3 activation due to the stimulation of immune response that favours th17 cell generation as the levels of th17 cell increase it brings a huge disadvantage to the intestinal tract due to the production of il17 furthermore il17 stimulates the production of il6 that is required to activate stat3 this indicates that the stat3 pathway activates for a long time longterm stat3 activation blocks antitumour immune response which supports the growth of cancer cells thus stat3 th17 and il17 are highly important in carcinogenesis concurrently the production of proinflammatory cytokines at the site of inflammation triggers the production of cox2 enzymes that release pge2 cox2 is also known for its carcinogenic abilities due to the production of pge2 that controls cell proliferation additionally bft affects signal transductions such as wnt wwwmjmsusmmy 0cmalays j med sci julaug nfκb and mapk signalling pathways and induces tumourigenesis considering that bft induces inflammation activates stat3 and alters signalling pathways it can be concluded that bft produced by etbf plays an important role in colon carcinogenesis boleij a hechenbleikner em goodwin ac badani r stein em lazarev mg et al the bacteroides fragilis toxin gene is prevalent the colon mucosa of colorectal cancer in patients clin infect dis httpsdoi101093cidciu787acknowledgementsnoneconflict of interestnonefundsnoneauthors contributionsconception and design hkkdrafting of the article with supervision cwtcritical revision of the article for important intellectual content fdfinal approval of the article hkk fdcorrespondencedr fabian davamaniphd microbiology university of madrasfaculty of biomedical scienceschool of health sciences international medical university kuala lumpur malaysia tel fax email fabian_davamaniimuedumyreferences snezhkina av krasnov gs lipatova av sadritdinova af kardymon ol fedorova ms et al the dysregulation of polyamine metabolism in colorectal cancer is associated with overexpression of cmyc and cebpβ rather than enterotoxigenic bacteroides fragilis infection oxid med cell longev httpsdoi10115520162353560 sears cl geis al housseau f bacteroides fragilis from carcinogenesis j clin symbiont invest httpsdoi101172jci72334subverts mucosal to biology colon lv y ye t wang h zhao j chen w wang x et al suppression of colorectal tumorigenesis by recombinant bacteroides fragilis enterotoxin2 in vivo world j gastroenterol httpsdoi103748wjgv23i4603 pierce jv bernstein hd genomic diversity of enterotoxigenic strains of bacteroides fragilis plos one 2016116e0158171 httpsdoi 101371journalpone0158171 sun j kato i gut microbiota inflammation and 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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 |
" although rnabinding proteins play an essential role in a variety of different tumours there are stilllimited efforts made to systematically analyse the role of rnabinding proteins rbps in the survival of colorectalcancer crc patientsmethods analysis of crc transcriptome data collected from the tcga database was conducted and rbps wereextracted from crc r software was applied to analyse the differentially expressed genes degs of rbps to identifyrelated pathways and perform functional annotation of rbp degs gene ontology go function and kyotoencyclopedia of genes and genomes kegg pathway enrichment analyses were carried out using the database forannotation visualization and integrated discovery proteinprotein interactions ppis of these degs were analysedbased on the search tool for the retrieval of interacting genes string database and visualized by cytoscapesoftware based on the cox regression analysis of the prognostic value of rbps from the ppi network with survivaltime the rbps related to survival were identified and a prognostic model was constructed to verify the model thedata stored in the tcga database were designated as the training set while the chip data obtained from the geodatabase were treated as the test set then both survival analysis and roc curve verification were conductedfinally the risk curves and nomograms of the two groups were generated to predict the survival periodresults among rbp degs genes were upregulated while were downregulated of which twelve rbpsnop14 mrps23 mak16 tdrd6 pop1 tdrd5 tdrd7 ppargc1a lin28b celf4 lrrfip2 msi2 with prognosticvalue were obtaineds the twelve identified genes may be promising predictors of crc and play an essential role in thepathogenesis of crc however further investigation of the underlying mechanism is neededkeywords colorectal cancer crc rnabinding protein rbp prognostic model construction survival analysisintroductionas a significant class of cellular proteins rnabindingproteins rbps can interact with rna by recognizingspecial rnabinding domains and are widely involved inmultiple posttranscriptional regulatory processes suchas rna shearing transport sequence editing intracellular localization and translation control it is estimatedthat there are up to different proteins that have the correspondence lgzhyd1962163comdepartment of neurology the first affiliated hospital of harbin medicaluniversity you zheng street harbin heilongjiang provincepeoples republic of chinapotential to bind rna in the human genome rbpsare characterized by the presence of an rnabindingdomain rbd that contains residues and usuallyadopts an α topology found in single or multiple copies these domains usually bind to rna depending onthe exact sequence or structure to date rbps havebeen reported to be associated with various human diseases such as spinal muscular atrophy and myotonicdystrophy there are various rbps involved intumourigenesis src associated with kda mitosissam68 is a member of the star signal transductionand rna metabolism activation family of rbps it is 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 0cfan world of surgical oncology page of involved in several steps of mrna metabolism such astranscription alternative splicing and nuclear export inaddition sam68 is associated with the signal transduction pathways required for the response of cells to stimuli cell cycle transition and viral infection tarbp2is overexpressed in metastatic cells and metastatichuman breast tumours and its abnormal activation canpromote the progression of breast carcinomas by affecting the stability of its target mrna colorectal cancer crc which includes colon andrectal cancer is a common digestive tract tumour themolecular pathogenesis of crc is a complex multistepprocess involving multiple acquired genetic and epigenetic abnormalities some rbps are known to be associated with colorectal cancer according to some studiesmuscleblindlike mbnl1 an rbp implicated in developmental control can significantly suppress crc cellmetastasis in vitro mbnl1 destabilizes snail transcriptsand thus inhibits the epithelialmesenchymal transitionemt of crc cells through the snailecadherin axisin vitro ras oncogenearecommonly seen in colon cancer activation mutationsinteractionsin this study an analysis was conducted of rbprelated genes in crc patients through differential geneexpression and protein moleculeinaddition a prognostic model was adopted to identifytwelve genes associated with the survival of crc patients we verified the model and performed survivalanalysis and risk assessment these results will help elucidate the underlying mechanism related to the survivalof crc at the molecular level thus providing a new direction for the prognosis of crc and clinical treatmentmethodsdata sourcethe fpkm transcriptome data of crc were obtainedfrom the tcga database website portalgdccancergov the total number of samples is of whichthere are samples in the tumour group and samples in the normal group then the rbp gene was obtained from the goa database website wwwebiacukgoa combined with the crc transcriptome sequencing map crc rbps were obtained the data ongene expression gse17536 in colorectal patients wereobtained from the geo database website wwwncbinlmnihgovgeo involving a total of cases allthe data were publicly available online this study requiresno experiments to be conducted by any author on humansor animals the flowchart of it is shown in fig between the two groups with the adjusted p andlogfc go and kegg pathway analysis of degsgo analysis represents a common method applied toconduct largescale functional enrichment study genefunctions can be categorized into biological processesbp molecular functions mf and cellular componentscc kegg is known as a commonly used databasewhere a large amount of data on genomes biologicalpathways diseases chemicals and drugsstoredthrough go and kegg analysis of degs barplot andbubble were drawn respectively all of the go and pathway terms were ranked by their log10 q valueisproteinprotein interaction ppi networkthe search tool for the retrieval of interacting genesstring database stringdb is designedto analyse the ppi information degs were input intothe string database to obtain ppi information subsequently the cytoscape software was applied to visualizethe ppi network the cytoscape plugin mcode wasused to obtain the most relevant subnetwork moduleand then the hub genes ofthe four modules wereenriched for go and kegg analysisconstruction and analysis of prognostic modelscox regression analysis was conducted on the prognosticvalue of rbps from the ppi network with survivaltime the rbps related to survival were identified and aforest map was generated then the samples of thetcga database were designated as the training set andthe samples of the geo database were treated as the testset to construct the best prognostic model based on thetraining set twelve survivalrelated genes were identified by the model based on which the correlation coefficient of each gene was obtained then the risk score ofeach patient in the training set and test set was calculated according to gene expression in additionthepatients were classified into highrisk and lowriskgroups by the median value of the risk score the patients in the training set and the test set were categorized into either the highrisk group or lowrisk groupa survival analysis was conducted an roc curve wasgenerated and then the risk curves were constructed forthe training and test sets furthermore with univariateand multivariate analyses nomograms based on thegenes obtained from the prognostic model were generated to predict the length of survival for the patientsdata processing of differentially expressed genes degsthe rbps were analysed using r software to identify thedifference between the tumour group and the samplegroup wilcoxon test was carried out to identify degsresultsidentification of rbps degstranscriptome sequencing data of rbps of crcwas obtained from the tcga database the differential 0cfan world of surgical oncology page of fig flowchart of systemic analysis of rnabinding protein in patients with crcexpression analysis was conducted to find out that therewere upregulated genes and downregulatedgenes based on which volcano and heat maps weredrawn as shown in fig functional enrichment analyses of degsthe up and downregulated genes of degs were analysed for go function and kegg pathway enrichmentwhile both barplot and bubble were plotted theenriched go terms were divided into cc bp and mfrelevantontologies the top mostitems wereselected as shown in fig with regard to the upregulated genome the results of go analysis indicated thatdegs were mainly enriched in bps including ncrnametabolic process ncrna processing ribonucleoproteincomplex biogenesis and ribosome biogenesis and so oncc analysis revealed that the degs were significantlyenriched in preribosome tutp complex smallsubunitprocessome and cytoplasmic ribonucleoprotein granuleand so on as for the mf the degs were enriched in 0cfan world of surgical oncology page of fig volcano and heat map of rnabinding protein degs a volcano map b heat map red nodes represent upregulated genes and greennodes represent downregulated genescatalytic activity thus influencing rna and ribonucleaseactivityin the downregulated genome bp analysisdemonstrated that the degs were significantly enrichedas reflected in the regulation of translation rna splicing the regulation of cellular amide metabolic processand so on cc analysis showed that the degs weresignificantly enriched in cytoplasmic ribonucleoproteingranule ribonucleoprotein granule cytoplasmic stressgranule etc as for the mf the degs were enriched intranslation regulator activity mrna ²utr bindingand so on regarding the results of kegg pathwayanalysis as shown in fig the degs in the upregulatedgenome were primarily enriched in the pathways inribosome biogenesis in eukaryotes and rna transportetcthe degs werelargely enriched in the pathways in spliceosome andrna transport etcin the downregulated genomeppi network constructionthe protein interactions among the degs were predicted using string tools a total of nodes and edges in the ppi network were obtained as shownin fig 5a then cytoscape software was applied to drawa network diagram of genes as shown in fig 5bbesides four key subnetworks with the mcode plugin were extracted go was performed table andkegg enrichment analysis was conducted table onthe genes of the four subnetworks respectively finallythe four subnetworks were visualized as shown in fig5ce the number of hub genes in these subnetworksis and respectivelyconstruction and analysis of prognostic modelscox regression analysis was carried out of the prognostic value of rbps interacting with survival time rbps related to survival were screened and a forestmap was drawn as shown in fig 6a then a prognosticmodel was constructed for the rbps related to prognosis and a prognostic marker gene comprised of rbps was established these twelve genes are nucleolarprotein nop14 mitochondrial ribosomal proteins23 mrps23 mak16 homolog mak16tudordomaincontaining tdrd6 processing of precursor pop1 tudor domaincontaining tdrd5 tudordomaincontaining tdrd7 peroxisome proliferatoractivated receptor1alphappargc1alin28 homolog b lin28b cugbpelavlike family member celf4 leucinerich repeatflightlessinteracting protein lrrfip2 and musashirnabinding protein msi2 then the corresponding forest map was drawn for these twelve genes asshown in fig 6b among them tdrd5 elf4 andlrrfip2 are classed as highrisk genes while the rest isclassed as lowrisk genes based on the establishedmodel the risk value of each patient was calculatedaccording to the median value the patients in thecoactivatamma 0cfan world of surgical oncology page of fig the gene ontology analyses of rnabinding protein degs a barplot shows go functional enrichment analysis predicted upregulateddegs including biological process cellular components and molecular functions the colour indicates the significance of the p value b bubbleshows go functional enrichment analysis predicted upregulated degs the size of the circle represents the number of genes enriched in theentry and the colour indicates the significance of the p value c barplot shows go functional enrichment analysis predicted downregulated degsd bubble shows go functional enrichment analysis predicted downregulated degstraining set and the test set were divided into either ahighrisk group or a lowrisk group among them thenumber of patients in the training set as well as thehighrisk group was the number of patients in thelowrisk group was in the test set the number ofpatients in the highrisk group was and that of patients in the lowrisk group was according to theresults the patients with highrisk scores had a shortersurvival time as shown in fig 6c d finally in terms ofsurvival prediction the roc curve showed a relativelydecent performance as shown in fig 6e f the aucvalue in the training set was and the auc valuein the test set was then the risk curves wereplotted for the training and test sets as shown in fig which reveals that their abscissas are the same theywere divided into high and lowrisk groups by the median value the patients were ranked by risk value inascending order the risk value of patients from left toright increased on a continued basis as did the risk offatalitythen independent prognostic analysis was conductedof univariate and multivariate for the training and testsets as shown in fig 8ad according to the results ofsinglefactor independent prognosis analysisfor thetraining and test sets age and tumour stage can betreated as independent prognostic factor for the survivalof colorectal patients p in the multivariate independent prognostic analysis age and stage can be takenas independent prognostic factor for crc in the test setp for the training set however only stage can 0cfan world of surgical oncology page of fig the kegg pathway enrichment analyses of rnabinding protein degs a barplot shows kegg pathway analysis predicted upregulateddegs the colour indicates the significance of the p value b bubble shows kegg pathway analysis predicted upregulated degs the size of thecircle represents the number of genes enriched in the entry and the colour indicates the significance of the p value c barplot shows keggpathway analysis predicted downregulated degs d bubble shows kegg pathway analysis predicted downregulated degsbe taken as independent prognostic factors for crc p not age p finally nomograms were plotted for these rbpprognostic genes in the training set to predict the survival time of the patients as shown in fig 8e the rnaexpression of rbps was applied as parameters todraw the point line in nomograms the scores wereadded to obtain the total score which can be used topredictthe 1year 2year and 3year survival ratesamong crc patientsdiscussionas one of the most common malignant tumours crc ischaracterized by a high recurrence rate and poor prognosis especially in developed countries it is the thirdmost common cancer among males and ranks secondamong females [ ] to date various methods havebeen applied to predict biomarkers of crc prognosis rbps can regulate mrna stability and contributeto cancerassociated pathways in this paper therbps of crc were analysed through a series of analyses marker genes related to the prognosis of crcwere identifiedtudor domaincontaining tdrd refers to a family ofevolutionarily conserved proteins in general piwi andtdrd proteins are recognized as the major influencingfactors in pirna biogenesis and the development ofgerm cells in a previous study it was found thatmethyl lysinebound tdrds are primarily involved inhistone modification and chromatin remodelling whilemethyl argininebound tdrds are usually associatedwith rna metabolism alternative splicing small rna 0cfan world of surgical oncology page of fig rnabinding proteins degs are used to construct proteinprotein interaction networks and subnetworks a ppi interaction network mapobtained from string website b cytoscape visualizes the genes of the interacting ppi network red nodes represent upregulated genes whileblue nodes refer to downregulated genes c four mcode modules visualization dg four most significant mcode components form theppi networkpathways and germ cell development [ ] tdrdshave now been detected in various cancers tdrd9 ishighly expressed in a subset of nonsmall cell lung carcinomas and derived cell lines through hypomethylationof its cpg island tdrd1 is closely associated witherg overexpression in primary prostate cancer according to the findings by jiang tdrdgenesphf20l1 arib4b setdb1 lbr tdrkhtdrd10 and tdrd5 showed high levels of amplification in more than of tcga breast cancer datasetstdrd5 has significant prognostic value for hepatocellular carcinoma hcc patients with higher tdrd5expression exhibit significantly poorer overall survivalthan patients with low tdrd5 expression an earlystudy revealed that tdrd5 was expressed in normalgastric and colonic mucosal tissues suggesting the possibility that the tdrd5 gene is modified in crc tdrd6 is capable of differentiating irradiated prostatecancer patients into early and late relapse groups inaddition tdrd7 may play a certain role in the migration of tumour cells in an analysis of crc mo discovered not only frameshift mutations butalso intratumoural heterogeneity of tdrd1 tdrd5and tdrd9 which in combination might alter tdrdgene functions and affectthe tumorigenesis of highmicrosatellite instability crc in our study it was foundthat tdrd5 tdrd6 and tdrd7 are differentiallyexpressed in crc and further studies on the role ofthese three genes in colon cancer are neededpop1 is a component of ribonuclease p which is aribonucleoprotein complex that generates mature trnamolecules by cleaving their ² end s[ ] in additionit is a component of the mrp ribonuclease complexwhich cleaves prerrna sequences in a previousstudy pop1 was found to be enriched in human prostate cancer celllines suggesting that it may besuitable as a potential marker for the diagnosis andprognosis of prostate cancer in addition pop1 is upregulated in crc and applicable as a prognostic factor forcrc nevertheless there is still no relevant research onthe mechanism of pop1 in crc so further studies arenecessaryppargc1a also known as pgc1α is a transcriptionalcoactivator of genes encoding proteins responsible forthe regulation of mitochondrial biogenesis and function derrico discovered that in the presenceof bax pgc1αinduced ros accumulation is one of themain apoptosisdriving factors in crc cells they also 0cfan world of surgical oncology page of table the go function enrichment analysis of four most significant mcode componentsontologyidsubnetwork descriptioncountp valuepadjustbpbpbpccccccmfmfmfgo0042254ribosome biogenesisgo0016072rrna metabolic processgo0006364rrna processinggo0030684preribosomego0034455tutp complexgo0032040smallsubunit processomego0140098catalytic activity acting on rnago0003724rna helicase activitygo0030515snorna bindingsubnetwork bpbpbpccccccmfmfmfgo0000377rna splicing via transesterification reactions with bulged adenosine as nucleophilego0000398 mrna splicing via spliceosomego0000375rna splicing via transesterification reactionsgo0071013catalytic step spliceosomego0000974prp19 complexgo0005682 u5 snrnpgo0090079translation regulator activity nucleic acid bindinggo0003743translation initiation factor activitygo0008135translation factor activity rna bindingsubnetwork bpbpbpccccccmfmfmfgo0000460 maturation of 58s rrnago0034427 nucleartranscribed mrna catabolic process exonucleolytic ²²go0043629ncrna polyadenylationgo1905354exoribonuclease complexgo0000176 nuclear exosome rnase complexgo0000178exosome rnase complexgo0017091go0000175go0016896aurich element binding²²exoribonuclease activityexoribonuclease activity producing ²phosphomonoesterssubnetwork bpbpbpccccccgo0051028 mrna transportgo0050657 nucleic acid transportgo0050658rna transportgo0000346transcription export complexgo0016607 nuclear speckgo0000784 nuclear chromosome telomeric region157e730e221e503e251e516e842e262e160e737e737e871e697e203e310e282e154e166e504e622e147e217e150e103e707e129e154e264e113e113e569e235e506e118e238e171e426e584e690e108e438e103e103e103e662e966e983e223e437e437e524e323e367e369e127e582e818e818e818e190e223e223e911e188efound that pgc1α induced mitochondrial proliferationand activation in human intestinal cancer cells shin demonstrated that pgc1α overexpression waseffective in upregulating the proliferation of hek293and ct26 cells in addition its overexpression was correlated with an enhancement of tumourigenesis in acasecontrol study heterozygous carriers of rs3774921 inpgc1α showed an increased risk of crc pgc1αplays an essential role in the pathogenesis of colon cancer in a clinical study the expression of pgc1α wasassessed in crc patients using realtime quantitativepcr and the mrna level of pgc1α was found to bedecreased in the tumours of most patients however immunohistochemistry has also been performed to 0cfan world of surgical oncology page of table the kegg function enrichment analysis of four most significant mcode componentslistsubnetwork1descriptionribosome biogenesis in eukaryotesidhsa03008countsubnetwork2subnetwork3hsa03040hsa03013hsa03015hsa03010hsa03018spliceosomerna transportmrna surveillance pathwayribosomerna degradationp value184e408e100e547epadjust368e285e351e128e475e475edetect the expression of pgc1α the results revealedthat of the crc samples were positive whileno or weak pgc1α expression was detected in the nucleiof normal mucosa cells pgc1α expression is demonstrated to be related to lymph node metastasis thus itcan serve as a possible prognostic marker our results also show that pgc1α can be used as an independent prognostic factor for crcit is thought that lrrfip2 functions as an activator ofthe canonical wnt signalling pathway which is associated with dvl3 a factor upstream of ctnnb1betacatenin it positively regulates tolllike receptor tlrsignalling in response to agonists probably by competing with the negative flii regulator for myd88 bindingwhich plays a crucial role in the progression of coloncancer [ ] in this study lrrfip2 was identified asa candidate gene for alternative splicing in colon andprostate cancer there were three splice variants thatdiffered in their inclusion or skipping of exons andor these exons contain five predicted putative serinephosphorylation sites and one putative oglycosylationsite and could modulate lrrfip2 protein function as a familial hereditary disease hereditary nonpolyposisis mainly caused by dnacrc lynch syndromefig rnabinding protein dges are used to construct prognostic models survival analysis and verification of geo data sets a the prognosticrelated rbps shown in the forest map red indicates highrisk genes and green denotes lowrisk genes b the rbps obtained byconstructing the prognostic model shown in the forest map c survival analysis curve of training set red indicates patients in the highrisk groupblue denotes patients in the lowrisk group d survival analysis curve of test set e roc curve of training set f roc curve of test set 0cfan world of surgical oncology page of fig risk curve of training and test sets a the risk score distribution of training set b the distribution of survival status for training set c intraining set the heat map of rbps for the high and lowrisk groups d the risk score distribution of test set e the survival status distributionfor test set f in test set the heat map of rbps for the high and lowrisk groupsmismatch mismatch repair in lynch syndrome morakand colleagues discovered a paracentric inversion onchromosome 3p222 between the dna mismatch repairgene mlh1 and the downstream lrrfip2 gene transcribed in the antisense direction this generates twonew stable fusion transcripts thus removing the mlh1gene and protein function in another study conducted on a lynch syndrome family it was found thatthe mlh1itga9 fusion allele caused loss of heterozygosity loh in five genes including lrrfip2 which resulted in the loss of mismatch repair capabilities thus lrrfip2 may play a critical role in the pathogenesis of crccelf4 is responsible for encoding a protein with threedomains that bind an rna recognition motif andregulate premrna alternative splicing some studiesshowed that celf4 was hypermethylated in endometrialcancer methylated celf4 may be suitable for endometrial cancer screening of cervical smears further research is still needed to determine the role of celf4 intumoursas a member of the musashi family msi2 belongs tothe family of drosophila melanogaster rnabindingproteins it has been identified as a critical regulator ofhaematopoietic stem cell hsc selfrenewal and fatedetermination [ ] in this study msi2 was found tobe a central component in an unknown oncogenic pathway to promote intestinal transformation via the pdkaktmtorc1 axis msi2 is highly expressed in avariety of cancers including hcc and lung cancer recent studies on colon cancer celllines havesuggested that both usp10 and msi2 proteins areupregulated in addition ubiquitinspecific protease usp10 could stabilize the oncogenic factor msi2through deubiquitination the expression of msi2was detected in crc and control specimens from patients by the tissue microarray technique and immunohistochemical staining msi2 was highly expressed in of crc samples in addition high msi2expression was related to liver metastasis in crc patients in other cancers guo found that msi2expression was markedly increased in both pancreatic 0cfan world of surgical oncology page of fig independent prognosis analysis and prediction of and years of nomograms of crc patients in the training and test sets a singlefactor prognosis analysis of training set b multifactor prognosis analysis of training set c singlefactor prognosis analysis of test set d multifactor prognostic analysis of test set e the nomograms for predicting 1year 2year and 3year survival probability of patients with crc fortraining setlines and humanductal adenocarcinoma pdac cellpdac specimens and high msi2 expression was associated with poor prognosis of pdac high expressionof msi2 mrna is associated with decreased survival inacute myeloid leukaemia furthermore msi2 mayact as a prognostic biomarker in patients with cervicalcancer bladder cancer and oesophageal squamous cell carcinoma it was also found that its expression is upregulated in crc which makesitapplicable as a prognostic marker gene for crclin28 an oncofoetal rnabinding protein modulatesstem cell maintenance somatic reprogramming metabolism anismal growthtissue development andtumourigenesis two paralogues of lin28 were included lin28a and lin28b it is well established thatlin28a and lin28b inhibit let7 family mirnas andderepress let7 targets including ras pi3kakt mychmga2 and igf2bps thus promoting oncogenesis in liver cancer stem cells fang found thatoverexpression of msi2 resulted in the upregulation oflin28a stemness and chemotherapeutic drug resistance induced by msi2 overexpression were dramaticallyreduced by lin28a knockdown moreover msi2 andlin28a levels positively correlated with the clinical severity and prognosis in hcc patients king found that lin28b overexpression is associatedwith reduced survival time and increased probability oftumour recurrence in patients constitutive lin28b expression promotes not only tumorigenesis but alsolgr5 and prom1 expression in colonic epithelial cells in addition lin28b promotes the proliferationcolony formation and tumourigenesis of colon cancercells by increasing bcl2 expression a clinicalstudy found that lin28a and lin28b were overexpressed in oesophageal cancer cells especially on the invasive front high expression of lin28a and lin28bcorrelated significantly with lymph node metastasis andpoor prognosis hu found that gastric adenocarcinoma gac patient survival time was negativelycorrelated with the lin28b expression level wherebyhigher lin28b expression correlated with shortersurvival time in pdac patients high lin28b expression was significantly correlated with high levels oflymphatic metastasis distant metastasis and a poor 0cfan world of surgical oncology page of prognosis in addition patients with increased lin28bhad markedly reduced overall survival compared tothose with low lin28b in hcc and oral squamouscell carcinoma oscc thus lin28b is highlyexpressed in crc and plays an important role in itspathogenesisindicating that it is suitable as a targetgene for crc prognosisnop14 is a stressresponsive gene required for 18srrna maturation and 40s ribosome production as indicated by zhou nop14 in pancreaticcancer cells promotes motility proliferation and metastatic capacity according to the findings by du nop14 induced tumour invasion and metastasisby improving the stability of mutp53 mrna by inhibiting the wntcatenin pathways nop14 suppressesbreast cancer in addition nop14 can reducemelanoma cell proliferation and metastasis by regulating the wntbcatenin signalling pathway inclinical studies of patients with ovarian cancer downregulation of nop14 was associated with a significantly worse survival rate this study showedthatthe expression of nop14 was upregulated incrc but its role in pathogenesis requires further research and confirmationthe mrps23 gene which is responsible for encodinga 28s subunit protein has been found to be overexpressed in breast cancer uterine cervical cancer hcc colorectal cancer and uterine leiomyoma as revealed by gao inhibitingmrps23 could lead to a significant reduction in breastcancer metastasis by inhibiting the emt phenotype pu found that high mrps23 levels can predict poorclinical outcomes in hcc although the expressionof mrps23 is increased in crc its specific pathogenesisremains unclearmak16 encodes a ribosomal protein and plays an important role in ribosome biogenesis throughout the cellcycle in this study it was found that mutations inmak16 can induce cell cycle arrest at g1 phase duringwhich the cell synthesizes mrna and proteins in preparation for cell division at present there is still nostudy of the role of mak16 in the pathogenesis of tumours which requires further research to confirmin this paper a discussion was conducted about therole of the identified genes in tumours althoughsome genes were found irrelevant to the pathogenesis ofcrc their biological functions and changes in their expression in crc suggest that they may play a role incrc to some extent and further experiments need to beconducted for verification this is also a limitation ofour study more research is needed to explore the pathogenesis of crcthe above genes are related to the prognosis of crcmore research especially experimental studies is neededto verify the specific function of each gene our findingsmay improve the understanding of the incidence andprognosis of crc thus providing a reference for furtherimprovement of the diagnosis and tr | Colon_Cancer |
pancreatic cancer is a devastating malignancy with a 5year relative survival rate of only dependenton the geographical location surveyed [] with these statistics exhibiting only modest improvementover the last four decades [] the median survival postdiagnosis ranges from just months forlocally advanced disease and months for metastatic disease it was estimated by the world healthanisation that pancreatic cancer is currently the 7th leading cause of cancerrelated death being responsible for over deaths worldwide in with incidence increasing pancreatic cancerhas been predicted to be the third leading cause of cancerrelated death in the european union by and the second leading cause of cancerrelated death in the united states of america and germanyby several factors contribute to the poor survival of pancreatic cancer patients a current lack of reliablediagnostic markers that would enable early screening coupled with largely nonspecific symptoms ofdisease results in over of patients presenting with metastatic disease at diagnosis this subgroupof patients have limited therapeutic options and are thus typically administered palliative chemotherapyaimed at prolonging survival and reducing symptoms during endoflife care [] moreover whilstapproximately of patients present with localised disease that is eligible for potentially curativesurgery disease recurs in over of patients postresection ultimately these factorsculminate in more than of patients diagnosed with pancreatic cancer succumbing to disease these harrowing statistics highlight that despite research efforts there remains a lack of understandingof the pathogenesis of disease which in turn limits the development of new therapeuticsreceived march revised july accepted 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 commonsattribution license cc byncnd 0cclinical science 101042cs20191211pancreatic ductal adenocarcinomapancreatic ductal adenocarcinoma is the predominant pancreaticmalignancypancreatic cancer can arise from either the endocrine or the exocrine region of the pancreas tumours arising fromthe exocrine compartment are termed pancreatic ductal adenocarcinoma pdac and account for over of allpancreatic cancers pdac develops via a stepwise progression from normal tissue through to invasive lesions which is associated withdistinct morphological characteristics [] it has been proposed that this process begins with a phenomenontermed acinartoductal metaplasia adm which is a normal homeostatic mechanism whereby acinar cells transdifferentiate into a ductal phenotype in response to particular stimuli however if compounded by an oncogenichit cells are pushed towards a pathogenic phenotype that develops into pancreatic intraepithelial neoplasia panin[] disease progresses through preinvasive stages termed panin1a panin1b panin2 and panin3 priorto invasive pdac this progression is associated with increasing nuclear atypia whereby the nuclei are no longerpositioned basally and loss of normal architecture as cells become more papillary in nature with panin3 lesionsdemonstrating increased mitoses as disease progresses to pdac cells become invasive and breach the basement membrane growing through the extracellular matrix and metastasising to distant ans figure less common precursor lesions include intraductal papillary mucinous neoplasms ipmns and mucinous cysticneoplasms mcns that also develop through multistep processes whilst they share some common featureseach lesion is morphologically and genetically distinct in contrast with panins that form within small ducts ipmnsdevelop within the primary or secondary branches of the main pancreatic duct whilst mcns lack ductal involvement an ammatory tumour microenvironment contributes to pdacpathogenesisan archetypal feature of pdac is the development of extensive stromal networks within the tumour microenvironment tme that can account for up to of the total tumour volume [] this unique characteristic drives theinflammatory nature of pdac that contributes to its aggressive phenotype desmoplasia is driven by pancreaticstellate cells pscs and cancerassociated fibroblasts cafs that upon activation produce a range of extracellularmatrix ecm components such as collagen laminin and fibronectin which in turn form a physical barrier preventing the penetration of therapeutics [] though pscs and cafs have been shown to support cancer metastasis and drug resistance they interact with cancer cells in a bidirectional manner with each promoting the survivalgrowth and proliferation of the other [] quiescent fibroblasts are able to differentiate into two unique subtypes termed myofibroblastic cafs mycafs and inflammatory cafs icafs these two subtypes are distinct whereby mycafs express high levels of αsmooth muscle actin αsma and are located adjacent to cancercells while icafs express low levels of αsma and instead secrete high levels of inflammatory mediators including il6 and are distributed distant from cancer cells within desmoplastic tumour regions broadly mycafsappear to have roles in epithelialtomesenchymal transition emt and ecm remodelling whilst icafs appear tobe involved in inflammation and ecm deposition a third less abundant subtype termed antigenpresentingcafs apcafs has more recently been defined these cells express low levels of both αsma and il6 andinstead express high levels of major histocompatibility complex class ii mhcii and related genes as such allthree subtypes are transcriptionally and functionally distinctthe wider tme contains a plethora of other cell types including endothelial cells tumourassociated macrophagestams and neutrophils tans mast cells regulatory tcells myeloidderived suppressor cells mdscs dendriticcells natural killer nk cells and nerve cells interactions between various cells within the tme can driveeither proor antitumorigenic functions of others for example cancer cells can induce pscs to secrete inflammatorycytokines that drive immune cells towards an immunosuppressive phenotype and also form a positive feedback loopby increasing the tumorigenic potential of cancer cells the ecm itself has also been suggested to modifypsc behaviour in particular that ecm stiffness promotes the mycaf phenotype indicated by increased αsmaexpression this results in substantial complexity that ultimately determines tumour phenotype the components of the microenvironment modify tumour behaviour through the production of cytokines growthfactors and other signalling molecules that predominantly drive a proinflammatory and immunosuppressive program that enhances pdac tumour growth and progression [] figure the ability of the tme toinhibit therapeutic efficacy through both molecular mechanisms and physical fibrotic barriers contributes to the 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 byncnd 0cclinical science 101042cs20191211figure our current understanding of the contribution of il6 family cytokines to panin and pdac developmentpreinvasive panin lesions develop from normal ductal epithelia through panin stages 1a 1b and to stage invasive andormetastatic pdac this process is associated with acinartoductal metaplasia adm early in disease combined with an accumulation of oncogenic mutations most common mutations are indicated a number of cells within the tumour microenvironment havebeen shown to secrete il6 family cytokines which in turn results in the activation of a protumorigenic signalling cascade a betterunderstanding of the relationship between each of these cells within the tumour microenvironment may reveal new therapeuticopportunities to manage cancer progressionintrinsic resistance of disease thus dual targeting of cancer cells and the tme may be required to induce afavourable therapeutic response although this poses a signficant scientific and clinical challenge []molecular basis of pathogenesispdac development is associated with accumulation of mutationsthe progression of tumorigenesis through panin and pdac stages is associated with the stepwise accumulation ofspecific genetic mutations that drive malignant transformation the most frequent genetic alteration is an activatingkras point mutation codon that occurs early on in tumour development and is detected in over ofpdac tumours [] mutations in kras have been shown to drive development of precursor panin lesions andwhen combined with an appropriate tumour suppressor mutation these lesions progress to invasive or metastaticpdac figure patient tumours harbouring wildtype wt kras often carry activating mutations indownstream effector molecules such as braf or pik3ca 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 byncnd 0cclinical science 101042cs20191211inactivation of a range of tumour suppressor proteins is also common including mutations in tp53 cdkn2aand smad4 in approximately and of tumours respectively whilst each mutation has uniquemechanistic outcomes all three proteins are either directly or indirectly involved in the regulation of the g1s cellcycle checkpoint analysis of patient tumours indicates that two or more of these mutations often occur together withcdkn2a mutation being combined with either tp53 smad4 or both usually in the background of kras mutation this suggests that by disrupting this checkpoint cancer cells are able to overcome inhibitory mechanismsallowing continued progression to invasive diseaseunbiased sequencing efforts have also enabled identification of low prevalence pdac mutations observed in lessthan of cases these include mutations in genes involved in chromatin modification kdm6a dnadamage repair atm and other tumourrelated processes such as growth tgfbr1 or tgfbr2 furthermore it is important to note that technical advances are continuously uncovering epigenetic mechanisms thatfurther modulate the pdac transcriptional landscape and ultimately influence disease heterogeneity and tumourprogression molecular subtypesthe pdac epithelial compartment is typically divided into two subtypes including a classical subtype exhibiting anepitheliallike expression profile and a squamous or mesenchymallike subtype an additional third exocrinesubtype is outlined in some analyses and is characterised by a gene expression profile related to digestive enzymeproduction the classical or epithelial subtype has also been further divided into a pancreatic progenitor andan immunogenic subtype whereby the immunogenic subtype is distinguished by significant immune infiltration andassociated gene programmes though there is no consensus on which classification system will allow the mostvaluable stratification of patients the mesenchymal subtype is invariably associated with a poor prognosis the stromal compartment has also been classified into either normal or activated subtypes reflecting the proandantitumorigenic capabilities of the tme with the activated subtype associated with reduced survival this isparticularly valuable as the extensive heterogeneity of pdac complicates clinically relevant stratification of patientsthus the identification of molecularly unique subtypes may enable development of tailored therapeutic regimensthat will provide improved clinical outcomescurrent treatment optionsregardless of disease stage at time of diagnosis patients have relatively limited treatment options for the majorityof patients disease will be locally advanced or metastatic disqualifying them from undergoing potentially curativesurgery in these cases patients are typically offered chemotherapy with palliative intent []surgery provides the only potentially curative treatmentsurgical resection remains the only potentially curative treatment option due to minimal efficacy of standardofcarechemotherapy and radiotherapy due to its aggressive nature the majority of patients present to clinic with locallyadvanced or metastatic disease with only of patients presenting with localised tumours that are eligiblefor surgical resection even for those able to undergo surgical intervention over of patients relapsepostresection with median survival improving to months and 5year relative survival rate increasingmodestly to the use of neoadjuvant therapy is generally reserved for borderline resectable disease inan effort to enable patients to become eligible for surgery however a range of recent trials have shown improved clinical outcomes including overall survival for neoadjuvant treatment of patients with resectable tumours following surgical resection patients are typically treated with adjuvant gemcitabinebased chemotherapy although a recent study showed improved diseasefree survival and overall survival with a modified folfirinoxtherapy combination of oxaliplatin irinotecan leucovorin and fluorouracil radiotherapy provides variable clinical outcomewhilst the use of radiotherapy and chemoradiotherapy combination chemo and radiotherapy in the neoadjuvantand adjuvant settings have been investigated there remains a lack of consensus regarding therapeutic benefit this is due to issues such as insufficient radiation dose and low participant numbers as well as low uptake of moderntechniques in the neoadjuvant setting preliminary studies reported reduced lymph node positivity and rates oflocal recurrence for chemoradiotherapy compared to surgery with adjuvant chemotherapy however the useof radiotherapy in the palliative setting was reported to modestly reduce overall survival more recent studiesusing ablative doses of radiation have shown a survival benefit highlighting that technological advancements mayprovide an avenue for improved clinical outcomes following radiotherapy these contrasting results highlight the 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 byncnd 0cclinical science 101042cs20191211need to determine which subset of patients may benefit from the inclusion of radiotherapy approaches in standardtreatment regimenschemotherapy remains the cornerstone of treatmentdespite modest improvements in overall survival palliative chemotherapy remains the standard treatment optionfor patients with locally advanced or metastatic disease gemcitabine monotherapy has been the mainstay treatmentfor pancreatic cancer since when it was demonstrated to improve median survival by just over month compared with fluorouracil within the last decade there have been some further improvements in clinical outcomewith combination chemotherapies gemcitabinenabpaclitaxel and folfirinox providing median overall survivalbenefits of and months respectively compared with gemcitabine alone although folfirinox treatment resulted in a lower percentage of patients experiencing reduced quality of life it also had increased toxicity andadverse events thus preventing its administration to patients with multiple comorbidities therapeutic resistance remains a signiï¬cant barrier to patient survivaldespite advances in chemotherapeutic options treatment efficacy and patient prognosis remain poor due to the inherent therapeutic resistance of pancreatic cancer it has been proposed that this drug resistance may be driven by thetme including changes to cytokine signalling and metabolic pathways [] this intrinsic resistance is demonstrated by patients experiencing similar overall survival for chemotherapy treatment months compared withbest supportive care months which encompasses the use of palliative surgery psychological support painmanagement and other methods of symptom control whilst a range of targeted treatments such as egfr orcheckpoint inhibitors have been trialled with or without chemotherapy they have shown limited success []emerging roles for the il6 family of cytokines in pdaccytokines are soluble molecular messengers that enable efficient communication between a range of cell types andhave been recognised to be major contributors to the growth and metastasis of cancers [] in pancreatic cancer cytokines mediate signalling between cancer cells and the cells of the tme including pscs cafs endothelialcells and a range of immune cells including macrophages mast cells neutrophils and regulatory tcells []it is the specific signalling pathways active within this community of cells that dictates the balance of pro andantitumorigenic functions the il6 family of cytokinesthe interleukin il6 family of cytokines includes il6 il11 leukaemia inhibitory factor lif oncostatin mosm ciliary neurotrophic factor cntf cardiotrophin1 ct1 cardiotrophinlike cytokine clc neuropoietin np il27 and il31 [] these cytokines are grouped as they share structural similarity forming a fourαhelical bundle termed helices ad with an upupdowndown topology il6 and il11 utilise a hexameric signalling complex consisting of two molecules each of the cytokine αreceptoreither il6r or il11r respectively and βreceptor glycoprotein gp130 [] il6 and il11 are ableto signal via two distinct mechanisms termed classic and transsignalling classic signalling involves the formation of a complex including membranebound il6r or il11r with gp130 and the respective cytokine converselytranssignalling utilises soluble il6r or il11r molecules which are able to form a signalling complex with gp130and the respective cytokine [] in this way classic signalling relies on the responding cells intrinsic expressionof il6r or il11r whilst transsignalling is able to activate any cell expressing gp130 lif osm il27 and il31 signal through trimeric complexes with a single cytokine molecule engagingthe respective receptor lifr osmr il27r wsx1 or il31r and either gp130 or osmr for il31[] cntf ct1 clc and np form tetrameric signalling complexes composed of one cytokinemolecule one lifr one cntfr and one gp130 receptor in each case the active signalling complex consists of two chains that are signalling competent with a combination of either gp130 lifr osmr il27r or il31r the requirement for multiple receptor subunits means that although gp130 is ubiquitously expressed the expression of other receptor subunits dictates the ability for any given cell to respond to cytokine as signalling initiationrequires the presence of cytokine and a compatible receptor complex figure 2asignalling complex assembly leads to transphosphorylation and activation of receptorassociated janus tyrosinekinases jaks largely jak1 and to a lesser extent jak2 and tyk2 in the case of gp130mediated signalling this results in phosphorylation of the cytoplasmic domain of gp130 at tyrosine y and phosphotyrosine py and of gp130 provide docking sites for signal transducer and the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commonsattribution license cc byncnd 0cclinical science 101042cs20191211figure il6 family cytokine signalling pathwaya schematic representation of the stepwise binding process for the il6 family members with il6 as an example the site interaction involves cytokine binding to the respective receptor with the site interaction generally between the cytokine and thecommon gp130 receptor chain finally site interactions involve formation of the final active signalling complex in this case formation of the il6il6rgp130 hexameric complex b general outline of the il6 family cytokine signalling pathway formation ofan active hexameric complex leads to activation of jaks with subsequent activation of the stat3 mapk and pi3k pathways leftthis results in upregulation of the negative regulator socs3 as well as a range of inflammatory and protumorigenic moleculesthe pathway is inhibited by socs3 pias3 and ptps via dephosphorylation ubiquitinmediated proteasomal degradation andsumoylation right 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 byncnd 0cclinical science 101042cs20191211activator of transcription stat molecules leading to their subsequent phosphorylation by jak1 and formation ofactive stat dimers [] phosphorylated stat pstat dimers then translocate to the nucleus and modulatetarget gene expression including upregulation of a range of genes involved in inflammatory and protumorigenicpathways [] figure 2b broadly these stat3regulated genes can be categorised into pathways associatedwith inhibition of apoptosis increased cell proliferation modulation of immunity and inflammation increased angiogenesis and increased invasive and metastatic potential []although jakstat signalling is the predominant pathway activated downstream of il6 family cytokines themitogenactivated protein kinase mapk and phosphoinositide 3kinase pi3k pathways can also be activated the mapk pathway has been suggested to be activated by a src homology domain 2containing phosphatase shp2mediated mechanism whereby shp2 is recruited to py759 on gp130 allowing jakmediated phosphorylation of shp2 this promotes association with the adaptor protein growth factor receptor bound protein grb2 leading to activation of the gprotein ras via son of sevenless sos with a subsequent phosphorylationcascade including raf mek and erk12 activity following this a mapkdependent phosphorylationevent leads to the recruitment of grb2associated binding protein gab1 to the plasma membrane where gab1 issuggested to act as a scaffold or adaptor protein to allow binding of pi3k and shp2 leading to activation of the pi3kand mapk pathways respectively figure 2bthe suppressor of cytokine signalling socs3 is largely responsible for regulation of signalling and is directlyupregulated by stat3 socs3 contains an sh2 domain allowing it to bind to py residues within the gp130receptor with preferential binding to y759 once bound socs3 recruits an e3 ubiquitin ligasecomplex containing cullin5 rbx2 and adaptors elongin b and c via its socs box domain this complexubiquitinates the gp130 receptor inducing its internalisation and targeting it for proteasomal degradation []and is also able to ubiquitinate jak2 in vitro socs3 also mediates direct inhibition of the kinase activityof jak12 via its kinase inhibitory region [] thus socs3 is able to downregulate il6 family cytokinesignalling pathways through two distinct mechanismsthe phosphotyrosine phosphatases ptps and protein inhibitors of activated stats piass also limit the strengthand duration of cytokine signalling a range of ptps including shp2 are responsible for dephosphorylatingtyrosine phosphorylated substrates including jaks stats and other shp2 molecules pias3 preferentially binds pstat3 and inhibits activity either by preventing stat3 interaction with dna by recruiting transcriptional repressors to stat3 target genes or by sumoylating stat3 to prevent its activity figure 2binterleukin in pdacelevation of serum il6 is a negative prognostic marker in human pdacserum il6 levels were increased in pdac patients compared with healthy patients [] or those withchronic pancreatitis and were also increased in patients with metastatic pdac compared to thosewith locally advanced disease [] moreover elevated serum il6 positively correlated with increased diseaseburden weight losscachexia and metastasis [] however there are conflicting observations inthe literature regarding il6 and cachexia although increased serum il6 levels correlate with increased disease stage and in metastatic patients correlates with poor overall survival as such it has been suggestedthat il6 may be a superior marker for diagnostic and prognostic purposes compared with the standard creactiveprotein crp carcinoembryonic antigen cea and carbohydrate antigen ca199 markers il6 is expressed within the tmell6il6 was overexpressed in human pdac tumours in comparison with adjacent normal tissue whilstthis tumourspecific elevation has been correlated with reduced survival in some studies othersshowed no significant correlation with survival similar to the data available in the cancer genome atlastcga dataset for both il6 and il6r figure 3ab the tcga comprise aggregate sequencing data which doeshave limitations regarding interpretation of contributions of individual cell populations to disease outcome howeverit remains a widely used resource for exploratory investigations however overexpression of il6 has been observedat the mrna and protein level in the pancreata of pdac mice with il6 expression increasing with agewhich is indicative of disease stage in these models despite the presence of il6 in tumours primary human and commercial pancreatic cancer cell lines have been reported to exhibit variable expression levels of il6 and secreted cytokine albeit consistently higher than normal pancreatic ductal epithelial cells in an anoid model minimal il6 was expressed by pancreaticcancer cells pccs or pscs in monoculture however in coculture pccs expressed only il6ra whilst icafs expressedhigh levels of il6 with this activating stat3 within pccs icafs also demonstrate an upregulation of 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 byncnd 0cclinical science 101042cs20191211figure il6 family cytokine expression in pdac patientsoverall survival for patients with high top quartile and low bottom quartile level expression of a il6 b il6r c il11 d il11re lif f osm g cntf h ctf1 ct1 i clcf1 clc and j il27 n per group data and graphs obtained fromoncolnc using data from the cancer genome atlas tcga statistical significance determined by mantelcox logranktest 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 byncnd 0cclinical science 101042cs20191211the jakstat pathway with expression of il6 being dramatically increased in vitro when incubated with pcc conditioned media indicating that soluble factors trigger il6 production more recently pccderived il1αhas been shown to induce autocrine lif secretion and thereby promote the icaf phenotype including activation ofthe jakstat signalling pathway and il6 production in addition tams have been identified as producers of il6 in pancreatic cancer by correlative immunohistochemistry and expression analysis of isolated cell populations production of il6 by tams was shownto influence tumour development via bonemarrow chimeras as mice reconstituted with il6 knockout ko il6myeloid cells developed lowgrade panins whilst those reconstituted with il6 wt cells developed panin3 lesions il6 is a driver of pdac pathogenesisboth in vitro and in vivo studies suggest that the presence of il6 in the tme can drive activation of stat3 with il6 inhibition reducing stat3 phosphorylation this il6stat3 program has been proposed tobe a driver of pdac pathogenesis by enhancing tumour initiation and progression angiogenesis regulation of cytokine expression and immune cell behaviour resistance to apoptosis and promotion of metastasis [] in aninducible krasdriven mouse model genetic deletion of il6 resulted in a reduction of adm and panin formationwhen kras mutation was initiated embryonically compared with controls suggesting a role for il6 in tumour initiation this was also observed in a constitutive kras mutant model where genetic deletion of il6 preventedtumour initiation in vivo with a reduction in the number of panin and lesions interestingly oncogenickras and hypoxic conditions both features of pdac tumours were shown to induce il6 production perhaps representing a feedforward pathway enhancing tumorigenesis however il6 is notabsolutely required for panin formation as induction of kras mutation at weeks of age in conjunction with anexperimental pancreatitis model drove formation of panin lesions that were not significantly different between il6wt and ko mice il6 mice exhibited reduced tumour progression with decreased proliferative capacity of both cancer and stromal cells enabling regression of precursor lesions furthermore this inhibition of tumour progression by il6deletion was due at least in part to the reversal of adm with ductal cells reverting to an acinarlike phenotype increased apoptosis of cancer and stromal cells was also shown to contribute to this reduced tumour progression as demonstrated by appropriate immunohistochemical analyses with upregulation of proapoptotic anddownregulation of antiapoptotic bcl2 family members this is mirrored by in vitro data whereby il6 stimulation increased the expression of antiapoptotic bcl2bcl2 and bcl2l1bclxl with blockade of il6signalling or stat3 activation inducing apoptosis collectively these data suggest that whilst il6 contributes it is not required for pdac initiation and progressionthe process of angiogenesis supports tumour growth and progression by enabling adequate blood supply whichis enhanced by il6 signalling upon il6 stimulation pdac cell lines upregulate key angiogenic factors such asvascular endothelial growth factor vegfvegf and neurophilin1 nrp1nrp1 with significant correlation observed between the expression of il6r and vegf on human pdac sections il6inducedupregulation of vegf correlated with a growth advantage in pccs with both features inhibited by treatment witha jak2 inhibitor another facet of the protumorigenic effects of il6 is the regulation of cytokine expression that enables modulationof the immune system in particular it has been shown that il6 is able to upregulate a type cytokine profile invitro that may inhibit antitumour immunity in disease il6 suppressed the differentiation of human cd14cells into dendritic cells dcs in vitro whilst combination treatment with il6 and granulocyte colonystimulatingfactor gcsf inhibited the ability of dcs to respond to alloantigen a process that is required for dc maturationand antigen presentation where these effects were reversed by blockade of il6 andor gcsf il6 has alsobeen implicated in driving increased apoptosis of type i conventional dcs cdc1s leading to cdc1 dysfunctionearly in tumorigenesis and thereby decreased cd8 tcell activation this notion is further supported by invivo studies whereby genetic deletion of il6 in a krasdriven pdac mouse model exhibited a significant decreasein the percentage of intratumoral cancerpromoting macrophages and mdscs utilising primary human pscsmdsc differentiation was shown to be driven by pscderived il6 in a stat3dependent manner the resultantmdscs were able to suppress tcell proliferation suggesting a role for il6 in promoting an immunosuppressivetme correlative analysis indicates that il6 through the generation of metabolic stress indirectly causes a reductionin the percentage of intratumoral natural killer nk cells cd4 and cd8 tcells in precachectic and cachecticmice this effect was coupled to a reduction in the expression of an array of genes involved in immune cell 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 byncnd 0cclinical science 101042cs20191211recruitment and tcell effector function indicating that il6 is able to directly and indirectly modulate immuneregulation to enhance tumorigenesisemt migration and invasion are prerequisite abilities that are required for tumour metastasis stimulation of pccswith il6 modulated the expression of a range of proteins that drive emt and migration including upregulationof snai1snai1 snail snai2 slug cdh2 ncadherin vim vimentin fn1 fibronectin col1a1 collagen and twist2 and downregulation of cdh1cdh1 ecadherin with these effects mitigated by il6 orstat3 inhibition il6treated pccs and preinvasive panins exhibited morphological cha | 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 |
" 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 |
" trophoblast cell surface antigen trop2 is overexpressed in many squamous cell carcinomas andpromotes tumor development and invasion the association between trop2 expression and occurrence anddevelopment of oral squamous cell carcinoma oscc remains to be understoodmethods we investigated the role of trop2 in oscc patients using a combination of biophysical approaches atotal of oscc patient specimens with varying degrees of differentiation were subjected to hematoxylin andeosin staining immunohistochemistry kaplanmeier survival curve analysis and atomic force microscopy to analyzetrop2 expression morphology and mechanical properties of oscc tissuesresults trop2 was overexpressed in of poorly differentiated oscc samples high levels of trop2 wereassociated with survival rate lower than and patient age odds ratio [or] p confidence interval [ci ] tumor size or p ci [] and tnm stageor p ci [] average surface roughness of low medium and highly differentiatedoscc tissues were ± ± and ± nm respectively the pearson coefficient revealed anegative association between tumor stiffness and trop2 expression r p overexpression of trop2 negatively associated with patient survival degree of tumor differentiationand tissue mechanics taken together our findings demonstrated that trop2 may be an indicator of osccdifferentiation leading to the altered mechanical properties of oscc tissueskeywords oral squamous cell carcinoma trop2 tissue stiffness differentiation survival oral squamous cell carcinoma oscc is a commonsubtype of head and neck and other malignant tumors[ ] the past few decades have shown increased incidence of oscc that is expected to rise further in the future thereforeimperative to determineisit correspondence zhangkllzu163com baoping zhang shuting gao and ruiping li contributed equally to thiswork1department hospital of stomatology lanzhou university donggang westroad lanzhou gansu chinafull list of author information is available at the end of the biological factors associated with the early diagnosis andtreatment of oscchuman trophoblast cell surface antigen trop2 alsocalled tumorassociated calcium signaltransduction2tacstd2 is a surface glycoprotein encoded by tacstd that has extracellular domains a single transmembrane domain and a short tail [ ] trop2 is overexpressed in many human cancers including ovarian [ ]gastric [ ] colorectal pancreatic and laryngealcancers inhibiting trop2 expression has shownpromise in clinical applications [ ] trop2 regulates the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0czhang bmc cancer page of tumorigenic properties including cancer cell adhesion invasion and migration tang have recentlyshown that trop2 impacts growth and metastasis byactivating pi3kakt signaling this phenomenon hasalso been observed in gallbladder cancer amongtheinvolved intumorigenesis the role of catenin has been studiedextensively [ ] this has shed light on the biological functions of trop2 and its use as a prognostic biomarker for osccvarious biochemical mechanismsatomic force microscopy afm is a powerful toolthat generates surface topographical images with magnifications that range between macro and nanoscalesafm has been used to determine the mechanical properties of tumor tissues in a variety of cancers such asthose of the breast liver and lung parameters for tissue stress such as mechanical phenotypeindex correlate with cancer development and invasion advancements in technology used for determiningbiophysical properties have facilitated the nanolevelanalysis of tumor tissuesthis study aims at investigating the correlation between trop2 expression and clinicopathological characteristics of oscc we have demonstrated the tissuemorphology and mechanics of oscc samples duringtumor development using afm we believe our findingswill help develop trop2 in accurately diagnosing osccin tumors with different grades of differentiationmethodstissue preparationthe protocols in this study were approved by the researchethics committee of lanzhou university tumor sampleswere collected from patients after obtaining written informed consent a total of patients with oral squamous cell carcinoma oscc were registered atthesecond hospital of lanzhou university between january and march among these samples sampleseach showed high moderate and low levels of differentiation the experimental group comprised males and females aged years average years all patientswere diagnosed with oscc based on surgery andfig paraffin pathological sections of tissues a d g 4fold b e h 10fold c f i 40fold 0czhang bmc cancer page of fig immunohistochemical staining was performed to detect the expression of trop2 at different stages of osccpathology patients did not undergo radiotherapy chemotherapy or immunotherapy before surgery pathologicalanalysis after tumor biopsy was performed by two experienced pathologists after which the diagnosis of other diseases including inflammation at other sites and secondarytumors were excluded cancer and cervical lymph nodetissues were collected after maxillofacial surgery all specimens were sampled from typical areas of the lesion andfixed with neutralbuffered formalin followed by conventional paraffin embedding among them and fig average optical density of trop2 poorly differentiated squamous cell carcinoma showed high expressionp 0czhang bmc cancer page of table correlation between trop2 expression and clinicopathological characteristicscharactersntrop2 expressionlow or nototalgendermalefemaleage¥ localizationmucosatonguedifferentiationwellmoderatepoortumor sizet1 cm cmt2 cmt34cmt4blymph node metastasesn0nxdistant metastasesm0m1tnm stagei iiiii ivperineural infiltrationnoyesvascular invasionnoyespearson x2p value highpatients exhibited no and cervical lymph node metastasesrespectively clinical tnm staging was performed according to the 7th edition tnm staging classification standardjointly developed by the international union for cancercontrol and american joint committee on cancer and world health anization guidelines hematoxylin and eosin he stainingoscc tissues were fixed overnight using neutralformalin solarbio beijing china paraffin embeddedsliced into 4μm thick sections dewaxed using xyleneand rehydrated using different concentrations of ethanol the sections were stained with hematoxylin for min and hydrochloric acidethanol and eosin for min followed by gradient dehydration transparentizationresin sealing solarbiobeijing china sections were visualized and imagedusing the olympus bx53 at magnifications of and sealing and neutral 0czhang bmc cancer page of immunohistochemistryhe sections were subjected to the sp method to detecttrop2 expression the sections were incubated overnight with the primary antibody against trop2 abcam usa at °c followed by incubation withbiotinlabeled goat antirabbitigg abcamusa at °c for h the sections were then developed using dab beijing zhongshan golden bridgebiotechnology china dehydrated transparentizationand film and neutral resin sealed the prepared sections were visualized using microscopy olympusbx53 japanafmfixed tissues were placed in petri dishes containingphosphatebuffered saline all analyses for mechanical properties were performed using the biologicalatomic force microscope bioafm nanowizard iiibruker usa silicon afm probes from the pointprobe®constant of nmcoating nanoworld usa werecontrreflexused the spring constant ofthe probe was calibrated using builtin thermal vibration before measuringandthickness of μm afm was performed using theseries with a khzforcetheresonancefrequencyofcontact model and a scan rate of hzs in airforcedistance curves are generated when the probecontacts the tissue following whichthe structuremorphology and mechanical properties of samplesare measured at μms six random sites wereselected for each sample and each site was measured times we used the modified hertz contact modelto analyze forcedistance curves and calculateyoungs modulus and roughness of oscc tissueswith varying differentiationstatistical analysisstatistical analyses were performed using spss statistical product and service solutions ibm forcespectrum data were expressed as mean ± standard errorand statistical comparisons were performed using oneway analysis of variance followed by the tukeykramerhsd test for pairwise comparisons pearson chisquaretest was used to analyze clinical features and trop2 expression based on the calculated odds ratios ors and confidence intervalci survival was evaluatedusing kaplanmeier curves and the difference was analyzed using the logrank test p was consideredstatistically significantfig trop2 total survival curve using kaplanmeier survival curves low blue line high green line 0czhang bmc cancer page of resultstissue morphology and trop2 expression across theclinical stages of oscctumor cells from poorly differentiated oscc samples exhibited characteristic atypia poor differentiation and irregular morphology fig howeverthe number volume atypia nuclear pyknosis andmitotic structures decreased in tumor cellsfromhighly differentiated oscc as compared to those inpoorly differentiated cells trop2 primarily localizedin the cytoplasm of tumor cells but not in adjacentnormal epithelial cells we observed that low differentiation and high malignancy of oscc was associated with higher trop2 expression fig theaverage optical density of trop2 among the lowmedium and highly differentiated oscc tissues were ± ± and ± respectively fig table trop2 expression risk factors with clinicopathological featurescharacteristicsntrop2 expressionlow or nototalgendermalefemaleage¥ localizationmucosatonguedifferentiationwellmoderatepoortumor sizet1 cm cmt2 cmt34cmt4blymph node metastasesn0nxdistant metastasesm0m1tnm stagei iiiii ivperineural infiltrationnoyesvscular invasionnoyesnote a well vs moderate b moderate vs poor c well vs poorp valueor cihigh 005a 0001b 0001c 0czhang bmc cancer page of association between trop2 expression and clinicalcharacteristics of osccwe analyzed the clinicopathological characteristics of patients with oscc with varying degree of differentiationdifferential expression of trop2 was associated with patient age tumor differentiation tumor size tnm stagepercutaneous nerveinvasiontable p patients with poorly differentiated tumors were more likely than patients with well and moderate differentiated tumors to have high trop2 expressionp however there was no association between theexpression of trop2 and patient gender tumor locationlymph node metastasis or distant metastases p and vascularfiltrationtrop2 expression and patient survivalusing kaplanmeier survival curves we observed that anincrease in trop2 expression negatively correlated withthe overall survival of patients fig and lowno oftrop2 expression groups 3years survival rate was a for high expression group and 5years ratewere and respectively trop2 expression wasassociated with patient age p or ci[] tumor differentiation well vs moderatep or ci [] moderate vspoor p or ci []well vs poor p or ci [] tumor size p or ci[] tnm stage p or ci[] vascular invasion p or ci [] and peripheral nerve invasionp or table high trop2 expressionwas detected in older patients with low degree of differentiation larger tumor volume higher tnm staging andvascular and peripheral nerve invasion thereby resultingin lower overall survival thus trop2 may be a prognostic indicator for survival in oscc patientsfig surface morphology of oscc tissue sections via afm detection irregular morphology appeared in the low differentiation 0czhang bmc cancer page of surface morphology and roughness of oscc tissuesthe surface morphologies of oscc tissues with varying degrees of differentiation were analyzed directtopographical imaging using bioafm figure showsthe representative image from each tissue acquiredduring the cantileverbased afm nano indentationtest the tissue interface varied with tumor differentiationindicating that highly differentiated oscc tissues had a regular and flat morphology oscc tissueswith low differentiation exhibited an overall irregularmorphology with distinct modulation and loose tissueanization figure summarizes the roughness ofoscc tissues with varying differentiation the average surface roughness of low medium and highly differentiated oscc tissues were ± ± and ± nm respectively roughness ofthe tissue surface was enhanced with increasing differentiation of oscc tissuesyoungs modulus of oscc tissueswe used bioafm to determine youngs modulusbased on the mechanical properties of oscc tissues with varying degrees of differentiation we randomly selected six contact points from each slice andeach contact point was measured times forcedistance curves were generated for each slice and thejpk data processing software version was usedto calculate youngs modulus figure shows theaverage variation in stiffness within individual tissuesin the range of kpa in the low differentiationsamples we observed low stiffness as compared tothat in high or medium differentiation samples p fig surface roughness results are express as mean ± sem nm 0czhang bmc cancer page of fig afm test average tissue stiffness youngs modulus e was thus confirmed to be a parameter of cell hardness for various cells and tissuepa p thus tissue differentiation was positively associated with its stiffness fig association between mechanical properties and trop2expression in osccthe pearson coefficient showed a negative associationbetween the stiffness of oscc tissues and trop2 expression fig r p thus we detectedan increase in stiffness with varying differentiation in thetumor samplesdiscussiontrop2 belongs to the family of genes involved in calcium signaling associated with tumorigenesis and foundin human trophoblast and chorionic cell lines studieshave shown that overexpression of trop2 is associatedwith tumorigenesis and malignancy []in thisstudy trop2 expression was observed to be a highlysensitive and specific marker of tongue squamous cellcarcinoma and tissue stiffness the relative thickness ofsamples helped accurately diagnose and determine thestaging of tongue squamous cell carcinomaimmunohistochemical analysis revealed that the expression of trop2 in poorly differentiated oscc tissueswas significantly higher than that in welldifferentiatedoscc tissues additionally trop2 upregulation wascorrelated with tumors of advanced tnm iii iv staging and poor differentiation than that in tumors withlow tnm i ii staging thus the abnormal expressionof trop2 may be associated with the occurrence anddevelopment of tongue malignancies furthermore hightrop2 expression predicted low survival as comparedto that in the tumors with low trop2 expression previous research has also demonstrated the correlation between shorter patient lifespan and high levels of trop2as compared to that in patients with laryngeal squamouscell carcinoma and low levels of trop2 trop2possesses sites for tyrosineserine phosphorylation thatregulate signal transduction or its expression and activity thereby rendering cancer cells resistant to apoptosis upregulated trop2 correlates with the poor prognosis of thyroid papillary carcinoma colon cancer liver cancer and other malignanciesthere have been an increasing number of studies on thebiological role of trop2 at the molecular level trop2induces the downregulationloss of pten thereby stimulating pi3kakt signaling and tumor development pten is a wellknown tumor suppressor that is a phosphatase and affects the pi3kpkbakt signaling axisduring the dephosphorylation of pip2 and pip3 pi3k signaling is important in regulating tumor cell proliferation migration and invasion [ ] thus pten is anegative regulator of cancer [ ] li have shownthat trop2 activates epithelialmesenchymal transitionvia pi3kakt signaling thereby promoting proliferationmigration and metastasis in gallbladder cancer similarly trop2 expression stimulates the proliferation migration and invasion of osteosarcoma cells hou demonstrated that trop2 regulates jak2stat3 signaling in glioblastoma cells 0czhang bmc cancer page of fig correlation analysis between changes in mechanical stiffness of oscc tissues and trop2 expression note changes have statisticalsignificance p and show a certain negative correlation r functional differentiation oftissues influences themicromorphology and mechanical stiffness of oscccells we detected low surface roughness on oscc tissues with loose structure reduced hardness and enhanced cell adhesion migration and invasion poorlydifferentiated oscc tissues are softer than highly differentiated oscc tissues pi3k is an important celladhesion molecule trop2 triggers the synthesis of proteins with homologous domains such as pleckstrinrac tiam and vav tiam and vav activate rac thatleads to reanization of the actin cytoskeleton cellrecognition and adhesion the underlying mechanisms involved in the alterationof micromechanical properties of oscc samples and occurrence development metastasis and invasion ofoscc tumors remain to be elucidated he staining isthe gold standard for tumor diagnosis with the development of biomechanics in the past two decades the mechanical properties of tissues need to be investigated based on biomedical and physical parametersin this study we have assayed the changes in mechanicalproperties at the micronanometer level using afm anddetermined the association between the tnm grademetastasis and stiffness of tumor samplesin we have demonstrated the association between differential expression of trop2 and patient agetumor differentiation tumor size tnm stage percutaneousnerve filtration and vascular invasion moreover high levelsof trop2 correlated with poor overall survival in patientshighly differentiated cancer tissues exhibited increasedsurface roughness and stiffness lastly high trop2 expression resulted in reduced tumor stiffness however thisstudy had some limitations first the cohort used in thisstudy was relatively small second we did not employ molecular methods of analysis such as western blotting orenzymelinked immunosorbent assay thus using a largerpatient cohort and multiple techniques in molecular andcell biology will help validate our findings and developtrop2 as a specific and efficient prognostic biomarker forosccthese findings could promote new methods for the earlyoscc diagnosis depend on the stage of cancer and developing screening methods with high sensitivity andspecificity more detailed studies are needed to determine the feasibility and therapeutic benefit of testing tissue stiffness in human diseaseabbreviationsoscc oral squamous cell carcinoma trop2 trophoblast cell surfaceantigen afm atomic force microscopyacknowledgementswe thank the individual who participated in this studyauthors contributionsbz sg and rpl are responsible for conception and design data wascollected by ytl rc jyc and ymg data was analyzed by ew and yh klzrevised the all authors have read and approved the manuscriptfundingthis work was supported by the fundamental research funds for thecentral universities no lzujbky2020cd03 baoping zhang doctoralmaster 0czhang bmc cancer page of students of the second hospital of lanzhou university sdkygg17 lan yangand key laboratory of mechanics on disaster and environment in westernchina the ministry of education of china no kailiang zhangavailability of data and materialsthe datasets used and analyzed during the current study are available fromthe corresponding author on reasonable requestethics approval and consent to participatewritten informed consent was obtained from each participant before samplecollection the study was approved by the committee for ethical affairs ofschool of stomatology lanzhou universityconsent for publicationnot applicablecompeting intereststhe authors have no conflicts of interestauthor details1department hospital of stomatology lanzhou university donggang westroad lanzhou gansu china 2institute of biomechanics andmedical engineering lanzhou university lanzhou chinareceived april accepted august 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thyroid j zhao p zhang z tnfα promotes colon cancer cell migration and invasionby upregulating trop2 oncol lett sin stk li y liu m yuan yf ma s guan xy downregulation of trop2predicts poor prognosis of hepatocellular carcinoma patients hepatolcommun zhang y zhang r luo g ai k long noncoding rna snhg1 promotes cellproliferation through pi3kakt signaling pathway in pancreatic ductaladenocarcinoma j cancer sai j owens p novitskiy sv hawkins oe vilgelm ae yang j pi3kinhibition reduces mammary tumor growth and facilitates antitumor immunityand antipd1 responses clin cancer res chen x pang b liang y xu sc xin t fan ht overexpression of zhang xr wang sy sun w wei c isoliquiritigenin inhibits proliferation andepcam and trop2 in pituitary adenomas int j clin exp pathol metastasis of mkn28 gastric cancer cells by suppressing the pi3kaktmtor signaling pathway mol med rep 0czhang bmc cancer page of wise hm hermida ma leslie nr prostate cancer pi3k pten and prognosisclin sci lond yuan b zou m zhao y zhang k sun y peng x upregulation of mir130b3p activates the ptenpi3kaktnfκb pathway to defense againstmycoplasma gallisepticum hs strain infection of chicken int j mol sci pii e2172li jw wang xy zhang x gao l wang lf yin xh epicatechin protectsagainst myocardial ischemiainduced cardiac injury via activation of theptenpi3kakt pathway mol med rep li x teng s zhang y zhang w zhang x xu k trop2 promotesproliferation migration and metastasis of gallbladder cancer cells byregulating pi3kakt pathway and inducing emt oncotarget gu qz nijiati a gao x tao kl li cd fan xp trop2 promotes cellproliferation and migration in osteosarcoma through pi3kakt signalingmol med rep hou j lv a deng q zhang g hu x cui h trop2 promotes theproliferation and metastasis of glioblastoma cells by activating the jak2stat3 signaling pathway oncol rep rivard n phosphatidylinositol 3kinase a key regulator in adherens junctionformation and function front biosci landmark ed pankova d jiang y chatzifrangkeskou m 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" breast cancer bc is the most commonly diagnosed malignant cancer in women bc is the main cause of cancerrelated death in women and seriously threatens the life and health of women worldwide micrornas mirnasmirs have been reported to regulate the development and progression of different types of cancer however the regulatory functions of mir1885p in bc have not been thoroughly demonstrated in this present research we identified that mir5p was downregulated in bc tissues and several bc cell lines downregulation of mir5p was significantly associated with advanced tnm stage moreover we identified that mir5p mimics significantly inhibited proliferation using cck8 assay colony formation and xenograft animal model suppressed invasion and migration detected by transwell invasion assay and increased the cellular apoptosis of bc cells as determined by cell apoptosis assay moreover mir1885p mimics also reduced the expression of nfκb p65rel to further investigate its regulatory mechanism transcription factor zinc finger protein zfp91 was predicted as the targeted protein of mir1885p by bioinformatic method we confirmed their specific binding by dual luciferase dlr assay we demonstrated that the overexpression of mir1885p significantly inhibited the expression of zfp91 in bc cell lines and reduced the expression of nfκb p65rel an inverse correlation was found between the expression of mir1885p and zfp91 in bc tissues importantly we demonstrated that the restoration of zfp91 was able to block the effect of mir1885p on the progression of mdamb231 cells therefore our study showed that mir1885p may be one of the important indicators and could inhibit the progression of human bc via targeting the zfp91nfκb p65rel signaling pathway suggesting that mir1885p may be a promising future target for bc treatmentcorrespondence to dr zhaoyu liu department of radiology shengjing hospital of china medical university sanhao street shenyang liaoning pr chinaemail liuzy1226126comkey words breast cancer mir1885p zinc finger protein zfp91 proliferation apoptosisintroductionbreast cancer bc is one of the most commonly diagnosed malignancy in the world the mortality rate for bc ranks first among all female malignant tumors globally the number of newly diagnosed bc cases reached approximately million in accounting for almost of cancer cases among women bc exhibits a complex pathogenesis and is a clinically heterogeneous disease with a wide range of clinical behaviors and treatment responses although many dysregulated molecular pathways have been discovered in bc the development of effective therapeutic methods has been limited it is urgent to discover novel molecules to suppress bc proliferation induce apoptosis and inhibit invasion and provide potential therapeutic strategies to improve the survival and quality of life of bc patients micrornas mirnasmirs are a class of endogenous noncoding rnas of approximately nucleotides in length which are generally located in unstable regions of the human genome and are usually dysregulated in malignant tumors to regulate gene functions mirnas regulate target genes through binding to the 'untranslated regions 'utrs of the target mrnas subsequently inhibiting gene expression through regulation of the targeted proteins mirnas play an important role in many tumor cellular processes such as proliferation cell cycle apoptosis invasion and metastasis and participate in almost all signaling pathways mir1885p has been reported to be an inhibitor of tumor growth and metastasis in prostate cancer and hepatocellular carcinoma however to the best of our knowledge the functions of mir1885p in bc remain elusivein the present study we detected the expression of mir1885p in tumor tissues of bc patient tissues and several bc cell lines furthermore we investigated its regulatory role in bc proliferation apoptosis and invasion we also predicted and confirmed the targeted protein of mir5p transcription factor zinc finger protein zfp91 elucidating the regulatory mechanisms of mir1885p in bcmaterials and methodspatients and tissues one hundred paired bc tissue specimens including malignant and normal tissues used in this study were obtained from shengjing hospital of china medical university shenyang liaoning china during the period 0cyang mirna1885p inhibits proliferation and induces apoptosis in bc via zfp91from january to december with the informed consent of patients the age range of the patients was from to years with a mean age of years all experiments were approved by the ethics committee of shengjing hospital of china medical university no 2016ps18j the samples were snapfrozen in liquid nitrogen and stored at Ëc tnm staging system was performed for tumor grading of bc and for evaluating and staging of patients respectively which was carried out according to the 7th edition of the american joint committee on cancer ajcc tnm classification system cell cultivation the bc cell lines mdamb231 atcc crmhtb26 bt549 atcc htb122 and mcf7 atcc crl3435 were cultured in rpmi1640 medium sigmaaldrich merck kgaa containing heatinactivated fetal bovine serum fbs mp biomedicals penicillinstreptomycin invitrogen thermo fisher scientific inc no the nonmalignant mammary epithelial cell line mcf 10a atcc crl10317 was cultivated in dmemf12 ham's mixture supplemented with equine serum hyclone ge healthcare egf ngml insulin µgml hydrocortisone mgml and cholera toxin ngml all from sigmaaldrich merck kgaa all cells were incubated at Ëc in a humidified co2 atmosphererna isolation and quantitative qpcr total rnas from tissues or cells were isolated using rnx¢plus reagent cinnagen and cdna was synthesized using the primescript¢ rt reagent kit takara according to the manufacturer's instructions qpcr was performed using a sybr premix extaq¢ kit takara with the following primer sets on the abi qpcr system applied biosystems actin was used to normalize the relative expression of the target genes mirnas were detected through a miscript ii rt kit qiagen in a fluorescence thermal cycler biorad laboratories inc the primers for mir1885p and the reference gene u6 were purchased from novland biopharm the thermocycling condition were Ëc for min followed by cycles of Ëc for sec and Ëc for 1min followed by a hold at Ëc the relative expression ratio of mir5p was quantified using the 2δδcq method the relative expression of mir1885p was normalized to u6 the primer sequences are listed in table iplasmid preparation the coding region of human zfp91 was amplified from human breast cancer cell mdamb cdna library by pcr then we cloned the prepared zfp91 fragment into pcmvtag2b stratagene to obtain pcmvtag2bzfp91 the primer sequences are listed in table icell transfection the mir1885p mimics and mirnc were purchased from thermo fisher scientific inc firstly lipofectamine transfection reagent thermo fisher scientific inc was used to transfect mir1885p mimics mirnc into mdamb231 cells in accordance with the manufacturer's instructionsafter the whole detection the pcmvtag2b vector pc was transfected into mdamb231 cells with mirnc ncpc or mir1885p mimics pcmir1885p pcmvtag2bzfp91 was transfected into mdamb cells with mir5p mimics mir5pzfp91 as the cells were grown to mir mimics or vector was transfected into cells according to the manufacturer's instructions then cells were cultivated for up to h finally the total rna and protein were extracted and properly stored for further researchcell proliferation assay cell counting kit8 cck8 kit dojindo was performed to detect cell proliferation based on the reduction of wst to wst formazan briefly the mdamb231 cells were seeded in a 96well plate at a density of 5x103 cellswell on the day of the experiment the cells were transfected with empty vector and mir1885p cck8 reagent was added into the culture medium at the indicated time and incubated for min the absorbance at nm was measured by a microplate readercolony formation assay mdamb231 cells from the different treated groups were seeded in a 60mm dish containing cells followed by a 14day cultivation at Ëc with co2 the supernatant was discarded and cells were washed twice with pbs the colonies were fixed in paraformaldehyde for min and then stained with giemsa staining solution solarbio science technology co ltd beijing china for min colonies were counted and images were captured under an inverted microscope nikon tokyo japan this assay was repeated timescell apoptosis assay mdamb231 cells were stained by annexin valexa fluor488propidium iodide pi staining to identify the apoptotic mdamb231 cells after transfection with mir1885p for h mdamb231 cells were stained with annexin valexa fluor488 for min on ice followed by the addition of pi solution for the secondary staining process all experimental procedures were strictly protected from lights the data were calculated by flowjo software v87 tree star after facs calibur bd biosciences analysiscell migration and invasion assays after the counting mdamb231 cells in the different groups were inoculated equally at a density of 5x105 cellsml in the upper compartments of polycarbonate membrane filters cell migration and invasion assays were performed uncoated for the migration assay and coated with matrigel bd biosciences for the invasion assay after h the migrated and invaded cells in the membrane were fixed with methanol and then stained with crystal violet for min at room temperature cells were observed under a light microscope with magnification x100western blotting protein samples extracted from tissues or cultivated cells were lysed in ripa buffer containing protease and phosphatase inhibitor cocktail and incubated at Ëc followed by the quantified measurement of protein using bca kit fujifilm wako pure chemical corp after protein samples µgeach sample were loaded and separated on sdspage gels for electrophoresis the proteins were then transferred onto a polyvinylidene difluoride pvdf membranes millipore usa the membranes were blocked in wv skim milk for h at room temperature and incubated at Ëc overnight with primary antibodies antizfp91 dilution 0concology reports bidirectional primer sequencetable i primer informationgene name f 'ccctctctcacatcccttgcat3'mir1885p r 'atcctgcaaaccctgcatgtg3' f 'tgagacctacaaaccccactt'zfp91 r 'ccttttgggtaaacgtggacttt3'homoactin f 'ttcctccgcaaggatgacacgc3'r 'ccttttgggtaaacgtggacttt3' f 'cgggtttgttttgcatttct3'u6 snrna f 'agtcccag catgaacagctt3'zfp91 zinc finger protein homolog f forward r reverse abcam ab30970 and antivimentin dilution cell signaling technology inc antiecadherin dilution cell signaling technology inc ncadherin dilution cell signaling technology inc matrix metalloproteinase mmp2 dilution cell signaling technology inc mmp9 dilution cell signaling technology inc nfκb p65 dilution cell signaling technology inc relb dilution cell signaling technology inc and gapdh dilution cell signaling technology inc as internal control on the following day all membranes were incubated with antirabbit igg hrplabeled secondary antibodies dilution cell signaling technology inc finally the signals were detected and analyzed with the application of luminata forte western hrp substrate millipore in the biorad chemidox xrs imaging system biorad laboratoriesluciferase reporter assay to further investigate the specific correlation between mir5p and zfp91 targetscan wwwtargetscanorgmamm_31 and miranda wwwmicrornaorgmicrornahomedo were performed the zfp91 was selected to be the predicted targeting of mir1885p the fragments of the 'utr of zfp91 containing mir5p binding sites and its mutants were amplified by pcr and then the pcr products were inserted into pmirglo dualluciferase mirna target expression vector promega corp the reporter and control vector were transfected into 293t cells using lipofectamine thermo fisher scientific inc after cultivation for h the relative luciferase activity was examined by the dualluciferase reporter assay kit thermo fisher scientific inc according to the manufacturer's instructionspreparation of tumor xenograft animal model and treatment with mir5p mimics thirtysix nude mice female weighing ± g were purchased from huafukang biotech beijing china the experiments were performed in the animal facility at the department of laboratory animal science of china medical university and approved by the animal ethics committee of shengjing hospital approval no nude mice were randomly divided into a control group n12 mir1885p group n12 and nc group n12 a density of 5x106 cells in logarithmic phase were transfected with 1x pbs control group nc or mir1885p then the different groups of cells were resuspended in 1x pbs and injected into the nude mice respectively then tumor size was measured every days using a slide caliper and the tumor volume v was calculated using the formula vlength x width22 after days the mice were euthanasia by cervical dislocation and the tumors were excised imaged weighed and stored properly for further investigationsstatistical analysis graphpad prism graphpad software inc was used to perform statistical analysis the results are represented as mean ± sd of at least independent experiments the comparisons between groups were evaluated by student' ttest oneway anova followed by tukey test was used to evaluate the differences for multiple comparisons the statistical significance of correlations between mir5p and zfp91 expression in bc tissue were analyzed by pearson's correlation coefficient p005 was considered to indicate a statistically significant differenceresultsmir5p is signif\ufeff\ufefficantly decreased in bc tissue and cell lines firstly we analyzed the expression level of mir1885p in cases of bc tissues and adjacent counterparts by rtqpcr the results showed that the level of mir1885p in bc tissues was significantly lower than that in the normal adjacent counterparts fig 1a p005 we also found that mir1885p was correlated with bc tnm stage fig 1b p005 the expression level of mir5p in advanced bc tumors was lower than that in early stage tumors suggesting that mir1885p is inversely correlated with the malignancy of bcwe also compared the expression level of mir1885p in the nonmalignant mammary epithelial cell line mcf10a and bc cell lines mdamb231 bt549 and mcf7 our data showed that the levels of mir1885p in the mdamb231 bt549 and mcf7 cells were lower than that in the mcf10a cells fig 1c p005 meanwhile the lowest mir1885p expression was detected in mdamb231 therefore mdamb231 cells were selected for further experimentsmir5p inhibits proliferation induces cell apoptosis and suppresses migration and invasion of bc cells as the expression of mir1885p in both bc cell lines and tumor tissues of bc patients were clearly downregulated we sought to investigate the effects of mir1885p on bc development by using both in vitro bc cell line cultivation and in vivo mouse tumor xenografts as shown in fig 2a transfection of mdamb cells with mir5p mimics significantly elevated the expression level of mir1885p when compared to the control and mirnc groups p005 importantly the increased level of mir5p in mdamb cells significantly inhibited the cell proliferation when compared to the control and mirnc groups fig 2b and c p005 it was also observed that the apoptotic mdamb231 cell numbers were significantly increased by the upregulation of mir5p when compared to the control and mirnc groups fig 2d 0cyang mirna1885p inhibits proliferation and induces apoptosis in bc via zfp91figure mir1885p is downregulated in bc tissue and bc cell lines a rtqpcr results showed that the level of mir1885p in bc tissues was downregulated compared with counterpart bc tissues the comparisons between groups were evaluated by student's ttest b relative expression level of mir1885p in patients at different clinical stages c relative expression level of mir1885p in bc cell lines mdamb231 bt549 and mcf7 relative to the normal human breast epithelial cell line mcf10a oneway anova followed by tukey's test was used to evaluate the difference for multiple comparisons in b and c all data are presented as means ± sd n3 bc breast cancerp005 importantly mir5p mimics significantly inhibited the invasion and migration abilities of the mdamb23 cells under transwell assay detection when compared to the control and mirnc groups fig 2e p005 moreover mir5p mimics significantly enhanced the expression of vimentin and ncadherin and reduced the level of ecadherin when compared to the control and mirnc groups fig 2f p005 the matrix metalloproteinases mmp2 and mmp9 mmp29 possess the ability to hydrolyze components of the basement membrane and stimulate tumor growth metastasis and epithelialmesenchymal transition emt mir5p mimics were demonstrated to significantly inhibit the expression of mmp2 and mmp9 fig 2f p005 these data provide robust evidence that mir1885p inhibits the tumor proliferation induces apoptosis reduces tumor invasion and migration and inhibits emt of bc which may be through the regulation of mmp29 expressionzfp91 is the downstream target of mir5p to further investigate the specific correlation between mir1885p and zfp91 targetscan wwwtargetscanorgmamm_31 and miranda wwwmicrornaorgmicrornahomedo were performed the results predicted that mir1885p possesses the binding sites of zfp91 fig 3a hence we sought to discover the regulatory mechanisms of mir1885p on bc development through targeting on zfp91 as hypothesized the upregulation of mir5p in mdamb cells decreased zfp91 mrna and protein levels when compared to the mirnc and control groups fig 3b p005 moreover the luciferase assay confirmed that mir1885p specifically binds to the 'utr of zfp91 fig 3c p005 it was also discovered that the injection of mdamb231 transfected with mir1885p mimics in tumor xenograft mice inhibited zfp91 expression fig 6b p005 these results suggested that zfp91 is the downstream target gene of mir1885p in bcmir5p regulates bc cell progression through targeting zfp91 to further investigate the biological functions of mir1885p in bc development we established a zfp91overexpressing mdamb cell line the expression of zfp91 was confirmed by rtqpcr fig s1 p005 with this system inhibition of zfp91 by mir5p mimics was reversed fig 4a p005 then it was found that the cotransfection of mdamb cells mir5pzfp91 group significantly enhanced the cell proliferation compared to that in pcmir5p group fig 4b and c p005 significantly suppressed cell apoptosis fig 4d p005 and significantly promoted invasion migration fig 4e p005 and emt fig 4f p005 in contrast with the monotransfection of mir1885p mimics in mdamb231 cells moreover the regulatory role of mir1885p on mmp2 and mmp9 was also reversed by overexpression of zfp91mir5p and zfp91 are correlated in tumor tissues of bc patients furthermore we examined the levels of zfp91 in the tumor tissues and adjacent normal tissues of bc patients the aberrantly high level of zfp91 was observed in the tumor tissues of the bc patients fig 5a p005 spearman's correlation analysis showed a significantly inverse correlation between mir5p and zfp91 in the bc patient tissues fig 5b p005 taken together these results further confirmed that the proliferation and apoptosis of bc is regulated by mir5pzfp91mir5p inhibits the proliferation of mdamb cells and reduces the expression of zpf91 in a bc xenograft mouse model moreover to evaluate the regulatory role of mir1885p in a bc xenograft mouse model we injected the mdamb231 cells transfected with mir1885p mimics or mirnc into nude mice the results showed that mir1885p mimics inhibited the tumor volume and weight compared to the mirnc group fig 6a p005 protein expression and the mrna level of zpf91 were also suppressed by mir1885p mimics in the tumor tissues of the xenograft mouse model when compared with the mirnc and control groups fig 6b p005mir5pzfp91 axis regulates nfkbp65 and relb expression numerous studies have reported that zinc finger protein zfp91 promotes proliferation and tumorigenesis 0concology reports figure effect of mir1885p on bc cell line mdamb231 mdamb231 cells were transfected with mir1885p mimics and mirnc a the mrna levels of mir1885p in mdamb231 cells were determined by rtqpcr p005 the proliferation of mdamb cells was examined by b cck kit post transfection and c colony formation p005 compared to control group p005 compared to mirnc group d the apoptotic mdamb cells were analyzed using annexin vpi staining and facs p005 e the invasion and migration capability of mdamb cells were detected by transwell assay p005 f expression of ecadherin ncadherin vimentin mmp2 mmp9 and nfκb p65rel were detected by western blotting the data are represented as the mean ± sd n3 p005 oneway anova followed by tukey's test was used to evaluate the difference for multiple comparisons bc breast cancer mmp matrix metalloproteinase nc negative controlof different cancer types via regulation of the nfκb p65 pathway therefore to further investigate the regulatory mechanism of the mir5pzfp91 axis we detected the expression of nfκbp65 and relb in bc cells the results showed that mir5p mimics significantly reduced the expression of nfκbp65 and relb together fig s2a p005 moreover the cotransfection of mir5p mimics and zfp91 also upregulated the expression levels of 0cyang mirna1885p inhibits proliferation and induces apoptosis in bc via zfp91figure zfp91 is a target of mir5p a zfp91 provides binding sites with mir5p b rtqpcr and western blotting were used for analysis of transcription and translation of zfp91 in vitro and in vivo p005 c the binding between mir5p and zfp91 was confirmed by luciferase assay p005 relative gene expression was normalized by gapdh expression the data are represented as the mean ± sd n3 oneway anova followed by tukey's test was used to evaluate the difference for multiple comparisons in b the comparisons between two groups were evaluated by ttest in c zfp91 zinc finger protein nfκbp65 and relb compared to the monotransfection of mir1885p mimics fig s2b p005 in summary these results illustrated that the mir5pzfp91 axis regulates the progression of bc via the noncanonical nfκb signaling pathwaydiscussionbreast cancer bc is one of the most common types of tumors diagnosed in women worldwide bc is the second leading cause of cancerrelated mortality worldwide in more than women were diagnosed with bc in china and almost percent of all newly diagnosed cancer cases were in women however the molecular mechanisms of bc still await elucidation and effective molecular targets for the diagnosis and treatment of bc are urgently required recently research has reported that mirnas are small noncoding rna molecules which regulate target protein expression to play critical roles as tumorpromotors or suppressors several studies have demonstrated that mir1885p promotes cell proliferation migration and metastasis in gastric cancer and hepatocellular carcinoma moreover iwakawa detected higher expression of mir1885p in stage iii breast cancer and tnbc wang reported that circulating mir1885p was upregulated in bc patients and associated with tnm of bc interestingly mir1885p was downregulated in bc mdamb231 and mcf7 cells moreover using gainof and lossoffunction analyses of mir1885p in breast cancer cells the authors demonstrated that mir1885p inhibited the proliferation and invasion of bc mdamb231 cells via targeting il6st however we demonstrated that the expression of mir1885p was drastically downregulated in bc tissue specimens which was also decreased in bc cell lines mdamb231 bt549 and mcf7 compared to normal breast epithelial cell line mcf10a moreover the downregulation of mir5p was significantly 0concology reports figure mdamb cell proliferation and apoptosis are regulated by mir5pzfp91 pcmvtag2b vector pc was transfected into bc mdamb cells with mirnc ncpc or mir5p mimics pcmir5p pcmvtag2bzfp91 was transfected into mdamb cells with mir5p mimics mir5pzfp91 a the mrna levels of mir5p in the cotransfected overexpressing zfp91mdamb cells were quantified by rtqpcr p005 cell proliferation was measured by b the cck kit and c colony formation assay p005 compared to control group p005 compared to mirnc group d cell apoptosis was detected by annexin vpi staining and facs p005 e the invasion and migration capability of mdamb231 cells were detected by transwell assay p005 f expression of ecadherin ncadherin vimentin mmp2 mmp9 and nfκb p65rel were detected by western blotting relative genes expression was normalized by gapdh expression the data are represented as the mean ± sd n3 p005 oneway anova followed by tukey's test was used to evaluate the difference for multiple comparisons bc breast cancer zfp91 zinc finger protein mmp matrix metalloproteinase nc negative control 0cyang mirna1885p inhibits proliferation and induces apoptosis in bc via zfp91figure mrna and protein levels of zpf91 in bc tissues a the mrna and protein levels of zpf91 in bc tissue and corresponding nontumor normal tissue were quantified using rtqpcr and western blot analysis the comparisons between two groups were evaluated by ttest n noncancer tissue c cancer tissue p005 b correlation between zpf91 mrna and mir5p was analyzed using pearson's correlation coefficient the data are represented as the mean ± sd n100 bc breast cancer zfp91 zinc finger protein figure the regulatory role of mir1885p in a bc xenograft mouse model bc mdamb231 cells were transfected with mir1885p mimics and mirnc and then injected into nude mice a the tumor volume and weight in the tumor xenograft mouse were compared among the mir1885p mimics group mirnc group and control group p005 compared to control group p005 compared to mirnc group b the protein expression and mrna level of zpf91 were detected by c western blotting and rtqpcr the data are represented as the mean ± sd n3 p005 oneway anova followed by tukey's test was used to evaluate the difference for multiple comparisons bc breast cancer zfp91 zinc finger protein nc negative controlassociated with advanced tnm stage however to investigate the relationship of mir1885p and bc patient prognosis we found that kaplanmeier analysis of mir1885p was limited due to the small sample size in tcga these results illustrated that the downregulation of mir1885p may be related with bc progression in the clinic suggesting that mir1885p may be a valuable bc diagnostic indicator moreover we confirmed that mir1885p mimics considerably inhibited the proliferation induced the apoptosis and inhibited the invasion of bc cells suggesting that mir1885p plays an inhibitory role in bc cellstranscription factor zinc finger protein zfp91 was firstly identified in the mouse in which was found to be overexpressed in colon liver prostate stomach and breast cancer zfp91 has a molecular mass of kda with amino acids containing five zincfinger motives a leucine zipper a coiledcoil structure and nuclear localization sequences zfp91 was confirmed to be a transcription factor located in the cellular nucleus ma reported that zfp91 functions as an oncogene in cancer development by activating hif1α transcription the overexpression of zfp91 0concology reports was also found to result in the promotion of nkκb signaling pathway activation through increasing nkκb inducing kinase nik whose activity and overexpression are related to cancer progression in melanoma pancreatic breast and lung cancer the inhibition of zfp91 was demonstrated to promote apoptosis in bc stomach cancer cells colon cancer and endometrial cancer in addition the overexpression of zfp91 was found to increase the cancer cell growth rate and metastatic capability zfp91 was also reported to interact with cyclindependent kinase inhibitor 2a cdkn2a which is an alternative reading frame arf tumor suppressor inhibiting the induction of p53dependent cell death to illuminate the molecular mechanisms of mir188 we predicted that il6st foxn2 zfp91 may be the targets of mir5p using targetscan and miranda furthermore peng reported that zfp91 is the target protein of mir5p in gastric cancer in addition overexpression of mir1885p was confirmed to inhibit the progression of breast cancer thus we chose the reported oncogene zfp91 for further investigation in the present study we confirmed that the 'untranslated region 'utr of zfp91 was bound by mir5p through dual luciferase assay moreover transfection of mir1885p mimics in mdamb cells reduced the zfp91 mrna and protein levels together mirnas usually bind to the 'utrs of target mrnas and do not reduce the level of mrnas however mirnas also were reported to decay the target mrnas and decease mrna level restoration of zfp91 largely reversed the decreased proliferation and induced apoptosis which were both regulated by mir1885p overexpression moreover in the tumor xenograft mouse model we observed that the expression of zfp91 was downregulated by an increased level of mir1885p furthermore the expression of mir5p and zfp91 were negatively correlated in bc patient tissues therefore our studies confirmed that mir5p can inhibit the progression of human bc via targeting zfp91zfp91 has been reported to promote proliferation in colon cancer prostate cancer and gastric cancer ma reported that zfp91 activates nfkappabp65 to promote proliferation and tumorigenesis of colon cancer paschke identified that zfp91 is a noncanonical nfκb signaling pathway regulator with oncogenic properties in prostate cancer in the present study we also confirmed that a decrease in zfp91 could significantly inhibit nfκbp65 and relb expression in bc cells therefore mir1885p overexpression reduced zfp91 via the noncanonical nfκb signaling pathway to inhibit the progression of bcin conclusion our data showed that mir1885p is downregulated in bc cell lines and tissues and the downregulated expression of mir1885p is associated with the poor prognosis of patients with bc we further investigated that overexpression of mir1885p could inhibit proliferation and induce the apoptosis of mdamb cells furthermore zfp91 was predicted and confirmed as a target gene of mirna5p and the effects of mir1885p on bc cells were dependent on the inhibition of zfp91 additionally a decrease in zfp91 significantly inhibited the nfκbp65 and relb expression in bc cells moreover the expression levels of mir5p and zfp91 were highly correlated with bc progression therefore we suggest that mir1885p can inhibit breast cancer progression via the zfp91nfκbp65 axis and may be a potential diagnostic indicator for bcacknowledgementsnot applicablefundingthis study was supported by the national natural science foundation of china grant nos and and talent projectavailability of data and materialsthe datasets used and analyzed during the current study are available from the corresponding author upon reasonable requestauthors' contributionszy and zl conceived and designed the study zy zc gy performed the experiments zy wrote the paper zy zl zc and gy reviewed the results and data and edited the manuscript all authors read and approved the manuscript and agree to be accountable for all aspects of the research in ensuring that the accuracy or integrity of any part of the work are appropriately investigated and resolvedethics approval and consent to participatetissues used in this study were obtained from shengjing hospital of china medical university shenyang liaoning china with the informed consent of patients and all experiments were approved by the ethics committee of shengjing hospital of china medical university no 2016ps18jpatient consent for publicationnot applicablecompeting intereststhe authors declare that they have no competing interestsreferences davidson ne armstrong sa coussens lm cruzcorrea mr deberardinis rj doroshow jh foti m hwu p k | Colon_Cancer |
"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 |
" healthcare is an essential service at any time more so in the crisis like covid with increase in numberof cases and mortality from covid the primary focus is shifted to the management of the covid crisis and otherhealth emergencies thus affecting normal health services and routine treatment of other diseases like cancermethods this reviews the published literature and guidelines on covid and cancer and discusses them tooptimize the care of cancer patients during covid pandemic to improve treatment outcomesresults the results of the review of published literature show a twofold increase in probability of getting cov2infection by the cancer patients and a fourfold increase in chance of death on the other hand if left untreated a increase in cancer death is expected data further show that none of the medicines like remdesivir hydroxychloroquin dexamethasone or azithromycin improves survival and response to covid in cancer patients surgicalresults too show similar outcome before and after the pandemic though most of these report on highly selectedpatients populationss the covid pandemic places cancer patients in a very difficult situation wherein if they seektreatment they are exposing themselves to a risk of developing cov2 infection and if they do not the probabilityof dying without treatment increases hence for them it is a choice between the devil and deep sea and it is forthe healthcare providers to triage patients and treat who cannot wait even though the data from the carefullyselected cohort of patients show no increase in mortality or morbidity from treatment during covidkeywords covid cancer cov2 coronavirus treatment chemotherapy surgeryintroductionwith the onset of covid19 pandemic the situation forcancer patients has become a nightmare most of the patients who were on treatment in march had to miss outtheir further treatment due to lockdown and closure ofhospitals and all modes of transport both public and private those who developed cancer during lockdown toocould not reach doctors for the same reason surgerieswere postponed radiotherapy was postponed and there correspondence manojpandey66gmailcomdepartment of surgical oncology institute of medical sciences banarashindu university varanasi indiawas uncertainty as far as chemotherapy and immunotherapy were concerned every society and anization cameup with their own guidelines however none of these addressed the logistic problems that patients faced reachingthe treatment centers this lead to an increased pool ofuntreated patients an analysis from england estimatesadditional deaths from cancer over next monthsthat is increase proposed due to covid pandemic those who were willing to travel and take the risk ofgetting infected with covid19 too faced challenges dueto travel restrictions though the indian government issued special passes to allow travel for cancer treatment 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 0cchakraborty and pandey world of surgical oncology page of however the rider was that he or she has to travel withhis own private transport and not more than two peoplewere allowed in a four wheeler this meant that apartfrom the driver only the patient could travel withoutany caregiver if the patient does not own the car andcaregiver does not know how to drive these passeswere issued district or state wise as there was a ban oninterdistrict and interstate travel so the patients comingto bigger institutes like ours faced special problemswherein despite having a travel pass some of them hadto pay fines most of our patients come from neighboring states of bihar and madhya pradesh and even fromwest bengal and nepalapart from that at the beginning of the pandemicthings were not clear and there was no data on the incidence and mortality from covid19 in cancer patientsfurther though there were several guidelines [] theywere not based on evidence as the evidence did not existat that time and it was not clear as to what treatmentmodalities are safe giving rise to uncertainties in themind of patients and health care workers the testing facilities were limited the availability of ppe was limitedand these were prioritized for suspected cases and contacts while other diseases were kept on hold or receivedlow priority this reviews the current literatureon various aspects of covid and cancermethodsa review of literature was carried out using the pubmedfor the s published using the string covid[allfields] and cancer s[all fields] or cancerated[all fields] or canceration[all fields] or cancerization[all fields] or cancerized[all fields] orcancerous[all fields] or neoplasms[mesh terms]or neoplasms[all fields] or cancer[all fields] orcancers[all fields] following filters were applied clinical study clinical trial comparative study controlledclinical trial metaanalysis observational study pragmatic clinical trial randomized controlled trialthe s that discussed the incidence prevalencemortality treatment of cancer during covid or morbidity of treatment were included in the review as the obtained data was very heterogeneous no attempt atpooling or metaanalysis was doneresultsa total of s were extracted the of allthe s was read and after excluding reviews s were included in this review including one metaanalysis as for the prevalence the initial data from amulticentric study comparing cancer with noncancerpatients infected with covid found higher incidence ofsevere outcome in cancer compared to normal the outcome was worse in lung cancer and metastatic disease a study evaluating fatality in patients withcovid found a times risk of death in cancer patientscontracting covid this fear and uncertainty had affected the quality oflife and metal health of cancer patients caregivers andhealth care workers there were no socializing no outlets no markets or movies or dining out even the placesof worships were closed an italian survey of young cancer patients showed that they feel more vulnerable tocontract cancer and risk of higher complications ahigher burnout has been reported in healthcare workersworking in covid hospitals over a period of time things have started clearing upand preliminary data on incidence severity and complications of covid19 is now available a retrospectiveanalysis of patients across mainland china reported incidence of severe cases of these had at least one comorbidity with hypertension being most prevalent followed by diabetes in thisstudy the cancer was seen only in patients ofwhom had severe illness another study from chinaon patients with covid had patients with cancer they too found higher severity in cancer patientshowever the number of cancer patients in this study toowas very small similarly the study of chen had patients in total of covid19 patients andonly three of these had a severe disease similar resultshave been reported by others [ ]a pooled metaanalysis of studies reporting oncovid prevalence in cancer patients found a pooledprevalence of prevalence was higher at insmaller series reporting on less than patients between march and june over patients wereseen at our center of which undergoing surgery orthe interventions were tested for covid of these patients were found to be positive the prevalenceof cancer is high among covid19 patients in europehowever these do not seem to convert into higher mortality of cancer patients a study from uk reporting on deaths did not find increased mortality in cancer except in patients receiving chemotherapy however ametaanalysis involving patients thatincluded cancer patients showed a significant increase inhazard of death in cancer patients infected with covid hr the study also showed increased admission to icu hr however when patients abovethe age of were considered no difference in deathrate was observed this increase in cancer mortalityin covid19 has been attributed to advance age additional comorbidities diagnosis of lung cancer use ofchemotherapy etc [ ]recent data from the usa canada and spain fromthe covid19 and cancer consortium ccc19 database reported on patients with cancer of these 0cchakraborty and pandey world of surgical oncology page of patients had died advance age smokingmale gender more than one comorbidity and ecogmore than were found to be associated with increasedmortality these results and those detailed above clearlyshow that prevalence of covid in cancer is higher andmortality in these patients too is higher especially in patients with additional comorbidities and active diseaseand one needs to exercise full precaution while takingtreatment decisionsdiscussionprecautions taken at our center to prevent spread incancer patientsas cancer patients are at a higher risk of contractingcovid19 than the general population special precautionsare taken when patients are seen in the outpatient clinicopd and surgery no patient or the caregiver is allowedto enter the opd without a face mask all patients arescreened for body temperature and those found to havefever are immediately sent to covid opd this is followedby filling up of a symptom checklist and questionnairebased screening for all patients those with covid symptoms are referred for rtpcr testing before being allowedto see doctor and get the treatment this is similar to allothers coming to the hospital for treatment as there areno separate guidelines for cancer apart from that stricthand hygiene is followed and ppe has to be worn byhealth care workers those planning to undergo procedures and surgery are screened for covid19 using rtpcr testing they are kept in the holding area and afterbeing reported are shifted to wards any patient foundpositive is treated as per guidelinesplanned surgeryelective surgeries for cancer patients are avoided ifpossible this is usually a collective decision taken bythe oncologist in consultation with the patient if it isdecided to do a surgery a special covid19 consent istaken all emergency surgeries are performed withproper written covid consent and after covid testingfor even asymptomatic patients other than emergencies patients who cannot wait for weeks for surgeryas the disease may progress and become inoperableare considered for surgery other than that patientswho were started on neoadjuvant therapy before theonset of covid and have now completed neoadjuvanttherapy are also candidates for surgery as the opportunity of window period should not be lost any patient where the multidisciplinary team think can waitfor weeks is postponed patients undergoing surgeryare reported to be at higher risk than those treatedby radiation planned radiotherapyin the beginning all patients were postponed after discussion with patients or caregivers however as it is notpossible to postpone radiation forever selected patientsare now being taken up for radiation patients are informed that whatever little evidence that is available suggest higher chances of covid infection in this subset ofpatients recent guidelines have suggested omitting radiation for patients older than years using fast andfast forward omitting boost and hypofractionation[ ] recent s also suggest modification of thedelivery technique to reduce treatment time [ ]immunosuppressive therapythe current evidence does not support changing orwithholding chemotherapy targeted therapy and immunotherapy in cancer patients some of the studiessuggested stopping of immunotherapy for patients whohave a complete response or prolonged response ofmore than years a report on immune check pointinhibitors suggested that as there is not much immunecompromise and minimal hematological toxicities theycan be used with caution as patients are not devoid ofits benefits [ ] at our center we have continuedwith chemotherapy and immunotherapy and have notfound any increase in covid19 infection or mortality inthis sub groupstem cell transplantationcurrent guidelines recommend delaying the plannedallogenic stem cell transplant [ ] this is a uniquesituation where the donor and recipient both are at riskand a careful decision making keeping all factors into account be made no stem cell transplants took place atour center during this periodcoronavirus therapy in cancerthere is no evidence of benefit of using various therapies to treat covid19 in cancer patients remdesivir hasbeen approved by the fda for treatment of covid casesunder emergency use authorization the ccc19 studyfailed to show any benefit of azithromycin hydroxychloroquine alone or in combination in fact use of acombination of the two was found to be an independentfactor predicting mortality with hazard ratio of which was statistically significant there are occasional reports on the use of lopinavirritonavir [ ]in lung cancer patient with covid another dataset fromccc19 study reported on patients treated withhydroxychloroquine n azithromycin n remdesivir n highdose corticosteroids n tocilizumab n andother therapy n showed no benefit of anytreatment apart from slight activity of remdesivir no 0cchakraborty and pandey world of surgical oncology page of other drug showed any benefit [ ] patients also relowmolecular weightceived dexamethasone aspirinheparinabovetreatmentsanticoagulants besidesand otherresearch on covid2019 and cancera recent bioinformatic study using the geo databaseshowed that angiotensinconverting enzyme ace2 iselevated in uterine corpus endometrial carcinoma andrenal papillary cell carcinoma this increase also correlated with immune infiltrate present in the tumor theauthors suggested that these tumors may be more proneto covid19 infections however the authors failedto present any evidence to suggest this hypothesis similar bioinformatics study by fu also showed similarresults and they suggested that testes may also get affected by covid19 infections other tumors thatmay be affected are pancreas and colon howeverall these studies are based on geo databases and thereis no experimental evidence presented till datesthe literature on cancer and covid is fast expanding withmore and more information pouring in the literature sofar is clear in indicating that cancer patients are at highrisk of developing covid and when developed the severityand mortality is higher than the normal population therecent data also suggests a possible role of remdesivir inthe treatment of covid19 in cancer patients however theevidence to support this is very weak this absence of conclusive evidences has led to development of strategies totreat cancer safely most of these are based on reducingthe hospital visits and avoiding immune compromise theevidence is still evolving and more practice changes areexpected in the days to come and that may continue evenin post covid eraacknowledgementsnoneauthors contributionsmc prepared the draft and mp conceived and designed the and edited the final version the authors read and approved the finalmanuscriptfundingnoneavailability of data and materialsnot applicableethics approval and consent to participatethis does not report on human and animal experiments ethicalapproval and publication of consent is not applicableconsent for publicationnot applicablecompeting intereststhe authors declare that there are no conflicts of interestreceived june accepted august references wise j covid19 cancer mortality could rise at least because ofpandemic study finds bmj 2020369m1735 httpsdoi101136bmjm1735m1735civantos fj leibowitz jm arnold dj stubbs vc gross jh thomas gr sargiz casiano rr franzmann ej weed d perez c samuels m goodman kwgoodwin wj ethical surgical triage of head and neck cancer patientsduring the covid19 pandemic head neck coles ce aristei c bliss j boersma l brunt am chatterjee s hanna g jagsir kaidar po kirby a mjaaland i meattini i luis am marta gn offersen bpoortmans p rivera s international guidelines on radiation therapy forbreast cancer during the covid19 pandemic clin oncol r coll radiol cortiula f pettke a bartoletti m puglisi f helleday t managing covid19in the oncology clinic and avoiding the distraction effect ann oncol elkaddoum r haddad fg eid r kourie hr telemedicine for cancer patientsduring covid19 pandemic between threats and opportunities futureoncol finley c prashad a camuso n daly c aprikian a ball cg bentley jcharest d fata p helyer l o'connell d moloo h seely a werier j zhongt earle cc guidance for management of cancer surgery during the covid pandemic can j surg 202063s2finley c prashad a camuso n daly c earle cc lifesaving cancer surgeriesneed to be managed appropriately during the covid19 pandemic can jsurg 202063s1hanna tp evans ga booth cm cancer covid19 and the precautionaryprinciple prioritizing treatment during a global pandemic nat rev clinoncol dai m liu d liu m zhou f li g chen z zhang z you h wu m zheng qxiong y xiong h wang c chen c xiong f zhang y peng y ge s zhen byu t wang l wang h liu y chen y mei j gao x li z gan l he c li zshi y qi y yang j tenen dg chai l mucci la santillana m cai h patientswith cancer appear more vulnerable to sarscov2 a multicenter studyduring the covid19 outbreak cancer discov deng g yin m chen x zeng f clinical determinants for fatality of patients with covid19 crit care casanova m pagani be silva m patriarca c veneroni l clerici ca spreaficof luksch r terenziani m meazza c podda m biassoni v schiavello echiaravalli s puma n bergamaschi l gattuso g sironi g massimino mferrari a how young patients with cancer perceive the covid19coronavirus epidemic in milan italy is there room for other fears pediatrblood cancer 2020e28318 wu y wang j luo c hu s lin x anderson ae bruera e yang x weis qian y a comparison of burnout frequency among oncologyphysicians and nurses working on the front lines and usual wardsduring the covid19 epidemic in wuhan china j pain symptommanag guan wj ni zy hu y liang wh ou cq he jx liu l shan h lei cl huidsc du b li lj zeng g yuen ky chen rc tang cl wang t chen pyxiang j li sy wang jl liang zj peng yx wei l liu y hu yh peng pwang jm liu jy chen z li g zheng zj qiu sq luo j ye cj zhu syzhong ns clinical characteristics of coronavirus disease in china nengl j med cai yc wang w li c zeng df zhou yq sun rh jiang h guo h wang sxjiang j treating head and neck tumors during the sarscov2 epidemic to sichuan cancer hospital head neck chen atc courafilho gb rehder mhh clinical characteristics of covid19in china n engl j med pii 101056nejmc2005203sa2 doi 1056nejmc200520310fong d rauch s petter c haspinger e alber m mitterer m infection rateand clinical management of cancer patients during the covid19pandemic experience from a tertiary care hospital in northern italy esmoopen 20205e000810 yu j ouyang w chua mlk xie c sarscov2 transmission in patients withcancer at a tertiary care hospital in wuhan china jama oncol 0cchakraborty and pandey world of surgical oncology page of zhang h xie c huang y treatment and outcome of a patient eith lungcancer infected with severe acute respiratory syndrome coronavirus2 jthorac oncol 202015e63 rivera dr peters s panagiotou oa shah dp kuderer nm hsu cy rubinsteinsm lee bj choueiri tk de lima lg grivas p painter ca rini bi thompsonma arcobello j bakouny z doroshow db egan pc farmakiotis d fecher lafriese cr galsky md goel s gupta s halfdanarson tr halmos b hawley jekhaki ar lemmon ca mishra s olszewski aj pennell na puc mm revankarsg schapira l schmidt a schwartz gk shah sa wu jt xie z yeh ac zhu hshyr y lyman gh warner jl utilization of covid19 treatments and clinicaloutcomes among patients with cancer a covid19 and cancer consortiumccc19 cohort study cancer discov 2020cd0941 yang j li h hu s zhou y ace2 correlated with immune infiltration servesas a prognostic biomarker in endometrial carcinoma and renal papillary cellcarcinoma implication for covid19 aging albany ny fu j zhou b zhang l balaji ks wei c liu x chen h peng j fu jexpressions and significances of the angiotensinconverting enzyme genethe receptor of sarscov2 for covid19 mol biol rep chai p yu j ge s jia r fan x genetic alteration rna expression and dnamethylation profiling of coronavirus disease covid19 receptor ace2in malignancies a pancancer analysis j hematol oncol publishers notespringer nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations desai a sachdeva s parekh t desai r covid19 and cancer lessons from apooled metaanalysis jco glob oncol httpsdoi101200go2000097557559lee lyw cazier jb starkey t turnbull cd kerr r middleton g covid19mortality in patients with cancer on chemotherapy or other anticancertreatments a prospective cohort study lancet giannakoulis vg papoutsi e siempos ii effect of cancer on clinicaloutcomes of patients with covid19 a metaanalysis of patient data jcoglob oncol httpsdoi101200go2000225799808 gosain r abdou y singh a rana n puzanov i ernstoff ms covid19 andcancer a comprehensive review curr oncol rep addeo a obeid m friedlaender a covid19 and lung cancer risksmechanisms and treatment interactions j immunother cancer e000892 yang k sheng y huang c jin y xiong n jiang k lu h liu j yang j dongy pan d shu c li j wei j huang y peng l wu m zhang r wu b li ycai l li g zhang t wu g clinical characteristics outcomes and risk factorsfor mortality in patients with cancer and covid19 in hubei china amulticentre retrospective cohort study lancet oncol mehta v goel s kabarriti r cole d goldfinger m acunavillaorduna apradhan k thota r reissman s sparano ja gartrell ba smith rv ohri ngarg m racine ad kalnicki s perezsoler r halmos b verma a casefatality rate of cancer patients with covid19 in a new york hospitalsystem cancer discov meng y lu w guo e liu j yang b wu p lin s peng t fu y li f wang zli y xiao r liu c huang y lu f wu x you l ma d sun c wu p chen gcancer history is an independent risk factor for mortality in hospitalizedcovid19 patients a propensity scorematched analysis j hematol oncolkuderer nm choueiri tk shah dp shyr y rubinstein sm rivera dr shetes hsu cy desai a de lima lg jr grivas p painter ca peters s thompsonma bakouny z batist g bekaiisaab t bilen ma bouganim n larroya mbcastellano d del prete sa doroshow db egan pc elkrief a farmakiotis dflora d galsky md glover mj griffiths ea gulati ap gupta s hafez nhalfdanarson tr hawley je hsu e kasi a khaki ar lemmon ca lewis clogan b masters t mckay rr mesa ra mans ak mulcahy mfpanagiotou oa peddi p pennell na reynolds k rosen lr rosovsky rsalazar m schmidt a shah sa shaya ja steinharter j stockerlgoldstein kesubbiah s vinh dc wehbe fh weissmann lb wu jt wulffburchfield exie z yeh a yu pp zhou ay zubiri l mishra s lyman gh rini bi warnerjl clinical impact of covid19 on patients with cancer ccc19 a cohortstudy lancet huang sh o'sullivan b su j ringash j bratman sv kim j hosni a bayleya cho j giuliani m hope a spreafico a hansen ar siu ll gilbert r irishjc goldstein d de aj tong l xu w waldron j hypofractionatedradiotherapy alone with gy per fraction for head and neck cancerduring the covid19 pandemic the princess margaret experience andproposal cancer vordermark d shift in indications for radiotherapy during the covid19pandemic a review of anspecific cancer managementrecommendations from multidisciplinary and surgical expert groups radiatoncol davis ap boyer m lee jh kao sc covid19 the use of immunotherapy inmetastatic lung cancer immunotherapy kattan j kattan c assi t do checkpoint inhibitors compromise the cancerpatients' immunity and increase the vulnerability to covid19 infectionimmunotherapy bersanelli m controversies about covid19 and anticancer treatment withimmune checkpoint inhibitors immunotherapy ardura mi hartley dm dandoy c lehmann l jaglowski s auletta jjaddressing the impact of the coronavirus disease covid19 pandemic onhematopoietic cell transplantation learning networks as means for sharingbest practices biol blood marrow transplant mahmoudjafari z alexander m roddy j shaw r shigle tl timlin c culosk american society for transplantation and cellular therapy pharmacyspecial interest group position statement on pharmacy practicemanagement and clinical management for covid19 in hematopoietic celltransplantation and cellular therapy patients in the united states biol bloodmarrow transplant 0c" | Colon_Cancer |
" leukotriene receptor antagonists ltras are broadly used for the management of allergic asthmaand have recently been indicated to inhibit carcinogenesis and cancer cell growth in colorectal cancer crcchemoprevention studies the occurrence of adenoma or crc itself is generally set as the trial endpoint althoughthe occurrence rate of crc is the most confident endpoint it is inappropriate for chemoprevention studies becausecrc incidence rate is low in the general population and needed for longterm monitoring aberrant crypt fociacf defined as lesions containing crypts that are larger in diameter and darker in methylene blue staining thannormal crypts are regarded to be a fine surrogate biomarker of crc therefore this prospective study was designedto explore the chemopreventive effect of ltra on colonic acf formation and the safety of the medicine in patientsscheduled for a poly resection as a pilot trial leading the crc chemoprevention trialmethods this study is a nonrandomized openlabel controlled trial in patients with colorectal acf and polypsscheduled for a polypectomy participants meet the inclusion criteria will be recruited and the number of acf inthe rectum will be counted at the baseline colonoscopic examination next the participants will be assigned to theltra or no treatment group participants in the ltra group will continue mg of oral montelukast for weeksand those in the no treatment group will be observed without the administration of any additional drugs at theend of the 8week ltra intervention period a polypectomy will be conducted to evaluate the changes in thenumber of acf and cell proliferation in the normal colorectal epithelium will be analyzeddiscussion this will be the first study to investigate the effect of ltras on colorectal acf formation in humanstrial registration this trial has been registered in the university hospital medical information network uminclinical trials registry as umin000029926 registered november keywords colorectal cancer chemoprevention leukotriene receptor antagonist aberrant crypt foci correspondence takuma_hyokohamacuacjpdepartment of gastroenterology and hepatology yokohama city universityschool of medicine fukuura kanazawa yokohama kanagawa japan the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0chigurashi bmc cancer page of cancer is a one of the major health issues and a leadingcause of death globally the incidence prevalence andmortality rates of colorectal cancer crc continue torise all over the world [ ] the majority of crc casesare derived from adenomatous polyps and their resection has been shown to reduce the risk of the futuredevelopment of advanced adenomas and crc [ ]thereby preventing crcrelated deaths however patients with polyps adenomatous polyps andor hyperplastic polyps represent a highrisk group for theoccurrence of metachronous colorectal polyps andorcrc then a paradigm is shifting from surveillancefor the early detection of advance adenomas or crcsand resection to novel tactics for prevention is requiredto reduce the mortality rate of this disease a number oflargescale epidemiologic andor clinicaltrials haveassessed the prophylactic agentsincluding vitamin dcalcium fiber and nonsteroidal antiinflammatory drugsnsaids such as aspirin and selective cyclooxygenase cox2 inhibitors in preventing against crc development we previously reported that the nsaidsulindac suppressed the development of sporadic colorectal adenomas to this point nsaids particularlycox2 inhibitors have been proven to offer the greatestpossibility for reducing crc risk either alone or in combination with other drugs while it has been reportedan elevated risk of serious cardiovascular events relatedwith the administration of cox2 inhibitors [ ] inview of these adverse cardiovascular effects and the lackof efficacy of other drugs that initially looked promisingthe development of novel agents that meet both safetyand efficacy in preventing crc is essentialleukotriene receptor antagonists ltras such asmontelukast and zafirlukast are commonly used for thetreatment of allergic asthma [ ] and tsai mj reported that ltras reduced cancer risk in a dosedependent manner in asthma patients it was alsoreported that the cancer incidence rate was significantlylower in ltra users than in nonltra users vs per patientsyear this means that apart fromits role in asthma ltra has also been associated withcarcinogenesis and tumormediated immunosuppression for example the overexpression of cysteinyl leukotriene receptor cyslt1r has been observed in crcand montelukast leads the apoptosis of crc cancer cells[ ] previous in vivo studies have shown the chemopreventive effect of ltras [ ] but the chemopreventive effects of ltra have not been studied in clinicalpractice therefore we designed this study to ivestigatethe chemopreventive effect of ltras in clinical practicein crc chemoprevention trials the occurrence of adenomas or crc itself is generally set as the trial endpointthough the occurrence of crc is the most confidentendpoint it is not recommended for chemopreventionstudies because crc incidence rate is low in the generalpopulation and needed for longterm monitoring furthermore there are ethical concerns about conductinglongterm trials to determine whether a test agent is effective or notaberrant crypt foci acf defined as lesions containing crypts that are larger in diameter and darker inmethylene blue staining than normal crypts [] areregarded to be a fine surrogate biomarker of crc our group has previously reported that acf is usefulbiomarker for crc [ ] and study endpoint for achemoprevention study [ ] the advantages of chemoprevention studies with the number of colorectalacf as the trial endpoint are that longterm observationis not needed to investigate the agent efficacy and thenumber of acf can be quantitatively estimated therefore we set the acf count as a good endpoint for thisstudy to the knowledge of us this is the first clinicalstudy investigating the use of ltras as chemopreventive agents against colorectal acf in humansmethodsstudy design and settingthis study was designed as a nonrandomized openlabel controlled trial to be conducted in patients withcolorectal acf it will be performed at the departmentof gastroenterology and hepatology yokohama cityuniversity ycu hospital japan the coordinating office will be at the ycu hospital and patient registrationwill be conducted at the ycu center for novel and exploratory clinical trials ynext and data collectionwill be done using electronic data caputureethical considerations and trial registrationthe trial protocol complies with the declaration ofhelsinki and the ethical guidelines for clinical research of the ministry of health labour and welfarejapan ethical approval of this trial was obtainedfrom the ethics committee of ycu hospital on may the study protocol and informed consent documents were approved by the ycu hospital ethics committee this trial has been approved in the clinical trialact in japan and registered in the japan registry of clinical trials jrct as jrcts031180094 and the universityhospital medical information network umin clinicaltrials registry as umin000029926 all study participants will submit a written study participation informedconsent formparticipation criteriawe will recruit the patients with colorectal acf and resectable polyps for this trial the inclusion criteria are asfollows patients with resectable polyps [adenoma 0chigurashi bmc cancer page of hyperplastic polyp and sessile serrated adenomapolypssap] patients with more than five rectal acfand submit written study participation informed consent formthe exclusion criteria are as follows patients withlesions suitable for early removal a history of ltrause within months before study participation a history of malignant disease within years before studyparticipation a history of heart renal liver failure orliver cirrhosis a history offamilial adenomatouspolyposis hereditary nonpolyposis crc or inflammatory bowel disease pregnancy or the possibility ofpregnancy prohibitions of montelukast allergiesto montelukast regular use of nsaids metforminand pioglitazone and participants considered as unsuitable for the study by the researchersinterventionall eligible participants will be assigned to the ltra orno treatment group because this is an openlabel trialpatients will be assigned to the no treatment group afterthe inclusion of patients in the ltra group participantsin the ltra group will receive mg of oral montelukast for weeks and those in the no treatment groupwill be observed without the administration of any additional drugs at the end of the 8week ltra treatmentperiod a polypectomy will be performed to evaluate thechanges in the number of acf and cell proliferation inthe normal colorectal epithelium will be analyzedoutcome measurementsthe primary endpoint will be the change in the numberof colorectal acf after weeks of treatment a magnifying colonoscope pcfq290azi hz290 olympus cotokyo japan will be used in all cases procedure preparation for the colonoscopy will begin day before theprocedure each participant will be informed to take alowresidue diet and mg oral sodium picosulfate onthe evening before the procedure on the day of the procedure each participant will be given ml polyethylene glycol peg if the stools are not clear enough anadditional ml peg will be given to ensure adequatebowel cleansing in most cases conscious sedation withmidazolam mg and pentazocine mg willbe use at the start of the colonoscopy subcutaneousscopolamine or glucagon will be administered for colonic movements reduction at the time of the first colonoscopy the endoscopists will insert into the cecumand the observe entire colorectum as the endoscope ispulled back one colonic mucosal sample will be collected the number of rectal acf will be counted usinga magnifying endoscope at the end of the 8week ltratreatment period the same endoscopists will performthe polypectomies and countthe number of acfendoscopists will record all procedures on a hard diskdrive and take photograph all acf the number of acfin each participant will first be counted by the endoscopists during the procedure to provide additional validation the number of acf will be recounted by threeblinded expert endoscopists aj ht and ak by observing the recorded hard disk drive cases that these evaluators deem colonoscopy to be inappropriate will beexcluded from the final analysisthe secondary outcomes will be as follows drugsafety adverse events aes will be graded according tothe national cancer institute common toxicity criteriafor adverse events ncictcae version all trialparticipants will be provided with a trial record for thedaily dose of the trial agent and aes participants whodevelop serious ae of grade or higher will be discontinued at that time in the study the effects ofltras on cell proliferation in the rectal mucosa onecolonic mucosal sample will be collected from the samestudy patient by performing a biopsy at the time of thebaseline colonoscopy and polypectomy a biopsy will beobtained from all participants cell proliferation will beevaluated by ki67 staining briefly we will randomly select six crypts and count the number of ki67positivecells per crypt in total cells will be counted at amagnification of à using a brightfield microscopethe results will be presented as the percentage of ki67positive cells all participants will receive laboratory testsand a physical examination at the point of the baselinecolonoscopy and polypectomydrug supplymontelukast capsules will be purchased from kyorincorporation ltd tokyo japan participants will be informed to take one tablet of the study drug every nightbefore bed medication adherence will be monitored bycounting the empty medication sheets returned by theparticipants at the time of their polypectomy the participants will also be interviewed and monitored to confirm thatthey have not used any prohibited drugsaspirin metformin andor other nsaids aes will bemonitored by the investigator and graded according tothe ncictcae version if serious aes or less than drug adherence are confirmed in a participant thisparticipant will be withdrawn from the trialsample size estimation and allocationwe previously reported the administration of mgmetformin per a day for weeks reduced the number ofacf in that trial the mean number of acf per patientdecreased significantly from ± at baseline to ± at weeks p although this study is exploratory research and the accurate chemopreventive efficacy of montelukastis unknown we assume that 0chigurashi bmc cancer page of montelukast may have an effect that is equivalent to of that observed for metformin on the reduction of acfnubmer therefore we estimate that the acf numberwill change by about to on average we determined that a sample size of individuals in theltra group was needed to detect a significant reduction in the number of acfs in the ltra group using apaired ttest with a twosided significance level of and power assuming some dropouts we propose to recruit participants in the ltra group to confirmthat the number of acf does not change during thestudy period we propose to recruit patients in the notreatment group after consecutively accumulating patients in the ltra group therefore we propose to recruit patientsstatistical analysisthe change in the number of acf which is the primaryendpoint will be compared before and after the 8weekstudy period between the ltra and no treatment groupsby the paired ttest drug safety will be assessed by thechisquare test and the remaining results will be compared by the ttest or mannwhitney u test between thetwo groups p will be defined as statistically significant all statistical analyses will be conducted using jmp® software sas institute inc cary nc usatrial steering committee and data monitoring committeethe trial steering committee and data monitoringthe ynext thecommittee will be located atmanagement team will perform central monitoring ofthe trial status and data collection every monthstudy flowa study flow is shown in fig discussionto the knowledge of us this is the first clinical trial proposed to investigate the efficacy of ltras on colorectalacf formation ltras are broadly used for the treatment of allergic asthma and rhinitis [ ] and ltrasare reported to decrease the risk of cancer in asthma patients in a dosedependent manner previous basicresearch has reported that cyslt1r is overexpressed incrc and that montelukast induces the apoptosis ofcrc cells [ ] cyslts have recently been focusedon as significant regulators of gut homeostasis with endogenous cyslt production mediating the proliferationand survival of gut mucosal cells recent evidence focuses on the effect of leukotrienec4 ltc4 in accelerating oxidative dna damage if notadequately repaired can contribute to increase mutationrates and genomic instability dna damage andgenomic instability are major drivers of carcinogenesis cyslts also acts as leukocyte chemoattractants inaddition cyslt1 mediates th17 cell migration the storage of which associates with the progression ofinflammationassociated cancers chronic inflammation is a risk factor for cancer initiation and progression as observed in patients with inflammatory bowelfig study flow chart 0chigurashi bmc cancer page of disease furthermore leukotriene d4 ltd4 antagonists suppress chronic inflammation in a rodent modelof acute enteritis and this may be effective in preventinginflammationassociated crc ltras are leukotriene pathway inhibitors and thus they may have potentialas chemotherapy andor chemoprevention agents to reduce the effect of leukotrienes previous in vivo studieshave elucidated the chemopreventive effect of leukotriene pathway inhibitors [ ] and showed the potentialuse of ltras for chemoprevention therefore we willconduct this trial to explore the chemopreventive effectof ltras in clinical settingthis trial may have some limitations as follow firstacf are believed to be a fine surrogate biomarker ofcrc though its biological significance in humans is stillcontroversial in crc chemoprevention studies typically set the occurrence of adenomas or the crc itselfas endpoint of the study though the occurrence of crcis the most appropriate endpoint it is inappropriate forchemoprevention studies because crc incidence rate islow in the general population and needed for longtermmonitoring our group has previously reported thatacf is useful biomarker for crc and conducted a chemoprevention study for colorectal acf [ ] therefore we designed this study using the number of acf asthe primary endpoint to investigate the chemopreventiveeffect of ltras second an intervention duration of weeks may be too short to reliably detect differences between two groups since our group reported in a previous trial that oral administration of metformin for weeks reduced the number of colorectal acf in humans an intervention period of weeks should beenough to assess the changes in the number of acf ifltras have a chemopreventive effectour group previously conducted a shortterm chemoprevention study of metformin for colorectal acfand reported the preventive effect of the agent on theformation of acf then we conducted a oneyearmetformin chemoprevention studycolorectalpolyps we propose to repeat the same steps as inour metformin study for the chemoprevention studyusing montelukastforif ltras were proved to have efficacy for crc prevention the impact would be significant therefore webelieve it will be very interesting to assess whetherltras inhibit the formation of human colorectal acfabbreviationscrc colorectal cancer nsaids nonsteroidal antiinflammatory drugs cox cyclooxygenase2 ltras leukotriene receptor antagonistscyslt1r cysteinyl leukotriene receptor acf aberrant crypt fociycu yokohama city university umin university hospital medicalinformation network ssap sessile serrated adenomapolyp ncictcae national cancer institute common toxicity criteria for adverseevents peg polyethylene glycol aes adverse events ltc4 leukotriene c4ltd4 leukotriene d4acknowledgmentsthe authors would like to thank the staff for their support in recruitingeligible patients and the patients who participated in this study and theirfamily we thank cathel kerr bsc phd and melissa crawford phd fromedanz group httpsenauthorservicesedanzgroupcom for editing a draftof this manuscriptauthors contributionsth ja and an conceived the study th and ja equally contributed to thismanuscript ja th ka nm ty tm af and ho will perform the baselinecolonoscopies and polypectomies ja th and ka will conduct the secondcount of acf using a hard disk drive recording to ensure validity tt nm tyaf and ho will recruit participants and followup with them at an outpatientclinic the analysis and interpretation of data will be conducted by ja thand ka all authors have read the final manuscript and approved its submission for publicationfundinga grant for this research from the kanagawa institute of industrial scienceand technology kistec was awarded to th we declare that the fundingbody has no role in the design data collection analysis interpretation andwriting of the studyavailability of data and materialsthe datasets used andor analyzed during the current study will be availablefrom the corresponding author on reasonable requestethics approval and consent to participateethical approval of this trial was obtained from the ethics committee of ycuhospital on may the study protocol and informed consentdocuments were approved by the ycu hospital ethics committee this trialhas been registered in the university hospital medical information networkumin clinical trials registry as umin000029926 all study participants willsubmit a written study participation informed consent formconsent for publicationnot applicablecompeting intereststhe authors declare that they have no conflicts of interestsreceived may accepted august referencestorre la bray f siegel rl global cancer statistics ca cancer jclin anderson wf umar a brawley ow colorectal carcinoma in black andwhite race cancer metastasis rev vogelstein b fearon er hamilton sr genetic alterations duringcolorectaltumor development n engl j med winawer sj zauber ag ho mn obrien mj gottlieb ls sternberg ss wayejd schapiro m bond jh panish jf prevention of colorectal cancer bycolonoscopic polypectomy the national polyp study workgroup n engl jmed citarda f tomaselli g capocaccia r barcherini s crespi m italianmulticentre study group efficacy in standard clinical practice ofcolonoscopic polypectomy in reducing colorectal cancer incidence gutzauber ag winawer sj obrien mj lansdorpvogelaar i van ballegooijenm hankey bf shi w bond jh schapiro m panish jf stewart et waye jdcolonoscopic polypectomy and longterm prevention of colorectalcancerdeaths n engl j med maisonneuve p botteri e lowenfels ab fiveyear risk of colorectalneoplasia after negative colonoscopy n engl j med author reply das d arber n jankowski ja chemoprevention of colorectal cancerdigestion epub oct review matsuhashi n nakajima a fukushima y yazaki y oka t effects of sulindacon sporadic colorectal adenomatous polyps gut 0chigurashi bmc cancer page of drazen jm cox2 inhibitorsa lesson in unexpected problems n engl jmed epub feb meyskens fl jr mclaren ce pelot d fujikawabrooks s carpenter pmhawk e kelloff g lawson mj kidao j mccracken j albers cg ahnen djturgeon dk goldschmid s lance p hagedorn ch gillen dl gerner ewdifluoromethylornithine plus sulindac for the prevention of sporadiccolorectal adenoma a randomized placebocontrolled doubleblind trialcancer prev res phila scott jp petersgolden m antileukotriene agents for the treatment of lungdisease am j respir crit care med shen z genomic instability and cancer an introduction j mol cell biolkim hs lee g the cysteinyl leukotriene receptor cysltr1 mediates th17cell migration j immunol bernstein cn blanchard jf kliewer e wajda a cancer risk in patients withinflammatory bowel disease a populationbased study cancer nishikawa m hikasa y hori k tanida n shimoyama t effect of leukotrienec4d4 antagonist on colonic damage induced by intracolonic administrationof trinitrobenzene sulfonic acid in rats j gastroenterol publishers notespringer nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations dahlen se dahlen b drazen jm asthma treatment guidelines meet the realworld n engl j med tsai mj wu ph sheu cc hsu yl chang wa hung jy corrigendumcysteinyl leukotriene receptor antagonists decrease cancer risk in asthmapatients sci rep wang d dubois rn eicosanoids and cancer nat rev cancer nielsen ck the leukotriene receptor cyslt1 and 5lipoxygenase areupregulated in colon cancer adv exp med biol burke l butler ct murphy a moran b gallagher wm osullivan j kennedybn evaluation of cysteinyl leukotriene signaling as a therapeutic target forcolorectal cancer front cell dev biol savari s liu m zhang y sime w sjolander a cyslt1r antagonists inhibittumor growth in a xenograft model of colon cancer plos one e73466 bellamkonda k satapathy sr douglas d chandrashekar n selvanesan bcliu m savari s jonsson g sjolander a montelukast a cyslt1 receptorantagonist reduces colon cancer stemness and tumor burden in a mousexenograft model of human colon cancer cancer lett roncucci l stamp d medline a cullen jb bruce wr identification andquantification of aberrant crypt foci and microadenomas in the humancolon hum pathol roncucci l medline a bruce wr classification of aberrant crypt foci andmicroadenomas in human colon cancer epidemiol biomark prev pretlow tp barrow bj ashton ws aberrant crypts putativepreneoplastic foci in human colonic mucosa cancer res pretlow tp oriordan ma pretlow tg stellato ta aberrant crypts in humancolonic mucosa putative preneoplastic lesions j cell biochem suppl takayama t katsuki s takahashi y ohi m nojiri s sakamaki s kato jkogawa k miyake h niitsu y aberrant crtpt foci of the colon as precursorsof adenoma and cancer n engl j med sakai e takahashi h kato s uchiyama t hosono k endo h maeda syoneda m taguri m nakajima a investigation of the prevalence andnumber of aberrant crypt foci associated with human colorectal neoplasmcancer epidemiol biomarkers prev epub jul ohkubo h takahashi h yamada e sakai e higurashi t uchiyama t hosonok endo h taguri m nakajima a natural history of human aberrant cryptfoci and correlation with risk factors for colorectal cancer oncol rep takahashi h yoneda m tomimoto a endo h fujisawa t iida h mawatarih nozaki y ikeda t akiyama t yoneda m inamori m abe y saito snakajima a nakagama h life stylerelated diseases of the digestive systemcolorectal cancer as a life stylerelated disease from carcinogenesis tomedical treatment j pharmacol sci hosono k endo h takahashi h sugiyama m sakai e uchiyama t suzuki kiida h sakamoto y yoneda k koide t tokoro c abe y inamori mnakagama h nakajima a metformin suppresses colorectal aberrant cryptfoci in a shourtterm clinical trial cancer prev res phila the world medical association wma declaration of helsinki ethicalprinciples for medical research involving human subjects the ministry of health labour and welfare ethics guidelines for clinicalresearch paruchuri s mezhybovska m juhas m sjolander a endogenous productionof leukotriene d4 mediates autocrine survival and proliferation via cyslt1receptor signaling in intestinal epithelial cells oncogene dvash e hartal m barak s meir o rubinstein m leukotriene c4 is themajor trigger of stressinduced oxidative dna damage nat commun 0c" | Colon_Cancer |
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 |
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 |
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 |
] we have filtered only research s published in english language and selected the following keywords air pollution and covid19 or sarscov2 particulate matter or pm and covid19 or sarscov2 nitrogen dioxide or no2 and covid19 or sarscov2 we choose as inclusion criteria all the available epidemiological studies aimed to identify any temporal and spatial association between reported covid19 cases andor deaths and air pollution data related to pm25 pm10 and no2 thus excluding any letter opinion commentary review or nonrelevant s we obtained a total of eligible published research s in their final version and paper in its preprint version for some of them we chose to include only principal findings that clearly fit the aim this review particulate matter and covid19 atmospheric particulate matter pm is originated by a wide range of anthropogenic and natural sources kim it consists of a heterogeneous mixture of solid and liquid ps suspended in air that varies continuously in size and chemical composition including nitrates sulphates elemental and anic carbon anic compounds biological compounds and metals who it has been associated with increased respiratory morbidity and mortality liu especially in susceptible people due to cardiorespiratory events including asthma chronic obstructive pulmonary disease and thrombosis li rhee in vitro and in vivo studies highlighted its role in the exacerbation of respiratory viral infections becker and soukup recently the research group of setti gave first preliminary evidence that sarscov2 rna can be present on outdoor particulate matter thus suggesting that in conditions of atmospheric stability and high concentrations of pm it could represent a potential early indicator of covid19 although it does not give information regarding covid19 progression or severity several observations report a significant association between ambient concentrations of pm25 adhikari and yin bashir fattorini and regoli frontera jiang li vasquezapestegui wu yao zhu zoran 2020a and pm10 bashir coccia 2020b fattorini and regoli jiang li yao zhu zoran 2020a with covid19 pandemic across the most affected countries china italy and usa see table first evidences on the temporal association between air pollution and covid19 were reported in china where the outbreak was first identified zhu explored the relationship between particulate matter and the viral infection caused by the novel coronavirus in cities in china the authors included over of dailyconfirmed new cases in the whole of china between january 23rd and february 29th they applied a generalized additive model gam to examine the effects of meteorological factors and air pollution on covid19 incidence applying a movingaverage approach to capture the cumulative lag effect of ambient air pollution and considering population size and density as potential confounders they observed that the effect of pm25 on daily confirmed cases was greater than pm10 in particular they found that a 10μgm3 increase lag0 in pm25 and pm10 was associated with a ci to and ci to increase in the daily counts of covid19 confirmed cases respectively jiang focused their attention on three most affected cities of china wuhan xiaogan and huanggang collecting data of daily cases and ambient air pollutant from jan 25th to feb 29th the authors by applying a multivariate poisson regression revealed a significant temporal association between pm25 increased and covid19 incidence in all the considered cities especially in huanggang wuhan rr ci xiaogan rr ci huanggang rr ci conversely an increase in pm10 concentrations was associated with a decrease of covid19 incidence these results were partially confirmed by findings of li who conducted a simple linear regression to compare covid19 incidence with pm concentrations in wuhan and xiaogan from jan 26th to feb 29th in they found that an increase in pm25 was correlated with an increase of covid19 incidence in both cities wuhan r2 p xiaogan r2 p while for pm10 only in xiaogan r2 p the spatial distribution of particulate matter and case fatality rate cfr of covid19 was studied by yao in cities of china including wuhan collecting data up to march 22nd first they found a significantly positive global spatial autocorrelation of covid19 cfr global morans index i p highlighting a high cfr clustering located in hubei province with a multiple linear regression they adjusted their results for several effect modifiers and confounder factors such temperature relative humidity gross domestic product gdp per capita hospital beds per capita local indicators of spatial association lisa map values city size and population or proportion of people older than years it was found that for every μgm3 increase in pm25 and pm10 the cfr increased by and respectively and the risk estimates increased to and with every μgm3 increase in average concentrations of pm25 and pm10 in respectively some studies describe the association between air pollution and covid19 across italy the second country of the world where the infection spread significantly at the beginning of the pandemic and suddenly has reached many other european countries the 28th of july italy recorded more than total confirmed cases and deaths who most of which were distributed in the regions of northern italy especially the lombardy it is recognized as one the most air polluted areas of europe eea where the frequent pm10 annual exceedances of the who threshold of μgm3 are responsible for attributable deaths per year corresponding to attributable community rates of deaths per inhabitants per year baccini bontempi 2020bfocused the attention on two of the most affected regions of northern italy lombardy and piedmont the authors based on pm10 daily exceedances and covid19 confirmed cases on march 12th thus before the italian sanitary crisis observed that pm10 concentration was exceeded only few times among the lombard cities that at the beginning of the epidemic were most affected on the contrary among some piedmont cities suffering of severe pm10 pollution events covid19 incidence was lower based on their results the authors concluded that covid19 diffusion by airborne pm10 is hard to demonstrate nevertheless several research revealed how pm in particular pm25 could had a role in accelerate and vast diffusion of covid19 in northern italy for example coccia 2020b by analyzed data on italian province capitals and data of infected individuals up to april 7th revealed a relationship between air pollution of cities measured with days exceeding the limits set for pm10 in previous years and covid19 diffusion in particular cities with more than days of pm10 exceedances showed a very high average number of infected individual about infected individuals on 7th april whereas cities having less than days of pm10 exceedances showed a lower average number of infected about infected individuals frontera gave also evidences on the role of pm25 as a contributing factor of covid19 outbreak in northern italy where environmentalresearch19120201101293 0cc copat table summary table reporting reviewed results on the association between covid19 casesdeaths and air pollution pm25 pm10 and no2 references zhu data analysis generalized additive model gam aim temporal association between daily confirmed cases and air pollution pm25 pm10 and no2 temporal association between daily confirmed cases and air pollution pm25 pm10 and no2 temporal association between daily confirmed cases and air pollution pm25 pm10 and no2 spatial association between fatality rate and air pollution pm25 and pm10 spatial association between deaths counts and air pollution no2 temporal association between total cases daily confirmed cases and total deaths and air pollution pm25 and pm10 temporal association between total cases daily confirmed cases and total deaths and air pollution no2 spatial description of pm10 exceedances versus covid19 cases multivariate poisson regression simple linear regression multiple linear regression descriptive analysis percentage of deaths in three no2 μmol m2concentration range pearson coefficient correlation pearson coefficient correlation descriptive analysis number of days of pm10 exceeding μgm3 and covid19 incidence area of study cities of china period from jan 23rd to feb 29th jiang li yao ogen zoran 2020a zoran 2020b bontempi 2020b from jan 25th to feb 29th from jan 26th to feb 29th in data up to march 22nd data up to the end of feb from jan 1st to apr 30th from jan 1st to apr 30th from feb 10th to march 12th wuhan xiaogan and huanggang china wuhan and xiaogan cities of china administrative regions in italy spain france and germany milan italy milan italy provinces of lombardy italy provinces of piedmont italy coccia 2020b data up to april 7th italian provinces fattorini and regoli data up to april 27th italian provinces pm25 a 10μgm3 pm25 increase lag0 was associated with a increase of daily confirmed new cases pm10 a 10μgm3 pm10 increase lag0 was associated with a increase of daily confirmed new cases wuhan rr ci1032 xiaogan rr ci huanggang rr ci wuhan r2 p xiaogan r2 p wuhan rr ci xiaogan rr ci huanggang rr ci wuhan r2 p xiaogan r2 p Ï2 p a μgm3 increase in pm25 was associated with a increase in fatality rate Ï2 p a μgm3 increase in pm10 was associated with a increase in fatality rate no2 a 10μgm3 no2 increase lag0 was associated with a increase in daily confirmed new cases wuhan rr ci xiaogan rr ci huanggang no association found wuhan r2 p xiaogan r2 p of fatality cases are associated with no2 μmolm2 r cid0 r r cid0 r cid0 r r cid0 r cid0 r cid0 r cid0 lombardy pm10 exceeding between and covid19 incidence between and piedmont pm10 exceeding between and covid19 incidence between and covid19 in north italy has a high association with air pollution of cities measured with days exceeding the limits set for pm10 r2 p r2 p continued on next page hierarchical multiple regression model pearson regression coefficient analysis r2 p spatial association between confirmed cases and air pollution pm10 spatial association between total confirmed cases and air pollution pm25 pm10 and no2 environmentalresearch19120201101294 0cc copat table continued references frontera frontera wu adhikari and yin bashir bashir vasquezapestegui vasquezapestegui vasquezapestegui period data up to 31st march data up to 31st march data up to april 04th from march 1st to apr 20th from march 4th to april 24th from march 4th to april 24th data up to june 12th data up to june 12th data up to june 12th area of study italian regions italian regions counties in the usa queens county new york usa california california districts of lima perù districts of lima perù districts of lima perù aim spatial association between total confirmed cases and air pollution pm25 spatial association between deaths and air pollution pm25 prediction of risk of covid19 deaths in the long term average exposure to fine particulate matter pm25 temporal association between daily confirmed cases and total deaths and air pollution pm25 association between confirmed cases and air pollution pm25 pm10 and no2 association between deaths and air pollution pm25 pm10 and no2 spatial association between total confirmed cases and air pollution pm25 spatial association between deaths and air pollution pm25 spatial association between case fatality rate and air pollution pm25 data analysis pearson regression coefficient analysis pm25 r2 p pm10 pearson regression coefficient analysis r2 p longterm exposure increase of μgm3 in pm25 is associated with a increase in the covid19 death rate estimate on cases values cid0 ci estimate on deaths value cid0 ci kendall r cid0 spearman r cid0 zeroinflated negative binomia models negative binomial regression model spearman and kendall correlation tests spearman and kendall correlation tests no2 kendall r cid0 spearman r cid0 kendall r cid0 spearman r cid0 kendall r cid0 spearman r cid0 kendall r cid0 spearman r cid0 kendall r cid0 spearman r cid0 multivariate regression model crude coefficient p multivariate regression model crude coefficient p multivariate regression model crude coefficient cid0 p mortality was found significantly higher than less polluted italian regions by collecting data up to march 31st for all italian regions and performing a pearson correlation analysis they found a strong positive association both with the total number of confirmed cases r and deaths r other than with hospitalized cases r the italian situation was further highlighted by the study of fattorini and regoli in italian provinces they explored the spatial association between air pollution and covid19 cases with data up to april 27th by applying the pearson regression coefficient analysis they revealed a positive association both with pm25 and pm10 r2 p and r2 p respectively a focus on the most affected city of italy milan was conducted by zoran 2020a this city is located in the po valley basin known hotspot for atmospheric pollution at the continental scale eea the authors performed a temporal association between covid19 total cases daily new positive cases and total deaths and particulate matter from jan 1st and apr 30th by applying a person correlation in accordance with other studied they found a positive association between daily confirmed cases and pm25 r and pm10 r although they did not consider any delay time from infection to covid19 onset nevertheless they found a negative association between total cases and total deaths and particulate matter but the assumption of a temporal linear correlation may be inaccurate because the above mentioned variables could have more complex nonlinear relationships to date the usa have more than million confirmed cases and thousand deaths who here ambient concentrations of pm and o3 were found responsible to cause between and premature deaths fann the association between air pollutants and covid19 cases and deaths was studied by bashir in the state of california from march 4th to april 24th corresponding to the beginning of the covid19 outbreak in usa based on their significant correlation found the authors state that a limited human exposure to these pollutants will contribute to defeating covid19 this conclusion seems unclear because they found a negative correlation with pm25 and pm10 environmentalresearch19120201101295 0cc copat by applying both the kendall rank correlation and spearmans one and it is not clear if they normalized covid19 cases by population size and if they performed a day by day association or a spatial association across the country a focus on the queen county new york usa was provided by adhikari and yin they retrieved data of pm daily concentrations from two ground monitoring stations and collected data of confirmed covid19 cases and numbers of related deaths from usafacts in the period from march to april the authors elaborated their data with a negative binomial regression model and considered the cumulative lag effect of pm25 on disease outcomes over the past days they found a significant negative association among pm25 and new daily confirmed covid19 cases cid0 ci and deaths cid0 ci low pm concentrations in this area of study mean μgm3 are likely to have played a less central role in the spread of infection than in other areas such as italy where pm25 monthly concentrations reached values higher than μgm3 fattorini and regoli frontera or in china where pm25 monthly concentrations reached values higher than μgm3 zhu jiang as said by the authors other gaseous pollutants such as no2 and so2 could have influenced transmission and pathogenesis of covid19 in the united states wu investigated whether longterm average exposure to fine particulate matter pm25 increases the risk of covid19 deaths by considering approximately counties in the united states of the population with an exposure prediction model the authors calculated the county level longterm exposure to pm25 averaged for to and collected covid19 deaths counts up to april 04th they conducted a strong and robust statistical analysis with zeroinflated negative binomial mixed models adjusting their results by several potential confounders such as sociodemographic socioeconomic behavioural and meteorological factors they found that a small longterm exposure increase of only μgm3 in pm25 is associated with a increase in the covid19 death rate confidence interval ci vasquezapestegui recently reported first evidences on the spatial relationship between particulate matter and covid19 outbreak from latin america the authors described the situation occurred in districts of lima located in the second most affected country of latin america peru in particular by applying a multivariate regression model they evaluated the association between the population exposure to pm25 concentrations in the previous years and cases deaths and casefatality rates of covid19 with data up to june 12th a significant association has been found both with cases and deaths crude coefficient with p and with p respectively but not with case fatality rate all these studies highlight the role of pm in triggers of the covid19 disease and how government measures targeting to sustainable growth such as the reduction of urban and industrial emissions could have a positive impact on the prevention of health outcomes reducing mortality rate as well the burden on health care systems nitrogen dioxide no2 and covid19 induced lung damage hence viral infection becomes more common after exposure to no2 zhu furthermore no2 is associated with other several health effects such as elevated risks for asthma allergic rhinitis and eczema in children to increase of outpatient visits and hospitalizations due to bronchitis and asthma exacerbation bahrami asl kowalska increase of chronic obstructive pulmonary disease copd ghanbari ghozikali pfeffer and increase of pulmonary heart disease related mortality chen a recent study explored the possible role of no2 in interference in angiotensin converting enzyme ace2 the expression of ace2 is high on lung alveolar epithelial cells and it is the human cell receptor of virus agent of covid19 alifano first observations report an association between ambient concentrations of no2 and covid19 pandemic across europe china and usa bashir fattorini and regoli jiang li et al ogen zhu et al zoran et al 2020b conversely to the other papers findings of zoran 2020b and bashir provides different findings reporting no association or a negative one between no2 and daily deaths counts in china zhu by applying the same method explained for pm observed that a 10μgm3 increase lag0 in no2 is associated with a ci increase in the daily counts of covid19 confirmed cases in cities of china these findings are confirmed by jiang and li et a who applied the same method described for pm jiang revealed a significant positive association between no2 and covid19 both in wuhan and xiaogan wuhan rr ci1053 xiaogan rr ci but did not found any significant association in huanggang li found a significant linear correlation both in wuhan r2 p and xiaogan r2 p ogen presented evidences on the relationship between exposure to no2 including the months of january and february shortly before the covid19 spread in europe and novel coronavirus fatality in the most affected european countries concluding that longterm exposure to no2 may be a potential contributor to mortality caused by sarscov2 he collected data concerning the number of fatality cases from administrative regions in italy spain france and germany and correlated mortality with tropospheric no2 concentrations measured by the sentinel5 precursor spaceborne satellite the major tropospheric no2 hotspot identified was located in the northern italy in all european regions considered gas concentrations ranged between and μmolm2 with airflows directed downwards results showed that out of the fatality cases by march were in five regions located in north italy and central spain furthermore by analysing mortality trends it was revealed that the highest percentage of deaths occurred in regions where the maximum no2 concentration was above μmolm2 with a significant decrease where the maximum concentration was between and μmolm2 and below μmolm2 the methodology used by ogen cannot support a longterm exposure investigation surely a validation of the satellite measure with those of the ground ones the adjustment of the results according to the different population size of each country could have made their results more robust nevertheless the study provide new insights for future investigation the italian situation was further studied by fattorini and regoli who collected data of covid19 incidence up to april 27th from italian provinces they revealed a strong spatial correlation with no2 mean levels concentrations pearson coefficient r2 p confirming the northern italy being a hotspot of no2 in addition to urbanized cities of central and southern italy such as rome and naples a focus on the temporal association between ground levels of no2 and covd19 cases total cases daily new positive cases and total deaths was performed by zoran 2020b for the city of milan italy in the period pre and postlockdown measures the authors nitrogen dioxide is a nastysmelling gas formed by reaction in the atmosphere of nitrogen oxides nox with other chemicals nox is naturally produced in atmosphere by lightning kang et al volcanoes oceans and biological decay thurston the major outdoor anthropogenic sources of nox are primarily emissions from transportation and fuel combustion in particular in urban areas they comes from vehicle exhaust gases and domestic heating grange maawa the nitrogen dioxide has mainly effect on the respiratory system because an increase of the outdoor concentration of no2 may significantly increase the risk of respiratory tract infection this phenomenon is particularly evident in children as they are more susceptible to no2 environmentalresearch19120201101296 0cacknowledgments c copat found no2 negative correlated with all the considered epidemiological data but the methodology used has some limitations as the delay time from infection to the covid19 onset or covid19 death was not considered as well the significant reduction of air pollution due to lockdown measures since midmarch in usa the association was also studied by bashir for the state of california as discussed above for pm the authors found a negative correlation also between no2 levels and covid19 cases and mortality nevertheless they stated that this pollutant contributes to the spread of the disease based on these scientific evidences in addition to confirming that exposure to no2 is harmful to human health and increases the risk of incurring respiratory diseases it can be stated that exposure to no2 may be one of the most important trigger for the spread and fatality caused by the covid19 disease declare references adhikari a yin j shortterm effects of ambient ozone pm25 and the authors declare no conflict of interest we have no funding to bontempi e 2020b first data analysis about possible covid19 virus airborne alifano m alifano p fez p iannelli a reninangiotensin system at the meteorological factors on covid19 confirmed cases and deaths in queens new york int j environ res publ health httpsdoi103390 ijerph17114047 heart of covid19 pandemic biochimie httpsdoi101016j biochi202004008 baccini m biggeri a grillo p consonni d bertazzi pa health impact assessment of fine p pollution at the regional level am j epidemiol httpsdoi101093ajekwr256 bahrami asl f leili m vaziri y salahshour arian s cristaldi a oliveri conti g ferrante m health impacts quantification of ambient air pollutants using airq model approach in hamadan iran environ res httpsdoi 101016jenvres201710050 bashir mf ma bj bilal komal b bashir ma farooq th iqbal n bashir m correlation between environmental pollution indicators and covid19 pandemic a brief study in californian context environ res https doi101016jenvres2020109652 becker s soukup jm exposure to urban air particulates alters the macrophage mediated inflammatory response to respiratory viral infection j toxicol environ health httpsdoi101080009841099157539 bontempi e 2020a commercial exchanges instead of air pollution as possible origin of covid19 initial diffusion phase in italy more efforts are necessary to address interdisciplinary research environ res httpsdoi101016j envres2020109775 diffusion due to air particulate matter pm the case of lombardy italy environ res httpsdoi101016jenvres2020109639 bontempi e vergalli s squazzoni f understanding covid19 diffusion requires an interdisciplinary multidimensional approach environ res httpsdoi101016jenvres2020109814 bremner sa anderson hr atkinson rw mcmichael aj strachan dp bland j m bower js shortterm associations between outdoor air pollution and mortality in london occup environ med httpsdoi 101136oem564237 cai qc lu j xu qf guo q xu dz sun qw yang h zhao gm jiang qw influence of meteorological factors and air pollution on the outbreak of severe acute respiratory syndrome publ health https doi101016jpuhe200609023 carugno m dentali f mathieu g fontanella a mariani j bordini l milani g p consonni d bonzini m bollati v pesatori ac pm10 exposure is associated with increased hospitalizations for respiratory syncytial virus bronchiolitis among infants in lombardy italy environ res https doi101016jenvres201806016 chen h chen y lian z wen l sun b wang p li x liu q yu x lu y qi y zhao s zhang l yi x liu f pan g 2020a correlation between the migration scale index and the number of new confirmed coronavirus disease cases in china epidemiol infect e99 httpsdoi101017 s0950268820001119 chen j zeng j shi c liu r lu r mao s zhang l associations between shortterm exposure to gaseous pollutants and pulmonary heart diseaserelated mortality among elderly people in chengdu china environ health httpsdoi 101186s1294001905008 chen s prettner k kuhn m geldsetzer p wang c b¨arnighausen t bloom de 2020b covid19 and climate global evidence from countries medrxiv prepr serv health sci httpsdoi1011012020060420121863 coccia m 2020a how high wind speed can reduce negative effects of confirmed cases and total deaths of covid19 infection in society ssrn scholarly paper no id social science research network rochester ny httpsdoi 102139ssrn3603380 coccia m 2020b factors determining the diffusion of covid19 and suggested strategy to prevent future accelerated viral infectivity similar to covid sci total environ httpsdoi101016jscitotenv2020138474 balakrishnan k brunekreef b dandona l dandona r feigin v freedman g hubbell b jobling a kan h knibbs l liu y martin r morawska l pope ca shin h straif k shaddick g thomas m van dingenen r van donkelaar a vos t murray cjl forouzanfar mh estimates and year trends of the global burden of disease attributable to ambient air pollution an analysis of data from the global burden of diseases study lancet lond engl httpsdoi101016s0140673617305056 conticini e frediani b caro d can atmospheric pollution be considered a co factor in extremely high level of sarscov2 lethality in northern italy environ pollut barking essex httpsdoi101016jenvpol2020114465 croft dp zhang w lin s thurston sw hopke pk van wijngaarden e squizzato s masiol m utell mj rich dq associations between source cohen aj brauer m burnett r anderson hr frostad j estep k conclusion the scientific evidences collected in the literature highlight the important contribution of chronic exposure to air pollution on the covid19 spread and lethality although the potential effect of airborne virus exposure it has not been still demonstrated in particular it seems that pm25 and no2 are more closely correlated to covid19 than pm10 the lower correlation of pm10 with covid19 incidence and mortality can be due to the impossibility of particulate matter greater than μm to reach type ii alveolar cells where is located the cell entry receptor ace2 for sarscov2 nevertheless differences between countries such as the implementation of different lockdown restrictions stage of infection topographic sociodemographic and socioeconomic characteristics level of air pollution and meteorological factors may have contributed to obtain some contrasting finding although most of the revised studies support the relationship between air pollution and covid19 the manifold limitations of this review are the small number of papers collected and the great diversity of methodologies used sometimes lacking in some parts which makes the results difficult to compare the authors who first investigated this association although with great effort and rapidity of analysis dictated by a global emergency sometimes do not include all confounding factors whenever possible such as control policy urbanization rate availability of medical resources population size weather lifestyles sociodemographic and socioeconomic variables in addition to date incidence data are underestimated in all countries and to a lesser extent mortality data for this reason the cases included in the considered studies cannot be considered conclusive more studies are needed to better clarify the role of air pollution during the covid19 pandemic particularly studies that consider the multiplepollutants to strengthen scientific evidences and support firm conclusions useful to implement pandemic application plans to adequately prevent new health emergencies for a long time we have known that reducing outdoor and indoor air pollution in cities or countries can have a significant effect on health almost immediately and the benefits can far outweigh the costs surely the health emergency that the world is experiencing right now highlights how environmental research is a fundamental reference point to improve the knowledge concerning diseases of infectious origin and how all the intellectual and economic resources are to be spent to accelerate actions aimed to implement environmental policies act to reduce air pollution and develop new urban planning interventions influences or multidisciplinary studies declaration of competing interest the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper environmentalresearch19120201101297 0cc cop | Colon_Cancer |
neutrophils account for of circulating leukocytes and are the ï¬rst immune cells recruitedto an ammatory site they play an important role in the innate immune response topathogens as patients with neutropenia are highly susceptible to bacterial and fungal infections neutrophils perform numerous functions that target microbes including phagocytosis the releaseof antimicrobial peptidesproteases and netosis interestingly neutrophils have garneredconsiderable interest for their emerging and prominent roles in modulating cancer growth andmetastatic progression the roles played by neutrophils in the cancer setting are diverse andcomplex leading to the concept of neutrophil heterogeneityplasticity and the notion that distinctneutrophil subsets might existgranulopoiesisdiï¬erentiationand mobilization of maturefrom the bone marrow intocirculation this process begins with the commitment of granulocytemonocyte myeloidsegmented neutrophilsregulatedprocessthatinvolvestheisatightlyedited bybrahm segaluniversity at buffalo united statesreviewed byye liuniversity of texas md andersoncancer center united statesconnie jackamancurtin university australiacorrespondencepeter m siegelpetersiegelmcgillcaspecialty sectionthis was submitted tocancer immunity and immunotherapya section of the frontiers in immunologyreceived may accepted july published august citationhsu be shen y and siegel pm neutrophils orchestrators of themalignant phenotypefront immunol 103389ï¬mmu202001778frontiers in immunology wwwfrontiersinaugust volume 0chsu neutrophils orchestrating the malignant phenotypegmps which progressthrough a series ofprogenitorsneutrophil progenitors myeloblast promyelocyte myelocytemetamyelocyte band cell untilthey become a matureneutrophil in cancer dysregulated granulopoiesis hasled to theidentiï¬cation of diï¬erent neutrophil subsets that play a role intumor progression preneus comprise a neutrophil precursorpopulation that retain their proliferative capacity and expandin the bone marrow and spleen of tumor bearing mice preneus diï¬erentiate into immature and mature neutrophilswith the former found to accumulate in growing tumors anearly stage committed unipotent neutrophil precursor nep hasalso been identiï¬ed and their adoptive transfer into humanizedmice promoted solid tumor growth by inhibiting t cellactivation two neutrophil subsets highdensity neutrophilshdns and lowdensity neutrophils ldns were identiï¬ed invarious tumor models by diï¬erential density centrifugation hdns represent mature segmented neutrophils whereas ldnscomprise a heterogeneous mixture of mature and immatureneutrophils increasing mobilization of ldns into theperipheral blood was associated with enhanced tumor growthand metastasis functionsand antitumorigenicin addition to the identiï¬cation of distinct neutrophil subsetsneutrophils exhibit plasticity in response to tumorderivedfactors in a manner similar to macrophages neutrophils havebeen classiï¬ed into two categories n1 and n2 to describetheir prorespectively in vivo evidence has shown that tumorassociatedneutrophils tans can change their function from a protumor phenotype n2 to an antitumor n1 phenotypewith the addition of a tgf inhibitor arguing that tgfis an important factor driving the n2 phenotype incontrast signals associated with an antitumor n1 phenotypeinclude type iinterferons and those propagated by themet receptor however this categorization is likelyto represent an oversimpliï¬cation of neutrophil diversityneutrophil polarization similar to macrophages could alsorepresent a continuum of diï¬erent neutrophil phenotypespresent in the tumor microenvironment these advancesregarding the degree of neutrophil heterogeneityplasticityobserved in the cancer setting have sparked an intense andrenewed interestin this cell population while there areongoing discussions in the ï¬eld regarding the relationshipsubsets we directbetween pmnmdscs and neutrophilto excellentthe readerthatfully discussthese we will brieï¬y discuss antitumorrelationshipsneutrophilreview will primarilyroles of neutrophils and neutrophildiscussassociated functionsgrowth andmetastatic progressionfunctions howeverin promotingthe recentreviewstumorthisantitumor neutrophil functionscan participateantitumorneutrophilsmechanisms thattumor growth or eliminate cancercells a wellstudied neutrophilassociated function isin a variety oflimittheir ability to generate reactive oxygen species ros tolimit tumor progression upon tumor cell contact mousederived neutrophils can release hydrogen peroxide to eliminatemetastatic cancer cells in vitro subsequentlyit wasdemonstrated that expression of trpm2 transient receptorpotential cation channel subfamily m2 on tumor cells increasedtheirsensitivity to neutrophilmediated h2o2dependentcytotoxicity this occurred through a mechanism that involveda transient increase in ca2 mobilization within cancer cells trpm2 upregulation in tumor cells occurred followingan epithelialtomesenchymaltransition emt and cancercells that have undergone an emt were more susceptible toneutrophilmediated killing more recently an interactionbetween the receptor for advanced glycation end productsrage which is expressed on tumor cells and cathepsin gpresent on murine neutrophils was shown to mediate in vitrotumor cell cytotoxicity in a h2o2dependent manner the release of neutrophil ros is also dependent on the tumormicroenvironment in hypoxic tumor microenvironments theability of murine neutrophils to kill tumor cells in vivo throughthe release of ros is greatly diminished thus neutrophilshave the capacity to mediate rosdependent direct tumorcell killingcausingthe interplay of neutrophils with otherimmune celltypes can also indirectly limittumor progression tumorassociated neutrophils suppress the protumorigenic role ofil17 secreting Îδ t cells by inhibiting their proliferationlow glutathione levels in Îδ17 t cells rendered them sensitiveto neutrophilderived rosenhanced oxidativestress and reduced proliferation in earlystage humanlung cancer a subset ofimmature neutrophils have beenidentiï¬ed as having antigenpresenting functions and act topromote antitumor immunity by stimulating the secretionofinaddition to neutrophilt cellinteractions communicationbetween neutrophils and monocytes can also elicit antitumoreï¬ects nonmetastaticifnÎproducing monocytes to the lungs ifnÎ release activatestmem173sting within neutrophils whichstimulatesneutrophilmediated killing of disseminated cancer cells in thelungs ammatory cytokinesfrom t lymphocytescan mobilizecancercellsneutrophils have been shown to ltrate deposits of prostatecancer cells within bone metastases importantly neutrophilsimpaired bone metastasis progression by inhibiting stat5signal transducer and activator of transcription functionwithin prostate cancer cells resulting in their apoptotic cell death recently neutrophils have been reported to be involvedin antibodymediated trogocytosis a process that mechanicallydisrupts the plasma membrane of antibodyopsinized cancercells leading to a lyticnecrotictype cell death iga antibodiesagainst receptors expressed by cancer cells her2 egfr couldenhance neutrophilmediated trogocytosis of cancer cells if thecd47sirpα innate immune cell checkpoint was simultaneouslyblocked taken together these results demonstrate thatneutrophils can impair tumor growth and metastasis using acombination of direct and indirect cancer cell killing mechanismssupplementary table frontiers in immunology wwwfrontiersinaugust volume 0chsu neutrophils orchestrating the malignant phenotypefigure neutrophil functions that promote tumor growth and metastasis to support primary tumor growth neutrophils can mediate t cell suppression and altermacrophage differentiation neutrophil release of timp1 enhances tumor cell invasion by inducing epithelialtomesenchymal transition once in circulation circulatingtumor cells interact with neutrophils which enables tumor cell proliferation secretion of various proammatory markers such as il8 il1 or mmps can mediateincreased tumor cell extravasation in addition neutrophils can inhibit intraluminal nkmediated killing of circulating cancer cells leading to increased extravasation atthe metastatic site various systemic and microenvironmental factors can promote neutrophil ltration neutrophils can awaken dormant cancer cells by promotingecm remodeling and angiogenesis lastly continued growth of the metastatic lesion is facilitated by key neutrophildependent mechanisms which includeangiogenesis proliferation immune suppression and immune exclusion csf1 colony stimulating factor timp1 tissue inhibitor of matrix metalloprotease pdl1programmed death ligand tgf transforming growth factor ros reactive oxygen species mmp matrix metalloproteinases gmcsf granulocyte macrophagecolony stimulating factor angptl2 angiopoetin like2 fgf2 ï¬broblast growth factor ltb4 leukotriene b4 inos inducible nitric oxide synthase net neutrophilextracellular trap caf cancerassociated ï¬broblastneutrophil functions thatpromote primary tumor growthneutrophils promote primary tumor growth by variousmechanisms figure netosis is a process that involvesthe extrusion of neutrophilderived chromatin structures thatare decorated with neutrophil granule constituents whichform extracellular structures called neutrophil extracellulartraps nets normally netosis and net productionhave been described in the context of a neutrophils ability tocapture and kill bacteria extracellularly however netshave been shown to play an important role in the growth of aprimary tumor tumor microenvironmental changes includingtumorassociated coagulation and enhanced thrombosishave been linked to enhanced tumor growth several recentstudies suggest that netosis may play an important role inthese processes lps stimulation was shown to increase c3arexpression within neutrophils enhance netosis and increasecoagulation these events were correlated with n2 neutrophilpolarization and increased tumor growth interestingly ithas recently been shown that immature neutrophils preferentiallyrespond to cancer cell derived c3a to promote their migration subsequently it was shown that breast cancer cells thatexpressed high levels of gcsf and il1 exhibited highneutrophil counts and tumorassociated thrombosis which wasdependent on net formation pharmacological blockadefrontiers in immunology wwwfrontiersinaugust volume 0chsu neutrophils orchestrating the malignant phenotypeof il1 receptor signaling reduced net formation attenuatedtumorassociated thrombosis and impaired tumor growth nets can also directly uence cancer cell proliferationneutrophil elastase ne present within nets activates tumorcells to increase mitochondria biogenesis and atp productionthereby further enhancing the growth of cancer cells in addition to the impact of nets neutrophils canalso interact with other immune cells through additionalmechanisms to promote tumor growth neutrophilderived roscan inhibit t cell proliferation creating an immunosuppressiveenvironment that is supportive of tumor growth phenotypiccharacterization and singlecell rna sequencing identiï¬ed aneutrophil subset that is cd84hi which exhibited potent t cellsuppressive activity and increased ros production in amodel of gastric cancer neutrophils were activated by tumorderived gmcsf that resulted in elevated programmed deathligand pdl1 expression these pdl1 neutrophils wereable to suppress t cell function and promote tumor growth secretion of mmp9 matrix metalloproteinase fromltrating neutrophils activates latent tgf and induces tcell suppression and tumor growth in a colorectal cancer model siglecfhigh neutrophils in lung adenocarcinoma createdan immunosuppressive environment by promoting macrophagediï¬erentiation causing the release of high levels of rosand enabling tumor progression together these ï¬ndingsindicate that neutrophils that ltrate diverse primary tumorscan modify the local environment in diï¬erent ways to favortumor growthneutrophil functions thatpromote metastasisthe ability of cancer cells to leave the primary tumor anddisseminate to distant ans represents the deadliest aspectof cancer progression indeed the emergence of metastaticcancer accounts for ¼ of cancer related deaths themetastatic cascade represents a series of barriers to cancercells and neutrophils have been found to assist cancer cells insuccessfully navigating several of these distinct steps figure supplementary table local invasionintravasationltrating neutrophils within primary tumors are associatedwith an increase in emt enhanced metastasis and pooroutcomes mechanisticallyof matrixmetalloprotease timp1 secreted by neutrophils induced anemt and consequently increased the migration and invasion oftumor cells cancer cells that had undergone an emt expressedcd90 which enhanced timp1 secretion by neutrophils in acontactdependent manner inhibitortissuesurvival in circulationextravasationthe ability of circulating tumor cells ctcsto surviveis criticalfor metastasis formation the formation ofheterotypic cancer cellneutrophil clusters was found to greatlyincrease metastatic ï¬tness using a 4t1 breast cancer modelit was demonstrated that ctcneutrophil interactions reliedon vcam1 dependent adhesion which enhanced cancercell proliferation and increased metastasis indirectlyneutrophils can also inhibit nk cellmediated tumor clearancein circulation thereby increasing the intraluminal survival ofdisseminated tumor cells in this study 4t1 breast cancer cellswere injected subcutaneously to mobilize murine neutrophilsly6gfollowing which d2a1 breast cancer cells wereinjected intravenously mice bearing 4t1 cells exhibited reducedclearance of d2a1 cells from the lungs when compared to micethat were not injected with 4t1 cells depletion of nk cellsresulted in enhanced d2a1 cancer cell accumulation in the lungswhile neutrophil depletion had the opposite eï¬ect cancer cells that have survived in circulation must exitthe bloodstream and extravasate into tissue parenchyma neutrophils have been shown to regulate the extravasationprocessthrough several mechanisms neutrophilderivedfactors can diminish the integrity of the endothelial barrierpermitting cancer cellsil8il1 and matrix metalloproteasesmmp8 and mmp9released from neutrophils activated endothelial cells reducedendothelialtransendothelialmigration and accelerated the rate of cancer cell extravasation to extravasate more easilyincreasedfunctionbarriersitesnetosis and net constituents can support cancer cellextravasation through enhanced trapping of ctcs withinmetastaticimportantly blocking netosisdecreases cancer cell adhesion and inhibits metastatic spread tothe lung and liver furthermore changes within speciï¬cmetastatic microenvironments such as exposure to ozoneor redox imbalance triggered netosis and led to increasedentrapment of cancer cells in the lung and enhanced metastasis collectively these studies show that neutrophils play animportant role in enhancing tumor cell survival and increasedextravasation which promote cancer metastasisrecruitmentearly seedingsurvivalsystemic and tumorderived factors have been implicatedin neutrophilin the premetastatic nichetumorderived il1 induces Îδ t cells to produce il17aand granulocytecolony stimulating factor gcsf whichresults in the recruitment of immunosuppressive neutrophilsto the lung gmcsf and il5 have been shown topromote the expansion and recruitment of prometastaticneutrophils in the lungs of obese mice which promotes lungmetastasis angiopoetinlike2 angptl2 secreted byosteosarcoma cells implanted in the tibia stimulates lungepithelial cells which led to the accumulation of neutrophilsin the lung and enhanced lung metastatic burden in the lung neutrophils secrete ltb4increases theinitiating cellsproliferation of ltb4rpositive metastasis activation of notch1 in colorectalcellsdrives tgf2dependent recruitment of immunosuppressiveneutrophils within the liver which enabled the formation of livermetastases cancerthatnets also support early cancer cell seeding and colonizationof metastases induction of nets by ovarian tumorderivedfactors has been shown to be important in promoting metastasisfrontiers in immunology wwwfrontiersinaugust volume 0chsu neutrophils orchestrating the malignant phenotypeto the omentum in the liver nets have also beenshown to promote metastasis by activating cancerassociatedï¬broblasts growth in the metastatic siteneutrophils have been shown to promote the growth ofmetastases after seeding minor subclones of breast cancer cellsthat secrete il11 and figf cfosinduced growth factor cansupport the formation of polyclonal metastases composed ofdriver and passenger subpopulations these il11 producingsubclones activated il11 responsive mesenchymal stromal cellswhich induced chemokine secretion and subsequent recruitmentof prometastatic neutrophils tumor cellderived gmcsfwas shown to stimulate neutrophils to synthesize and secretetransferrin an iron transport protein which has mitogenicactivity that promotes lung metastatic growth when taken up bycancer cells a recurring function of prometastatic neutrophils is theirability to create an immunosuppressive microenvironmentthat support metastasis within lung metastasesinduciblenitric oxide synthase inos producing neutrophils havebeen shown to limit cd8 t cell dependent antitumorresponses by promoting immune suppression recently p53deï¬cient cancer cells were found to increase the expressionof wnt ligands which in turn upregulated il1 productionfrom tumorassociated macrophages high il1 levelsengaged Îδ17 t cells which subsequently enhanced neutrophilrecruitment that promoted the formation of lung metastases furthermore loss of elf5 e74like transcription factorexpression in triplenegative breast cancer led to increased ifnÎ signaling resulting in the expansion of immunosuppressiveneutrophils in addition to tumorderived factors a lackof systemic testosterone levels can lead to an impairmentof antitumor neutrophil functions a shift toward immatureneutrophils was observed in castrated male mice leading toincreased neutrophilderived ros and suppression of nk cellactivation that promoted increased lung metastatic burden intwo melanoma models recently a role for net formationhas been described for the continued growth of establishedmetastases nets released during cancer progressionwas shown to limitthe ability of nk and cytotoxic tcells to eliminate cancer cells speciï¬cally net formationimpaired direct contact between the cancer cells and cytotoxicimmune cells nk and t cells inhibition of netosis with aprotein arginine deiminase pad4 inhibitor synergized withimmune checkpoint inhibitors to control tumor growth andmetastasis proangiogenic functions have long been ascribed forneutrophils which revealed that neutrophilderived proteasessuch as mmp9 could release stored angiogenic factors vegffgfs that were stored in the local environment to enable bloodvessel formation recently a diï¬erent mechanism bywhich neutrophils enhance angiogenesis has been describedthe synthesis and secretion of ï¬broblast growth factor fgf2 by neutrophils in the liver microenvironment drivesangiogenesis and growth of nascent colorectal cancerderivedhepatic metastases dormantresidual disease andtherapy resistanceneutrophils have also been implicated in awakening dormantcancer cells lpsinduced tissue ammation led to metastaticoutgrowth of dormant tumor cells in a neutrophildependentmanner mmp9 produced by neutrophils can trigger thegrowth of dormant cancer cells by remodeling extracellularmatrix and releasing potent angiogenic factors ne andmmp9 which are enzymes associated with nets can cleavethe extracellular matrix ecm leading to integrinmediatedsignaling which awakens dormant cancer cells and promotescancer cell growth severalstudies have shown that neutrophils promoteresistance to therapy doxorubicin and paclitaxel resistant breastcancer cells express more il17 and cxcr2 ligands whichincreases neutrophil recruitment a neutrophilenrichedsubtype characterized in triple negative breast cancer tnbcdetermined that neutrophils were largely immunosuppressiverendering these tumors resistant to immune checkpoint blockadetherapy in a genetically engineered mouse model ofsarcoma neutrophils promote resistance to radiation therapyby activating mitogenactivated protein kinase mapk pathway in addition cd177 neutrophil ltrates in colorectalcancer patients are associated with adverse outcome in patientsreceiving bevacizumab [antivascular endothelial growth factora vegf a] furthermore lysyl oxidaselike loxl4expressing neutrophils that ltrated colorectal cancer livermetastases were found to identify patients that were resistant toantiangiogenic therapy metabolic programming inneutrophilsrecentinteresthasbeenconceptin thethereofimmunometabolism and the realization that altered cellularmetabolism in ltrating immune cells can have a signiï¬cantimpact on tumor growth and metastasis neutrophilsare typically viewed as a cell type that is heavily reliant onglycolysis to perform their eï¬ector functions consistentwith this notion neutrophils have very few mitochondria andinhibitors of oxidative phosphorylation oxphos do notalter their rates of oxygen consumption howeverduring tumor progression neutrophils have been shown toundergo a metabolic switch which involves the upregulationof genes associated with oxphos fatty acid metabolism andglycolysis figure neutrophils isolated from lewis lungcarcinoma exhibit increased ï¬ux through oxphos glycolysisand increased atp production compared to naïve neutrophilssuggesting that multiple metabolic strategies are engaged intumor ltrating neutrophils recently upregulation offatp2 fatty acid transport protein in neutrophils was shownto increase lipid accumulation in these cells fatp2 regulated theuptake of arachidonic acid which was subsequently convertedto prostaglandin e2 neutrophilderived prostaglandin e2 wasfrontiers in immunology wwwfrontiersinaugust volume 0chsu neutrophils orchestrating the malignant phenotypefigure metabolic changes in cancerassociated neutrophils neutrophils which possess few mitochondria are reliant on glycolysis to generate atp to fueleffector functions such as phagocytosis generation of reactive oxygen species and netosis in cancer neutrophils upregulate oxidative phosphorylation oxphosand fatty acid transporters to mediate many neutrophil functions including migration and t cell suppression under nutrient limiting conditions such as low glucoseneutrophils can reprogram their metabolism to break down fatty acids or utilize certain amino acids glutamate proline to fuel protumorigenicprometastaticfunctions ppp pentose phosphate pathway glut glucose transporter mct monocarboxylate transporter tca tricarboxylic acid cycle fatp2 fatty acidtransport protein aa arachidonic acid pge2 prostaglandin e2found to be important or neutrophilmediated cd8 t cellsuppression and tumor growth metabolic ï¬exibility refers to the ability of a cell to shiftbetween one metabolic program to another in response tochanging metabolic demands or nutrient supply high metabolicï¬exibility increases the cells ability to survive various andeverchanging metabolic microenvironments neutrophilsubpopulations can also exhibit metabolic ï¬exibility figure in breast cancer splenic neutrophils can engage mitochondrialdependent fatty acid oxidation as a predominate fuel sourceto support ros production and maintain t cell suppression under glucoselimiting conditions similar to certain tumormicroenvironments immature ldns have been shown to utilizeoxphos to generate atp that is required to support theirprotumorigenic functions indeed immature ldns can supportnetosis under nutrient limiting conditions via mitochondrialdependent amino acid catabolism which is importantforeï¬cient breast cancer liver metastasis in addition thelongevity of neutrophils could also be altered due to the enhancedmetabolic ï¬exibility the ex vivo halflife of mouse circulatinghdns and ldns was and h respectively suchobservations raise the intriguing possibility that under certainconditions distinct neutrophil subsets may not be as shortlived as previously thought these studies argue that increasedmetabolic ï¬exibility in distinct neutrophil populations may beimportant for cellular functions that can uence tumor growthand metastatic progressionclinical importance futureperspectives on treatmentin keeping with their protumorigenicmetastatic functions thepresence of neutrophils across diï¬erent cancers was shownto be strongly associated with adverse patient outcomes frontiers in immunology wwwfrontiersinaugust volume 0chsu neutrophils orchestrating the malignant phenotypeamong certain subtypes of breast cancer er the presence ofa neutrophil ltrate in the primary tumor is also indicativeof worse patient outcomes furthermore in patients withadvanced cancers serum il8 levels and neutrophil ltrationare associated with worse overall survival and diminishedresponse to immune checkpoint inhibitors the mobilization of neutrophils into circulation also hasprognostic signiï¬cance the neutrophiltolymphocyte rationlr is an important risk stratiï¬cation and treatment selectiondiagnostic tool for cancer patients a high nlr is associated withpoor prognosis in many solid human cancers a highnlr is also associated with decreased overall survival in patientswith tnbc or metastatic breast cancer an important and unanswered question with respect to thenlr is the type of neutrophil that is being detected in thesepatients are they high or lowdensity neutrophils interestinglyldns have been identiï¬ed in patients with breast cancer lungcancer head and neck cancers urologic cancers and lymphoma in patients with advanced lung cancerit wasreported that higher proportion of ldns predictedpoorer survival these observations are in keeping withthe protumorigenic and prometastatic functions associatedwith ldnn2 neutrophils while most studies reveal a negativeprognostic impact of neutrophils in cancer there was one studythat associated the presence of a cd16high cd62dim neutrophilsubset with increased survival of head and neck squamous cellcarcinoma patients these observations highlight the needfor better markers that are capable of discriminating betweenneutrophils that exert antitumor vs those that mediate protumormetastatic eï¬ectsmechanistic insights have greatly advanced our knowledgeoftumorderived factorstumor growth andmetastasis in a neutrophildependent manner additional studiesimpactthatreferences sipsas nv bodey gp kontoyiannis dp perspectives for the management offebrile neutropenic patients with cancer in the 21st century cancer 101002cncr20890 kolaczkowska e kubes p neutrophil recruitment and function in healthand ammation nat rev immunol 101038nri3399inde visser ke neutrophilssb wellenstein md coï¬eltcancer neutral no more nat rev cancer 101038nrc201652 cowland jb borregaard n granulopoiesis and granules of humanneutrophils immunol rev 101111imr12440 evrard m kwok iwh chong sz teng kww becht e chenbone marrow neutrophilstraï¬ckinganalysisspecializedpopulationsexpansioninofal developmentaljetrevealsand 101016jimmuni201802002functionseï¬ectorimmunity79e8 zhu yp padgett l dinh hq marcovecchio p blatchley a wu r identiï¬cation of an early unipotent neutrophil progenitor with pro tumoralactivity in mouse and human bone marrow cell rep 41e8 101016jcelrep201807097 sagiv jy michaeli j assi s mishalian i kisos h levy l phenotypicdiversity and plasticity in circulating neutrophil subpopulations in cancercell rep 101016jcelrep201412039focused on characterizing the phenotypic and functional role ofneutrophils in cancer it may be possible to develop strategies thatspeciï¬cally target those neutrophil subsets that actively promotetumor growth and metastasis while sparing those neutrophilsthat possess antitumor and antimicrobial functions finallythe emerging concept of metabolic ï¬exibility that is exhibited bycertain neutrophil subsets may aï¬ord new ways of targeting theseprotumorigenicmetastatic neutrophilsauthor contributionsbh ys and ps wrote the review and prepared the ï¬guresall authors contributed to the and approved thesubmitted versionfundingwork from the authors laboratory cited in this review wassupported by an operating grant to ps from the cancer researchsociety and the terry fox research institute and québecbreast cancer foundation grant bh acknowledgessupport from the charlotte and leo karassik foundation phdfellowship and the rolande and marcel gosselin graduatestudentship ys holds an entrance studentship from thegoodman cancer research centre ps is a mcgill universitywilliam dawson scholarsupplementary materialthe supplementary materialfor this can be foundonline at httpswwwfrontiersins103389ï¬mmu202001778fullsupplementarymaterial coï¬elt sb kersten k doornebal cw weiden j vrijland k hau cs il17producing gammadelta t cells and neutrophils conspireto promote breast 101038nature14282cancer metastasis nature hsu be tabaries s johnson rm andrzejewski s senecal j lehuede c immature lowdensity neutrophils exhibit metabolic ï¬exibility thatfacilitates breast cancer liver metastasis cell rep 15e6 101016jcelrep201905091 fridlender zg sun j kim s kapoor v cheng g ling l polarizationof tumorassociated neutrophil phenotype by tgfbeta n1 versus n2tan cancer cell 101016jccr200906017 ohms m möller s laskay t an attempt to polarize human neutrophilstoward n1 and n2 phenotypes in vitro front immunol 103389ï¬mmu202000532jablonska j leschner s westphal k lienenklaus s weiss s neutrophilsresponsive to endogenous ifnbeta regulate tumor angiogenesis andgrowth in a mouse tumor model j clin invest 101172jci37223 finisguerra v di conza g di matteo m serneels j costa s thompsonaa met is required for the recruitment of antitumoural neutrophilsnature 101038nature14407 ostuni r kratochvill f murray pj natoli g macrophages and cancer frommechanisms to therapeutic implications trends immunol 101016jit201502004frontiers in immunology wwwfrontiersinaugust volume 0chsu neutrophils orchestrating the malignant phenotype brandau s moses k lang s the kinship of neutrophils and granulocyticmyeloidderived suppressor cells in cancer cousins siblings or twins semincancer biol 101016jsemcancer201302007 vols s sionov rv granot z always look on the bright side antitumor functions of neutrophils curr pharmac design granot z henke e comen ea king ta norton l benezra r tumorentrained neutrophils inhibit seeding in the premetastatic lung cancer cell 101016jccr201108012 gershkovitz m caspi y fainsodlevi t katz b michaeli j khawaled s trpm2 mediates neutrophil killing of disseminated tumor cells cancer res 10115800085472can173614 gershkovitz m fainsodlevi t khawaled s shaul me sionov rvcohendaniel l microenvironmental cues determine tumor cellsusceptibility to neutrophil cytotoxicity cancer res 10115800085472can180540 sionov rv fainsodlevi t zelter t polyansky l pham ct granotz neutrophil cathepsin g and tumor cell rage facilitate neutrophilanti8e1624129 1010802162402x20191624129cytotoxicity oncoimmunologytumor mahiddine k blaisdell a ma s crequergrandhomme a lowell caerlebacher a relief of tumor hypoxia unleashes the tumoricidal potentialof neutrophils j clin invest 101172jci130952 mensurado s rei m lanca t ioannou m goncalvessousa n kubo h etal tumorassociated neutrophils suppress protumoral il17 gammadeltat cells through induction of oxidative stress plos biol 16e2004990 101371 pbio2004990 singhal s bhojnagarwala ps obrien s moon ek garfall al rao as etal origin and role of a subset of tumorassociated neutrophils with antigenpresenting cell features in earlystage human lung cancer cancer cell 101016jccell201606001et hagerling c gonzalez h salari k wang cy lin c roblescooperationtumor prevents metastatic progressionaliinduced byof breast cancer proc natl acad sci usa 101073pnas1907660116eï¬ector monocyteneutrophilimmunethe primary costanzogarvey dl keeley t case aj watson gf alsamraae myu y neutrophils are mediators of metastatic p | Colon_Cancer |
" pharmacology and toxicology laboratory csirinstitute of himalayan bioresource technology preproof 0c preproofinfection f0b7 systemic oxidative stress and inflammation are significant outcomes of sarscov2 highlights f0b7 activated gsk3 following sarscov2 infection provoke the oxidative stress and inflammation in the host f0b7 gsk3 phosphorylates nucleocapsid protein of sarscov2 and helps in disease progression f0b7 inhibition of gsk3 can be a suitable target in curbing of covid19 pandemic 0cwith the host defence mechanism by the help of gsk3 protein the virally infected cells show the coronavirus disease covid19 outbreak caused by severe acute respiratory syndrome coronavirus sarscov2 had turned out to be highly pathogenic and transmittable researchers throughout the globe are still struggling to understand this strain's aggressiveness in search of putative therapies for its control crosstalk between oxidative stress and systemic inflammation seems to support the progression of the infection glycogen synthase kinase3 gsk3 is a conserved serinethreonine kinase that mainly participates in cell proliferation development stress and inflammation in humans nucleocapsid protein of sarscov2 is an important structural protein responsible for viral replication and interferes activated gsk3 protein that degrades the nuclear factor erythroid 2related factor nrf2 protein resulting in excessive oxidative stress activated gsk3 also modulates crebdna activity phosphorylates nfκb and degrades catenin thus provokes systemic inflammation preproofinteraction between these two pathophysiological events oxidative stress and inflammation enhance mucous secretion coagulation cascade and hypoxia which ultimately leads to multiple ans failure resulting in the death of the infected patient the present review aims to highlight the pathogenic role of gsk3 in viral replication initiation of oxidative stress and inflammation during sarscov2 infection the review also summarizes the potential gsk3 pathway modulators as putative therapeutic interventions in combating the covid19 keywords covid19 gsk3 nfκb nucleocapsid protein oxidative stress sarscovpandemic list of abbreviations ace2 angiotensinconverting enzyme ad alzheimers disease adp adenosine diphosphate aiibb3 glycoprotein iibiiia ards acute respiratory distress syndrome 0care antioxidant response elements asc apoptosisassociated specklike protein containing a card atp adenosine triphosphate balf bronchoalveolar lavage bzip basic leucine zipper cats catalase cbp creb binding protein covid19 coronavirus disease creb camp response elementbinding protein cul3 cullin gpx glutathione peroxidase gsh intracellular glutathione gsk3 glycogen synthase kinase3 damp death associated molecular pattern gcsf granulocyte colony stimulating factor hcv hepatitis c virus hdac3 histone deacetylase ho1 heme oxygenase1 ifnΠinterferongamma preproofnfκb nuclear factorκb nlrp3 nodlike receptors protein mcp1 monocyte chemoattractant peptide mip1α macrophage inflammatory protein 1α myd88 myeloid differentiation primary response nadph nicotinamide adenine dinucleotide phosphate hydrogen ikk ikb kinase il6 interleukin iraks interleukin il 1rassociated kinase iκb inhibitor of kappa b keap1 kelchlike ech associated protein licl lithium chloride nlrp3 nucleotidebinding domain nodlike receptor protein nox nadph oxidase nprotein nucleocapsid protein nrf2 nuclear factor erythroid 2related factor 0cntd nterminal domain o superoxide anion o2 oxygen molecule oxpls oxidized phospholipids pamp pathogen associated molecular pattern par proteaseactivated receptors pd parkinsons disease pedv porcine epidemic diarrhea virus ros reactive oxygen species sarscov2 severe acute respiratory syndrome coronavirus tak1 transforming growth factor tgfactivated kinase tf tissue factor tirap tirdomaincontaining adaptor protein sgmrna sub genomic messenger rna sods superoxide dismutase ppr pattern recognition receptor psgl pselectin glycoprotein ligand1 rigi retinoic acidinducible gene i preproofvwf von willebrand factor xo xanthine oxidase xor xanthine oxidoreductase tlr3 toll like receptor3 tnf tumor necrosis factor tnfr tumor necrosis factor receptor tnfα tumour necrosis factoralpha traf6 tumour necrosis factor receptor associated factor trs transcription regulating sequence introduction in late december wuhan china got attention worldwide after getting several patients diagnosed with pneumonia following a viral infection on 11th february the pathogenic strain of the virus was taxonomically designated as severe respiratory syndrome coronavirus sarscov2 by the international committee on taxonomy of viruses ictv the 0cassociated diseased condition was termed covid19 by the world health anization who the who announced sarscov2 virus infection a pandemic as it infected nearly million persons and engulfed more than worldwide sarscov2 is a member of coronaviruses consisting of kb singlestranded positivesense rna as genetic material it shows genetic similarity between another human coronavirus ie sarscov while similarity with bat coronavirus ratg1 and shares a high similarity index with pangolin coronavirus respiratory droplets are the primary source of viral transmission either through nasopharyngeal or oral route dry cough and high fever are the sarscov virusassociated respiratory disease replication within the host cell in disease progression the present review provides an inthe infected cells however in the case of sarscov2 infection aggressive inflammation significant symptoms observed in patients within days following viral infection the disease pathophysiology of covid19 also shows a close resemblance with previous reported and oxidative stress help in viral replication and damage the airway epithelium cell that results in acute respiratory distress syndrome ards which makes the condition worst glycogen synthase kinase3 gsk3 is a serinethreonine evolutionary conserved central molecule that the majority of respiratory viral infections are associated with the recruitment of immune cells the release of proinflammatory cytokines oxidative stress and finally phagocytosis of mainly participates in cell proliferation migration development apoptosis and immune regulation acquired and innate activation of gsk3 is associated with suppression of host immunity and inhibition of antioxidant response it is also supporting viral genome preproofdepth knowledge of oxidative stress inflammation and viral replication related to gsk3 during sarscov2 infection further the review highlights the gsk3 pathway modulators' gsk3 is a versatile serinethreonine kinase that regulates glycogen metabolism it consists of two isoforms gsk3α and gsk3 encoded by two separate genes both the isoforms share sequence similarity between kinase domains despite they never compensate for each other's' loss of function gsk3 has two prime functional domains a substratebinding domain which acquires substrates to gsk3 while the other kinase domain is responsible for phosphorylation of the substrate the nterminal region of gsk3 contains atp binding domain whereas the cterminal region consists of a large conserved activation loop responsible for the enzyme's full activation activation of gsk3 depends on the siteputative role as therapeutic interventions in combating the covid19 pandemic gsk3 structure 0cspecific phosphorylation that is controlled by various kinases gsk3 prefers prephosphorylate substrate by recognizing consequence sequences stxxxphosphost on substrate gsk3 is also involved in wntcatenin and sonic hedgehog cell signalling pathways mediating in cell proliferation differentiation maturation and cell adhesion transcription factors cjun creb stat3 cebpα nfat myc nfκb and p53 are the major substrate of gsk3 that can manipulate the expression of several other genes impaired activity of gsk3 has recognized in several clinical conditions such as metabolic disorders cancers alzheimer's disease ad parkinson's disease pd bipolar disorders and various other neurodegenerative diseases sarscov2 infection and inflammation covid19 patients' systemic cytokine profile shows a close resemblance with cytokine release syndrome characterized by macrophage activation an elevated level of cytokines like tumour necrosis factoralpha tnfα interleukin6 il6 and interferongamma ifnΠfurther elevated levels of these cytokines trigger ards characterized by a low level of oxygen in the severity of symptoms and death in sarscov2 infected patients depends on the viral infection and is greatly affected by the aggressive behaviour of the host immune system blood and difficulty in breathing leading to the death of the infected patients previous data on sarscov demonstrated that the virus predominately affects the endothelium cells of preproofin counterdefence the virus encodes numerous immunesuppressive proteins that help employs the same host receptor angiotensinconverting enzyme ace2 for infection like sarscov indicating that both the viruses target the same set of cells for infection the as an antagonist of interferon signalling interruptions in interferon signalling happened at various stages preventing the recognition of viral rna through pattern recognition receptor expression of the ace2 receptor is reduced in the lungs following sarscov infection disrupting the reninangiotensin system that affects fluidelectrolyte balance blood pressure it to evade from host immune response and helps in replication similarly to counter such problem sarscov2 evolves with numerous structural and nonstructural proteins that act the airway alveoli vascular system and macrophages in the pulmonary an sarscov2 increases the vascular permeability and inflammation in the airway ppr inhibiting the synthesis of type i interferon protein via interrupting the tolllike receptor1 tlr1 and retinoic acidinducible gene i rigi signalling disturbing stat signalling and initiating the host mrna degradation and interrupting host translation machinery fig1 0cat the time of replication cytopathic viruses including sarscov2 show a massive death and injury of the infected epithelial and endothelial cells triggering the excessive release of cytokines and chemokines in addition to this inflammationinduced cell deathpyroptosis also observed in sarscov2 patients that further provoke the systemic inflammatory response pyroptosis signalling proceeds via nodlike receptors protein nlrp3 present on the cell membrane activate caspase1 through asc apoptosisassociated specklike protein containing a caspase recruitment domain adaptor protein activated caspase1 further triggers the synthesis of proinflammatory cytokines such as il1 and il6 fig1 these cytokines further attract the other immune cells mostly tlymphocytes and monocytes at the site of infection bronchoalveolar lavage balf fluid from the sever lymphocyte and immune cells' requirement at the site of infection in most of the patients these recruited cells clear the infection recedes the inflammatory response and leads to recovery however some patients show cytokine storms because of an imbalance in the population of monocytederived fcn1 macrophage in addition to these responses sever cases of sarscov2 infection also disclose a significant expansion in the population of proinflammatory monocytes cd14 and cd16 in the peripheral blood as compared to mild covid19 patients showed ccl2 and ccl7 chemokines which require the recruitment of ccr2 monocytes further balf analysis also revealed a highly inflammatory around of sarscov2 infected patients show lymphopeniainfiltration of preproofsevere hospitalized covid19 patients' blood plasma exhibits a higher level of alleviation in the t cell population which is more noticeable in severe cases the level of helper t cell cd4 cytotoxic t cell cd8 and regulatory t cell were below the average level in severe cases of covid19 as compared to mild cases cd8 t cells directly attack and kill the virusinfected cells while cd4 participates in the production of cytokines to recruit other immune cells at the same time regulatory t cell maintains the normal immune homeostasis along with inhibition of proliferation the proinflammatory activity of maximum immune cascade that further inflames the lungs sarscov2 infected patients also show cases lymphocytes natural killer cells and bcells fig1 granulocyte colonystimulating factor gcsf il2 il6 il10 monocyte chemoattractant peptide mcp1 macrophage inflammatory protein 1α mip1α and tnfα the blood plasma of the infected patients shows a significantly higher level of il6 in severe cases compared to mild or nonsevere cases which further contributes to macrophage activation syndrome pulmonary infiltrationbased assessment in ards patients also revealed that a 0cmore significant portion of lung injury is associated with a higher level of il6 in peripheral blood all of this evidences suggest that sarscov2 infection is responsible for dysregulation of the host immune system with the abnormal synthesis of cytokines chemokines and a decrease in the level of lymphocytes that ultimately leads to cytokine storm responsible of multian failure role of nuclear factorκb in disease progression nuclear factorκb nfκb is the leading player that responds immediately following the a pathogenic stimulus provoked by a bacteria or a virus invasion exposure of mitogen proinflammatory cytokines growth factors and stress activates ikb kinase ikk which relb and crel are grouped in firstclass characterized by the presence of transactivation domain while nfkb1 p50 and nfkb2 p52 belongs to the second group that is devoid of transcriptionalmodulation activity so both the classes of proteins need to be heterodimerized with each other to perform their functions under normal physiological conditions rela and p50 the heterodimer's predominant form is inactivated in the cytoplasm by ikb protein pathogen's invasion by promoting inflammation controlling cell proliferation and survival nfκb is a heterodimeric transcription factor that belongs to the rel protein family there are 05rel proteins present in mammalian cells that further divided into two classes rela p65 preproofmembranelike tolllike receptor tlr pathogen associated molecular pattern pamp and death associated molecular pattern damp are inflammatory stimulating molecules suggested that the nucleocapsid protein of sarscov directly interacts with nfκb translocate it to the nucleus and finally upregulates il6 gene expression ample of shreds of evidence is there that shows sarscov directly or indirectly activates nfκb protein excessive cytokine release especially il6 plays a crucial role in sarscov2 infection and further progression of pathogenic conditions nfκb is a transcription factor that controls the expression of proinflammatory genes responsible for the cytokine storm a study following infection nfκb also activated by receptors present on the cell surface further phosphorylates and degrades ikb protein via ubiquitination process released by virusinfected cells which act as ligands for tlr subsequently activating nfκb protein via myd88dependent pathway oxidative stress is another important factor responsible for cytokine storm generation via crosstalk between nuclear factor erythroid related factor nrf2 and nfκb pathway nfκb suggested as a negative regulator of nrf2 driven genes either by recruiting histone deacetylase hdac3 which promote local histone hypoacetylation or deprive the cbp creb binding protein fig1 0c sarscov2 infection and oxidative stress oxygen is a crucial molecule in the aerobic system to maintain normal life processes under normal cellular conditions the oxygen molecule utilized to generate chemical energy in the form of atp in a very tight and controlled manner the oxygen molecule combustion generates a small number of reactive oxygen species ros which utilized for usual cell signalling cascades ros are oxygen molecules with an unpaired electron that behaves as free radicals and reactive metabolites several ros forms were discovered so far such as peroxidase oxygenfree radicals nitrogen oxide and singlet oxygen molecules generally ros associated cellular damage is processed via sophisticated antioxidant machinery involving both enzymatic catalase cats superoxide dismutase sods and glutathione peroxidase gpx and nonenzymatic glutathione and nicotinamide adenine dinucleotide phosphate mitochondrial dna get degraded under the influence of oxidative stress subsequently hydrogen [nadph] mechanisms in normal physiological conditions the antioxidant systems can work simultaneously to combat the exceeded levels of ros however in a pathological state ros overwhelmed the antioxidant mechanism and generated oxidative stress in cells all the crucial cellular components such as proteins lipids nuclear and the available literature of clinical and preclinical experiments proposed that oxidative preproofensures the clearance of the virus but due to imbalanced host immune system they also start to release excessive cytokines that further aggravate to cytokine storm the recruited phagocytic cell participates in ros generation along with inflammatory response nicotinamide adenine dinucleotide phosphate oxidases nadph oxidase and xanthine cov2 infection activates the host airway epithelium and alveolar macrophage further releasing cytokines to attract another immune cell from the blood neutrophils and monocyte that further differentiate into macrophage at the site of injury recruitment of these cells burst is another prompting factor for mortality following sarscov infection sarstriggering the process of cell death oxidase xo are the two wellknown enzymes responsible for oxidative stress in respiratory viral infections nadph oxidase nox is an evolutionary conserved membranebounded enzyme complex that catalyzes the molecular oxygen into superoxide humans nadph oxidase family consists of members nox15 duox1 and duox2 its cterminal region comprises nadph binding site flavin adenine dinucleotide binding domain while the nterminal region consists of transmembrane α helical domains with four conserved hemebinding sites nox2 is predominantly expressed in the recruited 0cphagocytes neutrophils and macrophages at the viral infection site and contributes to oxidative stress a study reported that alveolar macrophage depended oxidative stress is responsible for acute lung injury progression following h5n1 viral infection in mice mostly via oxidized phospholipid and superoxide however the same pathological events reduced following the suppression of p47phox a regulatory subunit of nox2 in a study influenza a virusinfected nox2y mice showed reduced oxidative stress improved alveoli epithelium condition less production of superoxide and reduced airway inflammation compared to wild type mice fig inflammation xor is converted into xo by oxidation of cysteine amino acid or calciumin superoxide synthesis via nox2 enzyme complex xanthine oxidase xo is another dependent proteolysis xo shows more affinity toward molecular oxygen resulting in the transfer of a univalent and divalent electron to oxygen that further generates superoxide and ros generating enzyme that participates in oxidative stress following respiratory viral infection in the mammalian system this enzyme is existing in interchangeable form between hydrogen peroxide respectively fig2 in vitro rhino viruss infection in primary bronchial and a549 respiratory epithelial cell lines decreased the intracellular glutathione xo to xanthine oxidoreductase xor xor is predominantly distributed in healthy tissues and reduces nad to nadh by utilizing electron form substrate while during similarly ex vivo influenza a virusinfected alveolar macrophage exhibited an increase preproofdecreased superoxide generation thus revealed that xo also participates in oxidative stress during infection in vivo analysis also revealed that xo is the main contributor to and serum analysis however allopurinol and chemical modified superoxide dismutase decreased the oxidative stress and mortality rate this evidences revealed that xo also superoxide synthesis during a respiratory viral infection mouse infected with influenza viral showed a higher mortality rate which found to be associated with xo and superoxide in balf gsh level leading to oxidative stress via enhanced superoxide production serine protease inhibitor or xo inhibitor oxypurinol treatment enhanced the intracellular levels of gsh and participates in the viral associated disease progression via oxidative stress a part of these activated phagocyte releases prooxidant mediators such as tnf and il1 which further enhances the oxidative stress in host cells during viral infection tnf binds with the complex ii of the mitochondrial respiratory chain hampering oxidative phosphorylation via restricting electrons transport as a result the electron transport chain becomes leakier and lastly it enhances superoxide production tnf also helps in detachment of nfκb protein from ikb complex resulting in suppression of antioxidant gene expression via binding to their 0c1keap1 binding domain keap1 is a cystine rich and cytoplasmic protein whose npromoter region following translocation from the cytoplasm to the nucleus fig during stress condition neutrophils also release lactoferrin along with lysosomal protein under the influence of il1 which further binds to iron and start to accumulate in the reticuloendothelial system when an ironbinding threshold reached superoxide ions combine with free iron to generate hydroxyl radicals via fenton reaction and enhances oxidative stress nrf2 a key regulator of antioxidant genes nrf2 is the main transcription factor that plays an important role to overcome oxidative stress it is a basic leucine zipper bzip protein that belongs to the cap n collar family of transcription factors nrf2 consist of highly conserved functional domain termed as neh nrf2ech homologies neh1 is a leucine zipper domain through which nrf2 interact with other transcription factors whereas neh2 is the kelchlike ech associated protein however during stress conditions nrf2 detached from the keap1 protein translocate to the terminal domain binds with cul3dependent e3 ubiquitin ligase complex while cterminal domain binds with nrf2 protein under normal physiological conditions keap1 protein nucleus heterodimerize with small musculoaponeurotic fibrosarcoma mafs proteins and finally initiate or supress the transcription of genes that consists of electrophile response elements ere or antioxidant response elements are in their promoters nrf2 regulates preproofbecause of its highly vascular nature and indirect contact with environmental oxidant which had already proven in numerous of respiratory disease it was found that lungspecific nrf2 conditional knockout rodents showed pulmonary protective behaviour in respiratory disorders more than genes expression belonging to oxidative stress inflammation autophagy metabolism and excretion the pulmonary system is more exposed to oxidative stress ubiquitinates the nrf2 resulting in its proteasomal degradation infection systemic oxidative stress and inflammation linked thrombus formation in sarscovabnormal coagulation a higher level of ddimers and low platelet count are the signs of poor prognosis and significant reasons for multiple an failure and death in severe cases of covid19 microthrombus had reported in the lungs the heart the kidneys and the brain of covid19 patients cytokine storm induces aberrant coagulation by expressing the tissue factor tf pathway tf is a member of cytokine receptor 0csuperfamily and type i integral membrane glycoprotein which is highly abundant in the vasculature subendothelium especially in the brain lungs gut skin as well as in the monocytes in response to proinflammatory cytokines especially il6 the expression of tf is upregulated in the monocytes and the perivascular cells resulting in tf exposure to circulation the exposed portion of tf forms a complex with circulating factor vii thus enhance its catalytic activity that further activates downstream circulating factors such as ix and x activated factor x participates in the transformation of prothrombin into thrombin that finally leads to the formation of blood clots by conversion of fibrinogen into fibrin fig main consequences of the cytokine storm that also provokes thrombin production via proteasediphosphate adp thromboxane a2 translocate cell adhesion molecule pselectin and cd40 ligand on the surface of platelet along with activation of the integrin aiibb3 receptor the released thromboxane a2 and adp trigger activation of neighbouring platelets via activated receptors par signalling pathway par is a unique gprotein coupled cell surface receptor that carries its ligand and remains inactive until unmasked by proteolytic cleavage by the tffactorviia complex thrombin mediated par activated platelets undergo a morphological transformation release platelet activators such as serotonin adenosine thromboxane receptor and p2y12 respectively activated aiibb3 on platelets' surface binds with von willebrand factor vwf and fibrinogen that contributes to platelet aggregation platelet activation different proinflammatory events and fibrin clot formation are the preproofalong with cytokine storm oxidized phospholipids oxpls also participate in the recognition receptors in an experimental model of acute lung injury oxpls triggered cytokine storm release via tlr4triftraf6nfκb pathway il6 further promoted tf platelets during viral infection adherent leukocyteplatelets interaction provides positive feedback to amplify the overall inflammatory response and procoagulation events these activated endothelium cells following par signalling also exposes cell adhesion molecules eselectin pselectin icam1 and vcam1 and expresses monocyte chemo coagulation cascade via the tlr4 receptors present on the monocytes and endothelium cells attractant proteini that facilitates recruitment adhesion and migration of leukocytes and events are prothrombotic which further contributes to blood clot formation fig oxpls concerned as pams patterns that are recognized by numerous conserved patternexpression on monocytes and activated the endothelium cell to express monocyte adherent protein for their requirement which finally participated in inflammatory events thrombotic complications can be reduced in preexisting metabolic and cardiovascular disorders in covid19 patients by interfering with the oxpls activated monocyte or 0cwhich consists of domains including the nterminal domain ntddomain cterminal domain ctddomain and linker region lkrdomain nterminal domain enriched with endothelial cell additionally during inflammation the natural anticoagulant pathways such as tf pathway inhibitors or antithrombin are nearly diminished subsequently facilitating coagulation cascade gsk3 and sarscov2 infection the virus has to undergo many complex processes that are tightly regulated to infect a host cell it begins with viral genomic rna entry into the host cytosol transcription and finally budding off as viral progeny these viral progenies are similar to their parent in morphology and function that consists of structural proteins spike s protein envelop e protein matrix m protein and nucleocapsid n protein the n protein of severe acute respiratory syndrome coronavirus is the most abundant protein existing in an infected host cell among all be responsible for nuclear localization signal the cterminal domain of the n protein is also responsible for protein dimerization both the domains of n protein ie domain and protein sequencing also revealed that n protein is highly conserved among the species preproofinterestingly to perform such activity nprotein should recognize the viral genomic nucleocapsid protection of viral genome timely replication and proper transmission is the capsid's primary function nprotein also inhibits host cell proliferation and cytokines by rna associate with it and finally oligomerize by selfassociation to form capsid or terminal domain mapped between and amino acids is enriched with lysin thought to arginine motif responsible for the multimerization of the nprotein and predicted as a hot spot region for phosphorylation in brief nprotein divided into three main domains that play diverse functions during different stages of the virus life cycle nprotein is a type of capsid protein whose primary function is to pack the virus's genomic rna into the protective positive charge amino acids which is responsible for binding with viral rna whereas cother proteins covering domain are linked to each other through linker region domain that consists of serineinteracting with elongation factors 1α resulting in a halt of translation mechanism moreover the nprotein of sarscov also hijacks the host innate immune system for the progress of new viral progeny and associated disease development posttranslational phosphorylation of the virus nprotein is essential for their activity which results in an increased affinity of nprotein toward virus rna rather than nonviral rna gsk3 found 0cto be an essential kinase responsible for the phosphorylation of nprotein on the serine residue in linkerregion fig sarscov infected veroe6 cells showed an reduction in viral titer and cytopathic effects following the treatment of gsk3 inhibitor kenpaullone and lithium chloride thus suggested phosphorylation by this kinase be strongly linked with the viral replication several subgenomic mrnas synthesized due to discontinued transcription mechanisms during the coronavirus replication which encodes major structural proteins transcription regulating sequence trs responsible for the discontinuous transcription process exists in front of each gene body trs and after the leader sequence leader trs templateswitching events happen via base pairing between the body trs and leader trs to synthesize the discontinuous minusstranded rnas discontinuous nested plusstrand this discontinuous transcription mechanism tightly controlled for the successful compilation participate in discontinues to a continuous process of virus replication moreover of the virus life cycle among all the structural proteins nprotein tightly regulates the discontinued transcription mechanism as the synthesis of subgenomic mrna is reduced subgenomic mrna transcribed from the previously generated minusstranded rnas phosphorylation of nprotein at the serinearginine motif also inhibits the tra | Colon_Cancer |
" immune checkpoint inhibitors that block programmed cell death1 pd1 and programmed cell death ligand1 pd l1 have improved outcomes for many cancer subtypes but do exhibit toxicity in the form of immune related adverse eventsobjective the aim of this study was to investigate the emerging toxicities of pd1 and pd l1 inhibitors including acute or reactivation of tuberculosis tb and atypical mycobacterial infection amimethods this study was completed as a retrospective review using the us food and drug administration adverse events reporting system faers for incidence of tb and ami due to pd1 and pd l1 inhibitors compared with other fda food and drug administration approved drugs the statistical methods included disproportionality signal analysis using the reporting or ror to compare cases the wald ci was reported to assess the precision of the rorresults out of the adverse events aes reported to faers for all drugs between january and march aes were due to the five fda approved pd1pd l1 inhibitors seventy two cases of tb were due to pd1pd l1 inhibitors specifically cases due to nivolumab due to pembrolizumab due to atezolizumab and due to durvalumab there were cases of ami due to nivolumab due to pembrolizumab and each due to durvalumab and atezolizumab avelumab was not attributed to any ae of tb or ami from analysis of the faers database the calculated ror for tb due to pd1pd l1 inhibitors was ci to p00001 and for ami was ci to p00001 pd1pd l1 inhibitors used in the treatment of cancer subtypes is associated with increased tb and ami risk although this complication is rare clinicians using pd1pd l1 inhibitors should be aware of the risksintroductionimmune checkpoint inhibitors icis that block programmed cell death1 pd1 and programmed cell death ligand1 pd l1 have transformed care for many cancer subtypes and have improved outcomes for patients with pd l1 overexpression1 through blockade of the pd1pdl1 axis the t lymphocyte mediated response against tumour cells is enhanced resulting in accelerated immune mediated destruction of key questionswhat is already known about this subject º case reports and case series suggest programmedcell death1programmedcell death ligand1 pd1pd l1 inhibitors are associated with acute tuberculosis tb or reactivation of tbwhat does this study add º this is the first large systemic effort to quantify the risk of tb due to pd1pd l1 inhibitors through retrospective analysis of faers food and drug administration adverse events reporting system a pharmacovigilance database pd1pd l1 inhibitors were not only associated with increased risk of tb compared with other drugs but atypicalmycobacterial infection as wellhow might this impact on clinical practice º although this complication is rare clinicians using pd1pd l1 inhibitors should be aware of thiscancer cells however facilitating immune mediated activation is not benign and patients receiving icis are known to exhibit unique toxicities that result in an damage known as immune related adverse events iraes3 the most common iraes with pd1 and pd l1 inhibitors are fatigue pruritus and diarrhoea4 some iraes can be fatal with pneumonitis hepatitis neurotoxicity and most commonly myocarditis reported5 while counterintuitive when the mechanism of action is considered an emerging and increasingly reported toxicity of pd1 and pd l1 inhibitors is acute tuberculosis tb and reactivation of tb6 the first case of tb due to the pd1 inhibitor was described in a patient with relapsed hodgkins lymphoma who developed pulmonary tb following treatment with pembrolizumab7 since then there have been other case reports of tb following initiation of pd1 or pd l1 inhibitors that make the development of tb a relevant concern8 in a preclinical mouse study pd1 deficient mice were found to be highly susceptible to tb with reduced survival compared with wild type mice12 however anand a0k et a0al esmo open 20205e000866 101136esmoopen2020000866 0copen accesstable adverse events of tb and ami due to pd1pdl1 inhibitors from january to march in faerstotal aes due to all drugs in faerstotal aes due to pd1pdl1 inhibitorstotal aes due to pd1 inhibitorstotal aes of tb in faerstotal aes of tb due to pd1pdl1 inhibitorstotal aes of ami in faerstotal aes of ami due to pd1pdl1 inhibitorsror calculation ror for tb due to pd1pdl1 inhibitors versus full database ror for ami due to pd1pdl1 inhibitors versus full database ci to ci to aes adverse events ami atypical mycobacterial infection faers food and drug administration adverse events reporting system pd1 programmed cell death1 pd l1 programmed cell death ligand1 ror reporting or tb tuberculosisthere is no current risk estimate describing the potential risk of developing tb or atypical mycobacterial infection ami from pd1 and pd l1 inhibitors in this study we retrospectively reviewed the us food and drug administration adverse events reporting system faers a pharmacovigilance database for the risk of tb and ami due to pd1 and pdl1 inhibitors compared with other fda foodand drug administration approved drugsmethodsthis study is a retrospective analysis that used data queries from the faers pharmacovigilance monitoring database faers is a public database that contains nearly million adverse event ae reports medication error reports and product quality complaints reported by healthcare professionals manufacturers and consumers from around the world since these reports are managed by fda and evaluated by clinical reviewers in the center for drug evaluation and research and the center for biologics evaluation and research date in each event report where applicable include individual case identification numbers for reference the suspected pharmaceutical agent treatment indication adverse reactions nature of the event ie serious outcomes eg hospitalised death other outcomes sex male female or unknown age weight event date initial fda receipt date latest fda receipt date pharmaceutical company reporter eg healthcare professional consumer pharmaceutical company unknown concomitant medications latest manufacturer received date country where the event occurred and manufacturer control number individual names and date of birth are excluded from these liststhe present study involved data queries of the faers database between january and march for aes secondary to pd1 inhibitors namely pembrolizumab and nivolumab and pd l1 inhibitors namely atezolizumab durvalumab and avelumab in all aes due to above five drugs we then searched for three aes specifically tuberculosis pulmonary tuberculosis and atypical mycobacterial infection tuberculosis and pulmonary tuberculosis were grouped together for analysis all other events that were reported in patients with tb or ami were characterised into subcategories including pulmonary infectious endocrine gastrointestinal hepatobiliary dermatological cardiac haematological neurological vascular infusion related rheumatological and otherstb and ami cases among patients treated with pd1 and pd l1 inhibitors were compared with all reported tb and ami events in the database due to other drugs by conducting a disproportionality signal analysis based on the reporting or ror the ror is a measure of the magnitude of association between an exposure to a pharmaceutical agent and the odds of a specific outcome occurring in the setting of an elevated ror it can be conferred that there is an elevated risk of an adverse event occurring with a specific medication the wald ci was used to assess the precision of the ror when lower limit of ror and ci did not cross ror was considered significant13 the likelihood of association between pd1pd l1 inhibitors and tbami were investigated using two sided Ï2 or fishers exact tests as warranted all analyses were conducted using sas sas institute inc cary north carolina usa and statistical significance was defined as p005resultsbetween january to march a total of adverse events report cases were generated in faers out of ae there were associated with the approved five pd1pd l1 inhibitors the majority of aes were reported with nivolumab and pembrolizumab at in faers there were reports of tb with any drug of which were reported with pd1pdl1 inhibitors the ror for tb due to pd1pd l1 inhibitors was elevated at ci to p00001 for ami there were reports associated with all drugs of which were due to pd1pd l1 inhibitors the anand a0k et a0al esmo open 20205e000866 101136esmoopen2020000866 0ctable details of tb ae due to pd1pdl1 inhibitorstable continuedopen access serious nivolumab pembrolizumab atezolizumab durvalumab lung cancer gastric cancer head and neck cancer hodgkins lymphoma malignant melanoma colon cancer neuroendocrine carcinoma ovarian carcinoma pancreatic carcinoma plasma cell myeloma renal cell carcinoma transitional cell carcinoma unknowntotal number of tb aespd1pdl1 inhibitor used indication for pd1pdl1 use type of reaction sex median age years range min maxoutcome reporter year initial report received region of origin of ae died hospitalised life threatening other outcome asia europe americas africa australia healthcare professional consumer male female n54 continuedsuspected drug pd1pdl1 inhibitor pd1pdl1 inhibitor ¥ aes adverse events pd1 programmed cell death1 pd l1 programmed cell death ligand1 tb tuberculosisror for ami due to pd1pd l1 inhibitors was elevated at ci to p00001 table out of cases of tb due to pd1pd l1 inhibitors were due to nivolumab followed by due to pembrolizumab and due to atezolizumab and durvalumab respectively there were no cases reported with avelumab the most common indication for which pd1pd l1 inhibitor was used was lung cancer median age of the whole cohort was years eighty per cent of the patients were men and were women out of cases cases had a reported outcome of death the most common region of origin in which tb was reported was asia sixteen cases had pd1pd l1 inhibitors plus one of more non checkpoint inhibitor drug listed as a suspect drug leading to ae table out of cases of ami due to pd1pd l1 inhibitors were due to nivolumab followed by due to pembrolizumab and each due to durvalumab and atezolizumab no report of ami attributable to avelumab was found the most common reason for use of pd1pd l1 inhibitor was lung cancer median age of the entire cohort was years seventy three per cent of patients were men and were women out of cases patient died the most common region in which ami was reported was asia one case had pd1pd l1 inhibitors plus one of more non checkpoint inhibitor drug listed as a suspect drug leading to ae table patients who had tb due to pd1pd l1 inhibitors also had additional reported pulmonary complications in of cases followed by other infectious complications in of cases similarly patients who had ami attributed to use of pd1pd l1 inhibitors had pulmonary complications in of cases followed by endocrine dermatological and others in of the cases table discussionin this retrospective pharmacovigilance database review pd1pd l1 inhibitors had a statistically significant positive signal with tb and ami with a proportion of these events associated with mortality nivolumab had the highest frequency of reported tb and ami whereas avelumab had no reported events most commonly affected patients were receiving treatment for lung cancer and the most commonly reported country of origin was japananand a0k et a0al esmo open 20205e000866 101136esmoopen2020000866 0copen accesstb has a high disease burden worldwide with the highest disease associated mortality of any infectious agent in there were million new cases globally and million reported deaths14 amis are estimated to occur in approximately to per persons with an increasing incidence in developed countries15 there is growing evidence that patients receiving icis can develop tb reaction while on treatment6 to date there are reported cases of tb secondary to icis none of which were attributed cytotoxic t lymphocyte associated protein ctla4 inhibitors median time to diagnosis from ici initiation was months range to months17 the mechanism by which a pd1pd l1 inhibitor results in tb is not clear in a murine study pd1 knockout mice had decreased survival compared with wild type mice following exposure to mycobacterium tuberculosis furthermore pd1 inhibition is needed to prevent cd4 t cells from promoting development of tb18 pd1 inhibition mitigates over production of interferon gamma ifnγ which is important for host resistance to tb19increased risk of tb and ami is also found in patients on tumor necrosis factor tnf alpha inhibitors and janus kinase jak inhibitor ruxolitinib20 patients treated with infliximab a tnf alpha inhibitor were and times more likely to develop tb and ami respectively22 in patients prior to start tnf alpha inhibitors screening for latent tb is recommended20 if the patient is found to have latent tb treatment with isoniazid is recommended as it substantially reduces the risk of developing tb reactivation23 however a recent study suggests that pd1 inhibition induced tb reactivation is actually driven by tnf alpha and use of tnf alpha inhibitor could reverse this complication24 there are currently no recommended screening guidelines for latent tb prior to starting pd1pd l1 inhibitors a single institution study in germany found that of patients had positive test for quantiferon gold tb plus qgt prior to starting icis however none of the patients who had a positive qgt test developed tb while on treatment with icis6 of the cases of tb reported in literature due to icis treatment with icis was stopped in all cases tb treatment was initiated and seven cases had re initiation of icis out of seven who had re initiation of ici five had response to therapy one had progression and in one case response was not available17 as tb reactivation may lead to treatment interruptions or discontinuation standardised recommendations for tb screening in patients with planned ici should be considered with substantiation of results from the current study in prospective studiesthis is the first study using faers to demonstrate the potential risk of developing tb and ami in pd1pd l1 inhibitor treated patients as pd1pd l1 inhibitors use becomes more prevalent on a global scale including regions with an elevated prevalence of latent tb clinicians need to consider the risk benefit and economic impacts of screening for latent tb and treatment initiation if the patient is positive these questions cannot be table details of ami ae due to pd1pdl1 inhibitors serious male female nivolumab pembrolizumab atezolizumab durvalumab lung cancer head and neck cancer malignant melanoma unknowntotal number of ami aespd1pdl1 inhibitor used indication for pd1pdl1 use type of reaction sex median age years range min maxoutcome reporter healthcare professionalyear initial report received region of origin of ae suspected drug died hospitalised life threatening other outcome pd1pdl1 inhibitor pd1pdl1 inhibitor ¥ asia europe americas n10 aes adverse events ami atypical mycobacterial infection pd1 programmed cell death1 pd l1 programmed cell death ligand1answered in this observational signal analysis and future prospective research studies should be conducted if a patient develops tb or ami while on treatment with pd1pd l1 inhibitors permanent discontinuation of therapy should be avoided if there is clear clinical benefit from ici and multidisciplinary discussions regarding treatment delay should be conducted with the treating oncologist and infectious disease specialists a majority anand a0k et a0al esmo open 20205e000866 101136esmoopen2020000866 0copen accesstable other aes grouped into major an systems in patients treated with pd1pdl1 inhibitorstb death events n13tb alive events n59tb totaln72ami death events n1ami alive events n12ami totaln13pulmonaryinfectiousendocrinegastrointestinalhepatobiliarycardiachaematologicaldermatologicalneurologicalvascularinfusion relatedrheumatologicalothers aes adverse events ami atypical mycobacterial infection pd1 programmed cell death1 pd l1 programmed cell death ligand1 tb tuberculosisof patients in whom tb or ami have reported are those with lung cancer it is worth pointing out that especially in patients with lung cancer there is significant difficulty in differentiating immune pneumonitis or radiation pneumonitis true disease progression or infectious causes prospective studies of iraes should include testing for tb or ami in diagnostic work upfrom pseudoprogression this study has limitations this analysis was a retrospective study of reported events in faers and as such baseline characteristics including presence of latent tb was not known moreover the actual incidence of tb or ami due to pd1pd l1 inhibitors cannot be determined because faers reports patients with aes not total number of patients taking the medication furthermore it is likely that not all cases of tb that occur in the clinical setting are reported within faers as such there are similar limitations in ror estimate ae reporting for a drug may be influenced by extent of use publicity and bias25 although the use of disproportionality analysis through pharmacovigilance databases to determine the increased risk of aes secondary to particular drug has been shown in various settings25 it is critical that any hypothesis generated by using pharmacovigilance databases are validated through prospective studiespd1pd l1 inhibitors used in treatment for cancer is associated with increased risk of tb and ami the most common drug in faers attributed to tb and ami is nivolumab in this study lung cancer was the most common indication for which use of pd1pd l1 inhibitor leads to tb or ami although this complication is rare clinicians using icis should be aware of this anand a0k et a0al esmo open 20205e000866 101136esmoopen2020000866possibility currently there is no additional data available to support or refute the need to screen patients for latent tb prior to initiation of icis prospective studies are needed to address these questions as well as indications to initiate prophylactic therapytwitter vivek subbiah viveksubbiahcontributors conceptualisation ka gs and spi data collection and analysis ka gs je and spi statistics je first draft preparation ka gs and spi review and final approval all authorsfunding the authors have not declared a specific grant for this research from any funding agency in the public commercial or not for profit sectorscompeting interests vs and spi coi can be found onlinepatient consent for publication not requiredprovenance and peer review not commissioned externally peer revieweddata availability statement data are available in a public open access repository all data relevant to the study are included in the article or uploaded as supplementary information faers used for this study is public databaseopen access this is an open access article distributed in accordance with the creative commons attribution non commercial cc by nc license which permits others to distribute remix adapt build upon this work non commercially and license their derivative works on different terms provided the original work is properly cited any changes made are indicated and the use is non commercial see a0http creativecommons licenses by nc orcid idskartik a0anand http orcid vivek a0subbiah http orcid references coit dg thompson ja albertini mr et a0al cutaneous melanoma version nccn clinical practice guidelines in oncology j natl compr canc netw ettinger ds wood de aggarwal c et a0al nccn guidelines insights non small cell lung cancer version j natl compr canc netw 0copen access postow ma sidlow r hellmann md immune related adverse events associated with immune checkpoint blockade n engl j med wang y zhou s yang f et a0al treatment related adverse events of pd1 and pd l1 inhibitors in clinical trials a systematic review and meta analysis jama oncol wang dy salem j e cohen jv et a0al fatal toxic effects associated with immune checkpoint inhibitors a systematic review and meta analysis jama oncol langan ea graetz v allerheiligen j et a0al immune checkpoint inhibitors and tuberculosis an old disease in a new context lancet oncol 202021e55 lee jjx chan a tang t tuberculosis reactivation in a patient receiving anti programmed death1 pd1 inhibitor for relapsed hodgkin's lymphoma acta oncol fujita k terashima t mio t anti pd1 antibody treatment and the development of acute pulmonary tuberculosis j thorac oncol chu y c fang k c chen h c et a0al pericardial tamponade caused by a hypersensitivity response to tuberculosis reactivation after anti pd1 treatment in a patient with advanced pulmonary adenocarcinoma j thorac oncol 201712e111 anastasopoulou a ziogas dc samarkos m et a0al reactivation of tuberculosis in cancer patients following administration of immune checkpoint inhibitors current evidence and clinical practice recommendations j immunother cancer picchi h mateus c chouaid c et a0al infectious complications associated with the use of immune checkpoint inhibitors in oncology reactivation of tuberculosis after anti pd1 treatment clin microbiol infect lázár molnár e chen b sweeney ka et a0al programmed death1 pd1 deficient mice are extraordinarily sensitive to tuberculosis proc natl acad sci u s a rothman kj lanes s sacks st the reporting odds ratio and its advantages over the proportional reporting ratio pharmacoepidemiol drug saf anization wh global tuberculosis report cassidy pm hedberg k saulson a et a0al nontuberculous mycobacterial disease prevalence and risk factors a changing epidemiology clin infect dis 200949e124 prevots dr shaw pa strickland d et a0al nontuberculous mycobacterial lung disease prevalence at four integrated health care delivery systems am j respir crit care med zaemes j kim c immune checkpoint inhibitor use and tuberculosis a systematic review of the literature eur j cancer barber dl mayer barber kd feng cg et a0al cd4 t cells promote rather than control tuberculosis in the absence of pd1 mediated inhibition j immunol sakai s kauffman kd sallin ma et a0al cd4 t cell derived ifnγ plays a minimal role in control of pulmonary mycobacterium tuberculosis infection and must be actively repressed by pd1 to prevent lethal disease plos pathog 201612e1005667 solovic i sester m gomez reino jj et a0al the risk of tuberculosis related to tumour necrosis factor antagonist therapies a tbnet consensus statement eur respir j anand k burns ea ensor j et a0al mycobacterial infections with ruxolitinib a retrospective pharmacovigilance review clin lymphoma myeloma leuk winthrop kl baxter r liu l et a0al mycobacterial diseases and antitumour necrosis factor therapy in usa ann rheum dis gómez reino jj carmona l ángel descalzo m et a0al risk of tuberculosis in patients treated with tumor necrosis factor antagonists due to incomplete prevention of reactivation of latent infection arthritis rheum tezera lb bielecka mk ogongo p et a0al anti pd1 immunotherapy leads to tuberculosis reactivation via dysregulation of tnfα elife 20209e52668 anand k ensor j trachtenberg b et a0al osimertinib induced cardiotoxicity a retrospective review of the fda adverse events reporting system faers jacc cardiooncology anand k ensor j pingali sr et a0al t cell lymphoma secondary to checkpoint inhibitor therapy j immunother cancer 20208e000104anand a0k et a0al esmo open 20205e000866 101136esmoopen2020000866 0c" | Colon_Cancer |
open accesscitation iburg km mikkelsen l adair t lopez ad are cause of death data fit for purposeevidence from countries at different levels ofsocioeconomic development one e0237539 101371 pone0237539editor brecht devleesschauwer sciensanobelgiumreceived april accepted july published august peer review history recognizes thebenefits of transparency in the peer reviewprocess therefore we enable the publication ofall of the content of peer review and authorresponses alongside final published s theeditorial history of this is available here101371 pone0237539copyright iburg this is an openaccess distributed under the terms of thecreative commons attribution license whichpermits unrestricted use distribution andreproduction in any medium provided the originalauthor and source are crediteddata availability statement the data underlyingthe results presented in the study are available inthe who mortality database appswhointhealthinfostatisticsmortalitywhodpms with the and objectivemany countries have used the new anaconda analysis of causes of national death foraction tool to assess the quality of their cause of death data cod but no crosscountryanalysis has been done to verify how different or similar patterns of diagnostic errors anddata quality are in countries or how they are related to the local cultural or epidemiologicalenvironment or to levels of development our objective is to measure whether the usabilityof cod data and the patterns of unusable codes are related to a countrys level of socioeconomic developmentmethodswe have assessed the quality of national cod datasets from the who mortality database by assessing their completeness of cod reporting and the extent pattern and severityof garbage codes ie codes that provide little or no information about the true underlyingcod garbage codes were classified into four groups based on the severity of the error inthe code the vital statistics performance index for quality vspiq was used to measurethe overall quality of each countrys mortality surveillance systemfindingsthe proportion of garbage codes varied from to across the countries countrieswith a high sdi generally had a lower proportion of high impact ie more severe garbagecodes than countries with low sdi while the magnitude and pattern of garbage codes differed among countries the specific codes commonly used did notsthere is an inverse relationship between a countrys sociodemographic development andthe overall quality of its cause of death data but with important exceptions in particularsome low sdi countries have vital statistics systems that are as reliable as more developedcountries however in lowincome countries where most people die at home the proportion one 101371 pone0237539 august one 0cfollowing selected variables country referenceyear icd10 code 5years age group sex andnumber of deaths data were also obtained fromthe world population prospect unpopulation division populationunwppdownloadstandardpopulation with the followingselected variables country reference year years age group sex and population numberfunding this study was funded under an awardfrom bloomberg philanthropies to the university ofmelbourne to support the data for health initiativewwwbloombergprogrampublichealthdatahealth grant number not applicablethe funders had no role in study design datacollection and analysis decision to publish orpreparation of the manuscriptcompeting interests the authors have declaredthat no competing interests existare cause of death data fit for purposeof unusable codes often exceeds implying that half of all causespecific mortality datacollected is of little or no use in guiding public policy moreover the cause of death patternidentified from the data is likely to seriously underrepresent the true extent of the leadingcauses of death in the population with very significant consequences for health priority setting garbage codes are prevalent at all ages contrary to expectations further researchinto effective strategies deployed in these countries to improve data quality can informefforts elsewhere to improve cod reporting systemsintroductiona key source of evidence for targeting health interventions to improve population health ishigh quality cause of death cod data that reliably document trends in mortality for differentdiseases and injuries however several studies have demonstrated that policy and practicein many countries are based on data that are far from accurate [] in order to target effortsto improve the utility of cod data for policy it is important to first understand what the keydiagnostic errors area major problem with cod data is poor cause of death certification practices that result ingarbage codes ie codes that provide little or no information about the true underlying causeof death garbage codes include what are often called illdefined causes but encompass alarger universe of uninformative diagnoses the major consequence of garbage codes is thatthey obscure the true mortality pattern in a population for example if a death certificate onlystates septicemia as the cause of death there is no way of knowing whether this resulted forexample from an infected wound following an accident from a postoperative amputationdue to diabetes or from meningitis or pneumonia each of which would require different preventive strategies if the underlying cause that led to septicemia is not indicated on the deathcertificate public policy to prevent these deaths would be misinformed potentially leading toinefficient resource allocation to prevent themcod data provide the essential health intelligence for health policies across countries atvarious levels of socioeconomic development our premise is that a better understanding ofgarbage codes ie their levels and patterns in countries at different stages of socioeconomicdevelopment will help to target improvements in cod reporting systems in this study weinvestigate whether the usability of cod data and the patterns of garbage codes are related toa countrys socioeconomic development using the anaconda software tool [ s1 file]to assess the quality of national cod datasets several countries have used this tool to assesshow fit for purpose their data are [] but there has not been any crosscountry analysis ofdata quality across a range of socioeconomic development levels and cod reporting systemsusing the common anaconda frameworkthe implication of our findings for public policy to improve population health is that if therelationship is found to be very weak or nonexistent then efforts to improve national civilregistration and vital statistics systems can expect to make significant progress towardsimproving the evidence base for policy without depending on further socioeconomicdevelopmentdata and methodswe carried out a crosssectional study using publicly available data from the who mortalitydatabase which contains cod data reported by its member states the countries one 101371 pone0237539 august one 0care cause of death data fit for purposewere selected on the basis that they used icd10 had provided data to who for a relatively recent year were located in all major regions of the world and differed in levels of socioeconomic development population data were taken from the un worldpopulation prospects with the youngest age group of years divided into and14years age groups using spragues interpolation we classified a countrys level of development using the socio demographic index sdiscore from the global burden of disease study gbd into three levels high middle and lowthe sdi is a summary measure of development expressed on a scale from to taking intoaccount the total fertility rate years of schooling and gross national income for the lowestsdi level the who mortality database only contained the few countries we selected for themiddle and high sdi levels we selected countries with recent data from different regions of theworld our study included low sdi countries middle sdi and high sdi countrieson the basis of the country specific icd10 codes used in gbd the most severecertification and coding errors that can mislead policy and public health planning were identified and categorized into four groups the fourtier garbage code typology used in anaconda is based on the premise that some garbage codes are worse than others depending onhow serious their impact is for guiding or misguiding policy debates and will thus likely impactdisease and injury control strategies differently ¢ level very highcodes with serious policy implications these are causes for which thetrue underlying cod could in fact belong to any of three broad cause group ie it is impossible to establish whether the true cause was a communicable disease a noncommunicabledisease or because of an injury a good example being septicaemia reported as the underlying cause of death these are the most serious misdiagnoses among the universe of unusable codes since they could potentially grossly misinform understanding of the extent ofepidemiological transition in the population¢ level highcodes with substantial implications for policy these are causes for whichthe true underlying cod is likely to belong to one or two of the three broad cause groupsfor example essential primary hypertension¢ level mediumcodes with important implications for policy these are causes forwhich the true underlying cod is likely to be within the same icd chapter for exampleunspecified cancer and thus are of some policy value¢ level lowcodes with limited implications for policy these are diagnoses for whichthe true underlying cod is likely to be confined to a single disease or injury category egunspecified stroke would still be assigned as a stroke death and not to some other diseasecategory the implications of unusable causes classified at this level will therefore be muchless important for public policy but a more specific code would have increased their utilityfor specific public health analysesa full list of the composition of specific icd10 garbage codes for each of the four severitylevels is given in s2 filegiven the considerable differences in population age structure between countries at highand low levels of socioeconomic development we age standardised the pattern of garbagecodes the point of agestandardising was to investigate whether countries with a comparatively old age structure and hence relatively high average age at death might expect to have agreater fraction of garbage codes simply because of the higher likelihood of multiple comorbidity in the elderly we used the global age distribution of deaths from the latest gbd studyas the standard one 101371 pone0237539 august one 0care cause of death data fit for purposein addition to diagnostic accuracy the ability of any dataset to describe the true mortalitypattern in a population also depends on how complete it is both in terms of capturing alldeaths that occur and in assigning each a cod completeness of the cod reporting ie thepercentage of actual deaths in a population that are assigned a cod for each of the countries was calculated using the adairlopez empirical method incorporated into anaconda the empirical method models the relationship between the crude death rate cdr andits principal determinants namely the age structure of the population and the overall level ofmortality as reflected by the level of child mortality the predicted cdr based on these inputvariables for a population is then compared with the observed cdr to estimate death registration completeness given that the model was built largely from historical data where the levelsof adult mortality and child mortality are closely correlated the predictions of completenessfor populations where this assumption is not valid such as those severely affected by hivshould be interpreted cautiouslydatasets that are both incomplete and have a high proportion of garbage codes provide limited insight into the true health status of a population we combined the proportion of unrecorded deaths with the amount of garbage codes to provide a summary measure of the utilityof the data for policy this indicator is particularly important when investigating data qualityin countries with low completeness where the data available may only come from hospitalsand other health facilities where diagnostic facilities and physician availability is greaterpotentially overstating the policy utility of the dataa key output of any mortality surveillance system is a table showing the leading causes ofdeath for the population in countries where garbage codes are commonly assigned they frequently appear among the or leading causes and can seriously impact the overall utilityof the cod data this is particularly the case when they permeate the top leading causesand are high impact providing little or no useful information for policythe anaconda software tool specifically developed for assessing quality of mortalityand cod data was used to investigate each dataset s1 file to identify the pattern and extentof garbage codes in the data their frequency among the leading causes the completeness ofthe dataset and to provide an overall summary index of the quality of the output of the mortality data system namely the vital statistics performance index for quality vspiq resultsthe proportion of garbage codes in the country datasets varied substantially by countryranging from to see fig while the relationship between sdi and amount of garbage codes in the data is broadly apparent the relatively low r2 arises from the presenceof outliers particularly uzbekistan kyrgyzstan nicaragua and colombia it is quite possiblethat specific certification and coding procedures have been introduced in these countries toavoid the use of garbage codes if these countries are omitted the strength of the inverse relationship is much more apparent further insights into the general characteristics of populationand mortality of the selected countries can be found in s2 filethe mean values for each sdi group indicate that on average countries of high sdi statushad a lower proportion of garbage codes in their data than middle and low sdi countries table interestingly there was large intercountry variation in theuse of garbage codes within each sdi level for example of the high sdi countries finlandhad the lowest amount of garbage codes in their data whereas in japan and france the level was five times higher affecting about one in three deaths for the middle sdigroup argentina thailand and tunisia had more than half of all cods coded to a garbagecode in thailand close to of these were high impact errors while for other countries in one 101371 pone0237539 august one 0care cause of death data fit for purposefig percentage of total garbage codes versus socio demographic index selected countries high sdi middle sdi low sdi101371 pone0237539g001this group notably uzbekistan turkey and colombia the use of garbage codes was much lessprominent surprisingly among the low sdi countries kyrgyzstan and nicaragua had comparatively low levels of garbage and respectively whereas in egypt at a similarsdi level twothirds of all deaths are coded to garbage codes and of these over were ofhigh impact table importantly the fraction of high impact garbage codes ranged from a low of finlandto egypt and for low impact codes from finland to brazil countries withhigh sociodemographic development had a lower proportion of high impact garbage codesmean compared with middle and low sdi countries table rankingcountries according to their percent of high impact garbage codes reveals a very substantialgap between the best and worst performing cod information systems and a surprising mixture of sdi levels fig kyrgyzstan has the secondbest performing system after finlandwith cod data in colombia of almost equal quality to the uk and that in nicaragua fallingbetween australia and canada uzbekistan turkey brazil and jordan all assign less causes tohigh impact garbage codes than both japan and francethe impact of population age structure on the overall level of garbage codes varied acrosscountries but was generally small contrary to what might have been expected table injapan and argentina the ageadjusted fraction of garbage codes was points lower than one 101371 pone0237539 august one 0ctable levels of garbage codes standardised by age and registration completeness for select countries ranked by their sociodemographic index sdiare cause of death data fit for purposecountryfinlandcanadaaustraliajapanfranceyearsdivalueunited kingdom high sdimeanturkeyargentinairanjordanthailandsouth africatunisiabrazilcolombiauzbekistanmiddle sdimeankyrgyzstanegyptnicaraguatajikistanlow sdi meantotal meansdi levelshigh impact gclow impact gca total gc ageadjb completeness of codc deaths of no policygc registration value sdi three levels collapsed from gbd five sdi levelshigh highmiddle high middle middlelow low low middlegarbage code gc levelshigh impact gc levels low impact gc level total gc high lowdeath of no value for policyc 1b a�bthis equation calculates unavailable deaths for policy ie unregistered deaths plus deaths with a garbage code101371 pone0237539t001the unadjusted fraction while in france and south africa it increased by a similar amountcolumn a agestandardisation therefore had no impact on the mean level of garbage codesin each development categoryagestandardisation however masks the age pattern of garbage coding particularly its relative importance at younger adult ages where accurate and specific diagnoses are critical fuiding policies designed to prevent premature deaths the perception that garbage codes arelargely confined to deaths among the elderly due to the presence of comorbidities at oraround the time of death is not confirmed by the agespecific fractions of garbage codes shown one 101371 pone0237539 august one 0care cause of death data fit for purposefig percentage of high impact garbage codes in total causes of death selected countries c high sdi middlesdi low sdi101371 pone0237539g002in fig with exact fractions reported in s3 file garbage codes are prevalent at all ages andoften in similar proportions to what is observed for the age group indeed in some highlydeveloped countries eg finland canada uk the proportion of garbage codes is significantly higher at ages for both sexes than at older ages indeed in tunisia for males andfig garbage codes as a percentage of all deaths by age selected countries101371 pone0237539g003 one 101371 pone0237539 august one 0care cause of death data fit for purposein jordan and kyrgyzstan for both sexes this pattern is already evident from age in uzbekistan garbage coding is more common for deaths of children and adolescents than at ages and abovegiven that the utility of a countrys mortality information system is reduced not only by theamount of garbage codes but also by how complete it is in terms of capturing all deaths wemeasured their combined impact for two countries namely jordan and tajikistan the consolidated indicator of registration completeness and fraction of garbage codes was close to suggesting that useful information on only about one quarter of all deaths that occur injordan and tajikistan is available for policy purposes col c table overall the combinedindicator showed the proportion of deaths for which information was either missing or of littleor no value for guiding health policy was much higher in low and middle sdicountries compared to high sdi countriestable shows the strong influence of garbage codes on the leading cause distribution whenthese are ranked with high impact garbage codes marked in red and low impact in orangethe presence of high impact garbage codes among the leading causes of death will substantially distort the true picture of what are the common cod that most people die from amonghigh sdi countries only japan and france have high impact garbage codes for males amongthe ten leading causes of death canada australia and uk had only low impact causes in thiscategory and finland had neither for the middle and low sdi countries there were manymore high impact garbage codes listed among the leading cod with egypt having seventajikistan five and thailand and tunisia each with four colombia and nicaragua had nonefor females egypt had seven iran tunisia and tajikistan five with the remaining countrieshaving between one and threenotwithstanding some variation in patterns and volume of garbage coding across countriesthe most common garbage codes were remarkably similar in particular other illdefined andunspecified causes of death senility heart failure unspecified neoplasm septicemia respiratory failure unknown cause of death hypertension and unspecified diabetes were observedacross all sdi levels in addition low sdi countries tended to report atherosclerosis hepaticfailure intracerebral hemorrhage and unattended deaths all garbage codes as leading causesfig ranks countries according to a single consolidated summary measure of system performance namely the vital statistics performance index for quality vspiq eleven countries scored and above a level where they could be considered as having wellfunctioningsystems six of the remaining countries achieved scores that would classify them as havingmedium performing systems with lower scores mostly arising from the high proportion ofgarbage codes that bias their cod distributions of the countries only jordan tajikistanand tunisia were classified as having poorly functioning systemsdiscussionin general one would expect that in high sdi countries where all deaths are medically certified with good quality clinical and diagnostic services comparatively few deaths would beassigned unusable garbage codes and certainly much less than in countries where such services are less common our findings based on an analysis of cod datasets from across theworld produced evidence both for and against this hypothesis france and japan do not seemto have more reliable cod data to guide policy than turkey colombia kyrgyzstan and nicaragua in france in deaths is assigned a garbage code with of all deaths being certifiedas due to other illdefined and unspecified causes of death r99 heart failure i509 orrespiratory arrest r092 similarly in japan old age senility r54 is a commonly assignedcause of death accounting for one fifth of all garbage codes other research has found that one 101371 pone0237539 august one 0cdeificepsnuesaesidtraehnoitcrafnilarberecdeificepsnusaercnapainomuenpdeificepsnuevitcurtsboyranomuplhtiwesaesidrewoletucayrotaripsernoitcefnideificepsnulnoocyrtnuocknarselamwolleynisenotcapmiwoldernidekramsedocegabragtcapmihgihxesdnayrtnuocybhtaedfosesuacgnidaelpotelbatcimeahcsicinorhcfoealeuqescinorhcsisohrriccilohoclarevilfoebolreppurosuhcnorbsremiehzlaesaesidgnuldeificepsnulaidracoymnoitcrafnideificepsnuetatsorptesnofomsalpoenetalhtiwesaesidesaesidtraehetucatnangilamsremiehzlacitorelcsorehtadnalnifgnignahybmrahdnanoitalugnartsfleslanoitnetniecalpdeificepsnunoitacoffusdeificepsnusaercnaptonekortssadeificepsegahrromeahnoitcrafnirocitorelcsorehtaesaesidtraehnoitcrafnilarberecfoealeuqesllecrevileudsitinomuenpdeificepsnunoitcrafnilarberecamonicractimovdnadoofotevitcurtsboyranomuplesaesiddeificepsnulaidracoymnoitcrafnideificepsnuetucaaitnemedfomsalpoentraehcimeahcsietatsorpdeificepsnuesaesidytilineseruliaftraehdeificepsnudeificepsnuhcamotslaidracoymnoitcrafnideificepsnurosuhcnorbdeificepsnugnuldeificepsnugnulainomuenpdeificepsnunapajcinorhcdeificepsnutnangilamcinorhcetucarosuhcnorbneilartsuaainomuenpohcnorbdeificepsnutonekortssadeificepsainomuenpdeificepsnuroegahrromeahnoitcrafniesaesidyranomuplrewoletucahtiwyrotaripsernoitcefnietatsorpdeificepsnuesaesidevitcurtsboesaesidtraehfomsalpoenaitnemedtraehcimeahcsilaidracoymnoitcrafnideificepsnudeificepsnugnulmodgnikcinorhccitorelcsorehtatnangilamdeificepsnucinorhcetucarosuhcnorbdetinudeificepsnusaercnapdeificepsnudeificepsnuesaesidesaesiddeificepsnudeificepsnutraehcimeahcsitserralaidracoymnoitcrafnideificepsnufomsalpoendeificepsnudnaetatsorpfosesuacytilatromdeificepsnugnulsremiehzlalnooceruliaftraehcinorhcyrotaripseretucatnangilamdenifedllirehtorosuhcnorbecnarfcinorhcralucsavorbereccitorelcsorehtacinorhceruliaftraehrosuhcnorbetucayekrutesaesidyranomuplroegahrromeahdeificepsnunoitcrafnievitcurtsbocinorhctonekortssadeificepsfomsalpoentnangilametatsorpdeificepsnucitorelcsorehtaaitnemedesaesidtraehlaidracoymnoitcrafnideificepsnudeificepsnugnuletucarosuhcnorbadanacdeificepsnulnoocdeificepsnuesaesidyranomupletatsorpevitcurtsbofomsalpoencinorhctnangilamrewoletucahtiwyrotaripsernoitcefniaimeacitpesdeificepsnuroegahrromeahnoitcrafnitonekortssadeificepsesaesidsremiehzlatesnoetalhtiwdeificepsnuhcamotsainomuenpdeificepsnuesaesidyranomupldeificepsnuevitcurtsboesaesidesaesidtraehevitcurtsboyranomuplesaesiddeificepsnudeificepsnudeificepsnugnullaidracoymnoitcrafnideificepsnudnadenifeddeificepsnufosesuacytilatromdeificepsnugnuldeificepsnullirehtorosuhcnorberuliaftraehetucaainomuenpanitnegraare cause of death data fit for purposeesaesidtraehdnasuhcnorbfodenifedllifomsalpoenesaesidtraehcimeahcsignulfosnoitpircsedhcamotsesaesidsetebaidsutillemdeunitnoceruliaftraehdeificepsnuainomuenpcitorelcsorehtayramirplaitnesserosuhcnorblarberecdeificepsnudeificepsnuesaesidtraehnoisnetrepyhdeificepsnugnulnoitcrafnideificepsnusnoitacilpmocsetebaidsutillemtuohtiwdeificepsnudnaevisnetrepyhesaesidtraehevitsegnoceruliaftraehhtiwfosesuacytilatrommsalpoentnangilamdnasnoitacilpmoctnangilamtraehevisnetrepyhcinorhcdeificepsnutserracaidracdenifedllirehtolaidracoymnoitcrafnideificepsnunoitcrafnilarberecdeificepsnutropsnarttnediccadeificepsnulaidracoymnoitcrafnideificepsnulaidracoymnoitcrafnietucanarietucanadroj one 101371 pone0237539 august one 0cniderunjinosrepnoitcrafnilarberecytilinestserrayrotaripserdeificepsnudeificepsnutnetniidenmretednuesaesidtraehtnevedeificepsnucimeahcsicinorhclaidracoymnoitcrafnietucalarberecartniegahrromeaheruliaftraehdnaaeohrraidlarivrehtonamuhfositiretneortsagtonsesaesidycneicifedonummifomsalpoentnangilamdnasuhcnorbgnultonekortssadeificepsnigirosuoitcefnideifissalcsesaesidcitisarapdnasuoitcefninignitlusernoitcrafnidemuserperehwesleesaesid]ivh[surivroegahrromeahaimeacitpesrehtofomsalpoentnangilamcitapehartnistcudelibdnarevilytilinesainomuenpdeificepsnumsinagrooterusopxedeificepsnurotcafdeificepsnuainomuenpyrotaripsersetebaidsutillemdeificepsnudemrifnocmsinagrotonlsisoucrebutlyllacigooiretcablyllacigootsihrodeunitnocelbatllirehtodnaliahtdnadenifeddeificepsnufosesuacytilatromdnadenifeddeificepsnullirehtofosesuacytilatromhtuosacirfarotomdeificepsnudeificepsnuoteudtnediccaelcihevronoisulccociffartlarberecfosisonetsseiretrahtaeddednettanuyramirplaitnessedeificepsnunoisnetrepyhsnoitacilpmocsetebaidsutillemtuohtiwetatsorpdeificepsnudeificepsnuegahrromeahmraerifnoitcrafnirofosesuacytilatromfomsalpoentnangilamrosuhcnorbgnulybtluassadnarehtoecalptonekortsdenifedllirehtosadeificepsdeificepsnudnaainomuenpdeificepsnulaidracoymnoitcrafnideificepsnuetucalizarbsutillemsetebaidtnevesnoitacilpmocdeificepsnurehtohtiwidenmretednuegahrromeahdeificepstnetninoitcrafnirotonekortssadeificepslaidracoymnoitcrafnideificepsnudeificepsnugnuldnadenifeddeificepsnufosesuacytilatrometucarosuhcnorbllirehtoaisinutdeificepsnueruliafdeificepsnudnagnullanercinorhcrehtoybtluassarosuhcnorbevitcurtsbocinorhcegrahcsidmraerifdeificepsnuesaesidyranomuplfomsalpoentnangilametatsorpsisorelcsorehtaeruliaftraehecalpdeificepsnutonekortssadeificepsegahrromeahnoitcrafnirorewoletucahtiwyrotaripsernoitcefnitraehcimeahcsietucarehtosesaesidlarberecartniegahrromeahgnignahybmrahdnanoitalugnartsemohnoitacoffusfleslanoitnetnideificepsnutraehcimeahcsiralucsavoidracdeificepsnuesaesidosesaesiddebircsedevitcurtsboyranomuplcinorhcesaesidhcamotscinorhccitorelcsorehtadeificepsrehtoegrahcsiddnateertsyawhgihdeificepsnuhcamotsdnasisorbiffosisohrricrevildnadenifeddeificepsnullirehtofosesuacytilatromainomuenpdeificepsnuevitcurtsbodeificepsnudnayranomuplmraerifesaesidteertsegrahcsiddeificepsnuyawhgihdnalaidracoymnoitcrafnideificepsnucinorhcrehtoybtluassaetucaaibmoloclaidracoymnoitcrafniesaesidtraehtraehcimeahcsiesaesidetucasirotcepanignaevisnetrepyhcinorhcnatsikebzunoitcrafnirevilfosisohrricroegahrromeahdeificepsnunoitcrafnilaidracoymetucadeificepsnudnarehtosadeificepsesaesidtraehtonekortscitorelcsorehtanatszygrykare cause of death data fit for purposemsalpoentnangilamnilusninonetatsorpfosetebaidtnednepedlaidracoymnoitcrafnifosisohrriccilohoclalanerhtiwsutillemsnoitacilpmocrevilrevilfosisohrricerehwesletonegahrromeaherehwesletonfosisohrricdeificepsnudeifissalcdnarehtoderunjinosrepdeificepsnunielcihevrotomciffarttnediccaevitcurtsboyranomuplcinorhcesaesiddeificepsnudeifissalctonekortssadeificepsegahrromeahnoitcrafnirorevilainomuenpdeificepsnusisorelcsorehtaytilinesetucaeruliafyrotaripserlarberecartnieruliafcitapehdnasisorbiftserracaidracnoisnetrepyhlanercinorhcdeificepsnueruliafyramirplaitnesseeruliaftraehtpygelaidracoymnoitcrafnideificepsnuetucaaugaracintraehfosnoitpircsedesaesidesaesidfosesuacytilatromnoitcrafnisesaesidroegahrromeahnoisnetrepyhnoitcrafnidenifedllisutillemsetebaidtraehcimeahcsideificepsnudnalaidracoymtraehcimeahcsidnasnoitacilpmocdeificepsnucinorhcsisorelcsorehtadenifedllirehtoetucaetucarehtoytilinestonekortssadeificepsyramirplaitnessenatsikijatdeunitnocknarselamefyrtnuoc one 101371 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0clarberecfoealeuqessremiehzlarehtolarbereccimeahcsicinorhcnoitcrafniesaesidnoitcrafnideificepsnuesaesidtraehdeificepsnudeificepsnuaitnemedlaidracoymnoitcrafnideificepsnuevitsegnoceruliaftraehhtiwdeificepsnuesaesidtraehesaesidesaesidtraehetucaevisnetrepyhsremiehzlacitorelcsorehtasremiehzlahtiwesaesidtesnoetaldnalnifdeunitnocelbatdeificepsnulnoocainomuenpdeificepsnudeificepsnutsaerbdeificepsnuhcamotsevitcurtsbocinorhcaitnemedralucsavesaesidyranomuplrewoletucahtiwnoitcefniyrotaripserdeificepsnudeificepsnurettulflairtadnasaercnapnoitallirbiflairtadeificepsnuevitcurtsboyranomuplcinorhcesaesiddeificepsnuevitcurtsboyranomuplcinorhcesaesiddeificepsnulaidracoymnoitcrafnideificepsnuetucaainomuenpdeificepsnusremiehzlaesaesidtonekortssadeificepsdeificepsnuroegahrromeahnoitcrafnideificepsnuesaesidtraehlaidracoymnoitcrafnideificepsnudeificepsnugnulaitnemedtsaerbcitorelcsorehtaetucarosuhcnorbdeificepsnuadanacsremiehzladeificepsnuesaesidnoitcrafnideificepsnularberecsremiehzladeificepsnuesaesiddeificepsnusadeificepstraehcimeahcsignulegahrromeahnoitcrafnirodeificepsnuesaesiddeificepsnulaidracoymnoitcrafnideificepsnuaitnemedtsaerbtonekortscinorhcrosuhcnorbetucadeificepsnuneilartsuanoitcrafnitimovdeificepsnufoealeuqessitinomuenprosuhcnorberuliaftraehlarberecdnadoofoteudgnuldeificepsnuainomuenpdeificepsnuytilinesnapajlaidracoymnoitcrafnideificepsnuetucatonekortssadeificepsegahrromeahnoitcrafnirodeificepsnuesaesiddeificepsnudeificepsnutraehcimeahcsignulaitnemedmodgniktsaerbcinorhcrosuhcnorbdeificepsnudetinudeificepsnulnooclaidracoymetucatonekortstserrayrotaripsereruliaftraehrosuhcnorbdeificepsnudenifedllirehtotsaerbsremiehzlaecnarfare cause of death data fit for 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| Colon_Cancer |
esophageal cancer ec is the eight commonest type of worldwide cancer and the sixth leading gastrointestinal malignancy with a poor survival rate which is caused by many factors such as tobaccosmoking heavy drinking lack of fruits and vegetables and there are two main histological types of ecesophageal adenocarcinoma eac and esophageal squamous cell carcinoma escc escc is characterized by weight loss difficulty in swallowing and a dry cough and accounts for of the casesof ec recent years in particular have seen major progress in the diagnostic and surgical techniquesradiotherapy and chemotherapy however the improvement of escc patients survival rates remains unsatisfactory hence identify reliable molecular biomarkers and targets for escc early diagnosis and prognosis indication are still urgently needed meanwhile an urgent need for fully comprehensive research 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 bsr20201727101042bsr20201727figure differentially expressed genes in esophageal squamous cell carcinoma escc and normal tissuea box plot of distributions of expression values of lncrnas b scatterplot of chips for assessing the variation of lncrnas cheatmap of lncrnas d box plot of distributions of expression values of mrnas e scatterplot of chips for assessing the variationof mrnas f heatmap of mrnasinto the crucial molecular targets mechanism of escc tumorigenesis and metastasis would provide new perspectivesinto the novel treatment strategynowadays long noncoding rnas lncrnas a class of rna transcripts with a length of nucleotides thathave little or no proteincoding capacity have drawn increased attention and accumulating studies demonstrated that lncrnas take part in multiple biological processes for example cell differentiation and proliferationposttranscriptional and transcriptional modifications epigenetic modulation chemotherapy resistance glucosemetabolism immune response furthermore a great many lncrnas are deregulated in escc making thempossibly as potential therapeutic targets or as diagnostic and prognostic biomarkers micrornas a kind of smallnoncoding rna characterized by nucleotides in length and play a significant role in gene transcriptionallevel act as regulators abnormal expression of mirna has been verified to be tightly associated with lncrnas andtranscription factors tfs and accumulating researches have indicated that lncrnas can act as cernas by binding and sequestering the 3cid3untranslated region utr of messenger rnas mrnas to regulate the mrna andprotein expression of target genes lncrnas and mirnas are becoming new kinds of diagnostic molecularmarkers of escc and chang reported that lncrna tusc7 suppressed chemotherapy resistance of escc bydownregulating mir224 to adjust desc1egfrakt pathway zhong revealed that lncatb promotesmalignancy of esophageal squamous cell carcinoma by regulating the mir200bkindlin2 axis tan found 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 bsr20201727101042bsr20201727figure venn diagram for overlapping demirnas and overlapping predicted genesa one demirnas was upregulated in both microarrays b two demirnas were downregulated in both microarrays c predicted genes for hsamir4093p d predicted genes for hsamir133b e predicted genes for hsamir1395plncrna h19 is overexpressed and promotes escc cell proliferation and metastasis tfs are also as significantfactors involved in controlling transcription and posttranscriptional regulation of genes through binding to particular dna sequences and involves in proliferation apoptosis and migration of tumor cells therefore further studyis required to elucidate the interrelationships of lncrnas mirnas mrnas and tfs in esccwith the rapid progress of highthroughput sequencing as well as computational techniques gene expression profiles analysis contribute to identifying immediately largescale genes that are differentially expressed at the very samemoment thus more and more lncrnas mirnas and mrnas have been discovered in the present study wemined the lncrna mirna and mrna gene expression data from the recent release of geo gene expression omnibus and tcga the cancer genome atlas database downloaded the noncoding rna expression microarrayof gse89102 to identify the differentially expressed lncrnas delncrnas and the mirna expression profile ofgse97051 and gse59973 to confirm the differentially expressed mirnas demirnas among escc and normalsamples the mirnarelated mrnas and connected lncrnas were predicted and then functional enrichment analysis of mirnarelated mrnas and differentially expressed genes degs in gse89102 were also conducted besideslncrnamirnamrna network proteinprotein interaction ppi network of mirnarelated mrnas and degscoexpression network of lncrna and degs regulatory network of transcription factors tfs and hub genes wereconstructed importantly the expression of hub genes was verified by tcga data furthermore we evaluated theprognostic value of hub genes in patients with ec via the kaplanmeier plotter database and also the diagnosticvalues of hub genes were assessed by receiver operating characteristic roc analyses as a group the current researchaimed to further explore potential therapeutic targets in the development and progression of esccmaterials and methodsmicroarray data collectiongeo httpwwwncbinlmnihgovgeo is a public functional genomics data repository to promote the understanding of gene expression profiles the long noncoding rna expression profiles gse89102 and twomirna expression data gse97051 and gse59973 were obtained from geo httpwwwncbinlmnihgovgeothe array data of gse89102 consisted of five paired escc tissues and normal tissues based on the platform of the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commons attributionlicense cc by 0cbioscience reports bsr20201727101042bsr20201727figure lncrnamirnamrna regulatory and ppi networksa lncrnamirnamrna network every node represents one gene and each edge represents the interaction between genesmrnas mirnas and lncrnas are indicated with ellipse rectangle and diamond shapes respectively red represents upregulated and green represents downregulated b ppi network of targets for demirnas nodes represent one gene indicated with arectangle and continuous mapping node color with brown using degree valuegpl16956 agilent045997 arraystar human lncrna microarray v3 agilent technologies inc santa clara causa deposited by bai in third military medical university gse97051 contained seven paired cancer tissue and adjacent normal tissue platform gpl21572 [mirna4] affymetrix multispecies mirna4 arrayaffymetrix inc santa clara ca usa and gse59973 based on the platform of gpl16770 agilent031181 unrestricted human mirna v160 microarray included three paired of human escc tissues and normal controlswere submitted by shi r from the first affiliated hospital of nanjing medical universitydata processinggeo2r httpwwwncbinlmnihgovgeogeo2r an interactive web tool that reprocessed the raw data in a geoseries also allowing users to evaluate the distribution of the values for the samples have been selected and viewedgraphically as a box plot was employed to identify delncrnas demirnas and degs by comparing escc andnormal tissue samples genes were identified based on the cut off criterion pvalues and log2fc ¥ theoverlapping demirnas were identified between gse97051 and gse59973 through an online tool for venn diagramanalysis httpbioinfogpcnbcsicestoolsvennyindexhtml 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 bsr20201727101042bsr20201727figure go terms displayed as an interaction network and kegg pathways shown as a bubble diagramyellow nodes nodes with pvalue and benjamini corrected pvalue a biological process b molecular functionc cellular component d kegg pathways 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 bsr20201727101042bsr20201727figure coexpression network ofrp111437a84 correlated downregulated degs p005the red and bottle green nodes represent lncrna genes pink and pale green nodes denote degs respectively the yellow nodesrepresent the lncrna coexpressed mrnaslncrna mast4it1 correlated upregulated degs p005ablncrnaprediction of target genes and lncrnas for overlapping demirnasthe database of mirpathdb targetscan and mirwalk with a score ¥ used as a cutoff criterion for the prediction analysis and was widely used to predict the mirnarelated mrnas interactions theinteraction between overlapping demirnas and lncrnas was predicted by using dianalncbase v20 wwwmicrornagrlncbase which provides a compositive integration of mirnalncrna interactions with thescore¥ as threshold criterion to perform the prediction in the prediction module no other than mirnarelatedmrnas included in all of mirpathdb targetscan and mirwalk databases were picked out for the further functional enrichment analysis and constructed the ppi network after the predicted lncrnas were intersected withdelncrnas in gse89102 cytoscape software version was utilized to visualize the regulatory network oflncrnamirnamrna 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 bsr20201727101042bsr20201727integration of proteinprotein interaction network and gene functionanalysis of selected mrnassearch tool for the retrieval of interacting genes string a widely used interactive tool stringdbthat included known interactions and predicted interactions was used to explore the interaction of selected mrnassubsequently ppi pairs that have a combined score¥ the value was used as a threshold criterion for statisticaldifference were identified to construct the network of ppi using cytoscape besides the selected gene with a degreevalue ¥ was identified as hub genes in the ppi network based on an earlier study finally gene ontology goenrichment analyses in terms for biological process bp molecular function mf and cellular component ccfor the selected genes were even further conducted with bingo tool in cytoscape and the metascape httpmetascape was employed to perform kegg pathway enrichment analysis the online tool omicsharehttpwwwomicsharecom was used to visualize the result of kegg pathway enrichment analysis by using a bubblediagramcoexpression analysis of selected lncrnas with degsthe gse89102 also explored the microarray of mrnas in escc the degs with the same criteria for lncrnas wereselected and then a pearsons correlation coefficients pcc between the selected lncrnas and degs was calculatedusing the matrix expression in excel microsoft corp redmond wa usa pcc with p005were used as a threshold value to concern significant correlations and then the genes were chosen to construct thecoexpression network with cytoscapewe also mined the genes associated with escc that proven by literature with palmist the lncrna coexpresseddegs in esccassociated gene set were screened out for go terms as well as kegg pathways enrichment analysisperformed by metascape and construction of ppi network also using string and visualized by cytoscape the termsand pathways with the thresholds were set at a p005 and the numbers of enriched genes ¥ were consideredas significant to product bubble diagrams the former five pivotal nodes in the ppi network with degree¥ wereidentified to further analysisprediction of transcription factor for hub degsto explore the interrelation of tfs and hub genes indepth tfs of hub genes with adj p005 were investigated bynetworkanalyst httpwwwnetworkanalystca an easytouse and highperformance webbased program for biological network analysis recorded using chip enrichment analysis chea then the intergenic interrelationwas visualized in cytoscape and analyzed with a topological property of degree distribution of the network using thetool networkanalyzertcga datasetmining analysisthe tcga dataset is a platform contains a large cohort of over human tumors for scientists to downloadand integrated multidimensional analyses cancergenomenihgov to verify the transcriptional expression of hub genes for improving the reliability of the current study two online webbased tools ualcanhttpualcanpathuabeduanalysishtml a userfriendly interactive web resource for analyzing cancer transcriptome data and oncomine wwwoncomine a cancer microarray database for contributing to discovery fromgenomewide expression analyses were employed to promote datamining the transcriptional expression levelof hub genes we conducted ualcan database analysis to assess hub genes expression level based on sample typesin tcga esophageal carcinoma datasets and transcriptional expression of hub genes for cancer samples comparedwith those in normal samples on oncomine database by using students ttest with pvalue 1e4 and fold changewere defined as for statistically significant and respectively data type and sample type were selected as mrna andclinical specimensurvival and roc analysis of hub geneskaplanmeier plotter km plotter httpkmplotcomanalysis is capable to appraise the effect of genes onthe survival of cancer samples and consists of gene expression profiles and survival information for ec patients was utilized to evaluate the prognostic value of these meaningful hub genes in ec the os of ec patientswas assessed using a km plot with patients divided into high and low expression groups according to the medianexpression of a specific query gene the hazard ratio hr with confidence intervals and log rank p values werecalculated to assess the relationship of gene expression with survival and also the numberatrisk is displayed below 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 bsr20201727101042bsr20201727figure gene ontology go and kyoto encyclopedia of genes and genome kegg enrichment analysisa the top most significant changes in the go biological process b the top most significant changes in the go cellularcomponent c the top most significant changes in the go molecular function d the top most significant kegg pathwaytermsthe curves besides we also performed a roc analysis with mrna expression value in gse89102 to investigate thediagnostic value of hub genes in escc using medcalc software cell culturenormal esophageal cell lines het1a and the escc cell lines kyse410 kyse150 eca109 t10 and te1 wereobtained from the shanghai institute of chinese academy of sciences cell collection and cultured in rpmi1640medium gibco grand island ny usa containing fetal bovine serum fbs gibco μgml streptomycinand uml penicillin at ¦c in a co2 incubator 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 bsr20201727101042bsr20201727figure the result of the ppi network with the lncrna coexpressed mrnasthe node represents the lncrna coexpressed mrnas indicated with a rectangle and continuous mapping node color with brownusing degree valuetable the speciï¬c primer sequences of hub genesgene namecdk1vegfaprdm10runx1cdk6hsp90aa1mycegr1sox2gapdhprimer sequence 5cid2 to 3cid2forward 5cid3ggaaggggttcctagtactgc3cid3reverse 5cid3ccatgtactgaccaggaggga3cid3forward 5cid3ctggagcgtgtacgttggt3cid3reverse 5cid3tttaactcaagctgcctcgc3cid3forward 5cid3catccaggtcagcgagccta3cid3reverse 5cid3cgaagtaaccgccttcacct3cid3forward 5cid3gggagcttgtccttttccga3cid3reverse 5cid3gagaggcaatggatcccagg3cid3forward 5cid3acagagcacccgaagtcttg3cid3reverse 5cid3gtatgggtgagacagggcac3cid3forward 5cid3ccgcccagagtgctgaatac3cid3reverse 5cid3acaccgaactggccaatcat3cid3forward 5cid3cgtcctcggattctctgctc3cid3reverse 5cid3gctggtgcattttcggttgt3cid3forward 5cid3gctggtggagaccagttacc3cid3reverse 5cid3tcatcgctcctggcaaactt3cid3forward 5cid3aaccagcgcatggacagtta3cid3reverse 5cid3gacttgaccaccgaacccat3cid3forward 5cid3gggaaactgtggcgtgat3cid3reverse 5cid3gggtgtcgctgttgaagt3cid3qpcr and western blot analysistotal rna was extracted from the cell lines using trizol reagent invitrogen and then total rna was reverse transcribed to complementary dna cdna by using the primescript rt reagent kit takara qpcr was performedusing sybr premix ex taq¢ takara on a cfx96 touch deep well detection system biorad usa following the manufacturers instructions gapdh was used as an internal control the relative levels of hub genes wereestimated using the ääct method the primer sequences of hub genes were listed in table 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 bsr20201727101042bsr20201727figure the network of transcription factors and hub genesnodes represent transcription factors indicated with diamond shapes red represents hub genes with an ellipse light blue represents transcription factors yellow and green indicated that the tfs regulating or hub genes respectivelyfor western blot analysis the cell lines were lysed with ripa lysis buffer kengen china and quantified witha bicinchoninic acid bca kit beyotime china equal amounts of cell lysates were separated by or sodium dodecyl sulfate polyacrylamide gel electrophoresis sdspage and then transferred onto polyvinylidene fluoride pvdf membranes millipore after blocking with nonfat milk the membranes incubatedwith primary antibodies against cdk1 ab32094 abcam vegfa ab183100 abcam prdm10 ab3787 abcam runx1 cell signaling technology cdk6 d120398 sangonhsp90aa1 d191063 sangon myc cell signaling technology egr1 ab133695abcam sox2 cell signaling technology gapdh r12101 huabio and βactin d110001 sangon overnight at ¦c subsequently membranes were incubated with appropriate hrplabeled goatantimouse or antirabbit secondary antibodies for h at room temperature after that the protein bands were visualized with ecl solution beyotime china and analyzed using imagej software national institutes of healthbethesda mdstatistical analysiscorrelation between delncrnas and degs were evaluated using pearsons correlation the kaplanmeier survivalcurves were used to show the differences of hub genes in patient os between the high expression group and lowexpression group and the statistical significance was obtained using the twosided logrank test the expression ofhub genes in gse89102 was used to conduct roc analyses the difference between the two groups was estimatedusing a twotailed students ttest p005 was considered statistically significantresultsidentiï¬cation of demirnas lncrnas and mrnasthe demirnas were screened using the geo2r tool as a result demirnas of gse97051 were identified between escc and adjacent normal esophagus samples including upregulated and downregulated demirnas 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 bsr20201727101042bsr20201727figure transcriptional levels of hub genes in different types of cancersthis information was attained from oncomine with the threshold of pvalue ¤ 1e4 fold change¥ and gene rank ¥top10and indicates the numbers of datasets with statistically significant p005 mrna highexpression red or lowexpression blueof cdk1 vegfa prdm10 runx1 cdk6 hsp90aa1 and myc different types of cancer vs corresponding normal tissue cellcolor was decided by the best gene rank percentile for the analyses within the cell and the gene rank was analyzed by percentileof target genes at the top of all genes measured by each studytable suppinfoxls about upregulated and downregulated demirnas of gse59973 also screened out forfurther analysis table suppinfoxlsmeanwhile the box plot view scatterplot and hierarchical clustering based on differentially expressed lncrnasfigure 1ac of lncrnas in gse89102 and the box plot view scatterplot and hierarchical clustering based on differentially expressed mrnas figure 1df of mrnas in gse89102 were also performed both of the box plotshown the distributions of expression values for the samples after normalization scatterplot assessing the variation 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 bsr20201727101042bsr20201727table the overlapping genes of selected mirnas in mirpathdb mirwalk and targetscan databasemirnasoverlapping geneshsamir4093phsamir133bhsamir1395pmrpl35 paqr5 ago2 zzz3 slc25a16 gab1 trim5 c1qtnf1 klf12ythdf3 gpr85 msrb3 l3mbtl3 zc3h12c adamts5 trim71 ipo7nr3c2 dennd2c mylk phc3 gpr61 scn1a cd96 nr4a2 lgsntmem242 larp1 papd5 znf423 tnni1 fam229b prmt6rab5caebp2 ank2 arrdc3 atp11a atp2b2 atxn1 camk2d ccnd2 csrnp2cttnbp2nl dpysl5 glcci1 heg1 hmgcr igf1r kcnd3 kiaa1549kpna4 map2 mbnl1 nme7 nrk pde3a pde4a pgm2l1 phf6 piezo1pitpna pmp22 ppargc1a prdm10 ptpn4 runx1 socs2 stx7stxbp5l syt14 taok1 tcf12 tet3 tmpo tspan3 uhmk1 xpo4updownupdowndownfigure tcga data portal analysis using ualcan and oncomine databaseag hub gene cdk1 vegfa prdm10 runx1 cdk6 hsp90aa1 and myc expression in ec hn hub gene cdk1 vegfaprdm10 runx1 cdk6 hsp90aa1 and myc expression based on oncomine for esccor reproducibility and hierarchical clustering reveal a differentiable microarray expression profiling among samples a total of delncrnas comprising upregulated and downregulated lncrnas were obtained table suppinfoxls also degs upand downregulated genes table suppinfoxls were identifiedin escc compared with normal controlsprediction of target genes and lncrnas for demirnasthrough analysis of microarray data gse59973 and gse97051 the updownregulated mirnas were identifiedthere are three overlapped mirnas including hsamir4093p upregulated figure 2a hsamir133b andhsamir1395p downregulated figure 2b in both microarrays based on the mirpathdb mirwalk andtargetscan database the relationships between these three demirnas and predicted genes were identified the intersection number of these predicted genes for hsamir4093p was figure 2c for hsamir133b was figure2d and for the hsamir1395p was figure 2e a total of predicted genes were listed in table next the interactions between the three differentially expressed mirnas and predicted lncrnas were obtainedusing lncbase v20 dataset as a result predicted lncrnas for hsamir4093p table suppinfoxls predicted lncrnas for hsamir133b table suppinfoxls and predicted lncrnas for hsamir1395p table suppinfoxls were screened out after deletion of redundant content then the intersection of predicted lncrnasand updown regulated delncrnas between escc and adjacent normal esophagus tissue were performed finallyas shown in table overlapped delncrnas for hsamir4093p overlapped delncrnas for hsamir133band overlapped delncrnas for hsamir1395p were identified 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 bsr20201727101042bsr20201727figure the receiver operating characteristic roc curves of the nine hub genes from ppi and tfs networksa cdk1 b vegfa c prdm10 d runx1 e cdk6 f hsp90aa1 g myc h egr1 i sox2roc curves were drawnusing medcalc software auc area under the roc curveconstruction of lncrnamirnamrna cerna and ppi networkthe aforementioned relationship between mirnamrna and lncrnamirna was used to construct a specific cerna lncrnamirnamrna network using cytoscape figure 3a we obtained a preliminary understanding of the mechanism of mirnas which had a multidimensional regulatory effect through pattern multiplelncrnasmultiple mrnas according to the information of ppi based on string databases then the ppi networkof the predicted genes was constructed figure 3b additionally the nodes with a higher value of degree wereidentified as hub genes the network consisted of nodes there is no interaction of 32nodes which is removedand edges the top hub genes included prset domain prdm10 and runt related transcription factor runx1 with the degree of connectivity was and respectively combined with the logfc with pvalue of overlapped delncrnas the lncrna rp111437a84 and lncrna mast4it1 were selected for coexpression analysiswith downupregulated degs respectively 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 0ctable the identiï¬ed lncrnas of selected mirnas in gse89102mirnagene symbollocationadjpval pvaluelogfcdownupbioscience reports bsr20201727101042bsr20201727hsamir4093phsamir133bhsamir1395prp111437a84rp11586k1210xloc linc00299rp11308d133xloc rmstuca1rp11214l131linc00475tmem161bas1xloc cldn10as1ac1047773rp11363g24thap9as1rp11385j12ac0965591rp11562f92sox2otrp1170c11dnm3osrp11657o91rp11366f62linc00467ctb89h124rp11392p76xloc rp11160o51mast4it1loc100130744hcg11sox2otxloc ac0099485rp11363g24xloc xloc chr163320377333206462chr163266392532666533chr19chr280077718324630chr4139618136139623232chr5chr129743165397565015chr191582896115836320chr185588168855920199chr99214146792160114chr58826902488436674chr20chr139547944495533910chr2150566134150568080chr132285177322854138chr48289300982900960chr3178526505178860352chr21257603812642912chr49226876792277075chr3180989783181699883chr34277061242773635chr1172136531172144794chr3135356067135439829chrx151904431151911455chr1211382803211435333chr5149494314149504670chr121292772612980307chr21chr176510081265111058chr56666233166662988chr51471269414716529chr62652345026526579chr3180989783181699883chr7chr2178413994178440243chr132285177322854138chr1chr2787e03172e02374e03393e05425e04191e03255e03534e04380e03297e07109e05610e03298e03115e04671e03130e02719e03185e05208e06339e03513e03686e03240e04130e04218e03599e03836e04171e06460e05349e04504e04624e05853e04787e10270e07157e03622e04780e06178e03420e03194e03572e07198e08738e04126e03183e03214e05918e06223e10787e10downdowndowndowndowndowndowndowndowndowndowndowndowndownupupupupupupupupupupupupupupupupupupupupupupupupfunctional analysis of the mirna target geneto understand the biological functions of predicted genes of demirnas go terms and kegg pathway analyses ofmrnas in the lncrnamirnamrna network were conducted in cytoscape plugin bingo the results of goanalysis on the bp level figure 4a such as positive regulation of macromolecule biosynthetic process pvalue 351e05 adjusted pvalue 152e02 regulation of transcription pvalue 117e04 adjusted pvalue 152e02 positive regulation of nitrogen compound metabolic process pvalue 150e04 adjusted pvalue 152e02 on the go terms of mf figure 4b the mrnas were mainly enriched in metalion binding pvalue 347e06 adjusted pvalue 461e04 cation binding pvalue 457e06 adjusted pvalue 461e04 andnucleic acid binding pvalue 939e05 adjusted pvalue 530e03 on the cc level figure 4c the mrnaswere markedly concentrated in the neuron projection pvalue 147e04 adjusted pvalue 309e02 neuronalcell body pvalue 452e04 adjusted pvalue 316e02 and nucleus pvalue 650e04 adjusted pvalue 316e02 the front most remarkable kegg pathway terms are displayed and the mrnas were primarily concentrating on the negative regulation of the pi3kakt network signaling by fgfr in disease and pi5p pp2a andier3 regulate pi3kakt signaling figure 4d 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 bsr20201727101042bsr20201727coexpression network of overlapped lncrnas and degsa coexpression network of updownregulated degs was built up underpinned by their pearsons correlation coefficients the coexpression analysis showed that the lncrna mast4it1 and upregulated degs figure 5alncrna rp111437a84 and downregulated degs figure 5b whose expressions are significantly correlated esccassociated genes were mined in palmist after esccassociated genes and coexpressed differentiallyexpressed genes intersecting the lncrna coexpressed mrnas were also screened out in the coexpression networkgo and kegg pathway analysis of the lncrna coexpressed mrnasto gain further understanding of the bioinformation of identified the lncrna coexpressed mrnas functional enrichment analysis was conducted via metascape again the front most important go terms of each type wereshown the lncrna coexpressed mrnas were mainly enriched in regulation of immune system process responseto external stimulus and positive regulation of biological process in biological processes figure 6a related to the extracellular space extracellular region part and cytoplasmic membranebounded vesicle in cellular component figure6b and also were mainly enriched in cytokine receptor binding chemokine receptor binding and protein bindingon the molecular function level figure 6c the lncrna coexpressed mrnas mainly involve in cytokinecytokinereceptor interaction cell cycle p53 signaling pathway micrornas in cancer and pathways in cancer the keggpathway terms of great significance are also shown in figure 6dconstruction ppi network for the lncrna coexpressed mrnasthe string online database and cytoscape software were also used to construct the ppi network of the lncrnacoexpressed mrnas a total of except cldn7 slpi and s100a14 of the lncrna coexpressed mrnas were mapped into ppi network complex figure the gca dst emp3 mgat2 htra1 ndufaf3atp2c1 arhgap24 cldn7 and slpi were isolated nodes and then excluded with degree value16 nodeswere identified as hub genes in the ppi network including vascular endothelial growth factor a vegfamyc protooncogene bhlh transcription factor myc heat shock protein alpha family class a member hsp90aa1 cyclindependent kinase cdk1 and cyclindependent kinase cdk6 these results suggestedthat the hub genes may significantly play a vital role in the progress of escctranscription factorshub genes pairsas shown in figure the transcriptionregulated network with nodes and edges and multiple hub genesassociated with tfs are shown in table interestingly we realized that sry sex determining region ybox sox2had been predicted to regulate cdk1 vegfa prdm10 runx1 cdk6 hsp90aa1 and myc hub genes andmyc runx1 cdk6 hsp90aa1 vegfa and prdm10 could be regulated by early growth response egr1dysregulated expression of hub genes in patients with esccseven hub genes were observed using the ualcan and oncomine database as a preliminary step we measuredthe expression of cdk1 vegfa prdm10 runx1 cdk6 hsp90aa1 and myc in different kinds of cancerand compared with normal individuals as shown in figure the mrna expression of cdk1 and runx1 wassignificantly raised in ec samples in multiple datasets tcga dataset mining was performed using ualcan toshown that abnormal expression of the hub genes including cdk1 vegfa prdm10 runx1 cdk6 hsp90aa1and myc were significantly upregulated between esca primary tumor and normal tissues figure similarlythe hub genes cdk1 vegfa runx1 cdk6 hsp90aa1 and myc with fold change and respectively in su esophagus dataset figure besides the hub genes cdk1 vegfarunx1 cdk6 hsp90aa1 and myc with fold change and respectivelyin hu esophagus dataset figure both sources report markedly increased expression of hub genes wereassociated wi | Colon_Cancer |
" drug resistance leads to tumor relapse and further progression during chemotherapy in lung cancer close homolog of l1 chl1 has been identified as a tumor suppressor in most malignancies however to the best of our knowledge whether chl1 mediates chemoresistance remains unknown the present study observed that chl1 was significantly downregulated in cisplatin ddpresistant cells a549ddp and paclitaxel ptxresistant cells a549ptx compared with a549 cells when treated with or without ddp and ptx silencing of chl1 in a549 cells promoted the cell survival rate and clone formation and decreased apoptosis whereas overexpression of chl1 in a549ddp and a549ptx cells impeded the cell survival and clone formation and promoted apoptosis additionally chl1 overexpression enhanced the chemosensitivity of a549ddp cells to ddp in vivo notably the chemoresistance induced by chl1 depletion was reversed by the akt inhibitor sc66 in a549 cells the results of the present study demonstrated that chl1 enhanced sensitivity of lung cancer cells by suppressing the akt pathway which suggested that chl1 may be a potential target for overcoming chemoresistance in lung cancerintroductionlung cancer is the most common human malignancy accounting for of all cancerassociated deaths worldwide during in addition its morbidity and mortality rank the highest among all malignant tumor types worldwide according to the differentiation degree and morphological correspondence to dr rimao huang department of cardiothoracic surgery xiangya changde hospital moon avenue west of langzhou north road changde hunan pr chinaemail xyhuangrm163comkey words lung cancer close homolog of cisplatin paclitaxel chemosensitivitycharacteristics of cancer cells lung cancer can be roughly classified into nonsmallcell lung cancer nsclc and smallcell lung cancer among patients with lung cancer nearly are diagnosed as nsclc which manifests with earlier diffusion and metastasis currently resection chemotherapy radiotherapy and targeted therapy are the primary treatments for lung cancer for patients with advanced nsclc or those who are clinically incapacitated for surgery chemotherapy is a remarkably important treatment cisplatin ddp is widely applied in the treatment of several malignancies and it exhibits a broad spectrum of antitumor effects by inducing dna damage and hindering dna damage repair paclitaxel ptx another commonly used chemotherapeutic agent in the clinic targets the microtubule cytoskeleton and impedes cell division the majority of patients have a good initial response to chemotherapy agents however subsequent relapse is common and largely due to the emergence of drug resistance thus chemoresistance is considered one of the main factors of poor prognosis in patients with advanced nsclc therefore there is an urgent need to investigate the target and mechanism of chemoresistance in nsclcclose homolog of l1 chl1 is a member of the l1 family of nerve cell adhesion molecules and is located on the 3q26 locus as a nerve cell adhesion molecule chl1 serves an important role in the development regeneration and plasticity of the nervous system the absence or mutation of chl1 can trigger 3p syndrome and schizophrenia the abnormal expression of chl1 may lead to reduced working memory and social behavior mental damage and abnormal behavior chl1 has been reported to be involved in carcinogenesis and progression in a variety of human cancers in esophageal squamous cell carcinoma escc chl1 downregulation is associated with invasion lymph node metastasis and poor overall survival functional studies revealed that chl1 has antiproliferation and antimetastasis abilities the expression of chl1 is downregulated by hypermethylation in human breast cancer and its negative expression contributes to breast tumorigenesis and progression in thyroid cancer and colonic adenocarcinoma chl1 impedes cell proliferation and invasion and acts as a tumor suppressor in lung cancer hÓ§tzel evaluated chl1 expression 0ccai chl1 enhances the chemosensitivity of lung cancer cellsin nsclc cases based on a tissue microarray and it was reported that chl1 expression is associated with t stage in adenocarcinomas as well as with metastatic lymph node status and improved survival additionally by analyzing the gene expression omnibus dataset gse21656 submitted by sun microarray results demonstrated that chl1 expression in ddpresistant h460 cells is significantly lower compared with that in parental cells suggesting that chl1 may be involved in nsclc chemoresistance however to the best of our knowledge the underlying mechanism remains unknownin the present study the expression of chl1 in ddp and ptxresistant a549 cells and the parental cells was assessed functional studies of chl1 were performed to investigate its potential role in chemoresistancematerials and methodsdata processing the human gse21656 microarray dataset was downloaded from the ncbi gene expression omnibus geo database wwwncbinlmnihgovgeo the available dataset gse21656 was based on the gpl6244 platform affymetrix human gene st array affymetrix thermo fisher scientific inc this data includes h460 cells and ddpresistant h460 cells sample and each cell has three repeats samples the online tool geo2r httpwwwncbinlmnihgovgeogeo2r was used to determine the differentially expressed genes in h460 and ddpresistant h460 cells p005 and log2foldchange¥ were set as cutoff standardscell culture the human nsclc cell line a549 the ptxresistant cell line a549ptx and the ddpresistant cells a549ddp were purchased from procell life science technology co ltd the cells were cultured in ham's f12k medium supplemented with fetal bovine serum both purchased from thermo fisher scientific inc uml penicillin and uml streptomycin cat no thermo fisher scientific inc in a Ëc humidified incubator with co2cell transfection the resistant cells a549ptx and a549ddp cells were transfected with µg chl1 recombinant expression plasmid cat no hg10143ny sino biological inc empty vector pcmv3spnha was used as the control a549 cells were transfected with pmol small interfering sirnas the sirna sequence for chl1 guangzhou ribobio co ltd were sirna 'gga gcu aau uug acc aua utt' sirna 'cag caa uau uag cga gua utt' and scrambled control 'uuc ucc gaa cgu guc acg utt' plasmids and sirnas were transfected into cells using lipofectamine® thermo fisher scientific inc following the manufacturer's instructions the time interval between transfection and subsequent experimentation was h for the rescue experiments the chl1 silenced a549 cells were treated with the akt inhibitor sc66 cat no s5313 selleck chemicals along with ddp µgml or ptx ngml both purchased from selleck chemicals for h at Ëcrna extraction and reverse transcriptionquantitative pcr rtqpcr assay total rnas were isolated using trizol reagent thermo fisher scientific inc according to the manufacturer's instructions and the mixed dnas were eliminated by dnase i new england biolabs inc firststrand cdna synthesis was conducted using the goscripttm kit promega corporation according to the manufacturer's instructions the reaction conditions for reverse transcription were Ëc for min Ëc for min and Ëc for min the sybr green realtime pcr master mix thermo fisher scientific inc was used to perform rtqpcr using a lightcycler480 system roche diagnostics gmbh the chl1 primer sequences were as follows forward 'ggc ttg gtc tct tgc ttt cc' and reverse 'atc ttc cct ccc ttt gca cg' and actin forward 'ttc ctt cct ggg cat gga gtc ' and reverse 'tct tca ttg tgc tgg gtg cc' the following thermocycling conditions were used for qpcr min at Ëc followed by cycles at Ëc for sec sec at Ëc and a final extension at Ëc for sec each reaction was conducted in triplicate relative expression levels were calculated using the δδcq method cell viability cell viability was detected by mtt assay a cell suspension µl was seeded into well plates at a density of 1x104 cellswell and incubated overnight at Ëc the concentrations of ddp used to treat a549 cells were and µgml while the concentrations of ptx used to treat a549 cells were and ngml the concentrations of ddp used to treat a549ddp cells were and µgml while the concentrations of ptx used to treat a549ptx cells were and ngml after treating with different concentrations of ddp or ptx for h at Ëc µl mtt mgml solution was added to each well and incubated for h at Ëc subsequently µl dmso was added to each well to dissolve the blue formazan crystals and the absorbance was measured using a microplate reader biotek instruments inc at nmclone formation assay a total of 1x103 cells were seeded into a mm dish in triplicate and maintained in f12k medium with or without ddp or ptx at Ëc for h a total of weeks later cells were fixed in paraformaldehyde for min at room temperature and stained with crystal violet dye at room temperature for min the rate of colony formation was calculated using the following equation number of coloniesnumber of seeded cells x100flow cytometry apoptosis was detected using a fitc annexin v apoptosis kit bd pharmingen bd biosciences according to the manufacturer's protocol cells 1x105 were collected and washed twice with pbs prior to being suspended in µl binding buffer subsequently cells were incubated with µl annexin vfitc and µl propidium iodide in the dark for min at room temperature and apoptosis was analyzed using a cytoflex flow cytometer beckman coulter inc data were analyzed using cytexpert software beckman coulter inc the ratio between early and late apoptosis was calculatedwestern blotting cells were collected washed twice with pbs and lysed with ripa lysis buffer thermo fisher scientific inc proteins were isolated from the cell lysis buffer and 0concology letters quantified using the piercetm¢ bca protein assay kit cat no thermo fisher scientific inc with bovine serum album as a standard equal amount of protein µg proteins were separated by sdspage gel next the proteins were transferred onto a polyvinylidene membrane thermo fisher scientific inc blocked with bsa thermo fisher scientific inc for h at Ëc and incubated overnight at Ëc with primary antibodies against chl1 cat no ap proteintech inc multidrug resistance gene mdr1 cat no ap proteintech inc multidrug resistanceassociated protein mrp cat no ig proteintech inc lowdensity lipoprotein receptorrelated protein lrp cat no ap proteintech inc phosphorylated pakt cat no ab38449 abcam and akt cat no ab227385 abcam after washing three times with pbs the membrane was incubated with horseradish peroxideconjugated goat antirabbit cat no ab6271 abcam_or rabbit antimouse cat no ab6728 abcam secondary antibodies for h at room temperature and the blots were detected with enhanced chemiluminescence reagent thermo fisher scientific inc protein expression was quantified using imagepro plus software media cybernetics incanimal experiments the animal experiments were approved by the medical ethics committee of xiangya changde hospital approval no and were performed in compliance with all regulatory institutional guidelines for animal welfare the national institutes of health publications no a total of male balbcnu mice weekold ± g hunan sja laboratory animals center of the chinese academy of sciences were used in this study all animals were kept at the spf level laboratory at Ëc a relative humidity of a h lightdark cycle and timesh of fresh air exchange all mice were given free access to food and water the bedding materials drinking water feeding cages and other items in contact with the animals were all autoclaved prior to use a549ddp cells 1x107 transfected with empty vector and chl1 overexpression vector using lipofectamine® reagent thermo fisher scientific inc were subcutaneously injected into the nude mice to establish xenograft models following anaesthesia with chloral hydrate mgkg xenografts were allowed to grow to mm3 over weeks and the mice were randomly divided into four groups n3group as follows i vector group a549ddp cells transfected with empty vector and treated with µl saline solution ii vectorddp group a549ddp cells transfected with empty vector and treated with mgkg ddp iii chl1 group a549ddp cells transfected with chl1 overexpression vector and treated with µl saline solution and iv chl1ddp group a549ddp cells transfected with chl1 overexpression vector and treated with mgkg ddp ddp was administered by intraperitoneal injection every days for weeks the mice were observed daily and the tumors were measured by a vernier caliper every days the tumor volumes were calculated as length x width22 a total of weeks postinjection mice were euthanized with co2 at volume displacement rate vdr per min using a programmable logic controller barrywehmiller design group inc mice were monitored continuously and once the mice were immobile except for breathing for min the vdr was provided at for min the animals remained in the euthanasia chamber for min and were then observed for an additional min breathing and heart rate were monitored to determine deathstatistical analysis all experiments were performed in triplicate and data are presented as the mean ± standard deviation all experiments were performed at least three times paired student's ttest was performed for comparisons between two groups and oneway analysis of variance followed by tukey's multiple comparison posthoc analysis was performed for comparisons between multiple groups spss ibm corp was used to perform the analysis p005 was considered to indicate a statistically significant differenceresultschl1 is downregulated in a549ddp and a549ptxresistant cells in order to investigate the mechanism of chemoresistance in lung cancer the lung adenocarcinoma cell line a549 the ddpresistant cells a549ddp and ptxresistant cells a549ptx were used in the present study cells were exposed to different concentrations of ddp µgml and ptx ngml and mtt assay was used to detect the cell survival rate a549ddp and a549ptx cells demonstrated higher resistance to ddp and ptx compared with a549 cells fig 1a the half maximal inhibitory concentration ic50 of ddp was significantly higher in a549ddp cells ± µgml compared with a549 cells ± µgml and the ic50 of ptx was significantly higher in a549ptx cells ± ngml compared with a549 cells ± ngml fig 1b in addition the expression levels of the drugresistant markers mdr1 mrp and lrp were significantly higher in a549ddp and a549ptx cells compared with a549 cells fig 1c additionally the mrna and protein expression levels of chl1 were significantly lower in a549ddp and a549ptx cells compared with those in a549 cells fig 1d and e and this was also observed in h460 ddpresistant cells obtained from the geo dataset gse21656 fig 1f these results suggested that chl1 may be involved in regulating ddp and ptx resistance in nsclcknockdown of chl1 enhances resistance to ddp and ptx in a549 cells as chl1 was upregulated in a549 cells chl1 was silenced in a549 cells using sirnas chl1 expression was significantly reduced in the chl1 sirna groups compared with that of the scrambled control group fig 2a as sirna demonstrated the greatest interference efficiency it was selected for use in the following experiments notably chl1knockdown enhanced the resistance to ddp and ptx in a549 cells fig 2b and c colony formation assay revealed that compared with the control group chl1knockdown significantly increased the rate of colony formation in the absence of chemotherapeutics and enhanced the resistance to ddp and ptx fig 2d flow cytometry results demonstrated significantly reduced apoptosis in chl1knockdown cells after ddp and ptx treatment compared with that of the control group fig 2e 0ccai chl1 enhances the chemosensitivity of lung cancer cellsfigure chl1 is downregulated in ddp and ptxresistant a549 cells a cell survival of a549 and a549resistant cells a549ddp and a549ptx treated with increasing concentrations of ddp and ptx as assessed by mtt assay b the ic50 values of ddp in a549ddp and a549 cells and the ic50 values of ptx in a549ptx and a549 cells p005 vs a549 cells c western blotting demonstrated the expression of drug resistancerelated proteins mdr1 mrp and lrp in a549 cells and a549resistant cells a549ddp and a549ptx p005 vs a549 cells the protein and mrna expression levels of chl1 in a549 cells and a549resistant cells a549ddp and a549ptx were analysed by d western blotting and e reverse transcriptionquantitative pcr respectively p005 vs a549 cells f the mrna expression of chl1 in h460 and h460ddp cells in the gse21656 dataset p005 vs h460 cells chl1 close homolog of l1 ddp cisplatin ptx paclitaxel mdr1 multidrug resistance gene mrp multidrug resistanceassociated protein lrp lowdensity lipoprotein receptorrelated protein ic50 half maximal inhibitory concentration chl1 overexpression enhances the sensitivity of a549 resistant cells to ddp and ptx as chl1 is downregulated in a549ddp and a549ptx cells the present study successfully overexpressed chl1 in these cells using chl1 recombinant expression plasmids fig 3a the results demonstrated that chl1 overexpression alleviated the resistance to ddp and ptx compared with that of the control group fig 3b and c in addition chl1 overexpression inhibited colony formation in the absence or presence of ddp and ptx fig 3d additionally flow cytometry results demonstrated that restoration of chl1 expression promoted apoptosis in resistant cells following ddp and ptx treatment fig 3eto further validate the effects of chl1 overexpression on ddp or ptx sensitivity xenograft mice model experiments were performed the results demonstrated that chl1 overexpression or ddp treatment significantly impeded the tumor growth fig 3f and decreased the tumor weight fig 3g in addition chl1 overexpression further aggravated ddpmediated repression on tumor growth fig 3f and g these data suggested that chl1 overexpression suppressed tumor growth and enhanced the chemosensitivity in nsclcchl1 mediates chemosensitivity by inhibiting akt activity recently studies have confirmed that chl1 inhibits akt activity in escc and neuroblastoma cell lines thus the present study investigated whether chl1 mediates chemoresistance via the akt pathway in nsclc in a549 cells compared with the scrambled group chl1knockdown elevated the expression of paktser473 fig 4a by contrast restoring chl1 expression in a549ddp and a549ptx cells inhibited the akt phosphorylation compared with the control group fig 4a suggesting chl1 mediates chemosensitivity via the akt pathway subsequently chl1silenced a549 cells were treated with the akt inhibitor sc66 and it was demonstrated that inhibiting akt activity significantly reduced the promotive effects on cell survival fig 4b and clone formation fig 4c and the inhibitory effects on apoptosis fig 4d induced by chl1depletion these results confirmed that chl1 mediates chemosensitivity in nsclc by inhibiting the akt pathway 0concology letters figure chl1knockdown increases a549 cell resistance to ddp and ptx a western blotting was performed to validate the efficiency of transfection with chl1 sirnas p005 vs scramble mtt assays were performed to determine the survival rate of chl1knockdown a549 cells treated with b µgml ddp or c ngml ddp d colony formation assay of a549 cells transfected with chl1 sirna in the presence or absence of µgml ddp or ngml ptx e flow cytometry analysis was used to detect apoptosis in a549 cells transfected with chl1 sirna in the presence or absence of µgml ddp or ngml ptx p005 p0001 chl1 close homolog of l1 ddp cisplatin ptx paclitaxel si small interfering discussionthe results of the present study demonstrated that chl1 was significantly downregulated in a549ddp and a549ptx cells compared with a549 cells the knockdown of chl1 in a549 cells facilitated the cell survival and clone formation and decreased apoptosis when treated with or without ddp and ptx whereas chl1 overexpression in a549ddp and a549ptx cells inhibited cell survival and clone formation and increased apoptosis the results of the present study also demonstrated that chl1 enhances nsclc chemosensitivity through inhibition of the akt pathway these data suggested that chl1 may be a promising target to improve the efficacy of chemosensitivity in nsclcchl1 belongs to the l1 family of nerve cell adhesion molecules it was initially cloned in mice and its expression in mouse development was analyzed by senchenko through cellcell interactions and mediating cellcell and cellmatrix interactions chl1 has an important effect on the development regeneration and plasticity of the nervous system previous reports have demonstrated that chl1 also participates in carcinogenesis chl1 was observed to be significantly downregulated in up to types of tumor tissues compared with their adjacent normal tissues in most tumors chl1 is a potential tumor suppressor gene whose silencing is associated with tumor growth invasion and metastasis for example knockdown of chl1 expression results in enhanced cervical cancer cell invasion and migration a low expression of chl1 in patients with neuroblastoma predicts a poor prognosis and enhancing chl1 expression suppresses tumor progression in contrast chl1 has been reported to promote cell proliferation metastasis and migration in human gliomas however to the best of our knowledge research on chl1 and tumor chemoresistance has rarely been reported 0ccai chl1 enhances the chemosensitivity of lung cancer cellsfigure overexpression of chl1 increases the sensitivity of resistant a549 cells to ddp and ptx a western blotting was performed to detect chl1 expression in a549ddp and a549ptx cells transfected with chl1 expression plasmids p005 vs vector effect of chl1 overexpression on resistant a549 cell survival rate when treated with b µgml ddp or c ngml ptx as determined by mtt assay d colony formation assays demonstrated the number of colonies of resistant a549 cells transfected with chl1 expression plasmids in the presence or absence of µgml ddp or ngml ptx e flow cytometry analysis was performed to assess apoptosis in resistant a549 cells transfected with chl1 expression plasmids in the presence or absence of µgml ddp or ngml ptx chl1 overexpression enhanced chemosensitivity of a549ddp cells to ddp in vivo which was demonstrated by the effect of ddp treatment or chl1 overexpression on the f growth and g weight of xenografts derived from a549ddp cells p005 p001 chl1 close homolog of l1 ddp cisplatin ptx paclitaxel the present study examined the differentially expressed genes in nsclc ddpresistant cells in a geo dataset chl1 was demonstrated to be upregulated in ddpresistant cells compared with parental cells suggesting that chl1 may be involved in nsclc chemotherapy resistance similarly a study that compared and analyzed the differentially expressed genes in chemosensitive tumors and chemoresistant ovarian adenocarcinomas tissues reported that the expression of chl1 in chemotherapysensitive tumor tissues is higher compared with that in drugresistant tissues suggesting that chl1 may help to predict the efficacy of chemotherapy for ovarian cancer in addition aberrant methylation of chl1 may be associated with the recurrence of colorectal cancer crc following chemotherapy azadc treatment restores flurouracil sensitivity in vitro which also suggests that chl1 may be involved in crc chemotherapy resistance the results of the present study demonstrated that chl1 was downregulated in a549ddp cells additionally as multiple drug resistance is a common characteristic another type of resistant cells a549tax cells were also used in the current study the results also demonstrated that chl1 was downregulated in a549ptx cells compared with control cells overexpression of chl1 significantly increased the sensitivity of cells resistant to ddp and ptx whereas knockdown of chl1 expression in 0concology letters figure chl1 mediates ddp and ptx sensitivity by inhibiting akt activity a western blotting was performed to detect the expression of pakt and total akt in chlsilenced and restored cell models p005 vs scramble or vector b mtt assays were performed to detect cell survival rates of a549 cells treated with chl1 sirna and akt inhibitor sc66 p005 c colony formation assays were performed in a549 cells treated with chl1 sirna and the akt inhibitor sc66 in the presence of ddp µgml or ptx ngml p005 vs sichl1 d apoptosis were measured in a549 cells treated with chl1 sirna and akt inhibitor sc66 in the presence of ddp µgml and ptx ngml p005 vs sichl1 chl1 close homolog of l1 ddp cisplatin ptx paclitaxel si small interfering p phosphorylated parent a549 cells displayed the opposite results to the best of our knowledge this study is the first study to suggest that chl1 may be involved in chemosensitivity in lung cancer the concentration of ddp used in vivo is mgkg however this may not be in line with the concentrations that would be used in a clinical setting in a clinical trial the human initial dose was calculated from the no observed adverse effect levels noaels verified in animal experiments noael is the maximum dose level without significant adverse reactions the noael verified in animal experiments can be converted to a human equivalent dose according to the body surface area conversion which is based on the area standardization mgm2 proportional among different species in the present study the concentration of ddp used in vivo was not the noael thus it was not consistent with the concentrations used in clinical settingsakt is a serinethreonine protein kinase that is activated by phosphorylation as a key molecule of the pi3kakt signaling pathway pakt regulates cell survival cell growth cell motility and angiogenesis and prevents apoptosis additionally akt activation is associated with tumor chemoresistance the results of the present study demonstrated that compared with the control groups the expression of pakt was increased in chl1knockdown a549 cells and its expression was reduced in chl1 overexpressed a549ddp and a549ptx cells when akt activity was inhibited by the akt inhibitor the sensitivity to ddp and ptx in chl1knockdown a549 cells was restored this finding suggested that chl1 enhanced the chemosensitivity of nsclc by inhibiting the akt pathway considering numerous studies have confirmed that the akt pathway mediates chemoresistance via regulation of atp binding cassette abc members the present study didn't further investigate the specific abc members and mechanisms which was a of the limitation to the present study thus this research should be further investigated in vivoin summary the present study demonstrated that chl1 was downregulated in resistant cells a549ddp and a549ptx and upregulation of chl1 enhanced the chemosensitivity of nsclc via inhibiting the akt pathway to the best of our knowledge this was the first study to confirm the function and 0ccai chl1 enhances the chemosensitivity of lung cancer cellsmechanism of chl1 in mediating chemosensitivity in cancer thus the development of chl1based therapeutic strategies may improve the efficacy of chemosensitivity in nsclcacknowledgementsthe authors of the present study would like to thank mr dingliang li xiangya hospital changsha china for his guidance and assistance in flow cytometric analysisfundingno funding was receivedavailability of data and materialsthe datasets used andor analyzed during the present study are available from the corresponding author upon reasonable requestauthors' contributionsrh conceived and designed the present study xc bh yh and pl performed experiments and collected the data sl zz and zh analyzed and interpreted the data ml and lz analyzed the data and prepared the figure xc ml and lz drafted the initial manuscript and revised it for intellectual content all authors read and approved the final manuscriptethics approval and consent to participatethe animal experiments were approved by the medical ethics committee of xiangya changde hospital changde china approval no patient consent for publicationnot applicablecompeting intereststhe authors declare that they have no competing interestsreferences parascandola m and xiao l tobacco and the lung cancer epidemic in china transl lung cancer res suppl s21s30 chen w cancer statistics updated cancer burden in china chin j cancer res oser mg niederst mj sequist lv and engelman ja transformation from nonsmallcell lung cancer to smallcell lung cancer molecular drivers and cells of origin lancet oncol e165e172 thatcher n faivrefinn c blackhall f anderson h and lorigan p sequential platinumbased chemotherapythoracic radiotherapy in early stage nonsmall cell lung cancer clin cancer res suppl s5051s5056 yano t okamoto t fukuyama s and maehara y therapeutic strategy for postoperative recurrence in patients with nonsmall cell lung cancer world j clin oncol fang z chen w yuan z liu x and jiang h lncrnamalat1 contributes to the cisplatinresistance of lung cancer by upregulating mrp1 and mdr1 via stat3 activation biomed pharmacother cai y dong zy and wang jy lncrna nntas1 is a major mediator of cisplatin chemoresistance in nonsmall cell lung cancer through mapkslug pathway eur rev med pharmacol sci han ml zhao yf tan ch xiong yj wang wj wu f fei y wang l and liang zq cathepsin l upregulationinduced emt phenotype is associated with the acquisition of cisplatin or paclitaxel resistance in a549 cells acta pharmacol sin liu j meisner d kwong e wu xy and johnston mr translymphatic chemotherapy by intrapleural placement of gelatin sponge containing biodegradable paclitaxel colloids controls lymphatic metastasis in lung cancer cancer res hassan wa yoshida r kudoh s kameyama h hasegawa k niimorikita k and ito t notch1 controls cell chemoresistance in small cell lung carcinoma cells thorac cancer tang h jiang l zhu c liu r wu y yan q liu m jia y chen j qin y loss of cell adhesion molecule l1 like promotes tumor growth and metastasis in esophageal squamous cell carcinoma oncogene liu h focia pj and he x homophilic adhesion mechanism of neurofascin a member of the l1 family of neural cell adhesion molecules j biol chem tassano e biancheri r denegri l porta s novara f zuffardi o gimelli g and cuoco c heterozygous deletion of chl1 gene detailed arraycgh and clinical characterization of a new case and review of the literature eur j med genet morellini f lepsveridze e kahler b dityatev a and schachner m reduced reactivity to novelty impaired social behavior and enhanced basal synaptic excitatory activity in perforant path projections to the dentate gyrus in young adult mice deficient in the neural cell adhesion molecule chl1 mol cell neurosci he lh ma q shi yh ge j zhao hm li sf and tong zs chl1 is involved in human breast tumorigenesis and progression biochem biophys res commun martÃnsánchez e mendaza s ulaziagarmendia a monrealsantesteban i blancoluquin i córdoba a vicentegarcÃa f pérezjanices n escors d megÃas d chl1 hypermethylation as a potential biomarker of poor prognosis in breast cancer oncotarget zhu h fang j zhang j zhao z liu l wang j xi q and gu m mir targets chl1 and controls tumor growth and invasion in papillary thyroid carcinoma biochem biophys res commun yu w zhu k wang y yu h and guo j overexpression of mir5p promotes proliferation and invasion of colon adenocarcinoma cells through targeting chl1 mol med hötzel j melling n müller j polonski a wolterseisfeld g izbicki jr karstens kf and tachezy m protein expression of close homologue of l1 chl1 is a marker for overall survival in nonsmall cell lung cancer nsclc j cancer res clin oncol sun y zheng s torossian a speirs ck sc | Colon_Cancer |
"pd1pdl1 blockade therapy is a promising cancer treatment strategy which has revolutionized the treatmentlandscape of malignancies over the last decade pd1pdl1 blockade therapy has been trialed in a broad range ofmalignancies and achieved clinical success despite the potentially curelike survival benefit only a minority ofpatients are estimated to experience a positive response to pd1pdl1 blockade therapy and the primary oracquired resistance might eventually lead to cancer progression in patients with clinical responses accordingly theresistance to pd1pdl1 blockade remains a significant challenge hindering its further application to overcomethe limitation in therapy resistance substantial effort has been made to improve or develop novel antipd1pdl1based immunotherapy strategies with better clinical response and reduced immunemediated toxicity in thisreview we provide an overview on the resistance to pd1pdl1 blockade and briefly introduce the mechanismsunderlying therapy resistance moreover we summarize potential predictive factors for the resistance to pd1pdl1blockade furthermore we give an insight into the possible solutions to improve efficacy and clinical response inthe following research combined efforts of basic researchers and clinicians are required to address the limitation oftherapy resistancekeywords pd1pdl1 blockade cancer immunotherapy resistance immunotherapy is a validated and significant cancertreatment strategy which eliminates tumors by normalizing the antitumor immune responses [ ] over thelast decade cancer immunotherapy has revolutionizedthe treatment landscape of malignancies and achievedclinical success especially in immune checkpoint inhibitors correspondence 189whueducn lschrjjs163com jinyu sun and dengke zhang are cofirst authors4department of general surgery the first affiliated hospital of nanjingmedical university nanjing china2key laboratory of imaging diagnosis and minimally invasive interventionresearch lishui hospital of zhejiang university the fifth affiliated hospitalof wenzhou medical university clinical medicine of center hospital of lishuicollege lishui chinafull list of author information is available at the end of the signalsandprogrammed death1 pd1 is a class of receptorexpressed on the t cell surface which could downregulate the immune system by abrogating t cellreceptorinducedantigenmediated t cell activation the interaction betweenpd1 and its ligand programmed deathligand pdl1 plays an essential role in maintaining selftoleranceand avoiding autoimmune diseases however pd1pdl1 could also prevent the activation of t cells in thetumor and thus result in immune resistance preventingpd1pdl1 blockade is a breakthrough in cancerimmunotherapy and it has been trialed in a broadrange of malignancies in the preclinical or clinicalincluding melanoma hodgkins lymphomastage breast cancer [ ] nonsmall celllung cancer as well as hepatocellular carcinomansclc the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0csun biomarker research page of [ ] despite the longterm potentially curelikeclinical benefits therapy resistance remains a significant challenge for the further application of pd1pdl1 blockade therapy only a minority of patientsin general are estimated to experience apositive response to pd1pdl1 blockade therapy[] and the primary or acquired resistance mighteventually lead to cancer progression in patients withclinical response [ ]in this review we provide an overview on the resistance to pd1pdl1 blockade and its underlying mechanisms moreover we summarize potential predictivefactors for the resistance to pd1pdl1 blockade furthermore we give an insight into the possible solutionsto improve efficacy and clinical response of pd1pdl1blockade therapyresistance to pd1pdl1 blockade therapycheckpoint inhibitors targeting pd1 or pdl1 coulddisturb the interaction between pd1 and pdl1 whichwould preserve antitumor properties of t cells withdraw immune escape and normalize their ability to induce tumor cell death currently pd1pdl1 blockadehas shown sustained survival benefits in multiple malignancies and is at the forefront of cancer immunotherapy howeverjust as tumor cells can avoid immuneevasion several cancers may evolve to resist pd1pdl1 blockade therapy clinical evidence indicated thateven for patients with tumors highly positive for pdl1more than of them might not respond to pd1pdl1 blockade due to tumor heterogeneity and manyother reasons clinical responses vary largely across different tumor entities the objective response rate was in melanoma in nsclc in head and neck carcinoma and in kidneycancer besides for most patients experiencing initial clinical response acquired resistance remains another problem which would lead to cancer progressionor relapse after a few years [ ]many studies have demonstrated that antipd1therapy can significantly improve survival outcomes forpatients with metastatic or unresectable melanoma however only a small number of patients approximately could achieve a complete response in arecent phase i trial of atezolizumab antipdl1 involving patients with metastatic melanoma the overall response rate was among efficacy evaluablepatients and the median response duration was months moreover in another study on the longtermoutcomes of melanoma patients receiving antipd1therapy complete responses were only observed in of patients after a median followup of months of patients were alive without additional melanoma therapy additionally in the retreatedpatients after disease progression the response was onlyobserved in retreated patients receiving singleagent pd1 blockade therapy and of patientsescalated to pd1 blockade plus ipilimumab therapy inthis cohort most complete responses were durable withthe treatment failure rate of at three years whilethe response to retreatment remained relatively infrequent with a response rate of for patients withsingleagent pd1 blockade therapy moreover in aphase ii study of pembrolizumab on patients withadvancedobjectiveresponse was observed in of patients with a diseasecontrol rate of after a median followup of months adrenocorticalcarcinomatheinterestingly the response rate of some malignanciesis relatively high in hematological malignancies for example for patients with relapsed or refractory classicalhodgkin lymphoma tislelizumab antipd1 achievedan objective response rate of and a completeresponse of in a phase ii singlearm multicenterstudy similarly the complete response rate of camrelizumab antipd1 was with a partial remission rate of mechanisms underlying the resistance to pd1pdl1 blockadesince therapy resistance remains a significant limitationof pd1pdl1 blockade in clinical practice interest isgrowing in understanding the mechanisms underlyingthe resistance the response to pd1pdl1 blockaderelies on a preexisting immune response and determinants of adaptive immunity currently multiple factorshave been discovered to be involved in the efficacy ofpd1pdl1 blockade therapy such as tumor immunogenicity t celltumormicroenvironment and so forthfunction pdl1 expressionthe lack of tumor antigensthe genetic alterations are centralin the oncogenicprocess which could lead to tumor immunogenicity andprovide an opportunity for cancer immunotherapy tumor immunogenicity is positively associated with theability of the t cell to recognize tumor cells which isessential for the antitumor effect of pd1pdl1 blockade however the lack of tumor antigen will significantlyimpede the recognition ability of t cells and eventuallyresult in the failure of immunotherapymicrosatellites are prone to dna replication errorswhich will usually be repaired in normal cells however in tumors with mismatch repair mmr deficiencythese errors will accumulate which eventually result in alarge number of mutations and lead to microsatellite instability msi importantly high msi positivelycontributes to increased neoantigen production greater 0csun biomarker research page of immunogenicity and a more robust immune response moreoverthe resultant high tumor mutationburden would contribute to tumor immunogenic andenhance the response to pd1pdl1 blockade therapy[ ]multiple studies have demonstrated that the tumormutation burden is positively correlated with neoantigenburden as well as response to immunotherapy [ ]for example in colorectal cancer with mmr deficiencywhich usually exhibits a high tumor mutation burdenantipd1 therapy showed a higher response rate andbetter survival outcome compared to other subtypeswith mmr proficiency [] yarchoan analyzed the objective response rates of pd1pdl1blockade therapy for the corresponding tumor mutationburden in various cancers and their results showed thatthe mutation burden was closely associated with the objective response rate moreover pancreatic cancer generally exhibits a lowermutation load compared with other solid tumors andtherefore pd1pdl1 blockade is usually ineffective forthose patients and fails to improve their survival outcomes nevertheless in pancreatic cancer patients harboring an mmr deficiency they appear to be responsiveto pd1pdl1 blockade therapy mmr deficiency significantly increases the somatic mutation rate whichcould be translated into neoantigens and recognized bythe immune system thus making these patients responsive to pd1pdl1 blockade therapy [ ] accordingly pembrolizumab has been approved for selectedcancer patients with mmr deficiencyt cell dysfunctioneffective pd1pdl1 blockade therapy relies on the tcell function and any disruption in the processes of tcell immune function will result in the failure of pd1pdl1 blockade therapy a recent review by ren has provided an indepth insight into the mechanisms underlying the t cell dysfunctionmediated resistance with a focus on t cell recognition activationdifferentiation infiltration depletion as well as chemotaxis identification byantigen presentation is a critical process for the tumorantigensinitial t cells beta2microglobulin b2m is a significant hla1 moleculewhose mutation will hinder tumor antigen presentationand result in therapy resistance [] zaretsky analyzed biopsy samples from patients with metastatic melanoma receiving pembrolizumab who exhibited disease progression after an initial tumor regressionand they found a truncating mutation in the b2m genein the following research gettinger identifiedacquired homozygous loss or downregulation of b2m inlung patients with resistance to pd1pdl1 blockadeto further explore the role of b2m in mediating resistance they knocked out the b2m gene in immunocompetent lung cancer mice by crispr technology and theloss of b2m resulted in the resistance to pd1pdl1blockade additionally b2m mutationinducedresistance primarily occurred in an environment ofactivated pd1 positive t cellinfiltration whichresistance to pd1pdl1 blockadesuggested thattherapy might be particularly common in patients withhigh pd1 positive t cell for example t cellmoreover t cell activation is another critical processfor pd1pdl1 blockade therapy after blocking pd1pdl1 tumor cells can still counteract the activity ofimmune checkpoints and activate additional inhibitorypathways via expression of other immune checkpointsand their ligands within the tumor immune microenvironmentimmunoglobulinmucin3 tim3 is another type of immune checkpointreceptor expressed on tumorinfiltrating lymphocytes inhuman head and neck squamous cell carcinoma tumorinfiltrating lymphocytes pd1 blockade was demonstrated to upregulate tim3 expression which inhibitedt cells activation and contributed to tim3mediatedescape from pd1 blockade in the tumor microenvironment via pi3kakt pathway pd1 or pdl1physiologicallyinteractions between pd1 and pdl1block t cell activation pathways related to the immuneresponse against specific antigens and the expression ofpd1 or pdl1 has gained importance as a significantplayerin regulating the response to pd1pdl1blockade therapy pd1 and pdl1 are upregulated inthe tumor immune microenvironment of various malignancies which is considered as a strategy to evadeimmunosurveillance and imposes a significant barrier ofthe antitumor immune response importantly pdl1 primarily exhibits two distinct expression patternson tumor cells or on tumorinfiltrating immune cellspdl1 expression on immune cells reflects the adaptiveregulation meditated by ifnγ which is accompanied byincreased effector t cells as well as tumorinfiltratinglymphocytes effector t cells differently the expressionof pdl1 on tumor cells is less prevalent and it indicates the epigenetically dysregulated pdl1 gene whichis correlated with reduced immune infiltration scleroticor desmoplastic stroma as well as mesenchymal molecular features multiple studies have revealed a significantly higherobjective response rate in tumor pdl1 positive patientsthan pdl1 negative subgroups together with an improved progressionfree and overall survival [ ]kowanetz observed that atezolizumab antipdl1 achieved an objective response rate of in 0csun biomarker research page of patients with high pdl1 levels on tumor cells alone andof in those with a high expression on immune cellsalone although these observations indicated that thefunctional importance of pdl1 expression in regulatingpd1pdl1 blockadeinduced t cellthemechanistic significance of pdl1 on tumor cells or immune cells remains vagueresponsenoncoding rnasa large amount of micrornas mirnas and some longnoncoding rnas lncrnas have emerged as players inregulating tumor immunity [] and resistance topd1pdl1 blockade therapy recently huber identified a panel of circulating mirnas mir146a mir155 mir125b mir let7e mir125a mir146b mir99b which wereassociated with phenotypic and functional features ofmyeloidderived suppressor cells mdscs in melanomapatients importantly mdscs are a subclass of immature myeloid cells pathologically associated with cancerand play an inhibitory role against antitumor t cell immunity the transcriptional analysis showed thatthese mirnas could facilitate the conversion of monocytes into mdscs by melanoma extracellular vesiclesand the expression level ofthese mirna was upregulated in circulating cd14 monocytes and tumorsamples which was associated with myeloid cell infiltration and could predict the resistance to pd1 blockadetherapy moreover hu revealed the role of oncogeniclncrna for kinase activation linka in losing antigenicity and evading immune checkpoints and demonstrated lncrnadependent antigenicity downregulationsuppression for patients withand intrinsic tumortriplenegative breast cancer and resistantto pd1blockade therapythey showed upregulated linkalevels and downregulated peptideloading complex components the analysis suggested that linka expressioncould attenuate protein kinase amediated phosphorylation of the e3 ubiquitinprotein ligase trim71 via facilitating the crosstalk between phosphatidylinositol []trisphosphate and inhibitory gproteincoupled receptor pathways consequently linka could contribute to the degradation of the antigen peptideloadingcomplex and upregulate intrinsic tumor suppressors gut microbiomethe gut microbiome is a complex system composed ofmore than trillion microanisms which has beendemonstrated to regulate the efficacy and toxicity ofcancer immunotherapy many studies have reported theinfluence of the gut microbiome on cancer immunotherapy and the therapeutic response of pd1pdl1blockade therapy can be improved or diminished via gutmicrobiome modulationin mice models with distinct microbiome a significantly different response to pd1pdl1 blockade therapy was observed for example melanoma mice with anincreased bifidobacterium species in the gut microbiomeexhibited an effective response to pd1 blockade therapy similarly antibiotic administration was reported toreduce the diversity and aggravate dysbiosis of the gutmicrobiome thus influencing the clinical response topd1pdl1 blockade in tumorbearing mice as well ascancer patients [] compared to patients withoutantibiotic treatment the oral antibiotic application couldsignificantly diminish the clinical benefit of pd1pdl1blockade therapy and decrease progressionfree survivaland overall survival therefore dysbiosis of the gut microbiome is considered as one of the putative mechanisms underlying poorresponse to pd1pdl1 blockade therapy and thedualdirectional modulation of the gut microbiome oncancer immunotherapy is increasingly revealed howeverit is still unclear how gut microbiome regulatestherapy response and whether a specific bacterial taxaor gut microbiome as a whole plays a primary role remains largely unclear further research is required toprovide a more indepth understanding of the underlying mechanismspredictive factors for pd1pdl1 blockadetherapydespite the clinical success achieved in pd1pdl1blockade across multiple cancers the knowledge concerning therapy selection criteria is relatively limitedconsidering the potential adverse events and high costof immune checkpoint inhibitor agents there is a substantial need to identify predictive factors to select patients likely to benefit from this therapy currently apartfrom the functional status of immune cells [] ortumor infiltrating lymphocytes multiple factorshave been identified to predict the response to pd1pdl1 blockade therapy such as pd1pdl1 expression antigen recognition gut microbiome and so forthtable pd1 or pdl1 expressioninhibiting the pd1 pathwaymediated immune suppression is the basis and premise of pd1pdl1 blockadetherapy accumulating research has suggested that pdl1 is a biomarker to predict therapeutic response to pd1pdl1 blockade across multiple tumor types forexample atezolizumab achieved overall survival benefitacross all pdl1 expression subgroups in nsclc patients while those with high pdl1 expression experienced a more substantial survival benefit currently 0csun biomarker research page of table predictive factors for pd1pdl1 blockade therapytumor typenonsmall cell lung canceragentatezolizumabmultiple cancerscolorectal cancerurothelial carcinomaurothelial carcinomaurothelial cancermelanomamelanomamelanomapembrolizumabnivolumabatezolizumabatezolizumabatezolizumabantipd1 therapyantipd1 therapyantipd1 therapymmr mismatch repair msi microsatellite instability tmb tumor mutation burdenpredictive factorpdl1pdl1mmr msitmbtmbtmbgut microbiomegut microbiomegut microbiomereference pdl1 testing is recommended as a predictive test fornsclc urothelial carcinoma or head andneck cancers and so forthott assessed the predictive value of pdl1expression in patients with advanced solid tumors receiving pembrolizumab and the analysis showed that tumors with higher pdl1 expression and tumor mutationburden were significantly associated with higher response rate and more prolonged progressionfree survival heat map analysis revealed a close correlationbetween pdl1 expression and a broader pattern ofcoregulated gene expression which involved cytokine recruitment of t cells t cell activation markers as well asantigen presentation also the regression metaanalysisdemonstrated that pdl1 expression level was positivelyassociated with objective response rate p aswell as progressionfree survival p moreover nct02853305 and nct02807636 evaluated the efficacy of pembrolizumab or atezolizumab asfirstline treatment and the current data showed reduced survival in patients with low expression of pdl1accordingly it is advised that pembrolizumab or atezolizumab should be used for adult patients with a relativelyhigh pdl1 expression pdl1 expression of ¥ foratezolizumab and a combined positive score of ¥ forpembrolizumab however the efficacy of pd1pdl1blockade therapy as firstline therapy for advancedurothelial carcinoma still remains unclear [ ]importantly pdl1 positive only is not a predictivefactor for the response to pd1pdl1 blockade sincemultiple factors are involved in the pd1pdl1 blockade therapy in a study on patients with metastaticmelanoma receiving pembrolizumab preexisting cd8t cells were demonstrated as a prerequisite for thetumor regression after pd1pdl1 blockade therapy besidesin advanced adrenocortical carcinomatumor pdl1 expression status was not associated withtherapy response additionally it was reported thatpdl1 expression on tumor cells was not associatedwith therapy response in resected head and necksquamous cell cancer additionalinvestigation isrequired to illustrate the mechanisms accounting for thedifferenceantigen recognitionantigen recognition plays a vital role in initiating theadaptive immune response while the lack of tumor antigens significantly impedes the response to pd1pdl1blockade therapycurrently the fda has approved pembrolizumab totreat unresectable solid tumors with high msi or mmrdeficiency in a study on recurrent or metastaticcolorectal cancer patients with mmr deficiency or highmsi nivolumab showed an objective response rate of and of the patients had a disease control rateof ¥ weeks which indicated that patients with highmmr deficiency or high msi might exhibit better responses to pd1pdl1 blockade therapy [ ] interestingly the responses of tumors with mmrdeficientare highly variable and approximately half are resistantto pd1pdl1 blockade therapy mandal revealed that msi and the resultant mutation load wereresponsible for the variable response to pd1 blockadetherapy in mmrdeficiency tumors and the responsedegree was significantly correlated with the degree ofinsertiondeletion mutation loadseveral studies have revealed the association betweentumor mutation burden and the response to pd1pdl1blockade therapy [ ] mariathasan examined samples from patients with metastatic urothelial cancer receiving atezolizumab treatment and identified highneoantigen and tumor mutation burden as major determinants of clinical outcome their results showed that thetumor mutation burden was closely correlated with the response in the excluded and inflamed phenotypes 0csun biomarker research page of gut microbiome compositionclinical experiments on the human gut microbiomehave identified several specific bacteria genres that playimportant roles in human immunity and can be used asprognostic biomarkers for clinical response to pd1pdl1 blockade therapy based on the gut microbiome analysis of melanomapatients receiving pd1 blockade gopalakrishnan found that patients with prolonged progressionfree survival showed a higher multiplicity of bacteriaand clostridiales ruminococcaceae and faecalibacterium were abundant in therapy responders moreovermatson evaluated the baseline stool samplesfrom patients with metastatic melanoma before pd1pdl1 blockade treatment and the results showed thatcommensal microbial composition was significantly associated with the clinical response bifidobacteriumlongum collinsella aerofaciensand enterococcusfaecium were more abundant in responders similarlyin patients with epithelial tumors routy revealed that akkermansiacea muciniphila and enterococcus hirae were significantly abundant in those withbetter clinical response progressionfree survival months all these results indicate that gut microbiomecomposition may be a potential determinant of therapyresponse and might be used as a predictive factor inthe following research more studies are needed to validate the predictive value of gut microbiome in largercohorts and explore their efficiency in the context ofvarious types of tumorsstrategies and it hasfuture perspectivesimmunotherapy is one of the most promising cancertreatmentrevolutionized thelandscape of cancer management over the last decadehowever together with the costly and timeconsumingtrialanderror approach the limited therapy responseremains a tricky problem which hinders the furtherapplication of pd1pdl1 blockade to overcome therapy resistance and potential adverse events substantialeffort has been made on developing novel antipd1pdl1 based immunotherapy strategies with better clinical response and limited immunemediated toxicityfigs tobetterclinicallikelyachievesystem issince the interaction between cancer and the immunecomplex and involves multiplefactors strategies in combination with multiple agentsareoutcomescompared with singleagent administration a largenumber ofcombinedtherapy is an effective therapeutic strategy againstcancers for example transforming growth factor βtgfβblocking agents concomitantly with combinedpd1pdl1 blockade combined provides a clinicallyrevealed thatstudies haveexperimentson mice withfeasible strategy to improve efficacy and reduce toxicity mariathasan revealed that metastaticurothelial cancer with upregulated tgfβ signalingbefore treatmentresponded poorly to pd1pdl1blockade therapy the tumors with dense collagenfibrils could trap t cells in the stromal compartmentthus preventing them from playing their functions inpreclinicalimmuneexcluded phenotype they demonstrated that the coadministration of pdl1 blockade and tgfβblockingagents could reduce tgfβ signaling facilitate t cellinfiltration and achieve active antitumor immunityand tumor regression similarly the combination ofpd1pdl1 blockade with tumor necrosisfactorinhibitor [ ] metformin antivegf agents or otherinhibitors egcxcr4 has been demonstrated as a clinicallyfeasible strategy with improved antitumor efficacyand reduced toxicityimmune checkpointinhibitor agentspd1pdl1 blockade usually acts on the whole hostimmune system instead ofsitespecifically targetingtumorspecific immune cells while nanomedicine technology provides a powerful tool to selectively deliverimmune checkpointto tumors orlymphoid ans using drugloaded nanops usually to nm in diameter recent studies suggest that the pd1pdl1 antibody could be conjugatedor modified on the surface of nanops which couldmaintain their stability enhance efficiency and minimizethe toxicity of pd1pdl1 blockade [ ] forexamplein gastric cancer cells the pdl1 blockadeconjugated nanops contributed to significantlyhigher cellular uptake and achieved more effective inhibition of pdl1 expression compared with the controlgroups moreover in patients with metastatic triplenegative breast cancer the coadministration of nabpaclitaxelatezolizumabprolonged progressionfree survival owing to thesuccess in previous research clinical trials on nanoimmunotherapysuch asnct03589339 and nct03684785 these clinical trialsshould provide substantial evidence for the combinationof nanomedicine and pd1pdl1 blockade in the nextfew yearscurrently underwayblockadepdl1plusarethe manipulation offurthermore accumulating evidence has demonstrated that gut microbiome significantly impacts theefficacy of cancer immunotherapy which in turn indithe gut microbiomecates thatcould latently affectthe response to pd1pdl1blockade therapy [] currently antibiotic applicationfecal microbiota transplantation fmt anddiet regulation are considered as practical approachesto manipulate gut microbiome for example fmtfrom patients with a positive response to germfree or 0csun biomarker research page of fig overview on the strategies to improve the resistance to pd1pdl1 blockade therapy multiple strategies have been proposed toimprove the resistance to pd1pdl1 blockade therapy including combined therapy nanoimmunotherapy gut microbiome manipulation andso forthin contrastantibiotictreated mice could improve tumor controlaugment t cell responses and ameliorate the antitumor effects of pd1 blockadethetransplantation from resistant patients did not resultin improvement similarly responses to pdl1blockade are distinctin mice with different commensal microbes and the positive response of micewith advantageous gut microbiome can be transplanted to mice with negative responses by fmt orcohousing conclusionsdespite the success across multiple types of cancersonly a minority of patients are estimated to exhibit apositive response to pd1pdl1 blockade therapy andthe primaryacquired resistance might eventually leadto progression in patients with clinical responses thelimitation in clinical response impairs the efficacy andhinders its further application since the understandingof the mechanisms underlying therapy resistance remains vague only a few therapeutic options areavailable for those patients currently illustrating thedeterminants of response or resistance is significant toaccelerate improving survival outcomes and developingimproved treatment options for cancer patients tobetter realize the therapeutic potential of pd1pdl1blockade therapyit is essential to identify predictivebiomarkers for therapy response develop novel therapeutic strategies and improve therapeutic strategies incombination with other agents in the following research combined efforts of basic researchers and clinicians are required to address the pd1pdl1 blockadetherapy resistanceabbreviationspd1 programmed death1 pdl1 programmed deathligand nsclc nonsmall cell lung cancer mmr mismatch repair msi microsatelliteinstability b2m beta2microglobulin tim3 t cell immunoglobulin mucin3mirnas micrornas lncrnas long noncoding rnas mdscs myeloidderived suppressor cells tgfβ transforming growth factor β fmt fecalmicrobiota transplantationacknowledgmentsnot applicableauthors contributionsjys dk z mx and xz wrote original draft preparation sq w jsj and xjprovided critical revision all authors read and approved the final manuscriptfundingthis study was supported by national key research and developmentprojects intergovernmental cooperation in science and technology of chinano 2018yfe0126900 to jiansong ji the key research and developmentproject of zhejiang province no 2018c03024 to jiansong ji the nationalnatural science foundation of china to xl 0csun biomarker research page of availability of data and materialsnot applicableethics approval and consent to participatenot applicableconsent for publicationnot applicablecompeting intereststhe authors declare that they have no competing interestsauthor details1the first college of clinical medicine the first affiliated hospital of nanjingmedical university nanjing medical university nanjing china 2keylaboratory of imaging diagnosis and minimally invasive interventionresearch lishui hospital of zhejiang university the fifth affiliated hospitalof wenzhou medical university clinical medicine of center hospital of lishuicollege lishui china 3college of medicine lishui college lishui china 4department of general surgery the first affiliated hospitalof nanjing medical university nanjing china 5department of radiologyaffiliated lishui hospital of zhejiang university lishui chinareceived april accepted august referenceshellmann md pazares l bernabe caro r zurawski b kim sw carcerenycosta e nivolumab plus ipilimumab in advanced nonsmallcell lungcancer n engl j med niglio sa jia r ji j ruder s patel vg martini a programmed death1or programmed death ligand1 blockade in patients with platinumresistant metastatic urothelial cancer a systematic review and metaanalysis eur urol sun jy lu xj cancer immunotherapy current applications and challengescancer lett andrews lp yano h vignali daa inhibitory receptors and ligands beyondpd1 pdl1 and ctla4 breakthroughs or backups nat immunol prestipino a zeiser r clinical implications of tumorintrinsic mechanismsregulating pdl1 sci transl med betof warner a palmer js shoushtari an goldman da panageas ks hayessa et | Colon_Cancer |
" 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 |
lenvatinib inhibits tyrosine kinases including vascular endothelial growth factor vegf receptor fibroblast growth factor receptor platelet derived growth factor receptor alpha ret proto oncogene and kit proto oncogene receptor tyrosine kinase we assessed the efficacy and safety of lenvatinib in patients with metastatic colorectal cancer after failure of standard chemotherapiespatients and methods this was an open label single centre single arm phase study eligible patients had unresectable metastatic colorectal adenocarcinoma refractory or intolerant to fluoropyrimidine irinotecan oxaliplatin trifluridinetipiracil anti vegf therapy and anti epidermal growth factor receptor therapy for tumours with wild type ras patients were treated with oral lenvatinib at mg one time a day in day cycles until disease progression or unacceptable toxicity the primary endpoint was centrally assessed disease control rate secondary endpoints included safety response rate progression free survival and overall survival the planned sample size was patients to expect a disease control rate of with a threshold disease control rate of one sided alpha of and power of results between october and january patients were enrolled and had received or ¥ lines of prior chemotherapy for metastatic disease respectively the median number of lenvatinib cycles was range the centrally assessed disease control rate was ci to one sided p00001 patients had a partial response and had a stable disease median progression free survival was months ci to median overall survival was months ci to the most common grade ¥ adverse events were hypertension thrombocytopenia increased alanine aminotransferase and anorexia eachs lenvatinib showed promising clinical activity and was tolerated in patients with metastatic colorectal cancer after failure of standard chemotherapiestrial registration number umin ctr umin000023446 and jamcct ctr jma iia00261introductionthe combination of cytotoxic chemotherapy with a molecular targeted agent has significantly key questionswhat is already known about this subject º no studies have previously reported the efficacy and safety of lenvatinib monotherapy in patients with metastatic colorectal cancer refractory to standard chemotherapieswhat does this study add º lenvatinib showed promising antitumour activity with acceptable toxicity for heavily pretreated patients with metastatic colorectal cancer refractory to standard chemotherapies º no unexpected safety signals were observed and toxicities were manageable with dose modification interruptions and supportive medicationshow might this impact on clinical practice º further prospective randomised studies are warranted to evaluate the efficacy of lenvatinib in patients with metastatic colorectal cancer refractory to standard chemotherapiesimproved the survival of patients with unresectable metastatic colorectal cancer1 from results of recent clinical trials trifluridinetipiracil and regorafenib are recognised as new treatment options for patients with metastatic colorectal cancer refractory or intolerant to standard therapies6 nevertheless the prognosis of patients which are refractory or intolerant to standard chemotherapies is poor and there are still an unmet medical needs for these patients especially for those who are in a good performance status and eligible for further therapieslenvatinib is an oral multitargeted tyrosine kinase inhibitor of the vascular endothelial growth factor receptor vegfr fibroblast growth factor receptors platelet derived growth factor receptor alpha ret and kit8 preclinical studies have shown that iwasa a0s et a0al esmo open 20205e000776 101136esmoopen2020000776 0copen accesslenvatinib not only interferes the interaction between cancer cells and endothelial cells but also inhibits tumour growth10 several phase trials of patients with solid tumours in the usa11 europe12 and japan13 showed that the optimum dosage of lenvatinib was mg one time a day in a day cyclea total of patients were enrolled in four phase studies of lenvatinib monotherapy of whom had colorectal cancer disease control rate dcr was achieved in out of patients including one with a partial response which continued for weeks mg two times a day for weeks of a week cycle grade palmar plantar erythrodysesthesia was reportedly much lower in of patients treated with lenvatinib for thyroid cancer in a japanese population of the select trial than that of reported in a japanese population of correct trial using regorafenib for metastatic colorectal cancer15 these results suggested that lenvatinib may have a potential for improving the outcomes of patients with unresectable metastatic colorectal cancer who have already received conventional chemotherapy with a fluoropyrimidine irinotecan and oxaliplatinwe conducted a single centre phase study to evaluate efficacy and safety in patients with metastatic colorectal cancer failing to standard therapiespatients and methodsstudy design and patientsthis study was a single arm phase study conducted at national cancer center hospital tokyo japan the inclusion criteria were histological diagnosis of colorectal adenocarcinoma excluding carcinoma of the appendix and the anal canal unresectable metastatic disease an eastern cooperative oncology group performance status of or an age of years no previous treatment with regorafenib or lenvatinib sufficient oral intake adequate an and bone marrow function at least one measurable lesion in accordance with the response evaluation criteria in solid tumors recist version refractory or intolerant to fluoropyrimidine irinotecan oxaliplatin therapy and antiepidermal growth factor receptor therapy for tumours with wild type ras and no systemic therapy for at least weeks weeks if any investigational drug had been administered before study enrolment the exclusion criteria were provided in the online supplementary materialtrifluridinetipiracil anti vegf all patients provided written informed consentprocedurespatients received lenvatinib at mg one time a day in day cycles orally until disease progression or unacceptable toxicity the dose was reduced to mg mg mg mg and mg if a patient had an intolerable grade or grade adverse event treatment was discontinued if a dose interruption was required for more than consecutive daystumour response was assessed by the independent radiological review committee based on the ct or mri performed at baseline every weeks for weeks and every weeks thereafter until confirmed objective disease progression safety assessments including laboratory tests were done at screening days and of cycle and days and of the subsequent cycles urinalysis thyroid function prothrombin time international normalized ratio pt inr and tumour markers both carcinoembryonic antigen and carbohydrate antigen were measured at screening and on day of each treatment cycle adverse events were recorded from the first day of the protocol treatment to days after the last dose of study medication and graded using the national cancer institute common terminology criteria for adverse events version blood sampling for biomarker analyses was done at baseline on days and and at the end of treatment plasma levels of angiopoietin2 were measured by the human angiopoietin2 quantikine elisa kit rd systems minneapolis usaoutcomesthe primary endpoint was centrally assessed dcr which was defined as the proportion of patients with a complete response partial response or stable disease persisting for more than weeks from the initiation of study treatment according to recist version a complete response and partial response were needed to be confirmedthe secondary endpoints were the objective response rate orr proportion of patients who had a complete response or partial response progression free survival pfs time from the enrolment until investigator assessed disease progression or death overall survival os time from the enrolment until death due to any cause and adverse events the incidence of adverse events was calculated based on the information of the worst grade of each adverse event experienced in each patient relative dose intensity which is unprespecified outcome was calculated as the proportion of the actual cumulative dose divided by planned cumulative dose mg times treatment daysstatistical analysisfor this single arm study the required sample size of patients provided power to reject the null hypothesis of dcr ¤ with expectation that of patients would have a disease control one sided α of considering the possibility of a few ineligible patients we planned to recruit patientsthe final analysis was planned approximately months after enrolment of the last patient we included all eligible patients in the efficacy analysis and all patients receiving a least one dose of lenvatinib in the safety analyses for the primary analysis binomial test was performed and the centrally assessed dcr was estimated with ci using the clopper and pearson method which corresponds to one sided α of we also estimated the investigator assessed dcr a supplementary analysis of the primary iwasa a0s et a0al esmo open 20205e000776 101136esmoopen2020000776 0ctable baseline patient characteristicscharacteristicstable continuedoverall n characteristicsoverall n open access median range continued intolerant wild type mutant ras mutational status braf mutational status wild type mutant unknownmsi status mss unkown there is an overlapping this number includes patients with the ras wild type and patient with mutant rasecog eastern cooperative oncology group egfr epidermal growth factor receptor msi microsatellite instability mss microsatellite stableendpoint and orr with cis using the same method we estimated the median time and month and year probability of os and pfs with the kaplan meier method the cis for the median time were calculated using brookmeyer and crowley method the cis of month and year survival probabilities were calculated based on the greenwoods formula hrs and cis were estimated by cox regression we did subgroup analyses divided by prespecified baseline patient and disease characteristic variables including ras status for dcr pfs and os we also did a prespecified exploratory analysis of potential predictive biomarkers in blood samples we did all analyses with sas v94resultspatient characteristicsbetween october and january patients with unresectable metastatic colorectal cancer were enrolled all patients were eligible and received the study medication table summarises the baseline characteristics of all enrolled patients the median number of previous lines of palliative chemotherapy was range and patients had received or ¥ prior lines of chemotherapy for metastatic disease respectively the data cut off date was january with median follow up of months iqr efficacythe centrally assessed dcr was ci to one sided p00001 two patients had a partial response and had a stable disease including unconfirmed pr table figure a total of patients had a reduction in target lesion size from baseline figure time on treatment for all patients is ¥ ¥ male female months ¥ months right sided colon left sided colorectum lung liver lymph node peritoneumage years sex ecog performance status primary site number of metastatic site metastatic an time from start of first line chemotherapy number of previous palliative chemotherapy previous chemotherapy and reason for discontinuation fluoropyrimidine refractory intolerantoxaliplatin irinotecan tas102 trifluridinetipiracil angiogenesis inhibitor anti egfr inhibitor refractory intolerant refractory intolerant refractory refractory intolerant refractory intolerantiwasa a0s et a0al esmo open 20205e000776 101136esmoopen2020000776 0copen accesstable best response to treatmentcomplete responsepartial responsestable diseaseprogressive diseasenot evaluabledisease control rate ciresponse rate cicentral assessmentn30 to to investigator assessmentn30 to to shown in online supplementary figures and events for pfs were recorded in all patients and median pfs was months ci to figure all deaths were recorded median os was months ci to with a month and year os of ci to and ci to figure safetypatients received the study treatment for four cycles at median range the median relative dose intensity was iqr dose interruptions and reductions were required in and patients respectively the major treatment related adverse events ¥ for dose reduction were proteinuria patients palmar plantar erythrodysesthesia patients diarrhoea patients hypertension patients fatigue patients and thrombocytopenia patients the reasons for treatment discontinuation of all patients were disease progression in patients and adverse events in patients gastrointestinal perforation and grade proteinuria in of each after treatment with lenvatinib patients received a subsequent treatment online supplementary table most patients only had mild grades adverse events table the most common grade ¥ adverse events were hypertension patients thrombocytopenia patients increased alanine aminotransferase and anorexia patients each no clear relationship was found between the incidence of lenvatinib associated adverse event of any grade and baseline body surface area online supplementary table serious adverse events occurred in four patients including figure waterfall plot analysis of maximum percentage change from baseline in measurable target lesions response evaluation criteria in solid tumors version central reviewiwasa a0s et a0al esmo open 20205e000776 101136esmoopen2020000776 0copen accessfigure kaplan meier curves of a progression free survival pfs by investigator assessment and b overall survival os in all patients n30five treatment associated events anorexia in two and gastrointestinal perforation central venous catheter related bloodstream infection caused by staphylococcus aureus and nausea in each one in each of four patients all patients recovered from these adverse eventssubgroup analysisin patients with wild type ras the median pfs was months ci to and that was months ci to in patients with mutant ras online supplementary figure in patients with wild type ras the median os was months ci ci to and months ci to in patients with mutant ras online supplementary figure plasma angiopoietin2 levels were decreased by lenvatinib treatment in almost all patients and increased at the iwasa a0s et a0al esmo open 20205e000776 101136esmoopen2020000776time of treatment discontinuation online supplementary table with a first quartile cut off point17 the eight patients with a first quartile or lower level of angiopoietin2 had a median os of months ci to months compared with months ci to in the patients with higher than a first quartile level of angiopoietin2 hr ci to online supplementary figure patients with a first quartile or less level of angiopoietin2 had a median pfs of months ci to compared with months ci to in the patients with more than a first quartile level of angiopoietin2 hr ci to online supplementary figure 0copen accesstable treatment related adverse events occurring in ¥ patients n30any gradegrade ¥treatment related adverse eventhypertensionproteinuriathrombocytopeniafatiguehypothyroidismweight losshoarsenesspalmar plantar erythrodysesthesia syndromeanorexiadiarrhoeamucositis oralserum ast increasedserum creatinine increasedast aspartate transaminase discussionpatients with metastatic colorectal cancer with disease progression after three or more lines of therapy have limited treatment options in this open label single arm phase study of patients with previously treated metastatic colorectal cancer lenvatinib demonstrated manageable toxic effects and promising antitumour activity a total of out of patients had disease control including with partial responses moreover patients experienced reduction in measurable tumour size the overall toxicity profiles were similar to that reported for lenvatinib across a spectrum of advanced malignant neoplasmstwo recent international phase studies reported that regorafenib or trifluridinetipiracil provided significant improvements in dcr pfs and os compared with placebo in patients with metastatic colorectal cancer after failure of standard chemotherapies dcr median pfs months median os months in the correct study and dcr median pfs months median os months in the recourse study6 interestingly the present single arm phase study of lenvatinib revealed favourable dcr and median pfs values in patients with metastatic colorectal cancer compared with those in the regorafenib or trifluridinetipiracil study moreover about half of the patients received post study treatment which led to a favourable osthe lenvatinib safety profile in this study was similar to the published safety profiles of lenvatinib for thyroid cancer and hepatocellular carcinoma in the japanese population18 moreover we found no unexpected or off target safety signals the most common adverse events were hypertension proteinuria thrombocytopenia and fatigue while the most case of grade or hypertension and proteinuria required treatment interruption and dose reduction while the target population for thyroid cancer or hepatocellular carcinoma that showed efficacy for lenvatinib was first line setting20 this study targeted patients receiving salvage line therapy most patients with metastatic colorectal cancer in the salvage line setting had grade or proteinuria and hypertension at baseline because of the long term prior treatment with anti vegfvegfr treatment whereas the occurrence of grade hypertension was significantly higher compared with that of regorafenib in a similar study population in the correct concur and consign trials7 it was manageable by dose reduction or interruption but it may be necessary to consider the starting dose in the future although palmar plantar erythrodysesthesia is a not life threatening toxicity these adverse events have a significant impact on treatment schedules and quality of life in treated patients grade ¥ palmar plantar erythrodysesthesia has been observed in and of patients treated with lenvatinib in this study and the select japanese population15 respectively while in patients treated with regorafenib in the correct japanese population16 to date the clear mechanism of palmar plantar erythrodysesthesia by vegf receptor tyrosine kinase inhibitors is not known but it has been reproduced that palmar plantar erythrodysesthesia by lenvatinib is well tolerated overall it is suggested that lenvatinib might be a favourable treatment option in terms of toxicitiesseveral preclinical studies demonstrated that vegf targeted treatment affects immune suppression by promoting the expansion of suppressive immune cell populations such as regulatory t cells and myeloid derived suppressor cells24 several clinical studies suggested that modulation of vegf mediated immune suppression via angiogenesis inhibition could potentially augment the immunotherapeutic activity of anti programmed cell death pd1 antibody26 regorafenib and nivolumab showed antitumour activity in patients with metastatic colorectal cancer including those with microsatellite stable tumours in a phase study28angiopoietin2 a relatively novel regulator of angiogenesis that acts through the tek tyrosine kinase endothelial tie2 receptor has been identified as a potential prognostic biomarker for some types of cancer although the baseline ang2 level was a predictive biomarker in patients with thyroid cancer in the select trial17 it did not become a reliable biomarker of lenvatinib response in this study prior treatment with anti vegfvegfr antibodies probably had an effect on baseline angiopoietin2 levels because the study population was refractory to standard treatment in this study the decrease in angiopoietin2 levels was observed after treatment therefore it may be an indicator of treatment responsethe limitations of our study include its small size which could limit the interpretation of the subgroup analyses iwasa a0s et a0al esmo open 20205e000776 101136esmoopen2020000776 0cand the absence of a comparison group however the level of clinical benefit in the form of confirmed responses observed in this study was remarkable in the historical context of other clinical trials done in heavily pretreated patients with metastatic colorectal cancer moreover most of the patients in our study had left sided tumours which were known to have a better prognosis compared with right sided tumoursin lenvatinib provided promising activity with prolonged survival relative to the anticipated median pfs in heavily pretreated patients with metastatic colorectal cancer the safety profile of lenvatinib was similar to that in other tumour types with no new safety signals recorded based on these findings further investigation of lenvatinib with anti pd1 antibody or other novel combinations with the potential to build on the benefit of lenvatinib is currently taking place nct03797326 and nct04008797acknowledgements the authors thank the patients and their families the members of the clinical research support office for their support with data collection and running the study and nai incorporated for editing a draft of this manuscriptcontributors all authors conceived and designed the study and drafted and revised the manuscript for publication si no hs yh at kk th nb and yy collected data ak go mk and kn analysed the data and managed data and study progress all authors interpreted the data and approved the final version of the manuscriptfunding the study was supported by the project promoting clinical trials for development of new drugs and medical devices japan medical association from the japan agency for medical research and development grant number jp18lk0201037 and by eisai cocompeting interests si has received research grants from eisai and merck biopharma th has received research grants from eisai and honoraria from merck serono yy has received honoraria from eisaipatient consent for publication not requiredethics approval the study was conducted in accordance with the declaration of helsinki and good clinical practice guidelines the study protocol was approved by the national cancer center institutional review board t4329provenance and peer review not commissioned externally peer revieweddata availability statement data are available upon reasonable request proposals should be directed to siwasa ncc go jp the data will be available for achieving aims in the approved proposalopen 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 idssatoru a0iwasa http orcid yasuhide a0yamada http orcid references saltz lb clarke s dÃaz rubio e et a0al bevacizumab in combination with oxaliplatin based chemotherapy as first line therapy in metastatic colorectal cancer a randomized phase iii study j clin oncol tabernero j yoshino t cohn al et a0al ramucirumab versus placebo in combination with second line folfiri in patients with metastatic colorectal carcinoma that progressed during or after first line therapy with bevacizumab oxaliplatin and a fluoropyrimidine raise a randomised double blind multicentre phase study lancet oncol open access van cutsem e tabernero j lakomy r et a0al addition of aflibercept to fluorouracil leucovorin and irinotecan improves survival in a phase iii randomized trial in patients with metastatic colorectal cancer previously treated with an oxaliplatin based regimen j clin oncol yamazaki k nagase m tamagawa h et a0al randomized phase iii study of bevacizumab plus folfiri and bevacizumab plus mfolfox6 as first line treatment for patients with metastatic colorectal cancer wjog4407g ann oncol price tj peeters m kim tw et a0al panitumumab versus cetuximab in patients with chemotherapy refractory wild type kras exon metastatic colorectal cancer aspecct a randomised multicentre open label non inferiority phase study lancet oncol mayer rj van cutsem e falcone a et a0al randomized trial of tas102 for refractory metastatic colorectal cancer n engl j med grothey a van cutsem e sobrero a et a0al regorafenib monotherapy for previously treated metastatic colorectal cancer correct an international multicentre randomised placebo controlled phase trial lancet matsui j yamamoto y funahashi y et a0al e7080 a novel inhibitor that targets multiple kinases has potent antitumor activities against stem cell factor producing human small cell lung cancer h146 based on angiogenesis inhibition int j cancer matsui j funahashi y uenaka t et a0al multi kinase inhibitor e7080 suppresses lymph node and lung metastases of human mammary breast tumor mda mb231 via inhibition of vascular endothelial growth factor receptor vegf r and vegf r3 kinase clin cancer res wiegering a korb d thalheimer a et a0al e7080 lenvatinib a multi targeted tyrosine kinase inhibitor demonstrates antitumor activities against colorectal cancer xenografts neoplasia hong ds kurzrock r wheler jj et a0al phase i dose escalation study of the multikinase inhibitor lenvatinib in patients with advanced solid tumors and in an expanded cohort of patients with melanoma clin cancer res boss ds glen h beijnen jh et a0al a phase i study of e7080 a multitargeted tyrosine kinase inhibitor in patients with advanced solid tumours br j cancer yamada k yamamoto n yamada y et a0al phase i dose escalation study and biomarker analysis of e7080 in patients with advanced solid tumors clin cancer res nakamichi s nokihara h yamamoto n et a0al a phase study of lenvatinib multiple receptor tyrosine kinase inhibitor in japanese patients with advanced solid tumors cancer chemother pharmacol kiyota n schlumberger m muro k et a0al subgroup analysis of japanese patients in a phase study of lenvatinib in radioiodine refractory differentiated thyroid cancer cancer sci yoshino t komatsu y yamada y et a0al randomized phase iii trial of regorafenib in metastatic colorectal cancer analysis of the correct japanese and non japanese subpopulations invest new drugs tahara m schlumberger m elisei r et a0al exploratory analysis of biomarkers associated with clinical outcomes from the study of lenvatinib in differentiated cancer of the thyroid eur j cancer takahashi s kiyota n yamazaki t et a0al a phase ii study of the safety and efficacy of a0lenvatinib in patients with advanced thyroid a0cancer future oncol yamashita t kudo m ikeda k et a0al reflect a phase trial comparing efficacy and safety of lenvatinib to sorafenib for the treatment of unresectable hepatocellular carcinoma an analysis of japanese subset j gastroenterol kudo m finn rs qin s et a0al lenvatinib versus sorafenib in first line treatment of patients with unresectable hepatocellular carcinoma a randomised phase non inferiority trial lancet schlumberger m tahara m wirth lj et a0al lenvatinib versus placebo in radioiodine refractory thyroid cancer n engl j med li j qin s xu r et a0al regorafenib plus best supportive care versus placebo plus best supportive care in asian patients with previously treated metastatic colorectal cancer concur a randomised double blind placebo controlled phase trial lancet oncol van cutsem e martinelli e cascinu s et a0al regorafenib for patients with metastatic colorectal cancer who progressed after standard therapy results of the large single arm open label phase iiib consign study oncologist iwasa a0s et a0al esmo open 20205e000776 101136esmoopen2020000776 0copen access ott pa hodi fs buchbinder ei inhibition of immune checkpoints and vascular endothelial growth factor as combination therapy for metastatic melanoma an overview of rationale preclinical evidence and initial clinical data front oncol kato y tabata k kimura t et a0al lenvatinib plus anti pd1 antibody combination treatment activates cd8 t cells through reduction of tumor associated macrophage and activation of the interferon pathway plos one 201914e0212513 taylor mh lee c h makker v et a0al phase ibii trial of lenvatinib plus pembrolizumab in patients with advanced renal cell carcinoma endometrial cancer and other selected advanced solid tumors j clin oncol makker v rasco d vogelzang nj et a0al lenvatinib plus pembrolizumab in patients with advanced endometrial cancer an interim analysis of a multicentre open label single arm phase trial lancet oncol fukuoka s hara h takahashi n et a0al regorafenib plus nivolumab in patients with advanced gastric gc or colorectal cancer crc an open label dose finding and dose expansion phase 1b trial regonivo epoc1603 jco iwasa a0s et a0al esmo open 20205e000776 101136esmoopen2020000776 0c' | Colon_Cancer |
] we have filtered only research s published in english language and selected the following keywords air pollution and covid19 or sarscov2 particulate matter or pm and covid19 or sarscov2 nitrogen dioxide or no2 and covid19 or sarscov2 we choose as inclusion criteria all the available epidemiological studies aimed to identify any temporal and spatial association between reported covid19 cases andor deaths and air pollution data related to pm25 pm10 and no2 thus excluding any letter opinion commentary review or nonrelevant s we obtained a total of eligible published research s in their final version and paper in its preprint version for some of them we chose to include only principal findings that clearly fit the aim this review particulate matter and covid19 atmospheric particulate matter pm is originated by a wide range of anthropogenic and natural sources kim it consists of a heterogeneous mixture of solid and liquid ps suspended in air that varies continuously in size and chemical composition including nitrates sulphates elemental and anic carbon anic compounds biological compounds and metals who it has been associated with increased respiratory morbidity and mortality liu especially in susceptible people due to cardiorespiratory events including asthma chronic obstructive pulmonary disease and thrombosis li rhee in vitro and in vivo studies highlighted its role in the exacerbation of respiratory viral infections becker and soukup recently the research group of setti gave first preliminary evidence that sarscov2 rna can be present on outdoor particulate matter thus suggesting that in conditions of atmospheric stability and high concentrations of pm it could represent a potential early indicator of covid19 although it does not give information regarding covid19 progression or severity several observations report a significant association between ambient concentrations of pm25 adhikari and yin bashir fattorini and regoli frontera jiang li vasquezapestegui wu yao zhu zoran 2020a and pm10 bashir coccia 2020b fattorini and regoli jiang li yao zhu zoran 2020a with covid19 pandemic across the most affected countries china italy and usa see table first evidences on the temporal association between air pollution and covid19 were reported in china where the outbreak was first identified zhu explored the relationship between particulate matter and the viral infection caused by the novel coronavirus in cities in china the authors included over of dailyconfirmed new cases in the whole of china between january 23rd and february 29th they applied a generalized additive model gam to examine the effects of meteorological factors and air pollution on covid19 incidence applying a movingaverage approach to capture the cumulative lag effect of ambient air pollution and considering population size and density as potential confounders they observed that the effect of pm25 on daily confirmed cases was greater than pm10 in particular they found that a 10μgm3 increase lag0 in pm25 and pm10 was associated with a ci to and ci to increase in the daily counts of covid19 confirmed cases respectively jiang focused their attention on three most affected cities of china wuhan xiaogan and huanggang collecting data of daily cases and ambient air pollutant from jan 25th to feb 29th the authors by applying a multivariate poisson regression revealed a significant temporal association between pm25 increased and covid19 incidence in all the considered cities especially in huanggang wuhan rr ci xiaogan rr ci huanggang rr ci conversely an increase in pm10 concentrations was associated with a decrease of covid19 incidence these results were partially confirmed by findings of li who conducted a simple linear regression to compare covid19 incidence with pm concentrations in wuhan and xiaogan from jan 26th to feb 29th in they found that an increase in pm25 was correlated with an increase of covid19 incidence in both cities wuhan r2 p xiaogan r2 p while for pm10 only in xiaogan r2 p the spatial distribution of particulate matter and case fatality rate cfr of covid19 was studied by yao in cities of china including wuhan collecting data up to march 22nd first they found a significantly positive global spatial autocorrelation of covid19 cfr global morans index i p highlighting a high cfr clustering located in hubei province with a multiple linear regression they adjusted their results for several effect modifiers and confounder factors such temperature relative humidity gross domestic product gdp per capita hospital beds per capita local indicators of spatial association lisa map values city size and population or proportion of people older than years it was found that for every μgm3 increase in pm25 and pm10 the cfr increased by and respectively and the risk estimates increased to and with every μgm3 increase in average concentrations of pm25 and pm10 in respectively some studies describe the association between air pollution and covid19 across italy the second country of the world where the infection spread significantly at the beginning of the pandemic and suddenly has reached many other european countries the 28th of july italy recorded more than total confirmed cases and deaths who most of which were distributed in the regions of northern italy especially the lombardy it is recognized as one the most air polluted areas of europe eea where the frequent pm10 annual exceedances of the who threshold of μgm3 are responsible for attributable deaths per year corresponding to attributable community rates of deaths per inhabitants per year baccini bontempi 2020bfocused the attention on two of the most affected regions of northern italy lombardy and piedmont the authors based on pm10 daily exceedances and covid19 confirmed cases on march 12th thus before the italian sanitary crisis observed that pm10 concentration was exceeded only few times among the lombard cities that at the beginning of the epidemic were most affected on the contrary among some piedmont cities suffering of severe pm10 pollution events covid19 incidence was lower based on their results the authors concluded that covid19 diffusion by airborne pm10 is hard to demonstrate nevertheless several research revealed how pm in particular pm25 could had a role in accelerate and vast diffusion of covid19 in northern italy for example coccia 2020b by analyzed data on italian province capitals and data of infected individuals up to april 7th revealed a relationship between air pollution of cities measured with days exceeding the limits set for pm10 in previous years and covid19 diffusion in particular cities with more than days of pm10 exceedances showed a very high average number of infected individual about infected individuals on 7th april whereas cities having less than days of pm10 exceedances showed a lower average number of infected about infected individuals frontera gave also evidences on the role of pm25 as a contributing factor of covid19 outbreak in northern italy where environmentalresearch19120201101293 0cc copat table summary table reporting reviewed results on the association between covid19 casesdeaths and air pollution pm25 pm10 and no2 references zhu data analysis generalized additive model gam aim temporal association between daily confirmed cases and air pollution pm25 pm10 and no2 temporal association between daily confirmed cases and air pollution pm25 pm10 and no2 temporal association between daily confirmed cases and air pollution pm25 pm10 and no2 spatial association between fatality rate and air pollution pm25 and pm10 spatial association between deaths counts and air pollution no2 temporal association between total cases daily confirmed cases and total deaths and air pollution pm25 and pm10 temporal association between total cases daily confirmed cases and total deaths and air pollution no2 spatial description of pm10 exceedances versus covid19 cases multivariate poisson regression simple linear regression multiple linear regression descriptive analysis percentage of deaths in three no2 μmol m2concentration range pearson coefficient correlation pearson coefficient correlation descriptive analysis number of days of pm10 exceeding μgm3 and covid19 incidence area of study cities of china period from jan 23rd to feb 29th jiang li yao ogen zoran 2020a zoran 2020b bontempi 2020b from jan 25th to feb 29th from jan 26th to feb 29th in data up to march 22nd data up to the end of feb from jan 1st to apr 30th from jan 1st to apr 30th from feb 10th to march 12th wuhan xiaogan and huanggang china wuhan and xiaogan cities of china administrative regions in italy spain france and germany milan italy milan italy provinces of lombardy italy provinces of piedmont italy coccia 2020b data up to april 7th italian provinces fattorini and regoli data up to april 27th italian provinces pm25 a 10μgm3 pm25 increase lag0 was associated with a increase of daily confirmed new cases pm10 a 10μgm3 pm10 increase lag0 was associated with a increase of daily confirmed new cases wuhan rr ci1032 xiaogan rr ci huanggang rr ci wuhan r2 p xiaogan r2 p wuhan rr ci xiaogan rr ci huanggang rr ci wuhan r2 p xiaogan r2 p Ï2 p a μgm3 increase in pm25 was associated with a increase in fatality rate Ï2 p a μgm3 increase in pm10 was associated with a increase in fatality rate no2 a 10μgm3 no2 increase lag0 was associated with a increase in daily confirmed new cases wuhan rr ci xiaogan rr ci huanggang no association found wuhan r2 p xiaogan r2 p of fatality cases are associated with no2 μmolm2 r cid0 r r cid0 r cid0 r r cid0 r cid0 r cid0 r cid0 lombardy pm10 exceeding between and covid19 incidence between and piedmont pm10 exceeding between and covid19 incidence between and covid19 in north italy has a high association with air pollution of cities measured with days exceeding the limits set for pm10 r2 p r2 p continued on next page hierarchical multiple regression model pearson regression coefficient analysis r2 p spatial association between confirmed cases and air pollution pm10 spatial association between total confirmed cases and air pollution pm25 pm10 and no2 environmentalresearch19120201101294 0cc copat table continued references frontera frontera wu adhikari and yin bashir bashir vasquezapestegui vasquezapestegui vasquezapestegui period data up to 31st march data up to 31st march data up to april 04th from march 1st to apr 20th from march 4th to april 24th from march 4th to april 24th data up to june 12th data up to june 12th data up to june 12th area of study italian regions italian regions counties in the usa queens county new york usa california california districts of lima perù districts of lima perù districts of lima perù aim spatial association between total confirmed cases and air pollution pm25 spatial association between deaths and air pollution pm25 prediction of risk of covid19 deaths in the long term average exposure to fine particulate matter pm25 temporal association between daily confirmed cases and total deaths and air pollution pm25 association between confirmed cases and air pollution pm25 pm10 and no2 association between deaths and air pollution pm25 pm10 and no2 spatial association between total confirmed cases and air pollution pm25 spatial association between deaths and air pollution pm25 spatial association between case fatality rate and air pollution pm25 data analysis pearson regression coefficient analysis pm25 r2 p pm10 pearson regression coefficient analysis r2 p longterm exposure increase of μgm3 in pm25 is associated with a increase in the covid19 death rate estimate on cases values cid0 ci estimate on deaths value cid0 ci kendall r cid0 spearman r cid0 zeroinflated negative binomia models negative binomial regression model spearman and kendall correlation tests spearman and kendall correlation tests no2 kendall r cid0 spearman r cid0 kendall r cid0 spearman r cid0 kendall r cid0 spearman r cid0 kendall r cid0 spearman r cid0 kendall r cid0 spearman r cid0 multivariate regression model crude coefficient p multivariate regression model crude coefficient p multivariate regression model crude coefficient cid0 p mortality was found significantly higher than less polluted italian regions by collecting data up to march 31st for all italian regions and performing a pearson correlation analysis they found a strong positive association both with the total number of confirmed cases r and deaths r other than with hospitalized cases r the italian situation was further highlighted by the study of fattorini and regoli in italian provinces they explored the spatial association between air pollution and covid19 cases with data up to april 27th by applying the pearson regression coefficient analysis they revealed a positive association both with pm25 and pm10 r2 p and r2 p respectively a focus on the most affected city of italy milan was conducted by zoran 2020a this city is located in the po valley basin known hotspot for atmospheric pollution at the continental scale eea the authors performed a temporal association between covid19 total cases daily new positive cases and total deaths and particulate matter from jan 1st and apr 30th by applying a person correlation in accordance with other studied they found a positive association between daily confirmed cases and pm25 r and pm10 r although they did not consider any delay time from infection to covid19 onset nevertheless they found a negative association between total cases and total deaths and particulate matter but the assumption of a temporal linear correlation may be inaccurate because the above mentioned variables could have more complex nonlinear relationships to date the usa have more than million confirmed cases and thousand deaths who here ambient concentrations of pm and o3 were found responsible to cause between and premature deaths fann the association between air pollutants and covid19 cases and deaths was studied by bashir in the state of california from march 4th to april 24th corresponding to the beginning of the covid19 outbreak in usa based on their significant correlation found the authors state that a limited human exposure to these pollutants will contribute to defeating covid19 this conclusion seems unclear because they found a negative correlation with pm25 and pm10 environmentalresearch19120201101295 0cc copat by applying both the kendall rank correlation and spearmans one and it is not clear if they normalized covid19 cases by population size and if they performed a day by day association or a spatial association across the country a focus on the queen county new york usa was provided by adhikari and yin they retrieved data of pm daily concentrations from two ground monitoring stations and collected data of confirmed covid19 cases and numbers of related deaths from usafacts in the period from march to april the authors elaborated their data with a negative binomial regression model and considered the cumulative lag effect of pm25 on disease outcomes over the past days they found a significant negative association among pm25 and new daily confirmed covid19 cases cid0 ci and deaths cid0 ci low pm concentrations in this area of study mean μgm3 are likely to have played a less central role in the spread of infection than in other areas such as italy where pm25 monthly concentrations reached values higher than μgm3 fattorini and regoli frontera or in china where pm25 monthly concentrations reached values higher than μgm3 zhu jiang as said by the authors other gaseous pollutants such as no2 and so2 could have influenced transmission and pathogenesis of covid19 in the united states wu investigated whether longterm average exposure to fine particulate matter pm25 increases the risk of covid19 deaths by considering approximately counties in the united states of the population with an exposure prediction model the authors calculated the county level longterm exposure to pm25 averaged for to and collected covid19 deaths counts up to april 04th they conducted a strong and robust statistical analysis with zeroinflated negative binomial mixed models adjusting their results by several potential confounders such as sociodemographic socioeconomic behavioural and meteorological factors they found that a small longterm exposure increase of only μgm3 in pm25 is associated with a increase in the covid19 death rate confidence interval ci vasquezapestegui recently reported first evidences on the spatial relationship between particulate matter and covid19 outbreak from latin america the authors described the situation occurred in districts of lima located in the second most affected country of latin america peru in particular by applying a multivariate regression model they evaluated the association between the population exposure to pm25 concentrations in the previous years and cases deaths and casefatality rates of covid19 with data up to june 12th a significant association has been found both with cases and deaths crude coefficient with p and with p respectively but not with case fatality rate all these studies highlight the role of pm in triggers of the covid19 disease and how government measures targeting to sustainable growth such as the reduction of urban and industrial emissions could have a positive impact on the prevention of health outcomes reducing mortality rate as well the burden on health care systems nitrogen dioxide no2 and covid19 induced lung damage hence viral infection becomes more common after exposure to no2 zhu furthermore no2 is associated with other several health effects such as elevated risks for asthma allergic rhinitis and eczema in children to increase of outpatient visits and hospitalizations due to bronchitis and asthma exacerbation bahrami asl kowalska increase of chronic obstructive pulmonary disease copd ghanbari ghozikali pfeffer and increase of pulmonary heart disease related mortality chen a recent study explored the possible role of no2 in interference in angiotensin converting enzyme ace2 the expression of ace2 is high on lung alveolar epithelial cells and it is the human cell receptor of virus agent of covid19 alifano first observations report an association between ambient concentrations of no2 and covid19 pandemic across europe china and usa bashir fattorini and regoli jiang li et al ogen zhu et al zoran et al 2020b conversely to the other papers findings of zoran 2020b and bashir provides different findings reporting no association or a negative one between no2 and daily deaths counts in china zhu by applying the same method explained for pm observed that a 10μgm3 increase lag0 in no2 is associated with a ci increase in the daily counts of covid19 confirmed cases in cities of china these findings are confirmed by jiang and li et a who applied the same method described for pm jiang revealed a significant positive association between no2 and covid19 both in wuhan and xiaogan wuhan rr ci1053 xiaogan rr ci but did not found any significant association in huanggang li found a significant linear correlation both in wuhan r2 p and xiaogan r2 p ogen presented evidences on the relationship between exposure to no2 including the months of january and february shortly before the covid19 spread in europe and novel coronavirus fatality in the most affected european countries concluding that longterm exposure to no2 may be a potential contributor to mortality caused by sarscov2 he collected data concerning the number of fatality cases from administrative regions in italy spain france and germany and correlated mortality with tropospheric no2 concentrations measured by the sentinel5 precursor spaceborne satellite the major tropospheric no2 hotspot identified was located in the northern italy in all european regions considered gas concentrations ranged between and μmolm2 with airflows directed downwards results showed that out of the fatality cases by march were in five regions located in north italy and central spain furthermore by analysing mortality trends it was revealed that the highest percentage of deaths occurred in regions where the maximum no2 concentration was above μmolm2 with a significant decrease where the maximum concentration was between and μmolm2 and below μmolm2 the methodology used by ogen cannot support a longterm exposure investigation surely a validation of the satellite measure with those of the ground ones the adjustment of the results according to the different population size of each country could have made their results more robust nevertheless the study provide new insights for future investigation the italian situation was further studied by fattorini and regoli who collected data of covid19 incidence up to april 27th from italian provinces they revealed a strong spatial correlation with no2 mean levels concentrations pearson coefficient r2 p confirming the northern italy being a hotspot of no2 in addition to urbanized cities of central and southern italy such as rome and naples a focus on the temporal association between ground levels of no2 and covd19 cases total cases daily new positive cases and total deaths was performed by zoran 2020b for the city of milan italy in the period pre and postlockdown measures the authors nitrogen dioxide is a nastysmelling gas formed by reaction in the atmosphere of nitrogen oxides nox with other chemicals nox is naturally produced in atmosphere by lightning kang et al volcanoes oceans and biological decay thurston the major outdoor anthropogenic sources of nox are primarily emissions from transportation and fuel combustion in particular in urban areas they comes from vehicle exhaust gases and domestic heating grange maawa the nitrogen dioxide has mainly effect on the respiratory system because an increase of the outdoor concentration of no2 may significantly increase the risk of respiratory tract infection this phenomenon is particularly evident in children as they are more susceptible to no2 environmentalresearch19120201101296 0cacknowledgments c copat found no2 negative correlated with all the considered epidemiological data but the methodology used has some limitations as the delay time from infection to the covid19 onset or covid19 death was not considered as well the significant reduction of air pollution due to lockdown measures since midmarch in usa the association was also studied by bashir for the state of california as discussed above for pm the authors found a negative correlation also between no2 levels and covid19 cases and mortality nevertheless they stated that this pollutant contributes to the spread of the disease based on these scientific evidences in addition to confirming that exposure to no2 is harmful to human health and increases the risk of incurring respiratory diseases it can be stated that exposure to no2 may be one of the most important trigger for the spread and fatality caused by the covid19 disease declare references adhikari a yin j 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guo q xu dz sun qw yang h zhao gm jiang qw influence of meteorological factors and air pollution on the outbreak of severe acute respiratory syndrome publ health https doi101016jpuhe200609023 carugno m dentali f mathieu g fontanella a mariani j bordini l milani g p consonni d bonzini m bollati v pesatori ac pm10 exposure is associated with increased hospitalizations for respiratory syncytial virus bronchiolitis among infants in lombardy italy environ res https doi101016jenvres201806016 chen h chen y lian z wen l sun b wang p li x liu q yu x lu y qi y zhao s zhang l yi x liu f pan g 2020a correlation between the migration scale index and the number of new confirmed coronavirus disease cases in china epidemiol infect e99 httpsdoi101017 s0950268820001119 chen j zeng j shi c liu r lu r mao s zhang l associations between shortterm exposure to gaseous pollutants and pulmonary heart diseaserelated mortality among elderly people in chengdu china environ health httpsdoi 101186s1294001905008 chen s prettner k kuhn m geldsetzer p wang c b¨arnighausen t bloom de 2020b covid19 and climate global evidence from countries medrxiv prepr serv health sci httpsdoi1011012020060420121863 coccia m 2020a how high wind speed can reduce negative effects of confirmed cases and total deaths of covid19 infection in society ssrn scholarly paper no id social science research network rochester ny httpsdoi 102139ssrn3603380 coccia m 2020b factors determining the diffusion of covid19 and suggested strategy to prevent future accelerated viral infectivity similar to covid sci total environ httpsdoi101016jscitotenv2020138474 balakrishnan k brunekreef b dandona l dandona r feigin v freedman g hubbell b jobling a kan h knibbs l liu y martin r morawska l pope ca shin h straif k shaddick g thomas m van dingenen r van donkelaar a vos t murray cjl forouzanfar mh estimates and year trends of the global burden of disease attributable to ambient air pollution an analysis of data from the global burden of diseases study lancet lond engl httpsdoi101016s0140673617305056 conticini e frediani b caro d can atmospheric pollution be considered a co factor in extremely high level of sarscov2 lethality in northern italy environ pollut barking essex httpsdoi101016jenvpol2020114465 croft dp zhang w lin s thurston sw hopke pk van wijngaarden e squizzato s masiol m utell mj rich dq associations between source cohen aj brauer m burnett r anderson hr frostad j estep k conclusion the scientific evidences collected in the literature highlight the important contribution of chronic exposure to air pollution on the covid19 spread and lethality although the potential effect of airborne virus exposure it has not been still demonstrated in particular it seems that pm25 and no2 are more closely correlated to covid19 than pm10 the lower correlation of pm10 with covid19 incidence and mortality can be due to the impossibility of particulate matter greater than μm to reach type ii alveolar cells where is located the cell entry receptor ace2 for sarscov2 nevertheless differences between countries such as the implementation of different lockdown restrictions stage of infection topographic sociodemographic and socioeconomic characteristics level of air pollution and meteorological factors may have contributed to obtain some contrasting finding although most of the revised studies support the relationship between air pollution and covid19 the manifold limitations of this review are the small number of papers collected and the great diversity of methodologies used sometimes lacking in some parts which makes the results difficult to compare the authors who first investigated this association although with great effort and rapidity of analysis dictated by a global emergency sometimes do not include all confounding factors whenever possible such as control policy urbanization rate availability of medical resources population size weather lifestyles sociodemographic and socioeconomic variables in addition to date incidence data are underestimated in all countries and to a lesser extent mortality data for this reason the cases included in the considered studies cannot be considered conclusive more studies are needed to better clarify the role of air pollution during the covid19 pandemic particularly studies that consider the multiplepollutants to strengthen scientific evidences and support firm conclusions useful to implement pandemic application plans to adequately prevent new health emergencies for a long time we have known that reducing outdoor and indoor air pollution in cities or countries can have a significant effect on health almost immediately and the benefits can far outweigh the costs surely the health emergency that the world is experiencing right now highlights how environmental research is a fundamental reference point to improve the knowledge concerning diseases of infectious origin and how all the intellectual and economic resources are to be spent to accelerate actions aimed to implement environmental policies act to reduce air pollution and develop new urban planning interventions influences or multidisciplinary studies declaration of competing interest the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper environmentalresearch19120201101297 0cc cop | 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 |
" chronic colorectal inflammation has been associated with colorectal cancer crc however its exact molecular mechanisms remain unclear the present study aimed to investigate the effect of tolllike receptor tlr9 on the development of colitisassociated crc cac through its regulation of the nfκb signaling pathway by using a cac mouse model and immunohistochemistry the present study discovered that the protein expression levels of tlr9 were gradually upregulated during the development of crc in addition the expression levels of tlr9 were revealed to be positively correlated with nfκb and ki67 expression levels in vitro inhibiting tlr9 expression levels using chloroquine decreased the cell viability proliferation and migration of the crc cell line ht29 and further experiments indicated that this may occur through downregulating the expression levels of nfκb proliferating cell nuclear antigen and bclxl in the findings of the present study suggested that tlr9 may serve an important role in the development of cac by regulating nfκb signalingcorrespondence to professor youxiang chen or dr chunyan zeng department of gastroenterology the first affiliated hospital of nanchang university yongwaizheng street nanchang jiangxi pr chinaemail chenyx102126com email zcy896163com abbreviations aif acute inflammation aom azoxymethane cac chronic inflammation crc colorectal cancer dai disease activity index dss dextran sodium sulfate ibd inflammatory bowel disease iecs immunohistochemistry pcna proliferating cell nuclear antigen tlr9 tolllike receptor uc ulcerative colitiskey words colitisassociated crc aom dss tlr9 nfκbintestinal epithelial cells ihc colitisassociated colorectal cancer cif introductionthe number of patients with colorectal cancer crc worldwide is increasing annually with an incidence rate of in chronic inflammation is the leading cause of immune cell infiltration and proliferation and it has been suggested to be a highrisk factor for colitisassociated crc cac inflammatory bowel disease ibd which encompasses both ulcerative colitis uc and crohn's disease was established as an important precursor to crc for example the incidence of ibdassociated crc in patients with uc was reported to have a cumulative risk rate of at years at years and at years of disease duration therefore further in vivo studies are required for researchers to gain an improved understanding of the molecular mechanisms of cac which may provide more exact molecular targets for the diagnosis and treatment of cac during the early stages tolllike receptor tlr9 a member of the tlr family is located in the cytoplasm and intracellular endosomes and can be activated by unmethylated bacterial cpg dna the activation of the tlr9 signaling pathway induces a type t helper cell immune response and stimulates the proliferation of b cells thus protecting the host from external microbial invasion multiple studies have revealed that abnormal tlr9 expression levels were involved in the pathogenesis and progression of uc in addition abnormal expression levels of tlr9 were also identified during the tumorigenesis and development of crc nfκb is an important transcription factor involved in various biological processes including inflammatory reactions immune responses apoptosis and proliferation in fact nfκb is regarded as a molecular hub that links inflammation and cancer it was previously suggested that nfκb may serve an important role in colorectal carcinogenesis by regulating matrix metalloproteinase expression previous studies have revealed that tlr9 was related to the biological characteristics of various types of cancer including bladder lung and prostate cancer such as cell proliferation invasion tumor growth and progression in fact one previous study reported that tlr9 regulated the expression levels of interleukin il through the myeloid differentiation primary response protein myd88 myd88nfκb signaling 0cluo tlr9 promotes colorectal carcinogenesispathway in myeloid cells to promote tumor recurrence after irradiation including in melanoma bladder carcinoma and colorectal carcinoma the current study aimed to investigate the effect of tlr9 on the development of cac through its regulation of the nfκb signaling pathway owing to the synergistic effects of azoxymethane aom a tumorinducing agent and dextran sodium sulfate dss a tumorpromoting agent the present study established cac model mice by coadministering aom and dss to analyze the expression levels of tlr9 nfκb and ki67 in cac tissuesmaterials and methodsreagents and antibodies aom cat no a5486 and chloroquine tlr9 inhibitor cat no c662825g were obtained from sigmaaldrich merck kgaa dss cat no 100g was purchased from mp biomedicals llc antitlr9 cat no ab134368 and antimyd88 cat no ab135693 primary antibodies were obtained from abcam antinfκb nfκb p65 cat no 8242s antibclxl cat no and antiki67 cat no 9449s primary antibodies were obtained from cell signaling technology inc the antiproliferating cell nuclear antigen pcna cat no sc primary antibody was obtained from santa cruz biotechnology inc and the antigapdh cat no ta309157 primary antibody was obtained from origene technologies inc horseradish peroxidase secondary goat antirabbit cat no zb and goat antimouse cat no zb antibodies used for western blotting and goat antimouserabbit antibodies cat no ta130001ta130015 used for immunohistochemistry ihc were obtained from origene technologies inccell lines and culture the human crc cell line ht29 was obtained from the american type culture collection and cultured in mccoy's 5a modified medium gibco thermo fisher scientific inc supplemented with fbs gibco thermo fisher scientific inc mgml penicillin and mgml streptomycin maintained in a humidified atmosphere of co2 at Ëcwestern blotting cells were lysed in ripa lysis buffer beijing solarbio science technology co ltd at Ëc for min the protein concentration was measured following centrifugation at x g for min at Ëc and quantified using a bicinchoninic acid protein assay kit beijing solarbio science technology co ltd an equal amount of protein µglane was separated via sdspage and electrophoretically transferred onto polyvinylidene difluoride membranes emd millipore the membranes were blocked with skimmed milk for h at room temperature and were subsequently incubated overnight at Ëc with the following primary antibodies antitlr9 antinfκb antibclxl antipcna antimyd88 and antigapdh following the primary antibody incubation the membranes were washed times with pbs for min each and incubated with the corresponding goat antirabbit or goat antimouse secondary antibodies at Ëc for h protein bands were visualized using an ecl reagent thermo fisher scientific inc on a gel doc xr system biorad laboratories inc and analyzed using image lab version software biorad laboratories incwound healing assay a wound healing assay was performed to analyze the cell migratory ability briefly ht29 cells 3x105well were seeded into sixwell plates and cultured to confluence subsequently a µl pipette tip was used to scratch a wound in the cell monolayer fresh serumfree mccoy's 5a modified medium containing different concentrations of chloroquine or µgml was added to each well and cultured for h in a humidified atmosphere of co2 at Ëc images of each well were captured at and h using a light phase contrast microscope olympus zkx olympus corporation with a magnification of x100 the woundhealing areas were assessed using imagej 152a software national institutes of health the migratory rate of cells wound area at hwound area at harea at hcolony formation assay ht29 cells 12x105well were cultured in well plates for h in medium containing different concentrations of chloroquine or µgml at Ëc and then seeded into cm cell culture dishes cellsdish and incubated in complete medium at Ëc after days the cells were fixed with paraformaldehyde for min and stained with crystal violet for min both at room temperature the number of colonies defined as cellscolony was counted manually using a light microscope with a magnification of x100 relative colony number number of colonies in observed groupnumber of colonies in control group all of the experiments were repeated ¥ timescell viability assay the viability of ht29 cells was analyzed using an mtt assay briefly ht29 cells were seeded at a density of cellswell in a well plate and treated for or h with chloroquine or µgml at Ëc and then analyzed using an mtt assay as previously described animal studies all animal experiments were approved by the ethics committee of the first affiliated hospital of nanchang university nanchang china a total of female balbc mice weight g age weeks were obtained from beijing vital river laboratory animal technology co ltd laboratory animal license no scxk the animals were maintained with a normal diet and tap water ad libitum at a temperature of ±Ëc and a relative humidity of and artificially illuminated on an approximate h lightdark cycle at the animal care facility in the medical college of nanchang university all of the mice experiments were approved by the animal care and use committee of nanchang university the total experiment lasted for weeks the mice were divided into four groups micegroup i group a aom dss which was intraperitoneally injected without anesthesia with mgkg aom once on the first day followed by dss given in the drinking water for week and then weeks of distilled water one cycle which was repeated for two additional cycles ii group b aom which was intraperitoneally injected with mgkg aom once on the first day and provided with distilled drinking water during weeks iii group c dss which was treated with dss 0concology letters as described for group a but without the aom treatment and iv group d blank control which received neither dss nor aom treatment and was provided with distilled drinking water for the first weeks all the mice were provided with a normal diet and tap water during weeks fig 1athe disease activity index dai was evaluated at the end of the experiment using the numerical system described by tian the dai parameters included total body weight loss none stool consistency wellformed pellets loose stool diarrhoea and the presence of fecal occult blood negative positive gross bleedingseveral mice were randomly sacrificed at certain times points following aom injection the 1st 2nd 3rd 6th 9th 12th 18th and 23rd weeks six mice were sacrificed at weeks and one mouse was sacrificed at week and four mice were sacrificed at weeks and in each group additionally six mice in group d were randomly sacrificed at week as control all remaining mice were sacrificed at week after the large bowels were resected and washed with pbs they were carefully examined photographed and fixed in formalin at room temperature for h further histological examinations were subsequently performedhistopathological analysis and immunohistochemistry ihc paraffinembedded colorectal sections µmthick were stained with hematoxylin for min and eosin for min at room temperature to analyze the degree of inflammation using a light microscope with magnifications of x40 and x100 briefly the severity of inflammation the thickness of inflammation the severity of epithelial damage and the extent of the lesions were each graded from to by two investigators who were blinded to the treatment groups as previously described the severity of inflammation was adapted from the grading system developed by truelove and richards as follows i grade no neutrophil infiltration in the lamina propria ii grade i infiltration of a small number of neutrophils [ cellshigh power field hpf] in the lamina propria involving a few crypts iii grade ii obvious neutrophil infiltration in the lamina propria cellshpf involving of the crypts iv grade iii infiltration of neutrophils cellshpf in the lamina propria with crypt abscess and v grade iv obvious acute inflammation in the lamina propria with ulcer formation grade i was classified as mild grade ii was classified as moderate and grades iii and iv were classified as severe the severity of inflammation ranged from to no inflammation mild moderate severe the thickness of inflammation ranged from to no inflammation mucosa mucosa plus submucosa transmural the severity of epithelial damage ranged from to intact epithelium disruption of architectural structure erosion ulceration and the extent of lesions ranged from to no lesions punctuate multifocal diffuseihc was performed as described in our previous study colorectal tissues were fixed in formalin for h at room temperature formalinfixed and paraffinembedded tissue blocks were cut into µmthick sections and mounted on glass slides slides were heated in an oven at Ëc for min and deparaffined in xylene twice for min each at room temperature rehydrated in a descending ethanol series and ethanol for min each at room temperature and incubated in h2o2 for min at room temperature to block endogenous peroxidase antigen retrieval was performed by heating in a microwave at Ëc in sodium citrate buffer mm ph for min slides were blocked with bovine serum albumin beijing solarbio science technology co ltd for h at room temperature to block nonspecific antibody binding and incubated overnight at Ëc with the following primary antibodies antitlr9 antinfκb and antiki67 following the primary antibody incubation the sections were washed three times with pbs and incubated with horseradish peroxidase secondary goat antimouserabbit antibodies ready to use at Ëc for min the sections were stained with 'diaminobenzidine at room temperature the duration of staining was based on the staining observed under a light microscope with a magnification of x100 and the reaction was terminated when the staining was yellowishbrown the sections were then counterstained with hematoxylin for min at room temperature the slides were observed under a light microscope with a magnification of x100 the widely accepted german semiquantitative scoring system was used to determine the staining intensity and area of staining according to the recommendations of remmele and stegner each specimen was assigned a score according to the intensity of the nucleic cytoplasmic andor membrane staining no staining not detected0 weak staining light yellow1 moderate staining yellowish brown2 strong staining brown3 and the extent of stained cells no staining the final immunoreactive score was determined by multiplying the intensity score with the extent of stained cells score ranging from the minimum to the maximumcoimmunoprecipitation assay ht29 cells were lysed in ripa lysis buffer beijin solarbio science technology co ltd at Ëc for min wholecell lysates were pelleted via centrifugation at x g for min at Ëc the supernatant was incubated with an antitlr9 antibody or goat antimouse igg cat no zb origene technologies inc together with protein ag plusagarose beads santa cruz biotechnology at Ëc overnight the beads were washed three times with the nonlubrol lysis buffer at x g centrifugation for min at Ëc then subjected to sdspage and subsequent western blotting analysis as aforementioned whole cell lysate was used as a controlstatistical analysis statistical analysis was performed using spss software ibm corp the data are presented as the mean ± sd all experiments were performed at least in triplicate a oneway anova followed by a tukey's post hoc test was used to determine the statistical differences between groups whereas an unpaired student's ttest were used to determine the statistical differences between groups a spearman's rank correlation test was used to determine the correlation between the expression levels of tlr9 nfκb and ki67 in crc tissues p005 was considered to indicate a statistically significant differenceresultsconstruction of the cac model mice in groups a aom dss and c dss the body weights of the mice decreased with 0cluo tlr9 promotes colorectal carcinogenesisfigure construction of the colitisassociatedcolorectal cancer model in mice a schematic diagram of the experimental protocol for colitisassociated colorectal cancer model mice in groups ad arrow indicates mgkg azoxymethane administration via intraperitoneal injection black square indicates administration of dextran sodium sulfate in drinking water for week continuous line indicates duration fed a normal diet and distilled water dotted line indicates duration fed a normal diet and tap water and triangle indicates timepoint of sacrifice b body weights of the mice in each group p0001 c disease activity index of groups a and c data are presented as the mean ± sd group a mgkg azoxymethane and dextran sodium sulfate water group b mgkg azoxymethane group c dextran sodium sulfate water group d blank controltime compared with group d blank control group fig 1b the average weight of the mice in group c declined from ± g at the beginning of the experiment to ± g by week while the average weight of the mice in group a decreased from ± g at the beginning of the experiment to ± g by week the differences between groups a and d and groups c and d were statistically significant at week p005 the dai which reflects the severity of colitis was also markedly increased in groups a and c after dss administration on the 1st 3rd and the 6th week fig 1c no weight loss or any signs of inflammation such as loose stool or diarrhea positive fecal occult blood or gross bleeding were observed in groups b aom and d blank control the dai of groups b and d was zero throughout the modeling process and is therefore not shown in fig 1c the lengths of the large bowels in groups a and c were decreased compared with groups b and d fig 2a notably the lengths of the large bowels were significantly decreased in groups a and c from week compared with the control group mice sacrificed in group d at week or with group dp005 fig 2b however the severity of inflammation was significantly increased in group a compared with in group c at week and the extent of inflammation was significantly increased in group a compared with in group c after week p005 while there was no significant differences in the thickness of inflammation and the severity of epithelial damage between groups a and c fig 2cby the 12th week colorectal tumors were only observed in the mice of group a fig 2d pathological results further revealed that the mice in group a presented with acute inflammation aif chronic inflammation cif adenoma and adenocarcinoma of the colorectum at weeks and respectively fig 2d and eexpression levels of tlr9 and nfκb are simultaneously upregulated during the development of chronic colitis in crc ihc staining revealed that tlr9 was located in the cytoplasm of the intestinal epithelial cells iecs and inflammatory cells in the lamina propria fig 3a tlr9 expression levels were significantly upregulated in aif cif adenoma and adenocarcinoma tissues compared with the corresponding tissues from control mice group d p00334 p00379 p00437 and p00008 respectively fig 3b furthermore the protein expression levels of tlr9 was significantly upregulated in the adenocarcinoma tissue compared with the aif p00077 cif p00278 and adenoma p00273 tissue fig 3bthe positive nfκb region was mainly confined to the cytoplasm of the iecs and inflammatory cells fig 3a the ihc results revealed that the expression levels of nfκb were significantly upregulated in the aif cif adenoma and adenocarcinoma tissues compared with the corresponding tissues from control mice p00061 p00043 p00019 and 0concology letters figure colorectal pathological results in mice a length of the large bowels of mice in each group at week representative bowels from independent animals are shown b mean length of large bowel of mice in each group p005 p001 p0001 vs control mice sacrificed in group d at week or group d bottom graph c histological score of severity thickness damage and extent of lesions of the colorectums from mice in groups a and c p005 d large bowels were retrieved at week and from mice in group a to determine the numbers of tumors formed p005 p001 e histological analysis of various pathological changes in the colons of mice in group a were determined using hematoxylin and eosin staining the control groups represents tissues taken at week from group d scale bars µm upper and µm lower inflammatory cells green arrows and intestinal epithelial cells red arrows are indicated group a mgkg azoxymethane and dextran sodium sulfate water group c dextran sodium sulfate water aif acute inflammation cif chronic inflammationp00001 respectively fig 3d moreover nfκb expression levels were significantly upregulated in the adenocarcinoma tissue compared with the aif p00001 and adenoma p00161 tissues fig 3dki67 expression levels were observed in the nuclei of iecs and inflammatory cells fig 3a the ihc results revealed that the ki67 expression levels were gradually upregulated across the intestinal lesions including in the aif cif adenoma and 0cluo tlr9 promotes colorectal carcinogenesisfigure tlr9 and nfκb are simultaneously upregulated as cac develops a ihc was used to analyze the expression levels and localization of tlr9 nfκb and ki67 in colorectal sections obtained from mice in group a inflammatory cells green arrows and intestinal epithelial cells red arrows are indicated ihc staining scores of b tlr9 c ki67 and d nfκb in sections of normal control group d aif cif adenoma and adenocarcinoma tissues which were obtained as described in the methods section scale bar µm p005 p001 and p0001 vs control p005 p001 and p0001 vs adenocarcinoma correlation analysis of e tlr9 and nfκb expression levels f ki67 and tlr9 expression levels and g nfκb and ki67 expression levels was conducted using spearman's rank correlation group a mgkg azoxymethane and dextran sodium sulfate water aif acute inflammation cif chronic inflammation tlr9 tolllike receptor ihc immunohistochemistryadenocarcinoma tissues p00331 p00092 p00241 and p00006 respectively compared with the expression levels in the corresponding tissues from the control mice fig 3c interestingly the expression levels of tlr9 and nfκb were discovered to be significantly positively correlated with other rho08236 p00001 fig 3e in addition a significant positive correlation was also identified between tlr9 and ki67 expression levels rho05515 p0001 fig 3f and between nfκb and ki67 expression levels rho05103 p001 fig 3gdownregulated tlr9 expression levels reduces the migration viability and colony formation of ht29 cells to further investigate the role of tlr9 in crc the human crc cell line ht29 was treated with chloroquine an inhibitor of tlr9 in vitro the results revealed that suppressing tlr9 with chloroquine at both doses inhibited the migration viability and colony formation ability of ht29 cells in a dosedependent manner fig 4ae additionally lysates were collected from ht29 cells treated with either different concentrations of chloroquine for h or µgml chloroquine for different time periods fig 4fi and western blotting was performed the analysis revealed that the expression levels of tlr9 nfκb pcna myd88 and bclxl were gradually downregulated in ht29 cells treated with increasing doses of chloroquine compared with the 0concology letters figure chloroquine inhibits the migration viability and colony formation of ht29 cells by inhibiting tlr9 chloroquine and µgml inhibited the migration of ht29 cells in a dosedependent manner at h compared with the control group which was determined using a wound healing assay a representative images were photographed magnification x100 and b migration rates were calculated proliferation rate of ht29 cells was reduced by chloroquine treatment and µgml which was determined using a colony formation assay c representative images were photographed and d the relative colony number was analyzed p005 p001 p0001 e viability of ht29 cells was decreased by chloroquine and µgml at and h compared with the control cells as determined using an mtt assay f expression levels of tlr9 nfκb pcna myd88 and bclxl in lysates obtained from ht29 cells treated with numerous concentrations of chloroquine or µgml for h were analyzed using western blotting g semiquantification of the expression levels of the proteins presented in part f was performed using imagej software gapdh was used as the loading control and for normalization h expression levels of tlr9 nfκb pcna myd88 and bclxl in lysates obtained from ht29 cells treated with µgml chloroquine for numerous durations or h were analyzed using western blotting i semiquantification of the expression levels of the proteins presented in part h was performed using imagej software gapdh was used as the loading control and for normalization j tlr9 interacted with the nfκb protein in ht29 cells which was determined using a coimmunoprecipitation assay goat antimouse igg antibody was used as the negative control all data are expressed as the mean ± sd of three independent experiments p005 p001 p0001 vs control0 h tlr9 tolllike receptor pcna proliferating cell nuclear antigen myd88 myeloid differentiation primary response protein myd88 ip immunoprecipitated ib immunoblotting 0cluo tlr9 promotes colorectal carcinogenesiscontrol group fig 4f and g a similar trend was observed in the expression levels of these proteins as the duration of chloroquine treatment increased fig 4h and i thus these results indicated that the expression levels of tlr9 nfκb pcna myd88 and bclxl in ht29 cells treated with chloroquine may be downregulated in both a dose and timedependent manner fig 4fi to verify whether tlr9 affected the colorectal carcinogenesis by interacting with nfκb coimmunoprecipitation assay was used to detect the interaction between tlr9 and nfκb in ht cells the results revealed that there was an interaction between tlr9 and nfκb fig 4jdiscussioncolorectal carcinogenesis is a multistep process starting from normal crypts to aberrant crypt foci then to polyps adenoma and eventually adenocarcinoma it has been reported that individuals with ibd may have an increased risk of developing crc which is directly proportional to the extent and duration of their disease however the exact mechanism and duration required for chronic colitis to develop into adenoma and then adenocarcinoma remains unclear it has been suggested that patients with ibd have an increased risk of crc following the inflammationdysplasiacarcinoma model including dysplasia and crc as primary consequences of chronic inflammation however there are currently still no defined molecular biomarkers or existing monitoring protocols for detecting the occurrence of a malignant tumor except for frequent colonoscopy examinationsin the present study an acute colitischronic colitisadenomaadenocarcinoma model was successfully constructed via aomdss induction using this model tlr9 expression levels were discovered to be upregulated as the severity of the colorectal lesions increased which indicated that tlr9 protein expression levels may be continuously activated during colitiscrc development tlr9 is a critical protein associated with innate and acquired immunity and it has been demonstrated to serve a significant role in the development of colitis and sporadic crc however the mechanism by which tlr9 regulates the development of crc remains to be elucidatedinterestingly ihc analysis revealed that the expression levels of nfκb and ki67 were simultaneously upregulated alongside tlr9 expression levels notably inhibiting tlr9 decreased the migration proliferation and viability of ht29 cells in vitro and tlr9 expression levels in vivo were identified to be significantly positively correlated with the expression levels of nfκb and ki67 a cell proliferation marker during the transition from colitis to crc the present study further revealed that the inhibition of tlr9 in vitro significantly downregulated the expression levels of nfκb myd88 pcna and the antiapoptotic protein bclxl in a dose and timedependent manner notably a previous study reported that tlr9 promoted the tumorpropagating potential of prostate cancer cells via nfκb signaling thus the findings of the present study indicated that tlr9 may promote cac through nfκb signaling however these findings may be controversial because other previous studies have revealed that tlr9 agonists exerted an antitumor effect in crc the majority of these studies primarily focused on the role of tlr9 in colitis or sporadic crc whereas the current study focused on the role of tlr9 in the pathogenesis of cac to provide novel targets for the treatment of cac for example alterations in microtubule endbinding protein were identified as a characteristic of sporadic but not ucassociated crc and in another previous study the immune profiling patterns of patients with cac were significantly different compared with the patients with sporadic crc chloroquine a nonspecific inhibitor of tlr9 is an old antimalarial drug which has recently attracted significant interest for its potential antitumor properties for example numerous studies have reported that chloroquine directly regulated cancer cells by inducing apoptosis inhibiting autophagy interacting with nucleotides eliminating cancer stem cells and enhancing the growth of cancer cells chloroquine also inhibited the expression levels of tlr9 by preventing the acidification and maturation of the endosomes and the trafficking of tlrs due to the multiple effects of chloroquine on tumor cells different concentrations of chloroquine were selected for use in the present study based on the lowest dose according to previous studies in order to obtain the best possible results with low toxicity to the cells in the future investigations using small interfering rna targeting tlr9 should be performed to determine the effect on the biological processes of crc cell lines to further verify the findings of the present study thus our future studies will focus on investigating the precise molecular mechanism by which tlr9 participates in the early occurrence of colorectal carcinogenesisin the present study developed a cac animal model the findings indicated that tlr9 may be closely associated with the process of inflammationdysplasiacarcinoma and may impact carcinogenesis by regulating the nfκb signaling pathway these results may provide promising potential to be developed into an early detection protocol or therapeutic molecular target for crcacknowledgementsnot applicablefundingthe present study was supported by grants from the national natural science foundation of china grant nos and the foundation of jiangxi educational committee grant no gjj170016 and the graduate student innovation funding program of nanchang university grant no cx2019119availability of data and materialsall data generated or analyzed during this study are included in this published article the original data are available from the corresponding author on reasonable requestauthors' contributionscz and ql designed the study and drafted the manuscript ql and lz performed the experiments ql ct and zz analyzed the data ql cz and yc revised the manuscript for important 0concology letters intellectual content cz and yc made substantial contributions to conception design and coordination of the study and gave final approval of the version to be published all authors have read and approved the final manuscriptethics approval and consent to participateall animal experiments were approved by the ethics committee of the first affiliated hospital of nanchang university nanchang china approval no pa | 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 |
bcl9 and pygo are bcatenin cofactors that enhance the transcription of wnt targetgenes they have been proposed as therapeutic targets to diminish wnt signaling output inintestinal malignancies here we find that in colorectal cancer cells and in developing mouseforelimbs bcl9 proteins sustain the action of bcatenin in a largely pygoindependent mannerour genetic analyses implied that bcl9 necessitates other interaction partners in mediating itstranscriptional output we identified the transcription factor tbx3 as a candidate tissuespecificmember of the bcatenin transcriptional complex in developing forelimbs both tbx3 and bcl9occupy a large number of wntresponsive regulatory elements genomewide moreover mutationsin bcl9 affect the expression of tbx3 targets in vivo and modulation of tbx3 abundance impactson wnt target genes transcription in a bcatenin and tcflefdependent manner finally tbx3overexpression exacerbates the metastatic potential of wntdependent human colorectal cancercells our work implicates tbx3 as contextdependent component of the wntbcatenindependenttranscriptional complexintroductionthe wnt pathway is an evolutionarily conserved cell signaling cascade that acts as major drivingforce of several developmental processes as well as for the maintenance of the stem cell populations within adult tissues nusse and clevers deregulation of this signaling pathway resultsin a spectrum of consequences ranging from lethal developmental abnormalities to several forms ofaggressive cancer nusse and clevers most prominently colorectal cancer crc is initiatedby genetic mutations that constitutively activate wnt signaling kahn secreted wnt ligands trigger an intracellular biochemical cascade in the receiving cells that culminates in the calibrated expression of target genes mosimann this transcriptionalresponse is orchestrated by nuclear bcatenin that acts as a scaffold to buttress a host of cofactorsto cisregulatory elements occupied by the tcflef transcription factors valenta among the cofactors the two paralogs bcl9 and bcl9l referred to as bcl99l and pygo12proteins reside within the wntbcatenin transcriptional complex and their concerted action isrequired to efficiently activate wnttarget gene expression kramps parker for correspondencekonradbaslerimlsuzhch kbandreasmoorbsseethzchaemclaudiocantuliuse cc these authors contributedequally to this workpresent address ¡division ofmolecular pathology thenetherlands cancer instituteamsterdam netherlandscompeting interests theauthors declare that nocompeting interests existfunding see page received april accepted august published august reviewing editor roel nussestanford university unitedstatescopyright zimmerli this is distributed under theterms of the creative commonsattribution license whichpermits unrestricted use andredistribution provided that theoriginal author and source arecreditedzimmerli elife 20209e58123 107554elife58123 of 0cshort reportdevelopmental biologyfigure the intestinal epitheliumspecific recombination of pygo12 does not recapitulate the effects of deleting bcl99l a schematic representationof the wntbcatenin transcriptional complex with emphasis on the socalled chain of adaptors components bcatenin bcl99l and pygo wntfigure continued on next pagezimmerli elife 20209e58123 107554elife58123 of 0cshort reportfigure continueddevelopmental biologyresponsive element wre the homology domains hd13 of bcl99l are shown b epithelialspecific pygo12 deletion via vilcreert2 pygo12ko does not lead to any obvious histological or functional defect neither in the small intestine nor in the colon as seen by hematoxylin and eosinstaining left panels the proliferative compartment detected via ki67 right panels seems also unaffected also refer to the count in figure figuresupplement 1de c quantitative rtpcr detecting lgr5 mrna extracted from colonic epithelium of control black bcl99l blue or pygo12 redconditional mutants ko d weekold male mice were treated with five tamoxifen tam injections ip mgday for five consecutive days days later mice were treated with dextran sodium sulfate dss ad libitum in drinking water for days while of control mice n wereseverely affected or died due to the dss treatment red lines of conditional bcl99lko n mice performed poorly in this test deletion ofbcl99l increased significantly the death rate after dss treatment pvalue000013 in fishers exact test no difference between pygo12ko andcontrol mice could be measured of control mice n and of pygo12ko n were affected upon dss treatment pvalue05626 infishers exact test e weekold female mice were exposed to a single dose of the carcinogenic agent azoxymethane aom followed by daysof dss administration in the drinking water this regimen results in the emergence of dysplastic adenomas that are collected for rna extraction andanalysis of the indicated targets via rtpcr wnt target genes and genes expressed during epithelialtomesenchymal transition emt associated withcancer metastasisthe online version of this includes the following figure supplements for figure figure supplement efficient epithelialspecific pygo12 deletion does not lead to obvious defectsfigure supplement intestinal epitheliumspecific recombination of pygo12 does not recapitulate the effects of deletingbcl99l van tienen figure 1a during vertebrate development their requirement in thebcateninmediated transcription appears to be contextdependent cantu li and they also have evolved bcateninindependentfunctions cantu cantu curiously however bcl9 and pygo always seem to act as a duetkennedy importantly bcl99l and pygo proteins were found to significantly contribute to the malignanttraits typical of wntinduced crcs deka gay jiang mani mieszczanek moor talla and brembeck theseobservations provided impetus to consider the bcl9pygo axis as relevant targetable unit in crclyou mieszczanek talla and brembeck zimmerli however here we noticed an apparent divergence between the roles of bcl99l and pygo proteins we found that genetic abrogation of bcl99l in mouse crc cells results in broader consequences than pygo12 deletion suggesting that bcl9 function does not entirely depend on pygo12among the putative bcateninbcl9 interactors we identified the developmental transcription factortbx3 intriguingly we show that also during forelimb development bcl99l possess a pygoindependent role in this in vivo context tbx3 occupies bcateninbcl9 target loci genomewide andmutations in bcl99l affect the expression of tbx3 targets finally tbx3 modulates the expression ofwnt target genes in a bcatenin and tcflefdependent manner and increases the metastaticpotential of human crc cells when overexpressed we conclude that tbx3 can assist the wntbcatenin mediated transcription in selected developmental contexts and that this partnership could beaberrantly reactivated in some forms of wntdriven crcsresults and discussionwe induced intestinal epitheliumspecific recombination of pygo12 loxp alleles pygo12ko thatefficiently deleted these genes in the whole epithelium including the stem cells compartment figure figure supplement 1a and b consistently with recent reports mieszczanek talla and brembeck and similarly to deletion of bcl99l deka mani moor pygo12ko displayed no overt phenotypic defects figure 1b figure figure supplement 1ce we were surprised in noticing that the expression of lgr5 themost important intestinal stem cell marker and wnt target gene barker was heavilydownregulated upon loss of bcl99l but unaffected in pygo12ko figure 1c to address the functionality of the stem cell compartment in these two conditions we subjected both bcl99l andpygo12 compound mutants koregeneration by dss treatmentkim figure 1d while bcl99lko mice showed a defect in regeneration after insultdeka pygo12ko proved indifferent when compared to controllittermatesfigure 1d while we cannot exclude that pygo12 also contributes to the wntbcateninto a model ofintestinalzimmerli elife 20209e58123 107554elife58123 of 0cshort reportdevelopmental biologydependent transcriptional regulation our results highlight that the bcl99l function in the intestinalepithelium homeostasis and regeneration does not entirely depend on pygo12 this was surprising since bcl99l proteins were thought to act as mere bridge proteins that tethered pygo tothe bcatenin transcriptional complex figure 1a fiedler mosimann both bcl9 and pygo proteins have been implicated in colorectal carcinogenesis gay jiang mieszczanek talla and brembeck we tested if the consequence of the deletion of bcl99l and pygo12 genes was also different in the context of carcinogenesis specifically we looked at the contribution to gene expression in chemicallyinduced aomdsscolorectal tumors figure 1e as previously observed bcl99lko tumors exhibit a massive decreasein wnt target gene expression epithelialtomesenchymal transition emt and stemness traitsdeka moor which was not observed in pygo12ko tumors figure 1efigure figure supplement 2a and b this phenotypic difference is consistent with a recentstudy in which bcl99l but not pygo12 loss reduced the activation of wnt target genes induced byapc lossoffunction mieszczanek we interpret this as an independent validation ofour observation all these experiments open up the question of how bcl99l imposes its functionindependently of pygosurprisingly the intestinespecific deletion of the homology domain hd1 of bcl99lfigure 2a that was previously annotated to interact only with pygo12 cantu kramps i suppressed the metastatic phenotype of the aomdss tumors while deletion of pygo12 did not figure 2b and ii induced a strong downregulation of wnt target emt andstemness genes figure 2c figure figure supplement the discrepancy between the geneexpression changes induced by recombining pygo12 or deleting the hd1 domain of bcl99l impliesthat currently unknown proteins assist bcl99l function we set out to identify new candidate bcl9partners that might be responsible for the different phenotypes to this aim we performed a pulldown of tumor proteins expressing either a fulllength or a hd1deleted variant of bcl9 followedby mass spectrometry figure 2d among the proteins differentially pulled down by control but notby mutant bcl9 we detected tbx3 figure 2d and e and selected it for further validation the invivo deletion of the hd1 domain in bcl99ldhd1 embryos leads to severe forelimb malformationswhile pygo12ko embryonic forelimbs are unaffected figure 2falso see schwab limb development thus represents another context where bcl99l appear to act independently ofpygo of note tbx3 plays a fundamental role in the development of this structure frank we confirmed cytological vicinity between transfected tagged versions of bcl9 and tbx3 byproximity ligation assay pla figure figure supplement 2ab however overexpressionbasedin vitro coimmunoprecipitation experiments could not detect any stable interaction between thesetwo proteins suggesting absence of direct binding or a significantly lower affinity than that betweenbcl9 and pygo figure figure supplement 2c hence we aimed at testing the functional association between tbx3 and bcl9 in a more relevant in vivo context to this aim we collected ca forelimbs from dpc wildtype mouse embryos and subjected the crosslinked chromatin toimmunoprecipitation using antibodies against bcl9 salazar or tbx3 followed bydeepsequencing of the purified dna chipseq figure 3a by using stringent statistical parameters and filtering with irreproducible discovery rate idr we extracted a list of high confidencebcl9 and tbx3 peaks figure 3b and c surprisingly we discovered that bcl9 occupies a largefraction ca 23rd of the tbx3bound regions figure 3d suggestive of a role for tbx3 within thewntdependent transcriptional apparatus motif analysis of the common tbx3bcl9 target loci identified statistical prevalence for tcflef and homeobox transcription factor consensus sequencesbut not for any tbx transcription factor figure 3e this suggests that tbx3 interacts with the dnain these locations via affinity to the wntbcatenin cofactors rather than via direct contact with dnaaccordingly tbxspecific motifs were detected within the group of tbx3 exclusive peaks which donot display bcl9 binding figure figure supplement notably tbx3 and bcl9 occupancywas detected at virtually all previously described wntresponsiveelements wre within known wnttarget genes figure 3fto test whether the in vivo abrogation of the simultaneous interactions mediated by bcl99lwould influence the expression of genes associated with tbx3 peaks we set out to mutate the bcl99l interaction domains while leaving tbx3 protein unaffected we combined different bcl99l allelesin which the hd2 bcatenininteracting and hd1 pygonew cofactorinteracting motifs arezimmerli elife 20209e58123 107554elife58123 of 0cshort reportdevelopmental biologyfigure identification of tbx3 as a putative bcl9 cofactor a the deletion of the hd1 pygointeracting domain of bcl9 and bcl9l induces avariation in the chain of adaptors causing the loss of pygo association with the wntbcatenin transcriptional complex cantu bimmunofluorescence staining of tumors collected from control or conditional pygo12ko and bcl99lko mice prox1 red and dapi blue are shownin in the top panels vimentin green and laminin red in the bottom panels c quantitative rtpcr of selected groups of targets compare it withfigure continued on next pagezimmerli elife 20209e58123 107554elife58123 of 0cshort reportfigure continueddevelopmental biologythe same analysis of pygo12ko in figure 1e of rna extracted from control or bcl99ldhd1 tumors d experimental outline of the tumor proteinspulldown and massspectrometry tbx3 was identified among the proteins potentially interacting with bcl9 but not with bcl9dhd1 e the ipproteins analyzed by mass spectrometry were in parallel subjected to sds page electrophoresis and probed with an antitbx3 antibody upper panelthe expression of tbx3 in control compared to bcl99ldhd1 tumors n was evaluated via qrtpcr bottom panel to exclude that differentialpulldown was due to lost expression in mutant tumors f dpc bcl99ldhd1 embryos display forelimb malformations and absence of digitsemphasized by dashed white lines a characteristic tbx3mutant phenotype upper panels the limb defect is absent in pygo12ko embryosbottom panels underscoring that bcl99l act in this context independently of pygo12the online version of this includes the following figure supplements for figure figure supplement qrtpcr of target genes associated with intestinal stem cell functionfigure supplement cytological proximity of bcl9 and tbx3deleted cantu double heterozygous animals for the hd1 bcl9dhd1 bcl9ldhd1 orthe hd2 bcl9dhd2 bcl9ldhd2 deletions are viable and fertile the cross between them leads to atransheterozygous genetic configuration in which both domain deletions are present bcl9dhd1dhd2bcl9ldhd1dhd2 referred to as bcl99ld1d2 figure 1a as in these mice bcl99l retain both thehd1 and the hd2 domains in heterozygosity this allelic combination is a way of testing the consequences of abrogating the tripartite complex mediated by the two interacting motifs of bcl99lwithout causing a full lossoffunction of these proteins bcl99ld1d2 embryos also display forelimbmalformations the cause of which cannot be due to pygo figure 2f but must be caused by thefailure of recruiting the new hd1interacting partner by bcl99l onto the bcatenin transcriptionalcomplex we collected forelimbs from control and bcl99ld1d2 mutant embryos at and measured gene expression via rnaseq figure 3g we found a significant enrichment hypergeometrictest p14e6 of tbx3 targets among the genes differentially expressed in bcl99ld1d2 mutantsfigure 3h the enrichment was particularly significant when considering downregulated genes inbcl99ld1d2 mutants indicating that the bcl9tbx3 partnership sustains transcriptional activationfigure 3h of note the design of our experiment directly implicates that these tbx3 transcriptional targets are also bcatenindependent the overlap list includes several regulators of limbdevelopment such as meis2 capdevila irx3 li and eya2 grifone figure 3h heat map on the right despite being of correlative nature this analysis supportsa model in which bcl99l and tbx3 cooperate to the activation of target genesso far we have presented genetic evidence that bcl9 proteins require additional cofactors andthat tbx3 associates with the bcateninbcl9 bound regions on the genome possibly influencing theexpression of target genes however the similarity of genomic binding profiles between tbx3 andbcl9 might be due to their binding in different cells and the decreased expression of genes withnearby enhancers bound by bcl9 and tbx3 might imply a requirement for bcl99l but not necessarily for tbx3 we reasoned that our hypothesis in which bcl9 functionally tethers tbx3 to the bcatenin transcriptional complex raises several testable predictions that will be addressed belowfirst our model implies that tbx3 could impact on wnt target gene expression and its activityshould be dependent on the main constituents of the wntbcatenin transcriptional complex second if tbx3 is tethered by bcl9 to its targets mutations in bcl99l should influence the ability oftbx3 to physically associate with wres finally as for bcl9 tbx3 should be capable of enhancingthe metastatic potential of colorectal cancer cellsto test our first prediction implying a potential role of tbx3 in the transcription of wnt targetgenes we overexpressed it in hek293t cells and monitored the activation status of wnt signalingusing the transcriptional reporter supertopflash stf consistent with its role as repressor tbx3led to a moderate but significant transcriptional downregulation that was importantly specific tothe stf but not the control reporter plasmid figure 4a upon wnt signaling activation achievedvia gsk3 inhibition tbx3overexpressing cells exhibited a markedly increased reporter activity whencompared to control cells in particular at nonsaturating pathway stimulating conditions figure 4aleft panel importantly tbx3 proved transcriptionally incompetent on the stf if the cells carriedmutations in tcflef d4tcf or ctnnb1 encoding for bcatenin dbcat doumpas strongly supporting the notion of its cellautonomous involvement in the activation of canonical wnttarget gene transcription figure 4a central and right panels respectively endogenous wnt targets showed a similar expression behaviour to that of stf upon tbx3 overexpression figure zimmerli elife 20209e58123 107554elife58123 of 0cshort reportdevelopmental biologyfigure tbx3 and bcl9 occupy wnt responsive elements wre in vivo a artistic representation of the chipseq experimental outline bc barplots showing the genomic distribution of highconfidence bcl9 peaks b total and tbx3 peaks c total d overlap of the highconfidence peak groups between bcl9 and tbx3 e selected result entries from motif analysis performed on the bcl9tbx3 overlapping highconfidence peaks significant enrichment was found for tcflef and homeobox motifs no tbx consensus sequence was detected in this analysis ffigure continued on next pagezimmerli elife 20209e58123 107554elife58123 of 0cshort reportfigure continueddevelopmental biologyselect genomic tracks demonstrating occupancy of bcl9 and tbx3 within the wnt responsive element wre of known wnttarget genes axin2ccnd1 nkd1 and lef1 and genes important in limb morphogenesis hand1 and hand2 the scale of peak enrichment is indicated in the topleftcorner of each group of tracks in light blue the bcl9 salazar and in orange the tbx3 replicates and in green the control track igggenomic tracks are adapted for this figure upon visualization with igv integrative genomic viewer igv two independent replicates forbcl9 and tbx3 chipseq experiments are shown g volcano plot displays all the differentially expressed genes degs in developing forelimbs uponmutation of bcl99l bcl99ld1d2 vs ctrl degs were a total of p005 with upregulated and downregulated n of individualmouse embryos for each condition were used for this analysis h a significant portion of degs exhibited overlap with tbx3 chipseq peaksthe overlap with tbx3 chipseq peaks appeared statistically significant in particular when the downregulated genes were considered hierarchicalclustering of samples ctrl versus bcl99ld1d2 right panel based on genes overlapping between degs and genes annotated for tbx3 chipseqpeaks normalized rnaseq read counts wards clustering method euclidian distance annotation added for genes associated by gene ontology townt signaling fgf10 ptk7 kremen1 zfp703 bmp2 and gli3 and genes known as regulators of limb development meis2 irx3 and eya2the online version of this includes the following figure supplements for figure figure supplement overlap of the highconfidence bcl9 and tbx3 peaks in developing murine limbs reveals the existence of bcl9 exclusive oneexample displayed in the genomic tracks on the left and tbx3 exclusive peaks one example in the genomic track on the rightfigure supplement while our experiments show that tbx3 can influence the expression of wnttarget genes the mechanisms by which this occurs remain to be elucidatedwe then addressed our second hypothesis in which bcl99l are required for tethering tbx3onto wres we performed chip of tbx3 in hek293t cells followed by quantitative pcr to detectenrichment on the wre present in the axin2 promoter jho consistent with an effecton transcription in the absence as well as in the presence of chir99021 chir figure 4a tbx3 wasbound to this region both in off and in on conditions figure 4b we then exploited ahek293t clone devoid of both bcl9 and bcl9l db99l van tienen and tested iftbx3 was capable of physical association with the wre of note enrichment of tbx3 in db99 l cellswas dramatically reduced to background levels figure 4b while we cannot exclude that tbx3might act independently of bcl99l on several of its targets this observation supports the notionthat bcl99l are responsible for tbx3 recruitment on classical wres figure 4c this also suggeststhat the previously identified targets of both bcl9 and tbx3 figure 3df must display simultaneous cooccupancy of these two factors in agreement with the notion that bcl99l are themselvesrecruited by the tcfbcatenin axis the physical association of tbx3 with the axin2 promoter wasalso lost in d4tcf and dbcat cells figure 4bfinally we evaluated the effects of tbx3 overexpression oe on growth and metastatic potentialof hct116 human colorectal tumor cells a representative model of crc driven by activating mutations in ctnnb1 mouradov using a in vivo zebrafish xenograft model rouhi approximately labelled control or tbx3oe hct116 cells were implanted in theperivitelline space of hours postfertilization hpf zebrafish embryos figure 4d three days afterinjection tbx3oe cells displayed a marked increase in number in the caudal hematopoietic plexusfigure 4ef the main metastatic site for cells migrating from the perivitelline space rouhi of note tbx3oe hct116 cells maintained consistently high expression of tbx3 within fishembryos throughout the experiment and this was accompanied by increased wntbcatenindependent transcription as measured by axin2 expression figure 4g while this experiment does notallow to exclude that tbx3 might also act independently of bcl9bcatenin in this context it showsthat increased expression of tbx3 enhances proliferation and migratory capability of human crccells bearing constitutively active wnt signaling and this is associated with simultaneous enhancement of the wntbcatenindependent transcription figure 4gtaken together our experiments show that in specific developmental and disease contexts thetranscription factor tbx3 can take active part in the direct regulation of wnt target genes by functional interplay with the bcateninbcl9dependent transcriptional complex our study suggests anew paradigm in which tissuespecific cofactors might be the key to understand the spectrum ofpossible transcriptional outputs observed downstream of wntbcatenin signaling nakamura moreover tbx3 has been linked to different cancer types willmer our observations suggest that tbx3 or its downstream effectors could be considered as new relevant targetsto dampen crc progressionzimmerli elife 20209e58123 107554elife58123 of 0cshort reportdevelopmental biologyfigure tbx3 controls the expression of wnt target genes a bcatenintcf luciferase reporter stf assay in parental left bcatenin knockout dbcat center and tcf knockout d4tcf right hek293t cells cells were treated with the indicated concentration of chir or dmso overnightoverexpression of tbx3 oe black bars compared to control ev empty vector white bars showed that tbx3 acts as a repressor on a wnttcfpathway reporter but switches to activator upon pathway induction only significant pvalues p005 are indicated three independent experimentsfigure continued on next pagezimmerli elife 20209e58123 107554elife58123 of 0cshort reportfigure continueddevelopmental biologyn are shown note that the logarithmic scale on the yaxis of the histogram on the left is different from the linear scale of the central and middlepanels b chip followed by qpcr in hek293t cells treated with dmso wntoff or chir wnton enrichment was identified on axin2promoter and the downstream enhancer note that the enrichment on the enhancer is only present upon pathway stimulation we interpret this asevidence for the enhancer looping onto the promoter occurring when the wntdependent transcriptional regulation is active the data are normalizedto immunoprecipitation performed in cells transfected with an empty vector ev and presented as the mean ± standard deviation of independentexperiments the fold enrichment of tbx3flag on axin2 promoter and enhancer n is lost upon mutations in bcl99l db99l n cnttb1dbcat n and tcflef d4tcf n c schematic representation of the axin2 locus indicates the position of the primers used black arrowsto test the binding of tbx3 despite the apparent absence of direct physical interaction between tbx3 and bcl99l the data support a model of tbx3recruitment by bcl99l onto the bcatenintcf transcriptional complex d schematic diagram of the human crc zebrafish xenografts model parentaland tbx3 overexpressing hct116 colorectal tumor cells were harvested and labeled with dii dye red the stained cells were injected into theperivitelline space of day old zebrafish embryos zebrafish were visualized with fluorescent microscopy at day post injection dpi and three dpi andprimary tumor cell invasion and metastasis were counted e representative images of hct116 tumor invasion and dissemination at and dpi inzebrafish xenografts for both control and tbx3 overexpressing cells the red asterisks indicate the position of the primary tumor red arrowheadspoint at clusters of disseminatingmetastatic cells f scatter plot representing the quantification of primary tumor growth and metastasis after hct116xenograft horizontal bars represent the mean value only significant pvalues p005 are displayed g quantitative rtpcr confirmed continuedincreased expression of tbx3 while hct116 disseminate through zebrafish tissue and that this is accompanied by enhanced wntbcatenintranscription as seen by axin2 expression each datapoint represents the extraction of total rna from pools of zebrafish embryos figure legendof figure supplementsthe online version of this includes the following figure supplements for figure figure supplement axin2 and nkd1 are here considered as representative wnt transcriptional targetsmaterials and methodstreatment of mice and histological analyseshomeostasis weekold male and female mice bcl9floxfloxbcl9lfloxflox vilcreert2 and bcl9floxfloxbcl9lfloxflox no cre littermates pygo1floxfloxpygo2floxflox vilcreert2 and bcl9floxfloxbcl9lfloxflox no cre were treated with five tamoxifen injections ip mgday sigma for five consecutivedays and the small intestine and colon were analyzed at different time points thereafter mouseexperiments were performed in accordance with swiss guidelines and approved by the veterinarianoffice of vaud switzerlandinduction of dss colitis weekold male mice were treated with five tamoxifen injections ip mgday for five consecutive days days later dss mg mp biomedicals catno was administered ad libitum in the drinking water for daysinduction of tumors weekold female mice were treated with five tamoxifen injections ip mgday for five consecutive days days later they were injected ip with mgkg body weightdmh 2hcl nn dimethylhydrazine dihydrochloride after another days later dss was administered ad libitum in the drinking water for daysmice were monitored clinically for rectal bleeding prolapse and general signs of morbidityincluding hunched posture apathetic behavior and failure to groomthe relative body weight in was calculated as follows x weight at a certain dayweight atthe first day of dss treatment epithelial damage of dss treated mice was defined as percentage ofdistal colon devoid of epitheliumto determine proliferation rates mice were injected ip with mgkg brdu sigma hr priorto sacrifice small intestines and colons divided into three equal segments to be named proximalmiddle and distal colon were dissected flushed with cold pbs cut open longitudinally and fixed in paraformaldehyde for hr at rt and paraffin embedded sections mm were cut and used forhematoxylineosin and alcian blue staining and for immunohistochemistry the primary antibodiesused were rabbit antisynptophysin dako rabbit antilysozyme dako mouse antiki67 novocastra mouse antibrdu sigma antibcatenin bd pharmigenantiactive caspase cell signalingthe peroxidaseconjugated secondary antibodies used were mouse or rabbit envision dakoor mouse antirat hrp biosourcezimmerli elife 20209e58123 107554elife58123 of 0cshort reportdevelopmental biologyrealtime pcr genotypingto determine the deletion rate the intestinal epithelium was separated from the underlining musclethe intestine was dissected flushed with pbs cut open longitudinally and incubated in mm ethylenediamine tetraacetic acid edta and mm dithiothreitol dtt in pbs for hr at rt on arotor the tubes were shaken vigorously the muscle removed and the epithelium centrifuged andused for genomic dna extraction sybr green realtime pcr assays were performed on each sample analyzedchromatin immunoprecipitationforelimb buds were manually dissected from ca rjorlswiss outbred dpc mouse embryoschromatin immunoprecipitation was performed as previously described cantu brieflythe tissue was dissociated to a single cell suspension with collagenase 1mgml in pbs for hr at Ëc washed and crosslinked in ml pbs for min with the addition of mm ethylene glycolbissuccinimidyl succinatethermo scientific waltham ma usa for proteinprotein crosslinkingsalazar and formaldehyde for the last min of incubation to preserve dnaprotein interactions the reaction was blocked with glycine and the cells were subsequently lysed in ml hepes buffer sds tritonx m nacl mm edta mm egta mmhepes chromatin was sheared using covaris s2 covaris woburn ma usa for min with the following set up duty cycle max intensity max cyclesburst max mode power tracking the sonicated chromatin was diluted to sds and incubated overnight at Ëc with mg of antibcl9abcam ab37305 or antitbx3 santacruz sc17871 or igg and ml of protein ag magneticbeads upstate the beads were washed at Ëc with wash buffer sds deoxycholate triton x100 m nacl mm edta mm egta mm hepes wash buffer sds sodium deoxycholate triton x100 m nacl mm edta mm egta mmhepes wash buffer m licl sodium deoxycholate np40 mm edta mmegta mm hepes and finally twice with tris edta buffer the chromatin was eluted with sds m nahco3 decrosslinked by incubation at Ëc for hr with mm nacl extractedwith phenolchloroform and ethanol precipitated the immunoprecipitated dna was used as inputmaterial for dna deep sequencing the pull downs | Colon_Cancer 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"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 |
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 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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 |
although autophagy plays a dual role in suppressing or promoting certain cancers the nature of its involvement in breast cancers remainsunclear here we investigated the function of stxbp6 a protein regulating theautophagyassociated snare complex in triple negative breast cancer tnbcresults we report that stxbp6 is profoundly downregulated in tnbc specimens in association with reduced overall patient survival notably we foundthat stxbp6 promoter was specifically hypermethylated in tnbc specimensectopic expression of stxbp6 inhibited tnbc cell proliferation in cellularand mouse models mass spectrometric analysis revealed physical interactionsof stxbp6 with a number of autophagyrelated proteins including snx27 amolecule involved in endocytosis of plasma membrane receptors and proteintrafficking overexpression of stxbp6 elicited autophagy through inhibitionof mtorc1 signaling reciprocally induction of autophagy rescued stxbp6expression by inhibiting ezh2 and altering stxbp6 methylation the mutualregulation between stxbp6 and autophagy was replicated in luminal breast cancer cells only when estrogen receptor er activation was abrogated ectopicexpression of stxbp6 significantly reduced tnbc cells migratory ability in vitroand tumor metastasis in vivos our results unveil a role of stxbp6 in tnbc that highlights anew paradigm in autophagy regulation our results significantly enhance theunderstanding of the mechanisms of tnbc aggressiveness which might help indesigning novel therapies targeting tnbcthis is an open access under the terms of the creative commons attribution license which permits use distribution and reproduction in any medium provided theoriginal work is properly cited the authors clinical and translational medicine published by john wiley sons australia ltd on behalf of shanghai institute of clinical bioinformaticsclin transl med 202010e147101002ctm2147wileyonlinelibrarycom ctm2 of 0ck e y wo r d sautophagy metastasis stxbp6 triple negative breast cancer tumor suppressor genelenka of tumor biology section researchdivision sidra medicine doha qatarcorrespondencedr lotfi chouchane genetic intelligencelaboratory weill cornell medicineqatarqatar foundation po box dohaqataremail loc2008medcornelledugovinda lenka and jingxuan shan contributed equally to this workfunding informationbmrp funding of weill cornell medicineqatar qatar national research fundgrantaward number nprp94593090 the biological and pathological diversity of breastcancer results in differences in prognosis and responsiveness to therapy and limited progress in reducing itssocietal impacts1 the heterogeneous nature of breast cancers is underlined by the variability of mutational patternresulting in differential expression of genes controllingcell growth and malignant potential23 triple negativebreast cancer tnbc accounts for approximately of all breast cancers despite intensive treatments usuallyassociating surgery radiation therapy and chemotherapytnbc tumors relapse early and often develop visceralmetastases hence tnbc is associated to the poorestprognosis among all breast cancers tnbc is biologicallydistinct from other subtypes due to the absence of estrogenreceptor progesterone receptor and her2 overexpression owing to a current lack of targeted therapies suchas hormone therapy or antiher2 therapy for tnbcnonselective chemotherapy remains the cornerstone oftreatment4 and there is an unmet need for novel targetedtherapies to improve the outcome for patients with tnbcamong the pathways essential to tumor growth theupregulation of the mtor pathway plays a pivotal rolein tnbc58 loss of pten homolog resulting in activation of the pi3kptenmtor pathway has been reportedin of metastatic tnbcs911 thus making mtorinhibitors also a potential target for treating tnbc in addition to cell cycle arrest growth retardationand reduced angiogenesis the cellular events mediatedby the inhibition of mtor activity include the activation of autophagy which is a natural process comprising orderly that is a tightly controlled process comprising lysosomal degradation and recycling of cellular proteins and anelles degradation and recycling of cellcomponents12 although decreased autophagy appears tobe a common hallmark of tumor cells autophagy has aparadoxical dual role of cytoprotection and cytotoxicitydepending on multiple factors including cancer type andstage tumor microenvironment and genetic context1314on one hand autophagy can maintain genome integrityprevent cell injury and inflammation by means of its protein and anelle quality control function as well as byits immunesurveillance capabilities in this capacity itprevents tumor initiation promotion and progressionthereby acting as a tumor suppressive mechanism especially in the early stage of tumorigenesis1516 on the otherhand autophagy can act as a tumor cells protective mechanism and enhances their survival and drug resistanceat the late stage of tumor progression1617 both oncogenes and tumor suppressor genes regulate autophagycommonly mutated oncogenes associated with antiapoptotic proteins and the pi3kaktmtor pathway possessautophagy inhibitory capacity so targeting them usingrapamycin analogues18 and bcl2 inhibitors1920 results inthe induction of autophagy similarly several tumor suppressive proteins such as pten tuberous sclerosic complex and tsc12 and the bh3only proteins induceautophagy while their loss reduces autophagy21 alsobeclin which is essential for autophagy induction acts asa haploinsufficient tumor suppressor protein22 thus farthe role of autophagy in breast cancers remains unclearunveiling the roles of autophagy throughout differentstages of carcinogenesis aids in developing novel therapeutic strategiessnares have been shown to regulate the initial stepsof autophagy including the autophagosome formation2324syntaxinbinding protein stxbp6 was identified asa regulator of the autophagyassociated snare complexformation25 by binding the syntaxin proteins recentlylenka 26 demonstrated a potential tumor suppressive activity for this protein in lung cancer cells however 0clenka of the mechanism underlying antitumor cell growth remainsunclearthe role of stxbp6 in snareregulation andautophagosome formation led us to hypothesize thatconstitutive expression of stxbp6 could influence breastcancer cell behavior through the modulation of autophagywe started with the assessment of stxbp6 expression inbreast cancer tumor specimen and cell lines since weobserved a specific methylationdriven downregulation ofstxbp6 in tnbc we extended our work with a myriadof biochemical and functional analyses to identify apotential mechanism by which stxbp6 may affect tnbcprogressionmaterials and methodsclinical tissue samplessixty pairs of malignant and adjacent nonmalignant tissues from tnbc patients were collected from the laboratory medicine and pathology and surgical departmentsof hamad medical corporation doha qatar all participating patients had primary breast cancer with unilateraltumors and without family history of cancer the diagnosisof cancer was confirmed by histopathologic analyses thestudy was approved by the weill cornell medicineqatarand hamad medical corporation ethics committees allpatients gave their written consent for participation in thestudytissue specimens were immediately placed in rnalaterinvitrogen solution and frozen at ¦c untilfurther use frozen tissues were sectioned for the immunohistochemical analysis dna and rna were extractedfrom tissues using all prep dnarnaprotein minikit qiagen the quantity and quality of dna andrna were measured by nanodrop¢ thermofisherscientificvirus pscell lines antibodies reagents andbreast cancer celllines mdamb231 mdamb468mcf7 and zr751 and nontumoral cell line hek293twere obtained from american type collection centreatcc preadipocyte cell line paz6 was kindly giftedby dr d strosberg27 all above cells were cultured indmemf12 medium gibco supplemented with fbs in a humidified incubator with co2antistxbp6 antibody hpa003552 was purchasedfrom milliporesigma antisnx27 antibody ab77799 waspurchased from abcam others were purchased fromcell signaling technology all chemicals were procuredfrom milliporesigma unless otherwise specified lentiviral orf ps of stxbp6 rc209598l3v and snx27rc218477l4v were purchased from origene technologies shrna lentiviral ps targeting stxbp6 sc61968v and snx27 sc88812v were purchased fromsanta cruz technologyquantitative pcrtotal rna was isolated with trizol reagent invitrogenand then was reversetranscribed to cdna with oligo 18tprimer gene expression were measured by quantitativepcr qpcr using the hypoxanthine phosphoribosyltransferase hprt1 gene as a reference all qpcr assayswere performed using sybr greenbased gotaq 2steprtpcr system kit promega on a quantstudio flexrealtime pcr system applied biosystems the primersequences are listed in table s3immunohistochemistryformalinfixed paraffinembedded ffpe tissue sectionswere cut into microns thick and were deparaffinized andrehydrated through three graded concentrations of alcohol endogenous peroxidase activity was quenched with h2o2 for min antigen was retrieved in mmph sodium citrate buffer for min at ¦c followed by min cooling at room temperature nonspecific binding was blocked with bsa for h sections were incubated with antistxbp6 overnightat ¦c and then with hrpconjugated secondary antibodies for h at room temperature hrp activity wasdetected by dab dako chromogen in between stepssections were washed three times with pbst each for min sections were counterstained with mayers hematoxylin and then dehydrated and mounted images weretaken by a bright field microscope carl zeiss sectionincubated with nonimmune serum acted as a negativecontrol western blotcells lysates were prepared in ripa buffer diluted inà sds sample buffer and heated for min at ¦cthe total proteins were separated on sdspagegel and transferred to a pvdf membrane membranewas blocked with milk for h at room temperatureand incubated with primary antibody overnight at ¦cfollowed by hrpconjugated secondary antibody for h 0c of lenka at room temperature in between steps membrane waswashed three times in tbst for min each the hrpactivity was detected by immobilon rcid2 crescendo millipore and images were acquired by chemidoc¢ mpbioradimmunofluorescence stainingmdamb231 cells expressing stxbp6 and its corresponding control cells were grown on culture slides bd falcon to identify stxbp6mediated autophagy inductionat h post treatment with nm everolimus cellswere fixed with paraformaldehyde for min atroom temperature and permeabilized in icecold methanolfor minutes cells were then blocked in normalhorse serum for h at room temperature and probedwith rabbit antilc3 and mouse antistxbp6 antibodies overnight at ¦c cells were incubated with antimouse iggalexafluor488 green and antirabbit iggalexafluor594 red secondary antibodies for h atroom temperature and then loaded with dapi for nuclearstaining the coverslips with the attached cells were finallymounted on glass slides with fluorescent mounting mediavector laboratories the fluorescence images were captured using a lsm710 confocal laser scanning microscopecarl zeisspyrosequencing assaybisulfite treatment of genomic dna was carried out usingan epitect fast bisulfite conversion kit qiagen followingthe manufacturers instructions then a predesigned pyromark cpg assay pm00057414 qiagen was used for quantification of cpg methylation of stxbp6 gene according tothe manufacturers instructionmethylationspecific pcrtotal cellular dna from autophagy induced tnbc cellsand their corresponding controls were isolated and purified using dneasy blood and tissue kit qiagen thedna was then subjected to bisulfite conversion usingan epitect fast bisulfite conversion kit with a programof cycles for s at ¦c and h at ¦c the converted dna was subjected to a methylationspecific pcrreaction using both unmethylated primer pairs for utgagtatgtttagaggtggtt and rev u aacttaaccaacccaaatac and methylated primer pairsfor m gagtatgtttagaggcggtc and revmacttaaccgacccgaatac cell proliferation assaycells in 96well plated were incubated with growthmedium containing mgml mtt for h at ¦c followed by incubation with μl dimethyl sulfoxide agitated on an orbital shaker for min covered with tinfoil absorbance was recorded at nm with clariostarbmg labtechcolony formation assayone thousand cells were seeded in a well of sixwell platesand cultured for weeks to form colonies then cellswere fixed with methanolacetic acid and stained with crystal violet finally the images of dried plates wereacquired with a digital cameracell migration assaycells were seeded and cultured to create a confluent monolayer in sixwell plate the cell monolayer was scratchedwith a μl pipet to produce a line washed one timewith growth medium to remove the debris and then incubated in growth medium to acquire the first image of thescratch using a phasecontrast microscope the cells werecultured in normal condition and the scratch was imagedevery hcoimmunoprecipitationcells were lysed using immunoprecipitation ip lysisbufferthermofisher scientific and centrifuged at à g for min at ¦c for each ip cell lysatescontaining μg total protein were first preclearedwith μl agarose slurry for h at ¦c on a rotatorprecleared lysates were mixed with μg antimyc for h at room temperature and then incubated with preblocked agarose slurry overnight at ¦c on a rotator beadswith ip product were washed five times with pbst andthree times with pbs resuspended in μl 2x laemmlisample buffer thermofisher scientific and boiled for min the supernatant was subjected to western blotanalysisgellcmsmsproteome analysis usingip samples were subjected to proteomics analysis according to ingel digestion method of rosenfeld 28 the ip 0clenka of samples were run in a 1dgel nupage bistris gel10well mm for approximately min until a clear single band was visible the band of each sample was thencarefully excised and cut into smaller pieces between and mm2 gel were transferred to clean tubes and coveredwith dithiothreitol and iodoacetamide buffers for reduction and alkylation of the proteins respectively the gelpieces were then covered with sufficient trypsin solutionfor overnight digestion into peptides at ¦c the peptideswere extracted from the gel pieces using various concentrations of acetonitrile and ethanol mixed with ammoniumbicarbonate buffer and dried down in a speedvac thepeptides were then purified using purespeed tips drieddown in the speedvac and acidified using formicacid to run in the qexactive hf thermo fisher scientific the mixed peptides were then subjected to nanoliquid chromatography lc coupled with mass spectrometry the analytical platform consisting of an easy nlc interfaced to a qexactive hf chromatography conditions were defined as follows a water with aceticacid b wateracetonitrile with acetic acidc nlmin flow rate μl injection volume anda maximal loading pressure of bars lcseparationwas run on cm long inhouse packed emitter columnsreprosilpur c18aq μm diameter beads kindlyprovided by dr maisch gmbh for the first trial sampleswere run using a gradient ranging from to mobilephase b over min followed by a 10min wash andcolumn reequilibration cycle data dependent mass spectrometric acquisition employed a top method brieflythe most intense ions excluding unassignedchargestates and singly charged ions detected in the preceding full scan were isolated th isolation width andfragmented using higher energy collisional dissociationhcd normalized collision energy precursor scanswere acquired at a resolution of scan range of mz and an agc target value of chargesmaximum ion injection time ms msms fragmentation spectra were acquired at a resolution of and anagc target value of charges maximum ion injection time ms with a fixed lower masstocharge cutoff of all scan events were recorded in profile modea dynamic exclusion list of 90s was employed and bothexclude isotopes and peptide match functionalities wereactivatedms data analysisraw ms data were processed using genedata expressionist software v1301 consisting of two modulesrefiner ms data preprocessing and analyst data postprocessing and statistical analysis briefly after noisereduction and normalization lcms peaks were detectedand their properties calculated mz and rt boundariesmz and rt center values intensity individual peakswere grouped into clusters and msms data associatedwith these clusters were annotated with msms ionssearch mascot using peptide tolerance ppmmsms tolerance da maximum missed cleavages and database uniprot swissprot taxonomyhomo sapiens results were validated by applying a threshold of corrected normalized false discovery rate fdrdue to the low complexity peptides with individual ionsscores only indicating identity or extensive homology p were also taken into account protein interference was done based on peptide and protein annotations redundant proteins were ignored according to theoccams razor principle and at least one peptide wasrequired for a positive protein identification protein intensity ranks for table s1 were computed using the hi3method29docking analysishomology models of the nterminal sec3pip2 domainfrom human stxbp6 and pdz domain from sortingnexin27 snx27 were prepared and evaluated withswissmodel30 using 3hle and 4z8j respectively astemplates while the crystal structure of 78kda glucoseregulated protein gpr78 was fetched from the proteindata bank pdb under the accession number 3iuc subsequently the proteins coordinates were docked usingcluspro implementing the mass spectrometry peptide sequences of snx27 and gpr78 as attraction restrainstop best poses corresponding to highly populated clusters from the four different contributions of the docking algorithm balanced electrostatic hydrophobic andvan der waals electrostatic were downloaded andthe solvationfree energy gain δig and interface areawere calculated with pisa3233 finally the intermolecular interactions were analyzed with scorpion34 and thefigures were rendered with pymol the pymol molecular graphics system version schrödinger llcthe primary sequences used for homology modelingare as followssec3pip2 from stxbp6qgeyltyiclsvtnkkptqasitkvkqfegstsfvrrsqwmleqlrqvngidpngdsaefdllfenafdqwvastasekctffqilhhtcqrypdz domaingprvvrivksesgygfnvrgqvseggqlrsingelyaplqhvsavlpggaadragvrkgdrilevnhvnvegathkqvvdliragekeliltvlsv 0c of xenograft mouse modelsthe orthotopic models à seven to weeks old female severe combined immunodeficiency scid mice weighting from to g wereobtained from shanghai slac laboratory animal co ltdand housed in the pharmalegacy laboratories vivariumthe adaptation to the environment for the mice was noless than days twelve mice were randomly assigned totwo groups for each experiment according to their bodyweight the procedures that were applied in this studyhave been approved by pharmalegacy laboratories institutional animal care and use committee all the includedmice were euthanized at their scheduled study terminationforstxbp6overexpressing mdamb2 cells stxbp6 knockdownmdamb231 cells or their corresponding mock cellsin pbs were injected into the mammary fat pad ofeach mouse and the tumor volume and mouse bodyweight were measured every days once palpabletumors appeared after weeks to evaluate the effectof stxbp6overexpression on tumor growth the tumorswere collected to evaluate effect of autophagy activityon xenograft tumor growth mgkg everolimus in pbswas injected intratumorally at 4day intervals for another weeks finally the xenograft tumors were excisedweight and examined by ihcfor metastasis models approximately à luciferaselabeled control mock and stxbp6lenti mdamb231cells suspended in pbs were injected via intracardiacroute35 to each mouse under anesthesia by isofluranethe distribution and mean fluorescence index mfi of thetumor were measured by the ivis in vivo imaging systemperkinelmer once a week for weekstcga expression analysiswe downloaded the tcga breast cancer rna sequencingexpression data portalgdccancergov using thegdc dtt desktop tool on february we identified the count data files htseqcount files and then filteredand scaled the count data using tmm normalization withedger the filtered and scaled expression datasetcontained genes the expression dataset was thenfurther analyzed in matlab with custom scripts to identifythe tnbc subset in the tcga dataset we used the tnbcsamples described in jiang 37 that resulted in identifying tnbc samples containing cancer samples andeight normal solid tissue we calculated the gene expression profiles of autophagy genes in tnbc and nontnbcsamples using the matlab 2019a boxplot function using thedefault settings we calculated the kendall tau rank corlenka relation between stxbp6 expression and other autophagygene expression using the corr function in matlab 2019awith default settingsstatistical analysisdata were expressed as the means ± sds from at leastbiological triplicates the statistical significance in different samples was analyzed by a ttest time course difference between two groups were analyzed using twowayanova all statistical tests were twosided pvalues were considered significant statistical analyses were performed using graphpad prism resultscorrelation of overall breast cancerpatient survival with stxbp6 expressionwe first examined the expression of stxbp6 at mrnaand protein levels in pairs of tnbc tissues and theircorresponding nontumor breast tissues we observed significant downregulation of stxbp6 mrna in the tumortissues relative to its adjacent nontumor breast tissuesp figure 1a we further showed the downregulation of stxbp6 expression at a protein level byimmunohistochemistry ihc in the same tumor tissuesstxbp6staining intensity in tumors compared with nontumor samples was found to be significant p figure 1b next we analyzed breast cancer patient survivalusing a kaplanmeier plotter wwwkmplotcombreastref as shown in figure 1c high expression of stxbp6was associated with an increased overall survival osin all breast cancer patients particularly in tnbcbasallike breast cancer patients we also analyzed patient survival using tcga datasets39 similarly high expression ofstxbp6 was associated with an increased overall survivalin tcga bc cohort figure s1 taken together these findings suggest a role for stxbp6 in tnbc biology and thepotential prognostic value of stxbp6 to predict tnbc disease progressioncontrol of stxbp6 expression viapromoter methylation in tnbc cellsto determine the mechanisms underlying the repressionof stxbp6 expression in breast cancer cells we first examined the endogenous expression of stxbp6 in tnbc subtype mdamb231 and mdamb468 luminal subtypemcf7 and zr751 and nonmalignant cells hek293t 0clenka of f i g u r e clinical analysis of stxbp6 in breast cancer a stxbp6 mrna levels in triple negative breast cancer tnbc tumor specimens and their corresponding nonmalignant breast tissues relative stxbp6 expression was calculated by δct the expression differencesbetween two groups were analyzed by a ttest p b the representative images of immunohistochemical staining of stxbp6 intwo tnbc specimens s1 and s2 and their corresponding nonmalignant breast tissues magnification à nonmalignant breast tissueswere also used as negative control in which the tissues were incubated without primary antibody left the mean values of the ihc quantification in arbitrary units au are shown in right p indicate the significant difference in stxbp6 immunoreactivity between tumor andnontumor breast tissues which was analyzed by a ttest n the number of patients included in the study error bars represent the standarddeviation of replicates c overall survival according to the stxbp6 expression levels high vs low using the median as cutoff in all typesof breast cancer patients bc and basallike breast cancer patients bl of kaplanmeier plotter dataset the differences between groups werecalculated by the logrank test represents each censored sampleand preadipocyte cell line paz6 in agreement withthe clinical specimen results stxbp6 was downregulatedin all breast cancer cells included in this study figure 2a notably the stxbp6 in tnbc cells is repressedmore than in luminal breast cancer cells in cancer thedownregulation of gene expression often ascribes to itspromoter methylation h3k27ac mark represents a hotspot where dna methylation often occurs40 accordinglystrong h3k27ac enrichment was found on the stxbp6 promoter region based on ucsc genome browser data figures2 indicating that stxbp6 expression might be silenceddue to promoter region methylation in cancer cells weverified the effect of methylation on stxbp6 expressionby treating breast cancer cells with the dna methyltransferase dnmt inhibitor 5aza2²deoxycytidine 5azastxbp6 expression was significantly upregulated only intnbc cells in a dosedependent manner p butnot in luminal subtype breast cancer cells figure 2bfurther pyrosequencing analysis revealed the stxbp6specific cpg island methylation a total of five cpg sitesincluding chr14 and in the ² untranslated region utr ofstxbp6 were examined this analysis revealed the highermethylation percentage of four of the five cpg sites inall tnbc tissues compared to luminal breast cancer typep and to nontumor breast tissue samples p included in this study figure 2c figure s3 in addition in silico methylation analysis using public data setsshowed also that the promoter of stxbp6 was hypermethylated in tnbc figure 2d together all the aboveresults strongly indicate that epigenetic control methylation of stxbp6 expression might be specific to tnbccellsproliferation by stxbp6inhibition of tumor cellthe clinical data analysis of stxbp6 indicated an antitumor role of stxbp6 to investigate the role of stxbp6in tnbc we developed overexpressing stxbp6 lentistxpb6 tnbc cells by infecting mdamb231 and 0c of lenka f i g u r e methylation analysis of stxbp6 in breast cancer a upper western blotting of endogenous stxbp6 using antistxbp6 antibodies in luminal mcf7 and zr751 and triple negative mdamb231 and mdamb468 breast cancer cells and nonmalignant control cellshek293t and the brown preadipocyte cell line paz6 equal loading was assessed by αtubulin lower normalized expression of stxbp6relative to αtubulin in the indicated cells the band intensity was measured by imagej b relative expression of stxbp6 mrna in breast cancer cells treated with and μm of methylation inhibitor 5aza2deoxycytidine 5aza for days relative expression levels of stxbp6 werenormalized against the untreated group of each cell line the expression difference was analyzed by ttest p error bars represent thesd of biological triplicates c quantification of dna methylation of stxbp6 in breast cancer tissues and their adjacent nonmalignant breasttissues each subtype of breast cancer tissue samples was compared to the corresponding nonmalignant tissue samples the methylation percentage difference was analyzed by ttest p d determination of methylation status of stxbp6 in tnbc samples of public datasetsgse78751 and gse78754 histograms of the cpg sites cg10301769 and cg10806811 in the utr of stxbp6 gene βvalue indicateshypermethylationmdamb468 cells with a lentiviral system carrying thestxbp6 gene we validated stxbp6 overexpression at bothmrna and protein levels figure s4 using mtt assay weshowed that stxbp6 overexpression caused a significantreduction in proliferation of both tnbc cells compared tocontrols p ¤ figure 3a and shrnamediated knockdown of stxbp6 had no significant effect on cell proliferation figure s5a we then performed a clonogenic assaywhich showed that stxbp6 overexpression significantlydecreased the rate of colony formation figure 3b conversely knockdown of stxbp6 promoted colony formation in both mdamb231 and mdamb468 cells figures5b to further confirm that stxbp6 inhibits tnbc cellgrowth lentistxbp6 cells and mock cells were tested ina xenograft mouse model we observed that tumors in thelentistxbp6 group were significantly reduced to fold compared to the control group figure 3cdstxbp6interacting proteins in tnbc cellsidentification ofto unveil the molecular mechanism by which stxbp6inhibits tumor cell growth we identified the interactingpartners of stxbp6 we immunoprecipitated all stxbp6interacting proteins in lentistxbp6 mdamb231 cells 0clenka of f i g u r e the effect of stxbp6 overexpression on breast cancer cell growth in vitro and in vivo a proliferation assays of tnbc cellsoverexpressing stxbp6 lentistxpb6 versus empty vector mock cells at a concentration of cells per well were seeded in the 96wellplate and incubated for indicated time the quantity of viable cells was determined by mtt assay the growth difference between lentistxpb6and mock groups was analyzed by twoway anova p b colony formation assays of mdamb231left upper panel and mdamb left down panel tnbc cells overexpressing stxbp6 lentistxpb6 compared with the control mock left the wholewell images ofclonogenic cells right panels the average number of colonies ± sd the difference of colony formation ability between lentistxpb6 and mockgroups was analyzed by ttest p c orthotopic tumor growth curve of stxbp6overexpressing mdamb231 cells lentistxpb6 andcontrol mock mdamb231 cells after palpable tumor appeared the tumor growth between lentistxpb6 and mock groups was comparedby a twoway anova p d left representative images of tumor size right average tumor weight of each group the tumor sizebetween lentistxpb6 and mock groups was compared by a ttest p error bars represent the standard deviation of biological triplicatesa b or six replicates c dand control cells immunoprecipitated proteins were gelexcised protein digested with trypsin and analyzed bynanolc coupled to mass spectrometry ms a completelist of identified binding partners of stxbp6 is providedin table s1 and the msms spectra for peptides matchingto stxbp6 are shown in figure s6 given its biologicalfunction in breast cancer we validated the interactionof snx27 with stxbp6 by coimmunoprecipitation figure 4a furthermore we performed docking of thisinteraction the proposed docking models of stxbp6 incomplex with the pdz domain of snx27 are shown infigure 4b left the interface formed between stxbp6and the pdz domain of snx27 accounts for of the total complex with a solvation free energy δigof kcalmol the interface is mainly composed ofhydrophobic interactions and additionally we counted hydrogen bonds and eight salt bridges three out of four ofthe best docking poses effectively matched the snx27pdzdomain epitope scorpion analysis revealed the presenceof seven hydrogens bond from several amino acids ofpdz domain for instance between the r19 guanidine sidechain and n54 d56 and s57 of stxbp6 similarly furthercontacts were observed from the co moieties of v18 andf16 with g55 and g50 moreover other hydrogen bondsare formed by n17 side chain amide group with both l79and g55 and finally between s12 side oh moiety and h87figure 4b right upper additional stabilizing interactionconsisted of cationÏ stacking and ionic contacts betweenguanidine r18 and g55 s57 and d56 of stxbp6 n17and f16 from the pdz domain provided further dipolarcontacts with g50 backbone carbonyl of pdz domainand finally seven van der waals interactions from r19n17 g13 s12 and e11 figure 4b right lower interestingly highest scorpion scores were found on the r19 sidechain of snx27pdz domain through four ionic contactsand one cationÏ stacking and additionally on the r53 0c of lenka f i g u r e stxbp6 interacts with snx27 a datadependent msms fragment spectra of peptide identifying snx27 b coip of stxbp6and snx27 proteins immunoprecipitated from total cell lysates of mdamb231 cells overexpressing stxbp6 lentistxpb6 versus emptyvector mock using an antimyc antibody bands of 61k corresponding to snx27 were identified in the ip product from cells overexpressingstxbp6 or in total lysate of cells but not in the ip product of mock cells c cartoon representation of the docking model of stxbp6 greycartoon in complex with pdz domain of snx27 cyan cartoon amino acidic sequence used for restraining mode in cluspro is colored ingreen protein interactions of stxbp6 green sticks and pdz domain of snx27 orange sticks and grey surface generated with scorpion consist 0clenka of guanidine group which interacts with the comoiety ofq91 of stxbp6 through t | Colon_Cancer |
"circadian clocks have important physiological and behavioral functions in humans and other anisms whichenable anisms to anticipate and respond to periodic environmental changes disturbances in circadian rhythmsimpair sleep metabolism and behavior people with jet lag night workers and shift workers are vulnerable tocircadian misalignment in addition non24h cycles influence circadian rhythms and cause misalignment anddisorders in different species since these periods are beyond the entrainment ranges in certain special conditionseg on submarines and commercial ships non24h watch schedules are often employed which have also beendemonstrated to be deleterious to circadian rhythms personnel working under such conditions suffer fromcircadian misalignment with their onwatch hours leading to increased health risks and decreased cognitiveperformance in this review we summarize the research progress and knowledge concerning circadian rhythms onsubmarines and other environments in which non24h watch schedules are employedkeywords circadian rhythm circadian clock entrainment range metabolism alertness submarine most anisms living on this planet possess circadianclocks that orchestrate their daily physiological and behavioral rhythms to the 24h rotation period of the earth however under special conditions in which thecycling periods do not extend over h the circadiansystems may be challenged in the laboratory manualnon24h conditions are established to study the basicfeatures of circadian rhythms although such conditionsare very rare in nature however in human societies asubstantial group of people lives or works under non24h schedules the non24h conditions were observedto lead to a decrease in adaptability to the environment correspondence guojinhumailsysueducn1key laboratory of gene engineering of the ministry of education state keylaboratory of biocontrol school of life sciences sun yatsen universityguangzhou chinafull list of author information is available at the end of the in all tested anisms across different kingdomscluding bacteria fungi plants and animalsinthe circadian clocks are well conserved across speciesthus we will first introduce the knowledge derived fromcircadian studies in model anisms and the next focuson summarizing studies in humans the former partwould help to elucidate circadian misalignment in circadian rhythms metabolism sleep and the cognitive andbehavioral consequencescircadian clock and circadian rhythmscircadian clocks govern life processes at multiple layersincluding the molecular cellular anism and integrallevels at the anism level circadian clocks are hierarchically composed of master clocks located in thepacemakers and peripheral clocks located in other tissues in mammals the pacemaker is the suprachiasmaticnucleus scn in the anterior of the hypothalamus inbirds and reptiles the eyes pineal gland or scn are 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 0cguo military medical research page of range circadian rhythms can be adjusted to match theenvironmental periods a process that is termed entrainment or synchronization circadian rhythms are both robust and flexible within a certain range tcycles thatdeviate from the natural 24h cycles can also inducerhythms with the same periodicity as the environmentin this scenario the endogenous circadian periodicitymay be masked by the non24h tcycles the factorsthat can entrain circadian rhythms are known as zeitgebers synchronizers or time givers [ ]to experimentally determine the entrainment rangesanisms are exposed to a series of fixed tcycles radually changing tcycles in addition the entrainment range to an environmental cue can be inferredby the phaseresponse curve prc information of thespecies given that according to the prc of a speciesthe maximum phase delay and maximum phase advanceare x hours and y hours respectively the entrainmentrange of this species to the indicated cue would rangefrom h y to h x hours prc representsthe nonparametric effects of zeitgebers on entrainmenthowever parametric components are also involved indetermining the entrainment range the parametricmodel assumes that light changes the velocity of the circadian rhythms for entrainment while the nonparametric model emphasizes the phase shift theentrainmentranges differ between specieshumans have a circadian clock with an endogenousperiod Ï of approximately h [ ] with upper andlower limits of and h respectively in conjack bean canavalia ensiformis displayed leaftrastmovement rhythmicity synchronized to lightdark ld but not ld suggesting that the lower entrainment limit is between and h fig 2a somespecies such as bakery mold neurospora crassa andhouse sparrow passer domesticus show very wide entrainment ranges in banding rhythms of conidia releaseconidiation fig 2b and locomotor rhythms respectively [ ]circadian rhythms run free when the t cycles are beyond the entrainment ranges under ld squirrelmonkeyscore bodytemperature cbt rhythm with a period of hsaimiri displaycebidaeacircadian pacemakers depending on the species []the peripheral clocks have their own circadian rhythmsbutthey can also be synchronized by pacemakersthrough the nervous and endocrine pathways at the molecular level circadian rhythms are generated by a set of core circadian genes which constitutetranscriptiontranslational negative feedback loops despite differences in circadian clock genes the regulatory mechanisms are highly conserved across kingdoms[ ] in mammals including humans positive circadianelements include brain and muscle arntlike protein bmal12 and clock and the negative elements include period per and cryptochromecry reverbs α and and retinoic acidreceptor rarrelated orphan receptor ror α and Î are additional regulators in mammalian circadiancircuits which act as ancillary loops to regulate bmail1expression reverb proteins inhibit while ror proteins activate the transcription of bmal1 [ ] at thetranscriptomic level circadian clocks regulate the expression of approximately of proteinencodinggenes in mice [ ] via the output pathways circadianclocks control most of the physiological and behavioralprocesses in mammals circadian clocks govern thecircadiandiurnal rhythms in sleepwake cycles feedingfasting control metabolism hormone secretion and immunity function fig although circadian rhythms are endogenous they areto the influence of environmental geneticsubjectphysiological and pathological factors fig [ ] ithas been demonstrated that disruptions in circadianrhythms impair adaptability in a variety of anisms in humans compromised circadian rhythms causechronic health consequences including an increased riskof gastrointestinal illness loss of bone mineral densitycoronary artery disease endocrine disruption metabolicsyndrome and cancer moreover disrupted circadianrhythms also affect emotions cognition and performance fig []entrainment range of circadian rhythmsthe cycling periods of the environment such as lightand temperature are defined as tcycles in a certainfig circadian rhythm disturbance and their consequences 0cguo military medical research page of fig circadian rhythms of different anisms in non24h conditions a leaf movement alternatively opening and closing rhythms ofcanavalia ensiformis c ensiformis b conidiation banding rhythms of n crassa neurospora is inoculated in a long and hollow glass tube withmedium inside which is called a race tube in the race tube neurospora grows towards the other end and releases conidiation bands coloredorange the release of conidiation is controlled by the circadian clock only the left part of the race tubes is shown c rectal temperature rhythmsof c saimiri neurospora conidiation rhythms are entrained in ld ld and ld while canavalia leaf movement rhythms andsubmarine crew rectal temperature rhythms were not entrained in ld therefore they have different entrainment ranges panel a was drawnaccording to the study by panel b was drawn according to the study by and panel c was drawn according to the study by instead of h suggesting the occurrence of a nearlyfreerunning cbt rhythm fig 2c an approachhas been proposed to measure the freerunning periodbased on the displayed periods under non24h cyclesbeyond the entrainment ranges [ ] importantly therhythms beyond the entrainment ranges should be calledsuperposition or superimposition [ ] superposition may result in a period different from the one measured in a constant condition despite the different entrainment ranges differentanisms exhibit rhythms with endogenous periodsafter transfer to constant conditions suggestingthe endogenous characteristics of the sustainability ofcircadian rhythms in a study the pregnant mice werekept in ld or ld and their progenies werealso raised in these tcycles until they reached puberty the progeniesshowed freerunning periodssoon after they were placed in constant conditions the sole recorded exception was that hydrodictyon maintains a period of h in the constantdark for at least days after having been retrievedfrom the ld condition different physiological and behavioral rhythms mayalso have different entrainment ranges within one species or even within individuals suggesting the existence of independent oscillators in addition to affectingthe pacemaker of the circadian clock zeitgebers alsobypass it directly impacting the overt rhythms whichcausesinternal desynchronization between differentphysiological processes as a consequence when t cyclesare out of the entrainment ranges eg the sleepwakecycles and the cbtthe molecular mechanisms determining the entrainment range have not been elucidated the only clue regarding this mechanism was that in mice clock mutantsexhibit a wider entrainment range ld to ld compared to wildtype mice suggesting that circadianclock genes play critical roles in regulating entrainment mutants with less robust rhythms may be moreprone to environmental entrainment although such passive rhythms have no anticipating property unlike circadian rhythmscompromised adaptive fitness in non24h cyclingconditionsthe circadian clocks derive from the past longterm evolution which confers the fitness of the cycling environment and it is plausible that lives on earth cannotadapt to non24h cycling conditions in a short periodof time two strategies were employed to test this hypothesis one strategy is to study the effects of abnormalcycles of environmental cues on anisms and theother strategy is to compare the competitiveness of mutants with different endogenous circadian period lengthsor with no circadian rhythmicity the adaptive fitness ofa number of model anisms has been investigatedconcerning the circadian clock for the environmenttable all the available evidence suggests that living undercircumstances with cycling periods beyond the entrainment ranges is harmful to reproductive fitness cyanobacteriaendogenouscircadian periods were mixed and after a month in culturethe strain with a period resonating with thepossessingstrainsdifferent 0cguo military medical research table selected studies changes in circadian rhythms and adaptation of model anisms under non24h conditionsspeciescyanobacteria synechococcus elongates ld ld adaptabilitydecreased growth competence and adaptabilitynon24h periods hpage of references[ ]bakery mold neurospora crassatemperature cycles t12conidiation rhythms ran freeld ld ld conidiation rhythms were entrainedld ld ld conidiation rhythms were entrainedtomatold ld significantly decreased growththale cress arabidopsis thalianald ld decreased leaf chlorophyll content decreased photosyntheticcarbon fixation decreased vegetative growth and survivalld ld the mutants showed a strong positive correlation in competitionunder the tcycle growth conditionsjack bean canavalia ensiformisld ld ld leaf movement rhythms were entrained under ld ran freeunder ld and ld fruit fly drosophila melanogasterld ld reduced life spanblowfly phormia terraenovaet26 t28decreased life spans under t26 and t28house sparrow passer domesticusld ld ld locomotion rhythms were at least partially entrained orshowed superpositioncommon chaffinch fringilla coelebsld ld ld ld activity rhythms were entrained to ranges from ld to ld mouse mus musculustemperature cycles t20 superposition of luciferase rhythms in lung and scn tissuespartially entrainednile grass rat arvicanthis niloticusrat rattus rattust22feeding cycles t20 t28locomotion rhythms were entrained between t and t decreased food intake and weight gain squirrel monkey cebidae saimiristudies under asymmetric tcycles are not includedl denotes light d denotes dark ld xy denotes cycling conditions with light for x hours and dark for y hours alternatively scn suprachiasmatic nucleuscore body temperature ran free lower amplitudeld ld environment prevailed while the strain with a perioddifferent from the environment failed in the competition [ ] several models have been proposedto explain the clockcontrolled fitness in cyanobacteria including the limited resource model diffusiblefactor model celltocell communication model andthe escapefromlight hypothesis which may alsohelp to characterize the enhanced fitness by the circadian clock in other anisms [ ] the escapefromlight hypothesis postulates that in the early stageof evolutionthe circadian clock acts to segregatelightsensitive reactions temporally to nighttime egcell division and dna damage repair to avoid thedeleterious effects of radiation and oxidation fromsunlight [ ]the growth ofgrowth and flowing time were extensively affected although the results varied in different species a comparison oftomato under different ldconditions demonstrated thattomato grows mostquickly in ld but very slowly in either ld or ld similar experiments have been conducted in a variety of species including house sparrowjack bean canavalia ensiormis n crassa and squirrelmonkey saimiri sciureus which indicate that light regimens with periods that deviated dramatically from hhampered growth or survival [ ] in a previousstudy several plants were tested in a series of ld cyclesranging from ld s5 s to ld for instance soybean soja max limper under ld min1 minshowed a markedly chlorotic phenotype and a notablereduction in size contained numerous spots of dead tissue and showed a tendency to die prematurely ofcourse the day length also contributes to the comprehensive influences on plantsit has been demonstrated that in many anismsnon24h cycles have extensive negative or deleteriouseffects on survival and competitive fitness lesion of thescn in antelope ground squirrels ammospermophilusleucurus led to the loss of circadian rhythmicity in locomotor activity and an increased ratio of loss caused bypredation the tau mutation in the enzyme caseinkinase 1ε ck1ε had a dramatic influence on circadianperiods in mice with the heterozygous mice showing atwohour shorter period while the homozygous miceshowed a fourhour shorter period in the seminaturalenvironment the relative frequency of the tau allele decreased to from the initial half in months due todisadvantages in both adult survival and the recruitmentof juveniles into the cohorts in addition to fitness misalignment of circadianrhythms with non24h cycling conditions impairs manyphysiological and metabolic processes moreover the 0cguo military medical research page of disruption of circadian rhythms leads to abnormalities incognition in animals the circadian control of cognitionand behavior has been demonstrated in many animalspecies disruption of mouse circadian rhythms by ldcycles led to extensive ramifications in metabolism brainfunction and behavior for instance in ld miceandchanges in emotionality which were consistent withchanges in neural architectureincluding shorter dendritic length and reduced complexity of neurons in themedial prefrontal cortex exhibited decreased cognitiveflexibilityin addition non24h cycles shorten life spans in manytested anisms for instance pittendrigh and minisraised drosophila under the conditions of t h andt h and the life spans of drosophila in both conditions were considerably shorter than those lived in t h a hypothesis proposed that when the cycling zeitgeberperiod is close to a demultiplicative period of the freerunning period the oscillator may also be entrainedwhich is known as frequency demultiplication however frequency demultiplication is more likely torepresent a passive response to cycling environmentalstimuli than circadian oscillations [ ]effects of non24h conditions on human healthand performancenon24h regimens affect circadian rhythms and sleepkleitman and richardson were the first persons who recorded their experiences under a 28h condition in acavern of mammoth national park in kentucky usain the constant dark of mammoth cave the sleepwakecycles and body temperature of richardson adapted tothe 28h cycles but kleitman failed in forced t21and t27 schedules simulated in spitzbergen the bodytemperatures in of subjects adapted immediatelywhile the excretory rhythms adapted in only three subjects suggesting the occurrence of desynchronization[ ] even periods with a slight difference from hcan be harmful to the growth or fitness of an individualfor instance a study of the sleepwake rhythms andsleep time in mars lander phoenixin which participants lived and worked on a 2465h schedule whichwas in accordance with the daily cycling period of marsrevealed inadaptation in the sleepwake rhythms in someof the subjects and loss of sleep in almost all of the subjects since the thirteenth century the 4h on8h off watchschedule has been employed in maritime crews which isdramatically different from a 24h day in the militarythe watch schedule of the us board vessel crews shiftedin the last century from the 4h on8h off routine tothe 6h on12h off routine to accommodate the threeshift sections [ ] approximately of enlistedmen serving aboard us navy submarines stand watchon the latter schedule in contrast most officers andsome senior enlisted men stand watch on a foursectionrotation ie h on and h off in studies of the circadian rhythms of submarine crewmembers some physiological variables including bodytemperature serum or urine melatonin cortisol andurine catecholamine have been used as hallmarks [] in a 4h on8h off schedule the oral temperatureof the subjects exhibited an 24h period instead of a12h period kelly investigated circadianrhythms of salivary melatonin in crew members during a prolonged voyage on a trident nuclear submarineand found that under 18h duty cycles the endogenousmelatonin rhythm of the crew members showed an average period of hin non24h cycles the circadian parameters including period amplitude and phase are all subject tochange during a prolonged voyage under a 4h on8h off schedule altered circadian rhythms in cbt occurred among the watchers the mostcommonphenomenon was the dampened amplitude one subject even showed a freerunning pattern naitoh found that a 6h on12h off routine duringa nuclear submarine patrol caused a loss of 24hrhythmicity in oral temperature which was due notonly to a decreased circadian amplitude but also to adispersion of the time of the peak a different watchtime in three shift groups who used alternative 4hon8h off regimens induced changes in the phase ofrectalandadrenaline excretion noradrenalinetemperatureexcretionchanges in circadian variables suggest that under non24h schedules circadian rhythms are prone to alteration and desynchronization may occur as a consequence in schaefer analyzed the rhythms ofbody temperature pulse rate and respiration rate of crew members under two different schedules an 18hwatch schedule 6h on12h off and a 24h schedule the results showed that crew members using theformer schedule but not the latter one exhibit superposition of 18h and 24h periods in all tested variables asimilar superposition was also observed in other studies[ ] as the schedule continues the impact oncircadian rhythms becomes more pronounced naitoh revealed that in a longterm voyage the groupsynchronization in oral temperature rhythmicity disappeared after approximately daysearly in it was noticed that sleep insufficiencywas caused by a 6h on12h off schedule in the submarine crew in schaefer measured theparameters of cbt heart rate and respiration rate every h for subjects living under different schedules some ofwhom employed a 6h on12h off the roster other 0cguo military medical research page of subjects employed the normal 24h schedule theformer schedule caused superposition on the rhythmsshowing both 24h and 18h periods sleep insufficiencywas also recorded in these subjects a survey of submarine crew members indicated that over ofthem had medical complaints and most complaintswere about sleeping difficulty however prevalentstudies have demonstrated that under non24h conditions both the quantity and quality of sleep are altered [ ] in real missions the sleep time ofthe submarine crew varies in a wide range extendingfrom h to h which may be influenced by manyenvironmental factors almost all studies indicated that non24h scheduleselicit misalignment with the environmental cycles anddeficiency in sleep quantity due to split sleep patternswhich means that sleep is separated into two or moreparts during a day usually both sleep quantity and sleepquality at night are better than those in the daytime [] in contrast kosmadopoulos reportedthat a split sleep pattern showed no detrimental effectson sleep quality and performance suggesting the necessity of conducting more extensive and systematicanalysesmelatonin regulates sleep in a circadian fashionfor subjects living in an 18h routine or a combinedroutine of h and hthe melatonin levels ofthese subjects showed periods between h and h suggesting thatinfree running occurred tothethiscyclesleepwakeendogenouscontrastphysiological parametersincluding melatonin andtemperature have considerably narrower entrainmentcausedesynchronization in physiology psychology and behavior at the systemic level discrepancy mayrangesin submarines apart from non24h adherence to arotating watch roster other physical and chemicalenvironmental cues in the cabin also influence the physiology and behavior of the crew members these cues include confinement deleterious atmospheres sunlightdeprivation and insufficient lighting hypercarbia noiseelectromagneticlightspectrum and intensity high temperature and humidityfig [ ] the effects of many of thesecues on human circadian rhythms have not been fullyelucidatedradiationradiationionizingmoreover in a real mission jet lag may further contribute to the effects of circadian desynchronizationcondon found that the sleep length of watchkeepers was shorter when they crossed four to five timezones eastbound compared to crossing the time zoneswestboundshowedslower adaptation on eastbound travel this findingmay be explained by the fact that the human freerunning period is h which facilitates westboundadaptation a 3day shift regimen is widely employedin submariners which may cause frequent shiftssuch as social jet lagsubjectiveand thealertnessfig disturbed circadian clock and impaired physiology and behavior in the submarine crew some connections between the components arenot shown 0cguo military medical research page of non24h regimens lead to changes in metabolismthe circadian clock governs metabolism by modulating the expression and function of essential geneswithin severalimportant metabolic pathways in atemporal fashion conversely misalignment in circadian systems disturbs metabolic homeostasis [ ]the homeostasis of metabolism is also tightly associated with sleep [ ] in mice decreased food intake and weight were observed in mice raised underld or ld suggesting that non24h cyclingconditions may influence metabolic homeostasis in an anchoredsleep study subjects were exposed toa schedule that enabled them to sleep for h fixedfrom to and another h unfixed duringthe day the subjects showed altered acrophases inincludingbody temperature and urinary variablespotassium and sodium suggesting thattheirmetabolisms were affectedthe level ofsleep consistentlyinsulin is under the control of thecircadian clock which peaks at nightin humans ma found that saliva insulin dramatically increased late at night and early in the morning in subjects under a schedule of h for work andrest and h forthe insulinlevels and insulin glucose ratios were increased inmice under ld conditions metabolic syndrome is a constellation associated with metabolicriskincluding abdominal obesity abnormal bloodlipid levels hypertension and impaired fasting glucose kang and song reported that submarine crew members had higher risks of metabolicsyndrome and changed levels oflow highdensitylipoprotein cholesterol and impaired fasting glucoseunder a non24h onwatch schedule taken together these data suggest that metabolism may bedisturbed which might be largely attributed to thenon24h schedulesin recent years the involvement of the gut microbiome in animal and human metabolism has beenincreasingly recognized changes in the category andthe abundance of gut and oral microbiota also display a circadian pattern [] changes in alimentary tract microbiota account for alterations in hostmetabolism relative to the fecal microbiomethesalivary microbiome has rarely been studied despiteits higher accessibility the oral microbiome containsbacteria viruses and fungi which associate and formbiofilms environmental changes might convert themto be pathogenic and trigger intestinal diseases orother inflammatory diseases recently ma found that disruption of the composition of theoral microbiome occurred in subjects living under anon24h schedule some bacterial species showedspecific change patterns eg tenericutes and sr1corresponding to sleep deprivation and circadiandesynchronization respectivelynon24h regimens affect mood cognition andperformancefatigue is an important cause of accidents and incidents at sea one requirement of safe submarineoperations is to ensure that sailors work at their highest level of readiness the non24h onwatch schedules cause misalignment in circadian rhythms whichin turnleads to sleep deprivation [ ] impairments in circadian rhythms and sleep have extensivephysiological and cognitive effects including increasedfatigue and changes in mood vigilance and workproductivity figs and it is critical to pay closeattention to the circadian rhythms and performancein submarineas mooreede pointed outthere should be some global concern aboutthehealth and performance of these men since they arethe ones with their fingers directly on the nuclearbutton crewsnaitoh assessed the mood changes in agroup of nuclear submariners by using subjectivequestionnaires of thayers activation mood activity ma and mood happiness mh and revealedthat during a 10week submerged patrol the 24hrhythmicities of mood activity and mood happiness werecontrolthe results from moodperiodscale questionnaires also indicated that compared tothe patrol period significantly more feelings of activity and happiness were observed in the postpatrol period abolished compared to thein another studyscheduletheresults ofin humansthe circadian nadir trough of cbtand alertness is late at night and early in the morning and changes in body temperature reflect the dynamics of metabolism which is also one ofthedeterminants of alertness [ ] under the 4hthe letteron8h offcancellation test and vector testfrom watchkeepers demonstrated in the case of the former thatthe best time is forenoon while in the latter it islate afternoon the slowest performance occurred either at the start of watches beginning immediatelyafter wakening or near in the morning which isthe nadir of alertness under submarine watchroutines both misalignments in circadian rhythmsand decreases in sleep length and quality imposenegative effects on mood cognition and performance[ ] during the existingwatch schedules used in canadian forces were evaluated revealing thatthe cognitive efficacy in thesubmarine crew dramatically decreased to a dangerously low level showed that ma the effects of 0cguo military medical research page of under a 4h on8h off routine the alertness of thesubjects decreased dramatically in the morning aschronic sleep deprivation may occur during non24h cyclesit is unclear whether misaligned circadianrhythms or sleep loss contributed more to the decreased alertnessthe fatigue avoidance scheduling toolfast¢program has been developed to optimize the scheduling and performance of the air force consideration and integration of moresuch asindividual differences might help to improve theprediction accuracy and practicability in not only theair force but also for personnel working under conditions with non24h periodsincluding submarinecrew members factorstheseand most ofto date many studies have documented the impairment of non24h schedules on circadian rhythms andcognitionstudies have beenconducted at the physiologicallevel by contrast themolecular mechanisms governing the effects of disruptedcircadian rhythms have not been elucidated which maythethuslimitphysiologicalofcountermeasuresunderstandingtheofdevelopmentindepthandthechangesin militaryand commercialcountermeasures conclusions and perspectivescircadian disruption profoundly affects health andperformancevesselcrews during a 90day voyage which is the longestvoyage recorded to date disturbance in circadianrhythms was reported in many chinese crew members most of the crew members experienced sleepdisordersflagging dysphoria and faintness somecrew members attempted to overcome these conditions by chewing dry peppers or daubing mentholcamphorate on their temples in maritime missions circadian misalignment causes serious consequences including high personnel turnover per voyage and requirements to train the new entrant howeverthe question remains of how we canovercome circadian misalignment and avoid deleterious consequences the entrainment range needs tobe considered if the environmental period is withinor close to the entrainment rangeit may be usefulto usecircadianrhythms if the environmental period is far beyondthe entrainment range it may be useful to constructa manmade 24hperiod microenvironmentisalso possible to combine both approaches someefforts have been made to lessen the deleterious effects of disrupted circadian rhythms and to improvehealth and performance which primarily involvedcountermeasuresto adjusttheitoptimization of the watch schedules and modifications of the zeitgebersoptimized watch schedules may improve circadianrhythms and performance duplessis compared the impacts of different watch schedules 6hon8h off and a compressed close6 h watch schedule and the wrist activity results showed that sleepduration was longer under the 6h on8h off schedule sleep discontinuity appears to be considerablyless severe on the 6h on12h off watch schedulethan that of the 4h on8h off watch schedule incontrast the 6h on12h off schedule allowed morecontinuous sleep which might be explained bythe fact that t12 is shorter than t18 compared tohuman frp the best approach is to use a 24h roster instead of a non24h roster a 24h schedule wasused durin | Colon_Cancer |
" variability in the health effects of dietary fiber might arise from interindividual differences in the gutmicrobiotas ability to ferment these substrates into beneficial metabolites our understanding of what drives thisindividuality is vastly incomplete and will require an ecological perspective as microbiomes function as complexinterconnected communities here we performed a parallel twoarm exploratory randomized controlled trial in adults with overweight and classi obesity to characterize the effects of longchain complex arabinoxylan n at high supplementation doses female gday male gday on gut microbiota composition and shortchainfatty acid production as compared to microcrystalline cellulose n nonfermentable control and integratedthe findings using an ecological frameworkresults arabinoxylan resulted in a global shift in fecal bacterial community composition reduced αdiversity andthe promotion of specific taxa including operational taxonomic units related to bifidobacterium longum blautiaobeum and prevotella copri arabinoxylan further increased fecal propionate concentrations p friedmanstest an effect that showed two distinct groupings of temporal responses in participants the two groups showeddifferences in compositional shifts of the microbiota p permanova and multiple linear regression mlranalyses revealed that the propionate response was predictable through shifts and to a lesser degree baselinecomposition of the microbiota principal components pcs derived from community data were better predictors incontinued on next page correspondence jenswalteruccie nguyen k nguyen and edward c deehan contributed equally to this work1department of agricultural food nutritional science university of albertaedmonton ab t6g 2e1 canada13department of biological sciences university of alberta edmonton abt6g 2e1 canadafull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cnguyen microbiome page of continued from previous pagemlr models as compared to single taxa indicating that arabinoxylan fermentation is the result of multispeciesinteractions within microbiomes this study showed that longchain arabinoxylan modulates both microbiota composition and theoutput of healthrelevant scfas providing information for a more targeted application of this fiber variation inpropionate production was linked to both compositional shifts and baseline composition with pcs derived fromshifts of the global microbial community showing the strongest associations these findings constitute a proofofconcept for the merit of an ecological framework that considers features of the wider gut microbial community forthe prediction of metabolic outcomes of dietary fiber fermentation this provides a basis to personalize the use ofdietary fiber in nutritional application and to stratify human populations by relevant gut microbiota features toaccount for the inconsistent health effects in human intervention studiestrial registration clinicaltrialsgov nct02322112 registered on july keywords arabinoxylan dietary fiber gut microbiota interindividual variability overweight adults shortchainfatty acids epidemiologic studies consistently associate dietary fiberconsumption with a reduced incidence of obesityassociated pathologies [ ] in largescale observationalstudies whole grains and cerealderived fibers eg arabinoxylan and glucan showed stronger associationswith reduced risk of developing cardiovascular diseasetype ii diabetes gastrointestinal cancers and of allcausemortality when compared to other fiber sources [ ] asubstantial body of animal research further consolidatedthe mechanisms by which fiber reduces metabolic pathologies despite these convincing associations findings obtained from human dietary intervention trialsaimed to improve metabolic risk markers by supplementing isolated dietary fibers remain inconsistent possibly due to an individualized clinical response [ ]owing to their chemical structure dietary fibers resistdigestion in the smallintestine and reach the colonwhere they become substrates for the gut microbiotathe microbial fermentation of fiber to shortchain fattyacids scfas has been implicated in the prevention ofobesityassociated pathologies propionate and butyrate are two scfas that are especially relevant as theyhave been linked to beneficial immunological and metabolic effects intervention studies with arabinoxylanisolated from wheat endosperm for instance have demonstrated increased fecal concentrations of both butyrateand propionate dietary fibers can further modulategut microbiota composition in a structuredependentway through the enrichment of bacterial taxa that utilizethe substrate and tolerate or benefit from the environmental changes caused by fiber fermentation [ ] forexample dietary interventions with shortchain fractionsof arabinoxylan resulted in an enriched abundance ofbacterial species that can either utilize arabinoxylan oliaxos directly eg bifidobacteriumgosaccharidesadolescentis and bifidobacterium longum or benefitfrom metabolic byproducts released during axos degradation eg anaerobutyricum hallii and faecalibacterium prausnitzii although fiberinduced alterationsto the gut microbiota are significant the effects are alsohighly individualized and this variability might haveclinical ramifications that could explain the individualized clinical responses to understand the individualized response of the gutmicrobiota to dietary fiber an ecological perspective isrequired as fiber fermentation is determined by complexinterspecies interactions between members of the gutmicrobiota the process is often based on a crossfeeding cascade where primary degraders that access thefiber provide breakdown products oligosaccharides disaccharides and monosaccharides to other microbesand metabolites that result from the fermentation ofthese products also serve as substrates interindividual variation in gut microbiota composition mayresult from the absence of keystone species that initiatethe degradation of recalcitrant fibers differences inunrelated species with similar ecological functions thatcompete for the same substrate or variation instrains of the same species that differ in their capacity tometabolize the substrate these compositional variations likely determine both the competitive and cooperative relationships between community membersthat form trophic networks some of which anize intoecological guilds that collaborate to degrade complexfibers although interindividual variation in the response of the gut microbiota to fiber can influence metabolite outputs relevant to health ie propionate orbutyrate this topic and the underlying ecologicalprinciples have received little attentionthe objective of this study was to apply an ecologicalframework to characterize the compositional and metabolic responses of the human gut microbiota to a longchain arabinoxylan isolated from corn bran compared to 0cnguyen microbiome page of a fiber that is not fermented by the gut microbiotamicrocrystalline cellulose mcc we further assessedwhether nutritional and microbiotarelated factors couldexplainamongindividualsresponsesobservedthevariableresultssubject characteristics and protocol adherenceto compare the effects of arabinoxylan and mcc weconducted a 6week parallel twoarm exploratory randomized controlled trial in individuals with overweightand classi obesity where females received gday andmales gday of either fiber fig of the subjectsenrolled and randomized to an intervention arm sevenwithdrew from the study in the arabinoxylan groupthree experienced challenges consuming the supplementand one reported constipation in the mcc group twowithdrew due to personal reasons and one due to constipation and weretherefore excluded from analysesadditional file fig s1 subjects that completed thestudy protocol n included females and males aged ± years with a body mass indexbmi of ± kgm2 no differences in age sex orbmi were detected between the intervention groups atbaseline additional file table s1 overall protocoladherence assessed by the amount weight of returnedsupplement was ± and ± in the arabinoxylan and mcc arms respectivelyeffect on the composition of the fecal microbiotafecal microbiota diversitynonmetric multidimensional scaling analysis of euclidean distances between subjects based on centered logratio clrtransformed operationaltaxonomic unitotu data showed that the two treatment groups harbored bacterialcould not becommunitiesthatdifferentiated at baseline p permutational multivariate analysis of variance [permanova] fig 2aoneweek supplementation with arabinoxylan alteredthe global fecal bacterial community which became significantly different from the fecal microbiota of subjectsreceiving mcc p this effect was maintaineduntil the end of the fiber intervention p thesechanges occurred by arabinoxylan inducing temporalshifts in fecal microbiota composition determined as theaverage diversity between the individuals treatmentand baseline samples which were significantly largerwhen compared to the mcc group p mannwhitney test fig 2b in addition while mcc increasedinterindividual differences diversity between subjectsp generalized estimated equation [gee] modelarabinoxylan reduced it p fig 2canalysis of αdiversity showed that arabinoxylan reduced fecal bacterial diversity shannons index p gee model fig 2d but not richness total otusafter weeks of supplementation overall these findingsshowed that while the nonfermentable mcc had no detectable effects on measures of bacterial diversity arabinoxylan altered the global bacterial community within week inducing temporal shifts in composition and a reduction of both interindividual variation and αdiversityeffect on the relative abundance of bacterial taxa and coabundance response groupsneither arabinoxylan nor mcc altered microbiota composition at the phylum level at lower taxonomic levelschanges in the relative abundance of two bacterial families were detected at weeks of arabinoxylan relative tobaseline and mcc namely an increase in bifidobacteriaceae q wilcoxon test fig 2e additional file table s2 and a decrease in erysipelotrichaceae q fig study design shaded study week blocks indicate a scheduled clinic visit the x indicates the task was completed during the study weekcdhq ii canadian diet history questionnaire ii stool characteristics selfreported stool consistency and bowel movement frequency 0cnguyen microbiome page of fig arabinoxylan alters the global composition of fecal bacterial communities and induces distinct shifts in taxa a nonmetricmultidimensional scaling nmds plot based on euclidean distance metrics of arabinoxylan and microcrystalline cellulose groups at each timepoint intersubject diversity showing changes in the distance between subjects over time euclidean distances b between fecal microbiotas ofsubjects at each study time point intersubject and c between each subjects fecal microbiota at baseline and during w1 and w6 of treatmentintrasubject d αdiversity displayed as shannon index and total otus of the fecal microbiotas of subjects at each time point e absolutechange δw6bl in relative abundance of bacterial taxa affected by the dietary intervention data analyzed using permanova for a gee modelswith bonferroni correction for b and d and mannwhitney tests for c for e data were analyzed using either wilcoxon tests to assess withingroup changes relative to baseline or mannwhitney tests to assess betweengroup changes ie ax vs mcc with fdr correction diversityand compositional data were reported as mean ± sd and centered logratio transformed prior to the statistical analyses bl baseline otuoperational taxonomic unit w1 week w6 week at the genus level arabinoxylan increased thegenera bifidobacterium and prevotella when comparedto both baseline and mcc and enriched blautia whencompared to mcc otu level analysis revealed that otus changed during arabinoxylan treatment relative tobaseline henceforth referred to as significant otusin particular otus related to bifidobacterium longumotu4 prevotella copri otu6 bacteroides plebeiusotu53 bacteroides sp otu56 bacteroides ovatussuccinatutensotu26otu38 blautia obeum otu85 subdoligranulum spotu11 clostridium leptum otu46 mollicutesotu32 and muribaculaceae otu79q phascolarctobacteriumbecame enriched while otus related to ruminococcusbromii otu5 eubacterium oxidoreducens otu41bacteroides uniformis otu7 and faecalibacillus sppotu21 declined in relative abundance supplementation with mcc only increased the family lachnospiraceaegenus parasutterella q numerically the dominant compositional effects of arabinoxylan were to a large degree specific to b longumotu4 and p copri otu6 as these taxa increased inrelative abundance by an average of 46fold and 4fold while other otus increased by and thein an attempt to identify groups of cooperating species that could function as ecological guilds in the 0cnguyen microbiome page of degradation of arabinoxylan we adapted a clustering approach conceptually similar to that described by tong instead of absolute proportions of taxa weused arabinoxylaninduced shifts to identify clusters ofspecies whose responses were intercorrelated this analysis revealed a total of seven coabundance responsegroups cargs fig 3a five of which showed statistically significant responses to arabinoxylan while noneresponded to mcc additional file table s2 thecarg that showed the largest increase in relative abundance was carg1 p wilcoxon test whichconsisted of six out of the eleven otus that increasedthrough arabinoxylan fig 3b among those six otusbshift andshowed significant connections to all but one member ofcarg1 rs q spearmans correlations usingpermutation tests suggesting arabinoxylan may be degraded through cooperative interactions between theselongum otu4 exhibited the largesttaxa in carg6 p copri otu6 exhibited the largestresponse but only showed one strong connection withanother member of the carg bacteroides massiliensisotu98 rs q which suggests that pcopri might act to a larger degree independently to degrade arabinoxylan fig 3b the majority of taxa thatdecreased during arabinoxylan consumption particularlyb uniformisotu7 clustered within carg7 andshowed negative correlations with taxa of carg1carg2 and carg6 suggesting competitive or antagonistic interactionstemporal responses of otus and cargsto determine if short and longterm treatment witharabinoxylan and mcc differed in their effects on thefecal microbiota we compared shifts from baseline toweek w1 with those from baseline to week w6however there were no detectable differences betweenfig identification of coabundance response groups cargs during arabinoxylan supplementation a heatmap shows the change δw6bl inrelative abundance of otus affected by arabinoxylan p wilcoxon test the hierarchical dendrogram shows clustering of centered logratio clr transformed otus rows based on spearmans correlation distances by the completelinkage clustering algorithm and then groupedon the dendrogram into seven cargs by permanova p subjects columns clustered based on euclidean distances colors from blue tored indicate the direction and magnitude of change b coresponse network analysis each node represents an otu where the size isproportional to the change δw6bl in relative abundance the shape indicates direction of change positive circle negative square and thecolor references the respective carg to which it was clustered lines between nodes represent significant positive red line or negative blueline spearmans correlations rs values ¥ or and q values bl baseline otu operational taxonomic unit w6 week 0cnguyen microbiome page of the two time frames q wilcoxon test data notshown in addition comparison of baseline w1 andw6 values by friedmans test indicated that the effectsof arabinoxylan occur rapidly within week with nofurther detectable changes at weeks fig 4a considering these findings analyses on compositional changeswere performed with w6 data unless otherwise statedinterindividual variation in responses to arabinoxylanbacterial shifts in response to arabinoxylan and theirmagnitude were highly individualized fig 4b for instance absolute increases in relative abundance rangingfrom to to 429fold change were detected inseven subjects for the otu classified as blongumotu4 while other subjects showed either a muchsmaller increase a decrease or the species was undetectable otus related to b obeum otu85 subdoligranulum sp otu11 b ovatus otu26 and c leptumotu46 were enriched by arabinoxylan in around twothirds of the subjects less frequently enriched wereotus classified as potu6 b plebeiusotu53 and bacteroides sp otu56 p copri otu6responded in only four subjects but effects were largewith the species expanding beyond to 7foldchange ofthe total bacterial community in threesubjectscoprito determine drivers of these individualized responseswe used multiple linear regression mlr analyses totest if responses in otus that showed numerically thelargest shifts p copri b longum b obeum and subdolifig temporal and individualized responses of the otus and cargs affected by arabinoxylan and microcrystalline cellulose a plots show thetemporal response of the ten most abundant otus detected in of subjects and the seven cargs centered logratio transformed datawere analyzed by friedmans test with dunns correction to assess withingroup changes between time points ie δw1bl and δw6w1 bbubble plot shows individualized differences δw6bl in relative proportions of the ten most abundant otus percentage of total microbiotacomposition and cargs sum of otus detected after weeks of arabinoxylan and microcrystalline cellulose supplementation the size of thebubble is proportional to the change in abundance relative to baseline while the color of the bubble represents the direction of the change redincrease black decrease the x indicates that the otu was either undetected or the change was relative abundance bl baseline cargcoabundance response group otu operational taxonomic unit w1 week w6 week 0cnguyen microbiome page of granulum sp and in cargs with significant responsescargs and could be predicted by baselinediet or microbiota composition baseline microbiota allotus and significant otus and diet variables werefirst reduced in their dimensionality by principal component analysis pca and then treated as predictors thisanalysis revealed that individualized responses of bacterial taxa and cargs to arabinoxylan and mcc could notbe predicted by baseline diet or microbiota compositionq additional file fig s2effect on stool characteristics and bowel movementswhile fecal moisture content was not changed by eitherfiber q wilcoxon test additional file table s3subjects consuming arabinoxylan reported softer stoolconsistencies when compared to subjects consumingmcc treatment effect p gee model additional file fig s3a both arabinoxylan and mcc ledto an increase in bowel movements relative to baselinep gee model additional file fig s3b withno difference detected between treatment groups treatment effect p effect on fecal ph and scfasfecal ph and scfa concentrations did not change after weeks of either fiber treatment q wilcoxon testadditional file table s3 considering that absoluteconcentrations of fecal scfas are affected by their absorption in the gut we additionally assessedchanges in the percentages of acetate propionate andbutyrate relative to total scfa concentrations at w6which has been previously shown to vary little across colonic regions this analysis revealed an increase inthe percentage of propionate produced through arabinoxylan when compared to mcc q mannwhitney test and a reduction in the percentage ofbutyrate relative to baseline q wilcoxon test although differences in butyrate were not detected whencompared to mcc q further investigation ofthe ratio between propionate and butyrate showed an increase in propionate relative to butyrate when comparedto baseline q and mcc q suggestingthat arabinoxylan supplementation directed the outputof scfas in favor of propionatecharacterization of the temporal response in the threeprimary scfas also showed an increase in fecal propionate concentrations by arabinoxylan at w1 p friedmans test fig 5a although propionate concentrations remained elevated at w6 this increase was notstatistically significant when compared to baseline p this loss of significance was caused by an increasein the interindividual variation at w6 fig 5b visualevaluation of the individualized temporal response ofpropionate to arabinoxylan revealed clear separation offig temporal and individualized output of fecal scfas in response to arabinoxylan and microcrystalline cellulose supplementation a line plotsshow the temporal response of acetate propionate and butyrate reported as mean ± sd b individualized temporal propionate response of w6responders red and w6nonresponder black grouped based on δw6w1 data analyzed for a and b using friedman test with dunnscorrection to assess withingroup changes between time points and for b using mannwhitney tests to assess differences betweengroup ateach time point bl baseline carg coabundance response group otu operational taxonomic unit scfa shortchain fatty acid w1 week w6week 0cnguyen microbiome page of subjects into two distinct patterns fig 5b based on thedirection of change from w1 to w6 ie positive ornegative subjects were grouped into w6respondersδ w6w1 and w6nonresponders δ w6w1 in general w6responders showed a higher outputof propionate at w6 p friedmans test butnot at w1 while the opposite isseen in w6nonresponders p the two groups differed bypropionate concentrations at w6 p mannwhitney testw6propionate responders and nonresponders differ intheir microbiota response to arabinoxylanmicrobiota compositionalbaseline and shifts anddiet data were ordinated using pca and then differencesandbetween w6propionaterespondersnonresponders were tested using permanova thisthe bacterial communities ofanalysis revealed thatw6responders wereindistinguishablefrom w6nonresponders at baseline but differed in their response to arabinoxylan δw6baseline fig thiswas detected if analysis was based on all otus p the significant dietresponsive otus p or the seven cargs p in contrastneither baseline microbiota composition fig nordietary factors additional file fig s4a separatedaccording to w6 response p in addition comparing w6respondersinterms of their baseline total grain whole grain andtotal fiber consumption or their stool consistency andbowel movement frequency during treatment did notreveal any differences either p mannwhitneyand w6nonrespondersfig the individualized temporal propionate response to arabinoxylan associates with compositional responses in the fecal microbiota principalcomponent analysis plots based on euclidean distance comparing the relative abundance of fecal microbiota both at baseline and arabinoxylaninduced shifts δw6baseline between w6responders red and w6nonresponders black microbiota variables ie otu or carg thatcontributed the most to intersubject variation were shown as vectors on the plot when statistical significances were determined by permanovap carg coabundance response group otu operational taxonomic unit w1 week w6 week 0cnguyen microbiome page of test additional files and fig s3c and fig s4btogether these findings indicate that the temporal response in fecal propionate concentrations is primarilyassociated with the shifts in the microbiota and notbaseline microbiota composition or dietindividualized scfa responses can be predicted by gutmicrobiota featuresas with compositional responses gut microbiota functional responses to fiber interventions have been shownto be individualized [ ] but what drives this variation is poorly understood we applied mlr to determine whether fecal scfa responses could be explainedby stool consistency and bowel movement frequencydiet or microbiotarelated factors and then comparedthe quality ofthe models using corrected akaikeinformation criterion aicc values where lower valuesmean higher quality these analyses revealed that thew6 scfa response to arabinoxylan could be predictedby the fecal microbiota fig additional file fig s5but not by baseline diet stool consistency or bowelmovement frequency reported during treatment additional file fig s6a and fig s6b the best modelswere achieved for propionate especially when principalcomponents pcs generated from w6 shifts of all otuswere used as predictors fig 7a additional file tables4 models were of lower quality when w6 shifts of significant otus cargs pcs of cargs or single otuswere used suggesting that global community measuresexhibited stronger linear relationships with the propionate response than single or groups of taxa although themodels that used baseline and w1 shifts of otus asfig individualized arabinoxylaninduced propionate responses could be explained by baseline gut microbiota composition and microbiotashifts a heatmap shows the linear associations between the individualized propionate response δw6bl dependent variable columns andmicrobiota profiles bl δw1bl δw6bl predictors rows cells represent individual multiple linear regression models with fdr correction thatassess whether the predictors explain the individualized propionate response multivariate microbiota data were simplified into principalcomponent pc variables pc1 pc2 and pc3 prior to analysis each model contained the best one or two predictors of pcs individual cargs orsignificant otus selected by stepwise regression all models were adjusted by fiber dosesex colors from white to red indicate relative aicchighest aicc value x lower aicc values red indicate higher quality models b scattercorrected akaike information criterion values calculated byplots show the linear relationship between propionate responses δw6bl and either the baseline contribution of all otus to pc1 or the shiftsof carg1 color and size of each point indicate propionate response magnitude and the shaded area specifies the confident interval thetop six otus that contributed the most to either pc1 of all otus or carg1 are further provided ax arabinoxylan bl baseline carg coabundance response group mcc microcrystalline cellulose otu operational taxonomic unit w1 week w6 week aicc value 0cnguyen microbiome page of predictors were of lower quality than those based on w6shifts they are still valid showing q values less than after benjaminihochbergs false discovery rate fdrcorrection linear relationships between propionate responses and significant predictors using baseline pc1 ofall otus and shifts carg1 were further visualizedusing scatter plots fig 7b reaffirming the quality ofthe analysis as a majority of subjects fall within the confidence regionssignificant models could also be designed for acetateand butyrate responses to arabinoxylan additional file fig s5 interestingly in contrast to propionate the bestmodels to predict butyrate responses were achieved usingshifts of a single otu e oxidoreducens otu41 aknown butyrate producer however overallthemodels for acetate and butyrate were of much lower quality than those for propionate in summary while individualized responses in scfas showed no association withdiet they could be predicted by microbiota shifts andbaseline composition in contrast to the analysis of the effects of arabinoxylan not one single mlr model wasfound to be significant for mcc indicating that the statistical approach based on mlr models did not detect anyassociations independent of fiber fermentationdetermining the role of bacterial taxa in propionateresponsemlr analyses were applied to determine connectionsbetween arabinoxylan responding otus within cargs and and fecal propionate concentrations fig 8athis analysis revealed that shifts in p copri otu6 didnot predict propionate responses while blongumotu4 and correlated taxa in carg1 showed strongerlinear relationships the highest quality models were obtained with b obeum otu85 b plebeius otu53and p succinatutens otu38 all of which encodemetabolic pathways for propionate production such analysis provides a potential explanation for themetabolic interactions between proposed primary degraders secondary fermenters and metabolite utilizersthat result in the promotion of propionate in responseto arabinoxylan fig 8bdiscussionin the present study we characterized the impact of a6week highdose corn bran arabinoxylan supplementation on the composition and function of the fecalbacterial community in healthy adults with overweightand classi obesity arabinoxylan treatment changedfig relationship between propionate responses to arabinoxylan and proposed primary degraders secondary fermenters and metaboliteutilizers a individual multiple linear regression models determine otu responses δw6bl that predict the fecal propionate response δw6blyaxis shows the coefficient for each predictor as in the average propionate response when otu relative abundance increases xaxis showsthe p value for each predictor all models were adjusted by fiber dosesex where bubble size represents the adjustedr2 b proposed model ofbacterial crossfeeding in the gut during degradation of complex soluble arabinoxylans otu operational taxonomic unit 0cnguyen microbiome page of showing two distinctcommunity structure and induced specific shifts inthe composition of the gut microbiota that manifestedthemselves after week of treatment without furtherchanges at w6 arabinoxylan induced increases inpropionate output both compositional and functionalresponses were highly individualized with propionateresponsestemporal patternscompositional responses to arabinoxylan could not bepredicted and functional responses were independentof stool consistency bowel movement frequency andbaseline diet however baseline microbiota composition and especially the compositional shifts correlatedwith propionateresponses the nonfermentablemcc showed virtually no effect on gut microbiotacomposition or functionan understanding of compositional and functional responses of the gut microbiota to changes in diet requiresan ecological framework arabinoxylan supplementation provides resources that can be used by microbesthat possess the traits to either access the chemicalstructures directly or utilize public goods released duringarabinoxylan degradation in our study the dominant effects of arabinoxylan were directed toward twobacterial species b longum and p copri while nine additional otus showed smaller increases including threebacteroides species eg b ovatus b plebeius and bacteroides sp this high degree of specificity toward blongum over other bifidobacterium species is in agreement with other studies testing longchain arabinoxylans[] and genomic analyses that showed that genesencoding arabinoxylandegrading glycosidase eg xylosidase and αarabinofuranosidase are conservedonly among b longum strains [ ] in contrast to thespeciesspecific enrichment of b longum arabinoxylanenriched several species within the phylum bacteroidetesthat possess the genetic and functional traits necessaryfor accessing arabinoxylan [] although arabinoxylan utilization is not universally conse | Colon_Cancer |
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 |
" heat shock transcription factor1 hsf1 was overexpressed to promote glutaminolysis and activatemtor in colorectal cancer crc here we investigated the mechanism for cancerspecific overexpression of hsf1methods hsf1 expression was analyzed by chromatin immunoprecipitation qrtpcr immunohistochemistrystaining and immunoblotting hsf1 translation was explored by polysome profiling and nascent protein analysisbiotin pulldown and m6a rna immunoprecipitation were applied to investigate rnarna interaction and m6amodification the relevance of hsf1 to crc was analyzed in apcmin and apcmin hsf1miceresults hsf1 expression and activity were reduced after the inhibition of wntcatenin signaling by pyrvinium orcatenin knockdown but elevated upon its activation by lithium chloride licl or catenin overexpression thereare much less upregulated genes in hsf1ko mef treated with licl when compared with licltreated wt mefhsf1 protein expression was positively correlated with catenin expression in cell lines and primary tissues aftercatenin depletion hsf1 mrna translation was impaired accompanied by the reduction of its m6a modificationand the upregulation of mir4553p which can interact with ²utr of hsf1 mrna to repress its translationinterestingly inhibition of mir4553p rescued catenin depletioninduced reduction of hsf1 m6a modificationand mettl3 interaction both the size and number of tumors were significantly reduced in apcmin mice whenhsf1 was genetically knockedout or chemically inhibiteds catenin suppresses mir4553p generation to stimulate m6a modification and subsequenttranslation of hsf1 mrna hsf1 is important for catenin to promote crc development targeting hsf1 could bea potential strategy for the intervention of catenindriven cancerskeywords colorectal cancer catenin hsf1 translation mir4553p m6a rna modification correspondence wangx118zjueducn jinhczjueducn1department of medical oncology cancer institute of zhejiang university sirrun run shaw hospital school of medicine zhejiang university hangzhouchina2labortary of cancer biology key lab of biotherapy in zhejiang sir run runshaw hospital school of medicine zhejiang university hangzhou 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 0csong molecular cancer page of introductioncolorectal cancer crc is the third most common cancer with high mortality rate globally the accumulation of various genetic and epigenetic changes activatesmultiple oncogenic signaling critical for the pathogenesisof crc such as wntcatenin signaling pathway its activation will eventually initiate a transcriptiondependent oncogenic process to promote cell cycle progression and apoptosis resistance while the mechanismfor activated wntcatenin signaling to promote crcdevelopment has been wellexplored no therapeuticstargeting this pathway has been successfully developedin addition to proliferation activation and apoptosisresistance metabolism reprogramming is one of important hallmarks of cancer cells for example cancercells favor glycolysis instead of oxidative phosphorylationfor glucose metabolism even in aerobic conditionswhich was wellknown as warburg effect [ ] pyruvate the last product of glycolysis is converted into lactate rather than acetylcoa acetyl coenzyme a fortca tricarboxylic acid cycle or the krebs cycletherefore targeting enhanced glycolysis has been proposed as novel options in the prevention and treatmentof human cancers including crc however as a metabolism hub tca cycle is important in both energyproduction and biosynthesis therefore it needs to bereplenished by anaplerotic reactions such as glutaminolysis previously we reported that heat shock transcription factor hsf1 stimulated glutaminolysis toactivate mtor and promote crc development by upregulating the expression of glutaminase gls1 thecritical enzyme in glutaminolysis hsf1 expressionwas increased in crc and had a positive correlationwith shorter diseasefree survival dfs however theupstream mechanism for hsf1 overexpression in crcwas still uncleargene expression can be controlled by multiple processesincluding transcription mrna degradation translationand protein degradation while gene translation and protein degradation have been extensively investigated moreand more studies focused on mrna translation by exploring the effect of noncoding rnas such as micrornasmirnas and new modifications of mrna including n6methyladenine m6a modification [ ] mirnas canform a mirnainduced silencing complex mirisc toposttranscriptionally regulate gene expression by inhibiting capdependent initiation and stimulating mrna deadenylation [ ] on the other hand as one of the mostabundant modifications in mrna m6a modification ofmrnas usually promotes translation by recruiting initiation factors such as eif3 to the ² end of the mrna while mirnas and mrna m6a modifications play a distinct role in mrna translation the interplays betweenthem were not clarifiedin this study we found that activated wntcateninsignaling stimulated hsf1 translation to promote crcdevelopment byrepressing hsf1 mrnatargetingmir4553p to increase m6a modification of hsf1mrna therefore targeting hsf1 translation could be anew strategy for the intervention of crc and other cancers driven by activated wntcatenin signalingmaterials and methodscell antibodies and chemicalshuman crc celllines sw480 sw620 dld1 rkowere obtained from the american type culture collection atcc all cells were routinely cultured in rpmi invitrogen or dmem invitrogen supplemented with fetal bovine serumall cells were incubated at °c with co2 and humidity the following antibodies were used for western blotting and ihc hsf1 12972s cell signalingtechnology cst ab52757 abcam catenin 8480scst actin l cst flag f1804 sigmacyclind1 ab134175 abcam cleaved parp1 9541scst mettl3 a8370 abclonal gls1 ap8809babgent pyrvinium p0027 licl cycloheximide r750107 chloroquine c6628 mg132 and pd150606 d5946 were purchased from sigmaaldrichinterfering rna sirnasirna mirna mimicsinhibitors transfectiontargeting cateninsmallmettl3 and micrornas were synthesized by genepharma shanghai china and ribobio guangzhouchina the sequence of these sirnas and mirnaswere listed in additional file table s1 sirnas andmirna mimicsinhibitors were transfected into cellsseeded overnight by lipo2000 invitrogen usa or lipofectamine rnaimax transfection reagentinvitrogenusa according to the manufacturers instructionsluciferase activity assaythe plasmid of catenin reporter was gifted from profximei wu zhejiang university for hsf1 activity assaya fragment containing x hse were synthesized andinserted into the pgl3basic vectors promega corporation usa the plasmid was cotransfected with prlrenilla and catenin sirna by using lipo2000 invitrogen usa or treatment with pyrvinium by xtremegene hp dna transfection reagent roche usa ²utr segment of the hsf1 was cloned by pcr andinserted into the vector pmirreporter promegathe mutation of mir455 binding sites in hsf1 ²utrwas generated by quick sitedirected mutagenesis stratagene usa the resultant plasmidswere cotransfected with prl renilla and mir455 mimicsby usinglipo2000 invitrogen usa h post 0csong molecular cancer page of transfection the luciferase activity was measured by thedualglo luciferase assay system promega corporation usachromatin immunoprecipitation chipchip analysis was conducted with the simplechip¢ enzymatic chromatin ip kit cst usa antibodies usedwere antihsf1 12972s cst tcf7l2c48h11 cstand flag f1804 sigma the primers used for thepcr analysis of precipitated dna were shown inadditional file table s2 for flagchip assay theflagcatenin vector was transiently overexpressed bytransfection after h the enrichment of flagcateninon col27a1 promoter was measured by the chip kitimmunoblotting and immunohistochemistryimmunoblotting and immunohistochemistry ihc assays were performed as previously reported for immunoblotting total proteins were extracted with ripabuffer supplemented with protease inhibitors rocheusa after heating the protein sample to °c for min celllysates were transferred to polyvinylidenefluoride pvdf membranes after the membranes wereblocked in milk primary antibody with gentle agitation overnight at °cfor ihc assay was performed in a tissue array containing cases of colonic tissues primary antibodies usedwere listed above the degree of immunostaining wasassessed by independent pathologists and evaluated byassigning a score of scores were defined as follows no staining faint staining moderate staining and strong staining final scores of and were regardedas low expression whereas scores of and consideredas high expressionapoptosis detectioncell apoptosis was measured by flow cytometry analysisand western blotting for flow cytometry analysis ofapoptosis cells were harvested and resuspended in μl x binding buffer μl fluorescein isothiocyanatefitc annexin v and propidium iodide pi bd biosciences usa were added to the cell suspensionand then incubated for min at room temperatureafter that the samples were attenuated with 400ul xbinding buffer and analyzed by acs caliburflowcytometerpuromycinlabellingto detect the change of nascent hsf1 synthesis x cells were plated in cm dish afterthe giventreatment cells were incubated with biotindcpuromycin nu925bios jena bioscience for hcells were lysed with np40 buffer mm trishclph mm nacl np40 glycerolcontaining 1x protease inhibitor cocktail after adequatecentrifugation the supernatant was incubated with 80ulstreptavidin sepharose beads ge17 sigma byrotating at °c for h to overnight the mixture waswashed by np40 buffer for times and subjected towestern blotting using hsf1 antibodyseparatestranslatingpolysome profilingpolysome profilingor nontranslating mrnas on a sucrose gradient according tothe number of bound ribosomes as previously described in brief cells were grown to confluence before collection cells were incubated with μgml ofcycloheximide for min then cells are lysed by polysome buffer [ mmoll kcl mmoll mgcl2 triton x100 μgml cycloheximide mmoll heparin and uml rnase inhibitor takara 1x cocktail] for min on ice lysates were centrifuged rpm for min and the supernatant was layered onto a to sucrose gradient gradients were then centrifuged at rpm for min at °c and polysomebound fractions were collected using an isco densitygradient fractionation system isco lincoln ne withcontinuous monitoring based on a260nm wavelengththe rna in each fraction was extracted using trizol reagent invitrogen and analyzed by realtime rtpcrbiotin pull down assaybiotin pull down assay was performed as described previously cells were transfected with biotinylatedmir4553p probes for h and resuspended using lysisbuffer mm tris ph mm nacl mmmgcl2 uml superasein mm dtt igepal protease inhibitors lysates were incubated withprepared streptavidin beadsge healthcare yeasttrna sigma was used for blocking lysates at °c for h then washed times with binding and wash buffer mm trishcl ph mm edta m naclfinally the bound rnas were extracted and purified forqpcrrna immunoprecipitation rip assayrip assay was performed by magna riptm rnabinding protein immunoprecipitation kitmilliporeno17 briefly à cells were lysed in μlrip lysis buffer and immunoprecipitated with antibodiesofinterest and protein g magnetic beads at °covernight followed by six times of washes in washingbuffer and protein digestion at °c total rna wasisolated and subjected to rtpcr analysis followingantibodies were used for rip n6methyladenosine synaptic mettl3 a8370 abclonal igg millipore 0csong molecular cancer page of rnasequencing2x106 wt mef and hsf1 ko mef were plated andcultured overnight following day cells were treatedwith mm licl for h cells were collected with trizol reagent the total rna was processed by nebnext®polya mrna magnetic isolation module to enrichmrna and the product rna was used for constructionlibrary via kapa stranded rnaseq library prep kitillumina sequencing libraries denatured by mnaoh to generate singlestranded dna as amplified insitu illumina cbot truseq sr cluster kit v3cboths gd4013001 illumina the ends of the generatedfragments were used to run cycles by the illuminahiseq sequencer all the experimental steps afterthe rna extraction were conducted in kangcheng biotechnology co ltd aksomics shanghai china rnasequencing was performed three timesanimal experimentsanimal care and experiments were conducted in compliance with institutional animal care and use committeeand nih guidelines the c57bl6 j mice and apcminmice were purchased from model animal research center of nanjing university marc nanjing chinahsf1 ko mice reported previously were used to generate apcmin mice hsf1 subsequently groupsof mice wild type apcmin apcmin hsf1 and apcmin treated with knk437 as previously reported were fed with highfat diet kcal fat for monthsthe intestine was dissected flushed with pbs and cutopen longitudinally along the main axis the number oftumors was counted and the sizes oftumors weremeasuredstatisticsall data were expressed as mean ± sd unless specifiedthe students ttest was performed for statistical significance analysis p value was considered as statistically significantresultscatenin activates hsf1 in crcin an effort to explore potential regulations of hsf1 wescreened chemicals generating a gene expression patternsimilar to hsf1 depletion by connective map httpportalsbroadinstitutecmapinterestingly a recently reported inhibitor of wntcateninsignaling pyrvinium had a similar effect on genomewide gene expression as hsf1 depletion fig 1aand additional file figs1ab moreover the expression signature related to wntcatenin signaling was positively correlated with the hsf1 signature fig 1b indicating a potential connection of hsf1 withwntcatenin signaling indeed pyrvinium attenuated[ ]the activity of a luciferase reporter driven by hsf1 binding sites hse heat shock response elements [ ]fig 1c and reduced the expression of wellknown transcriptional targets of hsf1 such as hsp90aa1 hspa4hspb1 and hsph1 fig 1d and additionalfile figs1c chromatin immunoprecipitation chip assayfurther confirmed the reduced interaction of hsf1 withits transcriptional targets fig 1e and additional file figs1d in consistence with pyrvinium knockdown ofcatenin by sirna also decreased hsf1 activity fig1f reduced the expression of hsf1 targets fig 1g andadditional file figs1e and attenuated the interactionof hsf1 with its targets fig 1i and additional file figs1f furthermore hsf1 targets were upregulated bythe potent gsk3 inhibitor licl in colorectal cancer cellline rko which had a low level of catenin expressionfig 1hto explore the biological relevance of hsf1 activationto catenin signaling we profiled gene expression ofwild type mouse embryonic fibroblasts wt mef andhsf1 knockout mef hsf1 ko mef before and afterlicl treatment ncbi geo gse151119 while only genes were upregulated in licl treatedhsf1 komef there were genes significantly upregulated inwt mef after licl treatment fig 1j among them genes displayed a dependence on hsf1 since theirexpression levels failed to be upregulated by licl treatment once hsf1 was depleted fig 1k in fact their expression levels had a high correlation with theexpression of a previously reported hsf1 signature fig1l furthermore genes had a hse heatshock response element hse within their promoter regions fig 1m and additional file meaning that theyare most likely bona fide targets of hsf1 indeed someof them such as tma16 dedd2 hspa9 and kif21a havebeen confirmed as the target of hsf1 by chipseqncbi geo gse57398 fig 1n and additional file figs1g taken together these results indicated that catenin can positively regulate hsf1catenin stimulates hsf1 protein translationto delineate how catenin regulates hsf1 we quantitated protein levels of hsf1 before and after inhibitingcatenin both pyrvinium and catenin depletion reduced the protein level of hsf1 fig 2a and b in contrast overexpression of exogenous catenin increasedhsf1 protein level fig 2c furthermore hsf1 proteinlevel was increased after activating wntcatenin signaling by licl treatment in both rko and mef cellsfig 2d in addition hsf1 expression correlates wellwith catenin expression in primary tissues fig 2e andf p chisquare test all of these data indicatedthat catenin upregulates hsf1 protein expression 0csong molecular cancer page of fig wntcatenin signaling activates hsf1 a chemicals influencing gene expression in a similar manner to hsf1 inhibition were screened byconnective map analysis b the correlation of wntcatenin signaling signature and hsf1 signature was detected by gepia c the effect ofpyrvinium on hsedriven promoter activity was explored by luciferase reporter assay d the effects of pyrvinium on the targets of hsf1 wereanalyzed by rtpcr e binding of hsf1 to the promoters of hsf1 targets in crc cells treated with or without pyrvinium was determined by chipf the luciferase assays of hse before and after catenin knockdown were shown as in c g and h the mrna levels of hsf1 targets with catenin knockdown or licl treatment were analyzed by rtpcr i binding of hsf1 to its targets promoter in crc cells before and after cateninknockdown was analyzed by chip j volcano plot displays differentially regulated genes in dhsf1 compared to wt parental cells with licl reddots indicate significantly regulated genes based on adjusted pvalue and logfold change logfc p log2fc k differential geneexpression analysis in wt and hsf1 mef treated with licl were performed by rnaseq numbers of upregulated genes in two cells wereshown in venn graph l the correlation of putative hsf1dependent genes from k with reported hsf1 signature was detected by gepia mnumbers of putative hsf1dependent genes with or without hse in their promoters were summarized n representative hsf1 chipseqtracks ncbi geo gse57398 for hsecontaining genes are shown asterisks indicate p 0csong molecular cancer page of fig catenin stimulates hsf1 protein translation a the effect of pyrvinium on the protein expression of hsf1 was explored by westernblotting b the effect of catenin knockdown on hsf1 protein level was analyzed by western blotting c the protein level of hsf1 before andafter catenin overexpression was analyzed by western blotting d the effect of licl on hsf1 protein level in rko and mef was analyzed bywestern blotting e the expression of catenin and hsf1 in colorectal tissue was analyzed by immunohistochemistry staining f the correlationbetween catenin expression and hsf1 expression in colorectal tissue was analyzed by chisquare test p g the effect of catenindepletion on hsf1 with puromycin labeling was determined by western blotting h amount of hsf1 mrna in various polysome fractions wasanalyzed by rtpcrp however there were no apparent alterations in hsf1mrna level after catenin knockdown or pyrviniumtreatment additional file figs2a and s2b meanwhile the halflife of hsf1 protein was also not changedbefore and after catenin knockdown additional file figs2c inhibitors of proteasome autophagy and calpains all failed to reverse hsf1 protein downregulationinduced by catenin knockdown additionalfile 0csong molecular cancer page of figs2d all of these results implied that catenin affects hsf1 protein expression mostlikely via translationregulation therefore puromycin labeling assay wasemployed to monitor the synthesis of nascent hsf1 protein as expected the puromycin labeling of hsf1was reduced by catenin depletion fig 2g to furtherconfirm it monopolysome fractions from cytoplasmicextracts of crc cells before and after catenin depletion were collected by sucrose gradient centrifugationthe subsequent rtpcr analysis revealed that catenindepletion considerably reduced the presence of hsf1mrna in the polysome fraction but increased in nontranslating ribosome fractions fig 2h in summary catenin upregulates hsf1 expression by stimulatinghsf1 protein translationhsf1 protein translation is regulated by mir4553pas micrornas mirnas play an important role inregulating the efficiency of protein translation we wondered whether hsf1 protein translation was regulatedby micrornas based on bioinformatics screening bytargetscan mirdb and starbase some micrornas including mir4553p mir2145p mir4315p mir184mir4903p and mir375 were proposed to target ²utr of hsf1 mrna fig 3a after functional validation by western blotting mir4553p and mir2145pbut not other micrornas were capable to suppress theexpression of hsf1 protein in crc cells fig 3b andadditional file figs3a however mir2145p but notmir4553p also reduced hsf1 mrna level additionalfile figs3b whats more an inhibitor of mir4553prather than mir2145p rescued the downregulation ofhsf1 protein by catenin knockdown fig 3c and additional file figs3c indicating that mir4553p mightbe relevant to catenininvolved regulation of hsf1protein translation indeed mir4553p inhibited the activity of luciferase driven by wild type hsf1 mrna ²utr but not its mutant unable to bind mir4553p fig3d the interaction of mir4553p with hsf1 mrnawas further confirmed by biotin pull down assay fig3e based on the analysis of tcga data httpmirtvibmssinicaedutwthe expression of mir4553p islower in colon adenocarcinoma than in normal tissuesadditional file figs3d similarly qpcr analysis revealed lower levels of mir4553p in crc tissues than inadjacent nontumor tissues fig 3f additionally wehad confirmed high expression of hsf1 in the same cohort of human crc tissues previously indeedmir4553p expression was negatively correlated with theexpression of hsf1 fig 3g on the other handmir4553p similar to hsf1 inhibition as we reportedrecently reduced the expression of hsf1 targets induced the viability inhibition and apoptosis activation ofcolorectal cancer cells fig 3hj and additional file figs3eg the seed sequence of micrornas was important for targeting mrna by basepairing indeed the seed sequence mutant of mir4553p could notdownregulate the protein level of hsf1 additional file figs4a confirming the importance of mir4553p totarget hsf1 protein expression in a word mir4553ptargets hsf1 mrna ²utr to inhibit its translationm6a modification of hsf1 mrna stimulates its proteintranslationin addition to microrna mrna modifications such asn6methyladenosine m6a play important roles in theregulation of hsf1 translation interestingly we noticedthat the matching sites of mir4553p seed sequence inhsf1 mrna ²utr contains a typical motif of m6amodification fig 4a which was supported by bioinformatic analysis httpwwwcuilabcnsramp fig 4b andadditional file figs4b moreover we have done themerip sequencing in sw620 and found that the ²utr region of hsf1 has one m6a modification site intriguingly this sequence is completely complementary tothe seed sequence of mir4553p additionalfile figs4c pcr analysis after merip m6a rna immunoprecipitation further confirmed m6a modification ofhsf1 mrna fig 4c whats more the activity of luciferase driven by the mutant hsf1 mrna ²utr whichwas unable to bind mir4553p but retains the m6amodification site sequence drach d a g or u r a or g h a u or c [] was higher than theactivity of luciferase driven by wild type hsf1 mrna²utr fig 3dindicating the importance of m6amodification to hsf1 expression as the main component of the methyltransferase writer complex [ ]mettl3 was also bound to hsf1 mrna fig 4donce its expression was depleted m6a modification ofhsf1 mrna was decreased fig 4e in consistencewith its potential roles in promoting protein translationsuch a reduction of hsf1 mrna m6a modification reduced hsf1 protein expression fig 4f and nascenthsf1 protein synthesis fig 4g furthermore mettl3depletion considerably reduced the presence of hsf1mrna in the polysome fraction but increased in nontranslating ribosome fractions fig 4h while hsf1mrna or the stability of hsf1 protein were not changed additionalfile figs4d and s4e moreoverhsf1 protein was decreased with the knockdown ofythdf1 which was the reader protein of hsf1 m6amodification fig 4i to sum up m6a modification ofhsf1 mrna was relevant to stimulate its translationcatenin suppresses mir4553p to increase hsf1 mrnam6a modificationnext we explored the interplay between mir4553p andm6a modification of hsf1 mrna both hsf1 mrna 0csong molecular cancer page of fig hsf1 protein translation is regulated by mir4553p a overlap of hsf1targeting micrornas predicted by targetscan mirdb and starbaseb the effect of mir4553p on hsf1 protein was analyzed by western blotting c the effect of mir4553p inhibitor on catenin knockdowninduced hsf1 downregulation was determined by western blotting d luciferase activity assay was used to analyze the effect of mir4553p onthe activity of ²utr with or without mir4553p binding sites p e the binding between biotinmir4553p and hsf1 mrna wasdetermined by biotin pull down assay p f expression of mir4553p in pairs of fresh crc tissues and adjacent nontumor tissues wasanalyzed by qpcr g the correlation of hsf1 protein and mir4553p in pairs of fresh crc tissues and adjacent nontumor tissues wasanalyzed h the effect of mir4553p on viability of crc cells was explored by mts assay i the effect of mir4553p on apoptosis of crc cells wasanalyzed using flow cytometry after pi and annexin vfitc double staining j apoptosis of crc cells treated with or without mir4553p wasdetermined by western blottingm6a modification and its binding to mettl3 were decreased by the overexpression of wild type mir4553pbut not its mutant unable to bind to hsf1 mrna ²utr figfigs5aand additional5afileinterestingly mettl3 depletion not only reduced m6amodification of hsf1 mrna fig 4e but also enhanced the interaction of mir4553p with hsf1 mrnafig 5b and additionalfile figs5b while the 0csong molecular cancer page of fig m6a modification of hsf1 mrna stimulates its protein translation a the sites of hsf1 ²utr binding to the seed sequence of mir4553pwas consistent with m6a rna modification elements drach b bioinformatic prediction of m6a modification in ²utr of hsf1 mrna c m6amodification of hsf1 mrna was analyzed by merip p d binding of mettl3 to hsf1 mrna was detected by rip p e m6amodification of hsf1 mrna with or without mettl3 depletion was analyzed by merip p f the protein level of hsf1 before and aftermettl3 depletion was detected by western blotting g the effect of mettl3 knockdown on hsf1 synthesis was determined by western blottingafter puromycin labeling h amount of hsf1 mrna in various polysome fractions was analyzed by rtpcrp i the effect of ythdf1knockdown on hsf1 protein level was analyzed by western blottingexpression of mature and primary mir4553p was notupregulated additional file figs5cd these resultsindicated that mir4553p may compete with mettl3for the m6a modification of hsf1 mrna thus inhibitinghsf1 protein translation furthermore the binding ofmir4553p to hsf1 mrna was not changed by ythdf1deletion additional file figs5e indicating that thetranslation repression of hsf1 mrna was more likely tobe mediated directly by the reduced m6a modification ofhsf1 0csong molecular cancer page of fig catenin suppresses mir4553p to increase hsf1 mrna m6a modification a m6a modification and mettl3 interaction of hsf1 mrnawith wt or mutant of mir4553p were analyzed by rip b the interaction between biotinmir4553p and hsf1 mrna with or without mettl3depletion was analyzed by biotin pull down c the effect of mir4553p inhibitor andor catenin knockdown on m6a modification of hsf1mrna was analyzed by merip d the effect of mir4553p inhibitor andor catenin knockdown on the interaction of mettl3 with hsf1 mrnawas analyzed by rip e the effect of catenin knockdown on interaction of mir4553p and hsf1 mrna was analyzed by biotin pull down f andg the levels of mature f and primary g mir4553p with catenin knockdown or licl treatment were determined by rtpcr h the correlationof col27a1 and mir455 was analyzed in linkedomics httplinkedomics i the effect of catenin depletion or licl on mrna level ofcol27a1 was analyzed by rtpcr j the interaction of catenintcf7l2 and hsf1 promoter was determined by chip k the correlation of catenin protein expression with the rna level of col27a1 was detected by linkedomics httplinkedomicsindeed mir4553pcatenindepletioninduced decrease of hsf1 mrna m6a modification fig 5c and additional file figs5f meanwhilethe interaction of mettl3 with hsf1 mrna wasinhibitorrescuedabrogated by depleting catenin fig 5d and additionalfile figs5g accompanied by the increased interactionof mir4553p to hsf1 mrna fig 5e and upregulationof mature fig 5f and additionalfile figs5h 0csong molecular cancer page of precursor additional file figs5i and primary mir4553p fig 5g and additional file figs5j in contrastwhen catenin was upregulated by overexpression or licltreatment both mature mir4553p fig 5f and additionalfile figs5h and primary mir4553p fig 5g and additional file figs5j were downregulatedprimary mir4553p was derived from a premirna hairpin encoded in intron of the collagen gene col27a1 additional file figs5k actually col27a1 expression was significantly correlated with the expression ofmir455 httplinkedomics fig 5h consistent withthis we observed col27a1 mrna levels were increasedupon catenin depletion while decreased after licl treatment fig 5i and additional file figs5l moreover catenintcf7l2 complex could interact with the promoterof col27a1 while the pair of primers negativechipprimer at a position far away from the promoter region couldnot enrich col27a1 fig 5j and additionalfile figs5m meanwhile the protein expression of cateninwas negatively correlated with rna level of col27a1httplinkedomics fig 5k these results indicatedthat the transcription of col27a1 was inhibited by wntcatenin signalingleading to decreased biogenesis ofmir4553p therefore catenin facilitates the shift frommir4553p binding to m6a modification in hsf1 mrnaby suppressing mir4553p expression eventually promoting hsf1 protein translationboth genetic and chemical inhibition of hsf1 attenuatecolorectal carcinogenesis in micein light of these in vitro findings we further exploredthe relevance of hsf1 to colorectal carcinogenesis inapcmin and apcmin hsf1 mice since the interaction of mouse mir4553p and mouse hsf1 mrnaseems to be well conserved mouse mir4553p seed sequence cagucca the binding site in mouse hsf1mrna ²utr tggactg the expression of hsf1 andits downstream target gls1 were increased whilemir4553p expression was reduced in intestine tissuesfrom apcmin mice compared with normal c57bl6mice fig 6a and b after fed with highfat diet for months these apcmin mice developed multiple tumorsin the intestine fig 6c however both the size andnumber of tumors were significantly reduced in apcminmice treated with a chemical inhibitor of hsf1 knk437and apcmin hsf1 mice fig 6d accompanied bythe downregulation of hsf1 targets fig 6e all of theseresults confirmed that hsf1 is a novel downstream target of wntcatenin signaling important to promotecrc developmentdiscussiongenetic changes in components of wntcatenin signaling such as deletion of the apc gene and ctnnb1mutations have been frequently detected in many typesof human cancers all of these muta | Colon_Cancer |
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